Las Antocianinas

INDICADORES QUMICOS CIDO-BASE I
Introduccin.
En 1664, Boyle escribi The Experimental History of Colours. En ella se inicia el reconocimiento de
cidos1 y bases a travs de los cambios de color de extractos de plantas2. A partir de Boyle, el cambio
de color del jarabe de violetas, sirvi para indicar la presencia de un cido; en este momento nacen los
indicadores qumicos. Sin embargo el primer reconocimiento, no lo fue con motivo de los cambios de
color, ya que 8 aos antes, Glauber haba definido la efervescencia del espritu cido como seal
inequvoca de su existencia3. En 1671, Ducls llama turnesol (litmus), a un indicador extrado de
lquenes, que le da un gran resultado4. Casi cien aos despus, James Watt, el inventor de la mquina
de vapor y nominador del caballo de vapor como unidad de potencia, descubre que la lombarda (col
roja) es uno de los mejores indicadores.
Indicadores qumicos cido-base.
Un indicador qumico es un cido o base dbil cuya forma disociada tiene diferente color que la forma
sin disociar5, ello es debido a que estn formados por sistemas resonantes aromticos, que pueden
modificar la distribucin de carga segn la forma que adopten. Esta alteracin por el desplazamiento
hacia una forma mas o menos disociada, hace que la absorcin energtica del sistema se modifique y
con ello el color.
Se podra establecer un equilibrio de disociacin para una forma de indicador cido HIn:
HIn
X
Color A
In+
Color B
H+
La aplicacin de la ley de accin de masas a este equilibrio, nos da que:
[In ][H ] , de lo que

=
Ka
[HIn ]
[ ]
Ka
In
=
.
H + [HIn ]
[ ]
Si el medio es cido, y aumenta la concentracin de H+, deber disminuir la relacin [In-]/[HIn]. Para
ello el equilibrio tendr que desplazarse hacia la izquierda, aumentando la concentracin de HIn, y
dominando su color. Si el medio es bsico, el cociente tendr que aumentar, desplazndose el
equilibrio hacia la derecha y dominando el color B. Naturalmente como se trata de un equilibrio,
coexisten las dos formas, y por ello el color que toma procede de la mezcla de colores y de su
proporcin. Como los indicadores tienen diferentes constantes de equilibrio, por eso cambian de color
en distintos intervalos de pH, esto suelo ocurrir aproximadamente a pH=pK"1 . Cuando coexisten
varios equilibrios entre formas tautmeras, hay varios pK, y por lo tanto ms de un cambio de color.
1
El concepto de cido segn Partington, ya aparece en el manuscrito indio Rasarnava, 1200 aos A.C. Las bases eran
conocidas como lcalis, debido a que el mas conocido (carbonato potsico) se extraa de las cenizas de la planta Kali (La
primera referencia se da en la obra de Abu Mansur Monafir, siglo X d.C.). Existen referencias ms antiguas, ya que en las
tablas sumerias las cenizas vegetales eran conocidas como Te-Gaz. En el primer diccionario de Qumica publicado por
Macquer en 1766, aparece como definicin de los lcalis: sustancias que vuelven verde el jarabe de violetas.El trmino
base, surge a mediados del siglo XVIII, y se debe al qumico francs Rouelle, ya que eran la base de la formacin de las
sales al combinarse con los cidos.
2
Escribe Robert Boyle: Take good syrup of violeta, impregnated with he tincture of the flowers, drop a little or it upon a
white paper (for by that means the change of colour will be more conspicuous, and the experiment may be practised in
smaller quantities) and on this liquor let fall two o three drops of spirit, either of salt or vinegar or almost any other
eminently acid liquor, and upon the mixture of these you shall find the syrup immediately turned red.
3
Actualmente sera el desprendimiento de dixido de carbono (antes gas silvestre, o gas fijo), cuando acta sobre un
carbonato.
4
El trmino tornasol, se conoca desde Plinio (I d.C.) y Dioscrides, aunque aplicado a determinadas plantas (heliotropo).
Como colorante aparece mencionado en el Art of Drawning, de Peachan, publicado en 1606.El litmus, o littmose,
aparece en uso desde 1518, derivado de lit (color) y mouse(aplicado a determinado tipo de plantas), por lo que vendra a ser
un colorante extrado de plantas, como lo es en realidad.
5
Esta definicin fue propuesta por Ostwald, en 1891, y publicada en 1894, en un contexto mucho mas amplio bajo el
ttulo:Die wissenschaftlichen Grundlagen der analytischen Chemic.
Indicadores qumicos cido-base naturales.

Se deben fundamentalmente a la proporcin que contengan de los pigmentos naturales conocidos
como antocianinas y antoxantinas. La antocianina es roja en medio cido, prpura en medio neutro y
azul en medio bsico, sin embargo la antoxantina es amarilla en medio bsico. La proporcin en que se
encuentre la mezcla de pigmentos hace que las flores tengan distintos colores y que se puedan
modificar segn el pH del medio.
Son glucsidos, con estructura parecida, modificndose la posicin de determinados grupos hidroxilo,
con carcter cido, que segn el medio producen diferentes formas encuadradas en una tautomera ceto-enlica. De su hidrlisis se extraen los pigmentos coloreados, las antocianidinas y antoxantidinas
As la forma ms genrica de las antocianidinas, y su transformacin sera:
Mientras que para las antoxantidinas, sera:
La mayora de los ptalos de las flores

contienen ambos pigmentos, por eso en
medio cido el jarabe de violetas produca
color rojo, mientras que en medio bsico
era verde, combinacin del amarillo y del
azul, tal como se muestra en la simulacin.
Si domina ms la concentracin de
amarillo, ser verde amarillento.
Prcticas de indicadores qumicos cido-base naturales.

1. Con el extracto de violetas.
Dado que histricamente fue el primer indicador, comenzaremos por l.
Se cortan las hojas de violeta con cuidado y se extrae en una termobatidora, en caliente con agua hasta
80C. (25g de ptalos de violetas/ 50g de agua).El extracto toma color violeta plido, casi incoloro
(segn la concentracin).
En el primer modelo de prcticas, observaremos a travs de qumica de la gota de extracto de violetas
en agua, la coloracin que toma un cido fuerte (H2SO4 6N), un cido dbil (actico 1M), una base
dbil (hidrxido amnico 1M) y una base fuerte (NaOH 6N). De esa forma se dispondrn en la caja
Petri 4 gotas de los compuestos citados, rodeando unas gotas de indicador (fig.1). Posteriormente se
pondrn en comunicacin las gotas (fig.2), continuando con las figuras 3 y 4.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Como se observa a pH cidos toma color rojizo, mientras que a pH bsicos el color es verde
amarillento, tal como se deca en los primeros estudios con indicadores naturales.
2. Con extracto de lombarda.

La lombarda se extrae en una termobatidora, en fro con alcohol absoluto (50 g de lombarda troceada/
100ml de etanol absoluto), y en caliente con agua hasta 80C. (250g de lombarda/ 500g de agua).El
extracto alcohlico toma color rojo plido, casi incoloro (segn la concentracin), mientras que el
acuoso lo hace violeta.
En el primer modelo de prcticas, observaremos a travs de qumica de la gota de extracto de lombarda
en agua, la coloracin que toma un cido fuerte (H2SO4 6N), un cido dbil (actico 1M), una base
dbil (hidrxido amnico 1M) y una base fuerte (NaOH 6N). De esa forma se dispondrn en la caja
Petri 4 gotas de los compuestos citados, rodeando unas gotas de indicador (fig.5). Posteriormente se
pondrn en comunicacin las gotas (fig.6).
Fig. 5
Fig. 8
Fig. 6
Fig. 7
En la sucesin de fotos (fig. 5, 6, 7 y 8), se observan los diferentes colores que toma la lombarda segn
el pH de medio. La difusin final separa en dos medios cido fuerte y base fuerte (fig. 8).
En medio alcohlico, debido a la diferente tensin superficial, la gota de indicador se desparrama y es

mucho ms difcil delimitar las fronteras, sin embargo los colores se intensifican. La sucesin de fotos,
se da en las figuras 9, 10, 11 y 12.
Fig. 10
Fig. 9
Fig. 12
Fig. 11
Los cambios de color corresponden a los que toma para los diferentes pH, que se obtienen de la
combinacin de las gotas de los reactivos respectivos, segn la tabla que se da:
Valores de
pH
Lombarda
en agua
Lombarda
en etanol
2
Rojo
Rojo
Rojo violceo
Desde Violeta a azul plido
Azul verdoso
Verde
10
11
Verde
Verde amarillento
12
13
Amarillo
Verde azulado
3. Con extractos de ptalos de ciclamen rojo, similar a los ptalos de rosa roja.
Se extrae con etanol absoluto, en fro, despus de trocear los ptalos. Aproximadamente 25 g de
ptalos con 50 mL de etanol). Se sigue el procedimiento habitual, obtenindose la sucesin de figuras
que se indica a continuacin.
Fig. 14
Fig. 13
Fig. 15
Fig. 16
Como se aprecia en la sucesin de fotos (fig. 13, 14, 15 y 16), existe una diferencia sustancial
frente a la lombarda, y es que en medio fuertemente bsico, el color es marrn oscuro, mientras que en
medio dbilmente bsico aparece violeta plido.
Valores de
pH
Ciclamen/
etanol
2
Rojo
plido
Desde rosa a violeta casi incoloro
10
11
Marrn amarillento
12
13
Marrn verdoso
4. Con extracto de fresas en etanol absoluto.

Siguiendo el procedimiento habitual de unir las gotas (fig.17 y 18), se observa que a pH bajos, toma
color naranja, pero si es algo es amarillo, con tintes violceos. Si los pH son intermedios casi no se
aprecia cambio de color (fig. 19 y 20).
Fig. 17
Fig. 20
Fig. 18
Fig. 19
5. Con tornasol (extracto de lquenes de los gneros Rocella, Variolaria y Lecanora).

Los colores son muy dbiles, en medio cido tiende al rosa, y medio bsico al azul grisceo.
Fig. 21
Fig. 22
Fig. 24
Fig. 23
6. Extracto de amapolas
Fig. 25
Fig. 26
En medio cido resulta incoloro, mientras

que en medio bsico aparece marrn.
Fig. 27
INDICADORES QUMICOS CIDO-BASE II
Indicadores cido-base sintticos

La segunda mitad del siglo XIX, fue el inicio de las grandes sntesis orgnicas, y como no poda ser
menos, tambin los indicadores cido base, que haban sido empleados como productos naturales,
iban a ser sintetizados a partir de 1868.
El primero indicador en ser sintetizado fue la fenolftalena1, conseguida por Baeyer
condensando el anhdrido del cido ftlico (ortobencenodicarboxlico), con fenol, en 1871. De la
fenolftalena salieron otros muchos indicadores, potenciando los cambios de absorcin al introducir
derivados sulfonados y bromados, estudiados por Lubs y Clark a partir de 1915. As aparecieron el
rojo fenol, el azul de timol, la timolftalena, el azul de bromotimol, azul de bromofenol y el cresol
entre otros.
Antes, en 1859, el francs Verguin, haba obtenido la fuchina2, oxidando por casualidad la
anilina con cloruro de estao(IV), que tambin fue obtenida por Hofmann poco despus. Este
compuesto sera el punto de partida de otros indicadores con estructura de trifenilmetano, como el
violeta de metilo, verde de metilo, el verde brillante, el verde malaquita etc, caracterizados por
tonalidades fuertes y brillantes a distintos pH.
Otra ruta de sntesis de indicadores fue de los colorantes azoicos, que dio lugar al
naranja de metilo (propuesto por Lunge en 1878). El segundo indicador cido-base de este tipo en ser
empleado, fue el rojo Congo3, descubierto por Bttiger en 1884. Despus se usaran el rojo de metilo
(introducido por Rupp y Loose en 1908), amarillo de alizarina etc. De estructura algo diferente entre
los colorantes azoicos y el tipo fuchina es el rojo neutro que tambin ser empleado en este trabajo.
Uso de los indicadores cido-base sintticos.

Por lo general se suelen emplear en forma de sales sdicas, por ser solubles en agua. En caso
contrario, se disolveran en etanol, lo cual tiene mas inconvenientes a la hora de usarse en la qumica a
la gota, ya que la gota de alcohol tiende a extenderse y desparramarse contactando antes de tiempo con
los diferentes medios.
Dado que se conocen los distintos pK, en los equilibrios tautomricos entre las formas con
distinto color y como se ha explicado anteriormente, en el tema Indicadores cido-base I, el cambio de
color o viraje se produce aproximadamente entre una unidad menos y otra mas del pK, se pueden
comprobar los distintos cambios de color. Muchas veces el color esperado no es el que aparece, pues
dado que se trata de formas en equilibrio, la combinacin de colores produce el que se aprecia.
1
El origen del nombre de la fenolftalena, parece sencillo si nos remontamos slo a su sentido qumico, sin embargo desde
el punto de vista remoto, el fenol deriva del griego phaino (nV4<T), con el significado de yo alumbro, hacindolo derivar
del benzol (C6H6),que haba sido descubierto por Faraday como residuo del gas del alumbrado de Londres, y la ftalena,
procede del trmino nafta, cuyo origen es muy remoto. Se podra considerar derivado del egipcio Na-Ptah, por que era
empleado en el culto del dios egipcio del fuego, Ptah (Ftha), equivalente al Vulcano latino, ya que era un lquido negruzco
trado de Persia, que arda muy bien (petrleo). De l derivarn la naftalina, obtenida en 1820, por Garden como residuo de
la destilacin del alquitrn de hulla, el naftaleno etc.
2
Su origen procede del de la planta de color rojo, fucsia, nombrada as por el francs Plumier, en honor del botnico
Leonhard Fuchs, que la descubri y que coincidir con el trmino alemn fuchs ( zorra), en francs, renard, nombre a su
vez de la casa comercial Renard, que fabric por primera vez la fuchina o fucsina en Lyon, en 1860.
Nombre impuesto por el marqueting de la casa alemana AGFA, que lo comercializ en 1888. Se eligi, debido a la
fascinacin que ejercan los trminos africanos en la sociedad berlinesa, en la incipiente colonizacin de aquel continente.
El trmino Congo es un hidrnimo de origen incierto, posiblemente portugus con alteraciones locales, ya que fue
descubierto por el navegante portugus Diogo Cam. No tiene lgica emparentarlo con el bant kong (montaa).
La tabla de indicadores sintticos empleados y sus cambios de color en funcin del pH, es la dada,
sealndose las regiones del viraje:
Problemas en su uso:
Los cambios de color que se dan anteriormente, pueden experimentar numerosas variaciones,
especialmente en la fotografa a la gota, que necesita de focos de luz, para poder captar desde muy
cerca las variaciones de color. Estos focos producen un cierto calentamiento del sistema, que alteran el
producto inico del agua4. De forma que al aumentar la temperatura, se desplaza hacia el lado alcalino
la coloracin cida del indicador sensible a las bases, por lo que un cambio de color tendr lugar a una
concentracin de OH- mayor que a la temperatura normal. Con indicadores sensibles a los cidos, esta
desviacin de la regin del viraje, se produce hacia el lado cido. De esta forma el anaranjado de
metilo que tiene una regin de viraje entre pH 3,1 y 4,4 a 18C, pasa a ser entre 2,5 y 3,7, si la
temperatura aumenta hasta los 80C.
Muchos de los colores indicados en la tabla en la regin descrita, se producen por mezcla de otros
colores, como por ejemplo en el caso del azul de bromotimol, cuyos cambios de color se deben a las
absorciones de la luz de las siguientes formas en equilibrio
El producto inico del agua es 10-14, a 25C, lo que produce un pKw=14. Si la temperatura disminuye, el pKw aumenta,
hasta 14,5 a 15C, y si la temperatura aumenta, el pKw disminuye hasta 13,5 a 40C, que son los mrgenes de temperatura
entre los que oscila las experiencias presentadas.
La aparicin del color verde en el medio de la regin, se debe a la combinacin de los colores azul y
amarillo de las formas en equilibrio entre pH 6 y 8. Lo mismo ocurre con las combinaciones azul y
rojo que produce una tonalidad violcea, a pH elevado.
En otros indicadores, como el anaranjado de metilo o naranja de metilo (del tipo diazoico), dado que el
cambio de color es menos radical (rojo-amarillo), no se producir este fenmeno.
Prctica con indicadores sintticos.

1. Fucsina o fuchina.
El mecanismo empleado es mismo descrito en Indicadores cido-base 1. O sea se disponen en una caja
Petri cuatro gotas, de cido actico 1N, cido sulfrico 6N, hidrxido amnico 1N e hidrxido sdico
6N, aproximadamente en los vrtices de un cuadrado, y en su centro la gota de indicador (fig.1).
Cuando ste est disuelto en alcohol, deber dejarse mas sitio dado que la gota por su menor tensin
superficial tiende a extenderse. Se unen las gotas (fig.2), y se observa la difusin de los diferentes
medios a travs del indicador (fig. 3 y 4)
Fig. 1
Fig. 3
Fig. 2
Fig. 4
2. Violeta de metilo.
Se sigue la misma tcnica anterior obtenindose la sucesin de fotos de las figuras 5 a 8.
Fig. 5
Fig. 7
Fig. 6
Fig. 8
El violeta de metilo destaca por los colores brillantes que toma, cambiando de color varias
veces en el intervalo de pH 0-14. Sin embargo estos cambios son una demostracin clara de la mezcla
de colores por ejemplo el verde por combinacin de azul y amarillo.
3. Verde de metilo.
Fig. 9
Fig. 12
Fig. 10
Fig. 11
4. Anaranjado de metilo.
Fig. 13
Fig. 16
Fig. 14
Fig. 15
5. Rojo de metilo.
Fig. 17
Fig. 20
Fig. 18
Fig. 19
6. Rojo Congo.
Fig.21
Fig. 24
Fig. 22
Fig. 23
7. Rojo neutro.
Fig. 25
Fig. 26
Fig. 27
8. Fenolftalena
Fig. 28
Fig. 31
Fig. 29
Fig. 30
8. Verde brillante.
Fig. 33
Fig. 32
Fig. 34
9.Azul de bromotimol
Fig. 36
Fig. 35
Fig. 37
Fig. 38
Obsrvese la aparicin del color verde en la fig 38

Combinacin del amarillo y azul , y del malva en
La 39, por combinacin de azul y rojo
Fig. 39
CESAR MILSTEIN, PREMIO NOBEL DE MEDICINA 1984

Autor: Dr. Csar Lorenzano
Profesor Titular de la Universidad de Buenos Aires
Coordinador de la Ctedra de Metodologa de la Investigacin
Director de la Maestra y Doctorado en Epistemologa e Historia de la Ciencia
de la Universidad Nacional de Tres de Febrero
Una versin de este artculo fue publicado en Mdico Interamericano,
publicacin oficial del Colegio Interamericano de Mdicos y Cirujanos con sede en
Nueva York, en un nmero monogrfico dedicado a cientficos notables de habla
hispana, en diciembre de 2000.
Ante la prdida irreparable de Csar Milstein, la Ctedra de Metodologa de la
Investigacin de la Facultad de Medicina de la Universidad de Buenos Aires, donde
obtiene su doctorado en 1957, rinde homenaje a su memoria dndolo a conocer a la
comunidad universitaria, en primer lugar, y todos aquellos que quieran saber quin fue
Csar Milstein, cmo vivi, y cules fueron sus aportes principales a la ciencia. Nos
deja el recuerdo de su sabidura, de su tesn como investigador, su integridad como
hombre y ciudadano del mundo.
Incluimos asimismo una copia de la carta que el Dr. Milstein me hizo llegar
comentando el articulo.
Introduccin
El Dr. Csar Milstein obtiene en 1984 el Premio Nobel de Medicina, junto con
su cercano colaborador, el Dr. George J. F. Khler, por el descubrimiento del principio
que rige la produccin de anticuerpos monoclonales, y con el Dr. Niels Jerne, autor de
al menos tres teoras fundamentales en inmunologa, que conciernen a la especificidad
en el desarrollo y el control del sistema inmune.1
Un currculum abreviado que muestre los principales hitos de su carrera hasta la
fecha en que realiza el descubrimiento que le vale el Premio Nobel, nos dice que:
i.
nace en Baha Blanca, Argentina, el 8 de agosto de 1927;
Los prrafos entrecomillados corresponden a la presentacin de los Premios que hace la Fundacin
Nobel. El Dr. Khler nace en la Repblica Federal de Alemania en 1946, y llega a trabajar en el
laboratorio de Milstein desde el Instituto de Inmunologa de Basel, en Suiza, muriendo tempranamente en
1995. El Dr. Jerne, originario de Dinamarca, donde nace en 1911, realiz su obra principalmente en el
Instituto de Inmunologa de Basel. Muere en 1994. Su teora acerca de la seleccin natural aplicada a la
formacin de anticuerpos indica que los individuos poseen anticuerpos para todos los antgenos hacia los
cuales puede responder; cuando un antgeno entra al organismo, selecciona el anticuerpo
correspondiente, unindose a l, lo que estimula su produccin especfica. Este punto de vista, que rompe
con la visin anterior de que antgenos hacen que se produzcan los anticuerpos correspondientes, es el
inicio de la moderna inmunologa celular. Su segunda teora nos muestra cmo el sistema inmunolgico
madura bajo la influencia de los antgenos endgenos, y cmo la rpida multiplicacin de las clulas
linfticas lleva a acumular mutaciones que dan cuenta de nuevas especificidades inmunolgicas. La
tercera teora predice cmo la respuesta inmune se encuentra regulada por una compleja red de
anticuerpos y anti anticuerpos. Vase: Press Release: The 1984 Nobel Prize in Physiology or Medicin.
Nobelfrsamlingen Karolinska Institute. The Nobel Assembly at the Karolinska Institute Octubre de
1984.
ii.
iii.
iv.
v.
vi.
vii.
viii.
ix.
en 1944 completa sus estudios de Bachiller en Baha Blanca;

en 1945 ingresa a la Facultad de Ciencias Exactas de la Universidad de
Buenos Aires, obteniendo en 1952 el ttulo de Licenciado en Ciencias
Qumicas;
en 1952 comienza a investigar en el Instituto de Qumica Biolgica de la
Facultad de Ciencias Mdicas de la Universidad de Buenos Aires, y culmina
esta etapa de su vida con el grado acadmico de Doctor en Qumica en 1957;
ese ao entra como miembro del grupo de investigadores del Instituto
Nacional de Microbiologa Dr. Malbrn, del que se ausenta en 1958 para ir
al Departamento de Bioqumica de la Universidad de Cambridge, gracias a
una beca del British Council;
en 1960 obtiene el Doctorado de la Universidad de Cambridge, donde
permanece un ao ms como investigador del Departamento de Bioqumica;
en 1961, regresa como Jefe de la Divisin de Biologa Molecular al Instituto
Nacional de Microbiologa de Buenos Aires, donde permanece hasta 1963;
ese ao, regresa a Cambridge como miembro del equipo de investigacin del
Laboratorio de Biologa Molecular, en el que realiza sus investigaciones
mayores;
en 1975 publica, junto con Khler, las investigaciones que lo llevaron a
obtener el Premio Nobel.2
En esta apretada sntesis no encuentra su lugar aquello que caracteriza a Milstein

como hombre y como investigador: sus orgenes, sus sueos, sus hallazgos y sus
tropiezos.
En las pginas que siguen trataremos de dar una visin de su trayectoria personal e
intelectual, desde sus comienzos hasta la obtencin del Premio Nobel.
Milstein, el hombre3
El padre de Milstein era un inmigrante judo de origen ruso, que llega -solo- a
Argentina con quince aos recin cumplidos, a realizar el viejo sueo que compartieron
millones de inmigrantes de diferentes orgenes, de vivir y progresar en una tierra nueva.
Se casa hacia 1923 con una muchacha que haba nacido en las colonias judas agrarias
de Entre Ros, Argentina. Viajante de comercio el padre, maestra de escuela la madre,
compartan ideales socialistas y libertarios, y desarrollaban actividades culturales en
medios judos y laicos. Tuvieron tres hijos -Csar era el segundo-, a los que con grandes
sacrificios enviaron a la Universidad.
Milstein reniega de su vida de pueblo chico en Baha Blanca durante largos
aos, al cabo de los cuales, retrospectivamente, comienza a apreciar recordando sus
lecturas en la Biblioteca Nacional, bien provista, y generosamente abierta, sus inicios
como integrante de un coro, que contina en Buenos Aires en el Collegium Musicum, y
que hizo de la msica parte de su vida. Y sobre todo, las clases de su profesor de
qumica inorgnica en cuarto ao del bachillerato, que le revelaron la belleza de la
qumica y de las frmulas que expresan su estructura.
2
Milstein, Csar. Curriculum Vitae. Manuscrito. 1989.

Milstein, Csar, Autobiography, en: Press Release. op. cit. 1984.
Milstein, Csar, Entrevista, en: Barn, A., del Carril, Mario y Gmez A. Por qu se fueron. EMECE.
Buenos Aires. 1995
3
Es posible que este sea el motivo por el cual, en su currculum vitae de 1989,
incluya entre los premios, las distinciones, las membresas a las ms altas sociedades
internacionales, el ser nombrado en 1985 miembro honorario de las modestas Sociedad
de Medicina Interna y Asociacin Mdica de su lejana Baha Blanca natal.
A los 18 aos se incorpora a la Facultad de Ciencias Exactas de la Universidad
de Buenos Aires, una institucin slida y con buenos profesores. Insiste Milstein en que
no era un estudiante brillante; sin embargo, las clases prcticas intensivas para pocos
alumnos que caracterizaban a la Facultad, lo formaron slidamente aunque concentraba
los perodos de estudio nicamente en las pocas de exmenes. Como muchos de su
generacin, se encontraba profundamente interesado en la sociedad, y en la posibilidad
de cambiarla. La Reforma Universitaria de 1918, ese intenso movimiento estudiantil
que cambi de raz a la universidad argentina, y que extendi su ideario generoso por
toda Amrica, estaba viva en los estudiantes que la reivindicaban. Entre ellos se cuenta
Milstein, que milita en el Centro de Estudiantes, llegando a ser su Presidente. El
idealismo libertario de aquellos das persiste en su ausencia de ambicin de poder, y en
el trato igualitario que lo caracteriza. En algn momento expres que si decidi ser
cientfico, es porque la ciencia no da poder. Es probable que detrs del cientfico
maduro que logra uno de los hallazgos con mayores consecuencias tecnolgicas, y sin
embargo deja que sea utilizado por todos aquellos que lo necesiten, sin trabas legales ni
patentes excluyentes -renunciando a las riquezas que implica- se encuentren, intactas
sus convicciones de juventud.
Una vez recibido, comienza a trabajar en la Facultad de Medicina con el Dr.
Andrs Stoppani, un brillante bioqumico que lo acoge a su lado -por otra parte, el nico
cientfico argentino que cita en su Autobiografa Nobel-, en las consabidas precarias
condiciones de los laboratorios argentinos de entonces, y que pese a ser el nico
profesor de dedicacin exclusiva, bordeaba la pobreza. La modestia era un sello de
marca de los cientficos argentinos: cuenta Milstein que cuando quiso comenzar a
investigar, fue a entrevistarse con el Dr. Federico Luis Leloir, quien aos despus
obtendra el Premio Nobel de Qumica; al llegar al laboratorio, no reconoce al cientfico
renombrado en ese individuo delgado, de guardapolvo gris, al que interpela como si
fuera el encargado del edificio en un estado lamentable, por otra parte-.
Se casa en 1953 con Celia, una compaera de estudios con la que comparte en
las pocas de estudiante interminables caminatas en los legendarios -entre los
universitarios argentinos- campamentos de Bariloche organizados por el Centro de
Estudiantes de Qumica, o las remadas por los riachos de Tigre. Realizan con muy
pocos recursos un viaje -inicitico- a Europa, viviendo en albergues de estudiantes, en
casas de amigos, con la mochila y la carpa a cuestas, hasta llegar a Israel, donde
trabajan en un kibutz.
Sin ningn apoyo econmico -trabajando l y su esposa en un laboratorio de
anlisis clnico para subsistir- prepara su tesis de doctorado sobre cintica de enzimas, a
raz de la cual publica varios trabajos, uno de ellos en una revista del exterior.
Casi simultneamente luego de doctorarse, gana un concurso en el Instituto
Nacional de Microbiologa Dr. Malbrn y una beca del British Council. Le guardan el
puesto en el Instituto -incluso pagndole el sueldo- mientras permanece en Cambridge.
Una vez en Inglaterra, un suceso inesperado cambia su perspectiva de la ciencia.
El supervisor que tena asignado, Malcom Dixon, le propone muy vagamente un tema
de investigacin, mas no lo supervisa en su trabajo. Milstein comienza a investigar los
mecanismos de la activacin por metales de la enzima fosfoglucomutasa (no es ocioso
recordar que las minuciosas investigaciones acerca una coenzima para la
fosfoglucomutasa inician la secuencia de trabajos que condujeron al Dr. Leloir al
3
premio Nobel). Esto hace que entre en contacto con un joven premio Nobel, Frederick
Sanger, quien trabaja en enzimas proteolticas, el que le insiste que repita un
experimento, aunque ya estuviera hecho, y su preparacin tardara largos quince das. La
insistencia de Sanger hace que invente un mtodo por el cual los resultados se obtienen
en un solamente un da, en vez de quince. Dice Milstein que en este momento abandona
la cintica y se dedic a la qumica de protenas. En corto tiempo termina su tesis de
doctorado, a la que aade sus nuevos hallazgos. Sus trabajos tienen repercusin
internacional, publicando sus resultados en seis o siete artculos en revistas de primera
lnea, dos de ellos en colaboracin con Sanger4.
Varios motivos inciden para que decida en 1961 volver a radicarse en la
Argentina. Al compromiso moral que tena con el Instituto se une al hecho de que luego
de 1955 se funda el Consejo Nacional de Ciencia y Tecnologa -CONICET- bajo la
presidencia del Dr. Bernardo Houssay, el gran investigador argentino, inicindose una
etapa de un fuerte impulso para la investigacin cientfica.
Adems, el Instituto Nacional de Microbiologa le ofrece la oportunidad de
encabezar su propio grupo de investigacin como Jefe de Divisin de Biologa
Molecular. Tiene 33 aos, y trae al pas una intensa experiencia, y un incipiente, pero
slido prestigio cientfico.
Ya en el Instituto Malbrn realiza con su equipo trabajos de primer nivel; tanto,
que Fritz Lippman, un premio Nobel, que investiga dentro del mismo campo de
estudios, le escribe desde los Estados Unidos que quera ver lo que hacan en el
laboratorio, pues estaban obtenido resultados que sobrepasaban los suyos. Cuando en
1963 llega Lippman a Buenos Aires a dar un seminario en el Instituto, ste se encuentra
intervenido, un comit de investigaciones persigue a su director, el Dr. Piroski, y haba
gente dejada cesante. Milstein niega toda autoridad a quienes interrogan
inquisitorialmente a los investigadores, y renuncia cuando dejan cesante a cuatro
miembros de su equipo. Pese a contar con todo el apoyo de Houssay y Leloir, la
renuncia no tuvo ningn efecto sobre las autoridades. Es bueno recordar -para su
vergenza- que el Dr. Tuburcio Padilla, Ministro de Salud Pblica del gobierno civil
controlado por los militares del Dr. Jos Mara Guido, hace esperar ms de cuarenta
minutos en su antesala al Dr. Leloir cuando lo visita para pedirle que no dejara ir a
Milstein, pues si no se desarmaba un importantsimo equipo de investigaciones. El
ministro solo contesta -obtuso- que pase lo que tenga que pasar. Antes de eso, en una
visita al Instituto, sugiri que los investigadores se fueran del pas, ya que ac no se
puede hacer nada.
Milstein, que no tena la piel de elefante que hay que tener para aguantar lo que
viniera en esa universidad, en ese pas -como se lo dijo el eminente cientfico Dr. De
Robertis5- y conservaba la dignidad de sus pocas de militante estudiantil, regres a
Cambridge, donde se reencuentra con Frederick Sanger, quien haba sido nombrado Jefe
de Divisin de Qumica de las Protenas en el recin formado Laboratorio de Biologa
Molecular del Consejo de Investigaciones Mdicas. El reencuentro es crucial, pues lo
pone en la lnea de investigacin que lo lleva al premio Nobel, abandonando el campo
de la bioqumica para adentrarse en la inmunologa.
Milstein, C., Sanger, F. The amino acid sequence around the serine phosphate in phosphoglucomutase.
Biochim. Biophys 1960. Acta 42. 173-174.
Milstein, C., Sanger, F. an amino acid sequence in the active center of phosphoglucomutase. Biochem. J.
1961; 79: 456-469.
5
La cita es textual, y se encuentra en Milstein, C. Entrevista. op. cit. 1995.
A partir de ese momento, el Milstein hombre se identifica con el Milstein

cientfico. Esta etapa de su vida es la que vamos a narrar a continuacin. Ya un
cientfico maduro, la estabilidad acadmica de Cambridge le brinda una tranquilidad por
la cual, pasados los aos mozos, su existencia transcurre sin sobresaltos.
Puede, por fin, dedicarse exclusivamente a investigar en Inglaterra, donde en su
juventud supuso se haca la mejor ciencia del mundo. El lugar por el que transitaron
antes que l quien fue el primero en ensearle lo que es la ciencia, el Dr. Stoppani, y el
Dr. Leloir, que trajeron consigo la manera inglesa de hacer ciencia, que tan bien se
adapta a las austeras condiciones argentinas, y que consiste en profundizar en un
puado de temas elegidos, con pocas herramientas tcnicas -en contraste con tradiciones
que privilegian el ataque de los problemas con una inversin grande en tecnologa-.
Leloir, adems, recomendaba eludir los temas que se hubieran trabajado extensamente,
donde para avanzar era necesario dominar una literatura inmensa, y buscar aquellos
problemas sobre los que no se haba escrito mucho, y en los cuales se pudiera trabajar
directamente.
Con todo, no olvida a ese pas en el que transcurri su juventud, en el que
aprendi las canciones de cuna6; ese pas que maltrata y expulsa a sus cientficos; que
los forma aunque restrinja luego sus oportunidades de investigar, y vuelve, una y otra
vez, a brindar su consejo y su apoyo cada vez que es convocado por sus amigos, sus
colegas o un gobierno democrtico.
Los anticuerpos monoclonales
Para el Instituto Karolinska de Suecia, en su sucinta presentacin de los Premios
Nobel de 1984, el reconocimiento a la obra de Milstein y Khler se debe a, como
mencionramos, el descubrimiento del principio que rige la produccin de anticuerpos
monoclonales, que se concreta en la tcnica del hibridoma, que representa el avance
metodolgico ms importante en el campo de la biomedicina durante los setentas, con
importantes consecuencias tecnolgicas para el tratamiento y el diagnstico.
Sin embargo, el lenguaje tcnico con el cual se refieren a las investigaciones de
Milstein no puede opacar el deslumbramiento que produce un hallazgo que entronca
con al menos dos de las lneas de investigacin ms caras dentro de la historia de la
biologa en general, y de la inmunologa en particular. Tampoco ocultar que se realiza
en el seno de investigaciones bsicas relevantes y originales, y que a su vez revierte
sobre los temas ms bsicos de la ciencia. No es una simple herramienta tecnolgica, ni
una manera de hacer ms fcilmente lo que la naturaleza hace por s sola, sino que es un
resultado inscripto en conocimientos bsicos avanzados, que redunda en un dispositivo
tecnolgico -artificial, como toda tecnologa- que a su vez hace avanzar an ms las
investigaciones.
A fin de presentarlas, voy a relatar primeramente el problema terico que se
intentaba resolver, y para cuya solucin se crean esos instrumentos tcnicos que son los
anticuerpos monoclonales y su produccin; a continuacin, mostrar en qu consiste
dicho hallazgo y cules son sus consecuencias tecnolgicas y cientficas, para concluir
indicando las grandes tradiciones de investigacin a las que contina, y que despertaron
La frase la pronuncia Milstein en la entrevista publicada en el libro Los que se fueron, refirindose a que
nunca conocer las canciones de cuna inglesas, en respuesta a la pregunta sobre su sentimiento de
pertenencia a Inglaterra, luego de tantos aos de residencia en Cambridge.
la adhesin de sus colegas, al coincidir con problemas hondamente sentidos por la

comunidad de investigadores biomdicos.7
Los anticuerpos y su produccin natural
Es sabido desde el principio de las investigaciones inmunolgicas que los
animales reconocen un cuerpo extrao antgeno-, sea una bacteria, un virus o una
sustancia aislada, y lo neutralizan o lo destruyen con sus anticuerpos. Se sabe asimismo
que esta respuesta es especfica para cada sustancia, y que la misma es elaborada por los
linfocitos, esas clulas blancas de la sangre, bazo, timo y ganglios, tambin de manera
especfica. Dado que los antgenos son numerosos a decir verdad, millones de
sustancias pueden comportarse como tales-, los anticuerpos son igualmente numerosos,
y por lo tanto, hay millones de linfocitos que difieren slo en el anticuerpo que
producen. Cuando una sustancia ingresa al organismo, aquellos linfocitos que se
encuentran capacitado para producir el o los anticuerpos que la neutralizan, comienzan
a reproducirse y a producir cantidades cada vez mayores del anticuerpo especfico.
Puesto que todas son hijas idnticas de un mismo linfocito -pertenecen a una misma
lnea de linfocitos- se denominan clones, y el anticuerpo, producido por una nica
variedad de clones, monoclonal.
La forma natural de reaccionar que tiene un organismo ante la invasin de una
sustancia extraa, es reproduciendo la estirpe especfica de linfocitos, a fin de que
produzcan anticuerpos monoclonales.
Cmo es posible que en el organismo exista tal diversidad de linfocitos,
preparados para responder a sustancias frente a las cuales el organismo nunca se
enfrent anteriormente? Una respuesta posible consiste en decir se encuentra
predeterminado por el cdigo gentico, y que en consecuencia, existen numerossimos
segmentos de ADN destinados a darles origen, quizs tantos como el nmero de los
linfocitos que difieren por su secrecin de anticuerpos.
Cuando Milstein comienza a investigar, esta respuesta no es viable, puesto que
se conoce que los anticuerpos poseen una porcin constante para todos los anticuerpos,
y una porcin variable formada por dos cadenas de sustancias, una liviana y otra
pesada- unidas por puentes de disulfuro. Al menos la porcin constante provendra de
una nica secuencia de ADN.8
Ahora bien. Hacia 1970, Milstein pens que si exista una porcin constante, y
las porciones variables o hipervariables podan ser reducidas a unas pocas familias o
grupos de secuencias9, provenientes de un nmero relativamente pequeo de genes
individuales, la respuesta correcta a la diversidad de los anticuerpos deba consistir en
mutaciones somticas, ocurridas durante el proceso mediante el cual se generan los
linfocitos. A fin de investigar esta hiptesis, decide estudiar la posibilidad de
7
Se tomaron como referencia para elaborar esta sntesis de la labor de Milstein bsicamente los
siguientes trabajos:
Milstein, Csar. From Antibody Structure to Inmunological Diversification of Inmune Response, en:
Science. 14 March 1986. Vol. 231: 1261-1268.
Milstein, Csar. Main points of my research in Immunology. Manuscrito 1989.
Milstein, Csar. Monoclonal antibodies, en: Scientific American. 1980. 243. No. 4: 56-64.
8
La sugerencia de que la unin los elementos de las porciones que forman un anticuerpo lo haca
molculas de disulfuro fue la primera intuicin de Milstein en este campo. Vase: Frangione, B.,
Milstein, C., y Pink, J.R.L., Structural studies of inmunoglubin G, en: Nature 1969; 221: 145-148. En
este artculo se establece la estructura de los puentes de disulfuro.
9
Milstein, C. Linked groups of residues in inmunoglobin chains, en: Nature 1967; 216: 330-332. En
este artculo se establece la existencia de familias bsicas o subgrupos en la regin variable e
hipervariable.
mutaciones en un cultivo de clulas de mieloma, un tumor cuyas clulas poseen la

particularidad de producir una protena, una inmunoglobulina, que aunque posee la
estructura de un anticuerpo, no acta frente a ningn antgeno. Precisamente esta
cualidad hizo que fueran elegidas para los estudios, una vez que fueron excluidos los
propios linfocitos como clula experimental por su conocida incapacidad de mantenerse
y reproducirse en un medio de cultivo el tiempo necesario para encontrar en la sucesin
de las generaciones a las supuestas mutaciones.
Sin embargo, a partir de este momento aparece un interesante problema
metodolgico, que puede ser planteado de la siguiente manera. La forma de saber si se
ha producido una mutacin, es detectando cambios en la protena que producen los
mielomas. Pero la complejidad de su estructura qumica hace inviable su anlisis
detallado, por lo que la respuesta no poda provenir nicamente de la qumica de
protenas. Tampoco poda estudiarse su accin como anticuerpo, pues no la poseen.
Dadas estas condiciones, se impona la necesidad de contar con una lnea de
clulas que segregue una inmunoglobulina que exhiba una actividad de anticuerpo, y
que sta sea fcilmente detectada, a fin de investigar las mutaciones en su sector
variable. Era evidente que no exista tal lnea de clulas.
Si recapitulamos, la preocupacin terica esencial consiste en averiguar cmo es
posible la diversidad de la respuesta inmunolgica, a cargo de anticuerpos especficos y
cmo se generan los linfocitos que los producen. El camino para probar la existencia de
mutaciones que den cuenta de la variabilidad inmunolgica, llev, inesperadamente, a
plantear la necesidad de un mtodo de deteccin de los mutantes que no fuera el anlisis
qumico de los anticuerpos que producen. Este mtodo fue el de los hibridomas, y la
consecuente produccin de anticuerpos monoclonales a voluntad.
Hibridomas y anticuerpos monoclonales
La propia lgica de la investigacin de mutantes en cultivos de clulas
mielomatosas, cuyo paso posterior fue fusionar dos tipos de clulas de mieloma y
mostrar que el hbrido resultante produce inmunoglubulinas de ambas clulas
progenitoras10, lleva gradualmente a su mayor descubrimiento.
Corre el ao 1973, cuando al conocer estos resultados, George Khler se
incorpora como becario al equipo de Milstein. El camino que finalmente encuentran
para proseguir las investigaciones, es el de producir ellos mismos la lnea de clulas que
buscan. Una tarde en que Milstein y Khler conversan acerca de unas experiencias en
las cuales diversos virus infectan a los linfocitos, transformndolos en clulas
tumorales, pensaron si no fuese posible que una fusin entre un linfocito y una clula
tumoral diera por resultado esa clula que necesitaban.
Esa misma noche Khler, siguiendo las indicaciones de Milstein, puso en
marcha el experimento. A la maana siguiente, comprueba que se haba producido un
hbrido de mieloma y de linfocito, un hibridoma como se denomina al hbrido de
mieloma- que elabora anticuerpos.11
El hibridoma toma del mieloma la posibilidad de reproducirse indefinidamente
en un medio de cultivo, y del linfocito, la de producir anticuerpos.
10
Cotton, R.G.H., Milstein, C. Fusion of two inmunoglobulin-producing myeloma cells, en: Nature
1973, 197; 3: 244: 42-43. En este artculo se presenta la primera hibridacin exitosa de clulas de
mieloma.
11
Los resultados fueron comunicados en: Khler, G., Milstein, C., Derivation of specific antibodyproducing tissue culture and tumour lines by cell fusion, en: European Journal of Immunology 1976;
6: 511-519.
Se abre la posibilidad de producir anticuerpos a voluntad, y en cantidades

considerables, si se parte de la hibridacin con una lnea de linfocitos dada, puesto que
la capacidad de producir anticuerpos la comparte toda su descendencia monoclonal.
El procedimiento, a grandes rasgos, es el siguiente: se inmuniza a un ratn con
un antgeno dado. Se toman a continuacin de su bazo los linfocitos que producen los
anticuerpos correspondientes, y se los fusiona con clulas mielomatosas, para originar
un conjunto de hibridomas, que se cultivan en delante de manera indefinida.
Fig. Primera Fase del procedimiento
Pero todava no es un conjunto monoclonal. Esto es as, puesto que un agente

antignico dado -sea simple o complejo- provoca la produccin de diversos anticuerpos
dirigidos contra l. Como cada uno de estos anticuerpos es segregado por una lnea
diferente de linfocitos, el primer cultivo de hibridomas resulta una mezcla de lneas
diversas.
El siguiente paso consiste en aislar hbrido por hbrido -detectndolos por sus
anticuerpos- y cultivarlos separadamente. La estirpe que resulta de cada uno de ellos, es
efectivamente monoclonal, y produce un slo tipo de anticuerpo, que puede recolectarse
en el medio de cultivo. Si el clon se inyecta a un ratn, ste desarrolla un tumor que
produce cantidades grandes de anticuerpo, susceptible de ser aislado del suero del
animal, o del lquido de ascitis.
Sorprendentemente, el resultado que se obtiene mejora las condiciones en las
que se desenvuelve la anterior seleccin natural, pues mientras que slo el uno por
ciento de los linfocitos que se usan en la fusin segregan anticuerpos, cerca del diez por
ciento de los hibridomas lo hacen, mejorando notablemente su seleccin.
Si bien es cierto que se seleccionan hbridos que fabrican anticuerpos de ciertas
propiedades deseadas, si el animal del que se obtienen los linfocitos no los produce
aunque se lo haya inmunizado previamente, no hay forma de inmortalizar su
elaboracin. Afortunadamente, puntualiza Milstein, se puede ir ms all. La ingeniera
gentica permite modificar los anticuerpos, y fabricar nuevas variaciones que son, a su
vez, elaborados por los hibridomas, construyndolos a medida de los antgenos que se
desea neutralizar.
Fig. 2. Segunda Fase del procedimiento
Finalmente haban obtenido una lnea de clulas que produca un anticuerpo

especfico, y podan utilizarla para explorar los mutantes de la regin hipervariable de
los anticuerpos. Sin embargo, la lgica propia del descubrimiento llevara rpidamente a
explorar otros horizontes.
Las consecuencias tecnolgicas y cientficas
Casi inmediatamente fue evidente que la capacidad de producir anticuerpos a
voluntad poda utilizarse para fines diagnsticos, de purificacin de sustancias,
teraputicos, o de investigacin bsica.
El primero de los caminos explorados por Milstein, tiene que ver con la
produccin de reactivos estndar tales como los antgenos para anti-histocompatibilidad
y para grupos sanguneos.12 Luego se desarrollaran tcnicas adecuadas para
diagnosticar diversas enfermedades virales, bacterianas, parasitarias, inmunolgicas,
etc.
Otra particularidad de la tcnica consiste en que hace posible desentraar la
composicin exacta de una mezcla sucia de elementos, aislando cada uno de ellos.
Una de las pruebas ms exigentes a las que se la someti fue el aislamiento y
purificacin de interfern, una sustancia de difcil identificacin con medios
12
Galfre, G., Howe, S. C., Milstein, C., Butcher, C.W., Howard, J.C. Antibodies to major
histocompatibility antigens produces by hybrid cell lines, en: Nature 1877; 266: 550-552.
Voak, D., Sacks, S., Anderson, T., Takei, F., Lennox, E.S., Jarvis, J.M., Milstein, C., Darnborough, J.,
Monoclonal anti-A from a hybrid mieloma: Evaluation as a blood grouping reagent, en: Vox. Sang.
1980; 39: 134-140.
convencionales, y que luego fue utilizada en gran escala por la industria

biofarmacutica.
En cuanto a las posibilidades teraputicas que se abren a partir de las
investigaciones de Milstein, cabe mencionar las que hacen al tratamiento de tumores, y
a los problemas de rechazo observados en transplantes.
Es posible, sin embargo, que pese a las enormes consecuencias tecnolgicas,
utilitarias, de la mquina biolgica de producir anticuerpos monoclonales, una de sus
mayores contribuciones sea para la investigacin bsica.
Podramos citar la posibilidad de establecer la estructura de las membranas
celulares; de arrojar nueva luz sobre la teora de cmo los anticuerpos se ligan a los
antgenos y originan un precipitado en los tubos de ensayo, as como nuevas
interpretaciones acerca de la reaccin antgeno-anticuerpo; de utilizarlos en estudios
embriolgicos, de receptores para hormonas, o de neurotransmisores.13
En el camino de las mejores tradiciones
Es probable que en la visin del jurado de la Academia de Ciencias que otorg
el premio Nobel a Milstein y a Khler hubiera algo ms que el reconocimiento al
descubrimiento de la produccin de anticuerpos monoclonales, de indudables valores
intrnsecos. El mismo lenguaje con el que se describe la tcnica, o se encabeza su
descripcin general empleando trminos por otra parte ya utilizados por el propio
Milstein en sus artculos- es altamente sugestivo de un valor simblico adicional. Se lee,
as que ellos inmortalizaron clulas que producen anticuerpos fusionndolas con
clulas tumorales, o que el hibridoma es una tcnica para la eterna produccin de
anticuerpos monoclonales en clulas cultivadas. Milstein en reiteradas ocasiones
menciona los trminos inmortal, inmortalizar o inmortalidad para referirse a los
anticuerpos o a los hibridomas, en expresiones tales como: inmortalizar las clulas del
ratn, inmortalizar la expresin de las clulas plasmticas, aparentemente
estbamos consiguiendo selectividad junto con inmortalidad, el hbrido resultante era
una clula inmortal capaz de expresar la actividad de producir anticuerpos de la clula
paterna, y la inmortalidad adquirida del mieloma, inmortalizamos el anticuerpo en
forma de un hbrido de mieloma, etc.14
Para la biologa, la ciencia de la vida, su perduracin no es un asunto trivial. Ya
Alexis Carrel, premio Nobel de medicina en 1912 por sus avances tcnicos en ciruga
vascular, haba sorprendido al mundo cientfico manteniendo en cultivo por tiempo
indefinido un tejido embrionario de pollo. La posibilidad de inmortalizar la capacidad
de producir anticuerpos entronca con esta tradicin de investigacin biolgica, que
abreva adems en terrenos preparados por mitologas ancestrales.
La segunda tradicin que contina Milstein se encuentra en los orgenes de la
inmunologa, expuesta posiblemente por vez primera por Paul Ehrlich, cuando expresa
que las sustancias inmunes, a la manera de balas mgicas, buscan al enemigo. La
asombrosa capacidad de la tcnica de anticuerpos monoclonales para identificar y
producir todo tipo de anticuerpos, lleva consigo el viejo sueo de la bala mgica, la
bsqueda interminable del remedio preciso que da en el blanco de la enfermedad,
dejndola fuera de combate, pero que al mismo tiempo respeta los componentes sanos
del organismo, a su ms alto nivel.
13
Cuello, A.C., Galfre G., Milstein, C., Detection of substance P in the central nervous system by
monoclonal antibody, en: Proc. Natl. Acad. Science 1979, USA; 76: 3532-3536.
14
Estas expresiones figuran en:
Milstein, C. (1980), Milstein, C. (1986), y Karolinska I. (1984)
10
Cuando Ehrlich habla de esta manera, no se refiere nicamente a la especificidad

de los anticuerpos, que la recin fundada teora inmunolgica explica tan bien. Se
refiere asimismo a esa bala mgica que es la quimioterapia, de quien fue el iniciador,
cuando utiliza la afinidad que tienen los colorantes con las diversas estructuras
celulares, una propiedad que posibilita su tincin en los preparados histolgicos,
distinguindolas, para buscar sustancias asimismo colorantes en un comienzo- que
posean la misma selectividad con respecto a los microorganismos y a sus toxinas. As se
descubrieron el rojo Trypan, destinado a destruir al tripanosoma de la enfermedad del
sueo, o el Salvarsn, para la sfilis: utilizando sistemticamente las afinidades de los
colorantes qumicos para fabricar artificialmente sustancias que actan como antgenos,
hasta llegar al Prontosil, la primera de las sulfamidas.
Comenta agudamente George Canguilhem que una nueva tcnica sustituye la
extraccin de sustancias por la produccin de productos: no hay quimioterapia sin una
sociedad cientfica, sin una sociedad industrial, y a su vez cita a Bachelard, quien dice
que quien fabrica la anilina (el colorante por antonomasia de entonces) conoce la
realidad y la racionalidad de los colores.15 Ya Pasteur habra conocido la modificacin
experimental de los productos naturales como un recurso terico de anlisis de lo real,
con los problemas tcnicos que involucra.
Canguilhem nos muestra dos hechos que subyacen al descubrimiento de la
quimioterapia. En primer lugar, las estrechas relaciones entre el desarrollo de la
sociedad, y el descubrimiento cientfico. La sociedad industrial lleva en su lgica a la
produccin de las anilinas, y de all utilizndolas- a la produccin artificial de
antitoxinas. Sin las condiciones de posibilidad que le brinda, esto es imposible. En
segundo lugar, y de manera ms provocativa, que para desentraar los secretos de un
fenmeno natural, es central reproducirlo artificialmente, mediante estructuras que no
existen en la naturaleza.
De la misma manera, es a travs de una estirpe artificial de clulas monoclonales
que fabrican anticuerpos a pedido, como se posee la clave para comprender la
complejidad de los anticuerpos, y de las estructuras antignicas que la genera.
En este sentido, ms profundo que el de la metafrica bala mgica, la obra de
Milstein contina, por otros medios, la tradicin de los fundadores de la inmunologa.
En su Conferencia Nobel, expresa acorde con la misma: Mientras que la seleccin es la
estrategia de la respuesta de anticuerpos de un animal, la inmunoqumica del futuro
volver a una clase de aproximacin similar a la de la teora de la instruccin (la que
deca que el anticuerpo era fabricado a medida del antgeno) en la que el antgeno nos
dir cul es la estructura del anticuerpo que construiremos16.
Son palabras en las que se trasparenta un viejo anhelo que proviene de los
albores de la inmunologa.
BIBLIOGRAFA
15
Canguilhem, George (1986), pp. 70-71.

Bachelard, Gaston (1953) p. 202.
Vase asimismo: Silverstein, A. (1989)
16
Milstein, C. (1986) op. cit. p. 126.
11
Bachelard, Gaston, (1953), Le matrialisme rationnel, PUF. Paris.

Canguilhem, George (1986), Leffet de la bacteriologie dans la fin de Thories Mdicales u XIXe
sicle, en Canguilhem G., Ideologies scientifiques et medicales, PUF, Pars.
Cotton, R.G.H., Milstein, C. (1973), Fusion of two inmunoglobulin-producing myeloma cells, Nature,
197, 3, pp. 244: 42-43.
Cuello, A.C., Galfre G., Milstein, C. (1979), Detection of substance P in the central nervous system by
monoclonal antibody, Proc. Natl. Acad. Science, 76, pp. 3532-3536.
Frangione, B., Milstein, C., y Pink, J.R.L. (1969), Structural studies of inmunoglubin G, Nature, 221,
pp. 145-148.
Galfre, G., Howe, S. C., Milstein, C., Butcher, C.W., Howard, J.C. (1977), Antibodies to major
histocompatibility antigens produces by hybrid cell lines, Nature, 266, pp. 550-552.
Khler, G., Milstein, C., Derivation of specific antibody-producing tissue culture and tumour lines by
cell fusion, en: European Journal of Immunology 1976; 6: 511-519.
Milstein, C. (1967), Linked groups of residues in inmunoglobin chains, Nature, 216, pp. 330-332.
Milstein, C. (1980) Monoclonal antibodies, Scientific America, 243, No. 4, pp. 56-64.
Milstein, C. (1984), Autobiography, Press Release, op. cit.
Milstein, C. (1989), Main points of my research in Immunology, manuscrito.
Milstein, C. (1995), Entrevista, en Barn, A., del Carril, Mario y Gmez A., Por qu se fueron, Buenos
Aires, EMECE.
Milstein, C. (14 March 1986), From Antibody Structure to Inmunological Diversification of Inmune
Response, Science, vol. 231, pp. 1261-1268.
Milstein, C., Sanger, F. (1960), The amino acid sequence around the serine phosphate in
phosphoglucomutase, Biochimistry. Biophys, Acta 42, pp. 173-174.
Milstein, C., Sanger, F. (1961) An amino acid sequence in the active center of phosphoglucomutase,
Biochemtry Journal, 79, pp. 456-469.
Nobelfrsamlingen Karolinska Institute. The Nobel Assembly at the Karolinska Institute (Octubre 1984),
Press Release: The 1984 Nobel Prize in Physiology or Medicin.
Silverstein, A. (1989), Magic Bullets and Poisoned Arrows: The Uses of Antibody, en: Silvertein A. A
History of Immunology. Academic Press. San Diego. 1989.
Voak, D., Sacks, S., Anderson, T., Takei, F., Lennox, E.S., Jarvis, J.M., Milstein, C., Darnborough, J.
(1980), Monoclonal anti-A from a hybrid mieloma: Evaluation as a blood grouping reagent, Vox.
Sang., 39, pp. 134-140.
12
13
Teora_de_la_red_idiotpica
Teora de la red idiotpica (1984) Niels Kaj Jerne (1911-1994)
HIPOTESIS DE JERNE: Teora de la red idiotpica.
Algunas porciones variables del Ac (IDIOTIPOS) pueden comportarse como determinantes antignicos.
Los Ac anti-idiotipo actan de reguladores de la actividad del idiotipo y de los TCR (linfocitos T)
Segn la teora de la red de Jerne, frente a los idiotipos se formaran anticuerpos que al unirse a los mismos
formaran un entramado (?red?) de anticuerpos unidos a otros anticuerpos que tendran como accin final la
regulacin del proceso de sntesis de nuevas inmunoglobulinas. Cada uno de los idiotipos se encuentra
representado en tan pequea cantidad que pasa desapercibido para el sistema inmune, sin embargo, cuando un
determinado clon de clulas B reconoce su antgeno especifico, prolifera, se diferencia a clula plasmtica y
produce una gran cantidad de inmunoglobulinas de una misma especificidad, sus determinantes idiotipicos
pasaran a encontrarse en mucha mayor cantidad y ahora s darn lugar a una respuesta de anticuerpos contra ellos,
anticuerpos anti-idiotipo, que podrn unirse a las inmunoglobulinas que ocasionaron su generacin. La unin de
los anticuerpos anti-idiotipo al idiotipo que los origino podr dar lugar al bloqueo de las inmunoglobulinas
solubles que compartan ese idiotipo o unirse a las inmunoglobulinas de membrana presentes en linfocitos B de la
misma especificidad, o incluso a las regiones hipervariables del receptor para el antgeno de la clula T que
reconocen ese mismo antgeno, con efectos en cada uno de los casos inhibidores o estimuladores.
Los idiotipos se encontraron mediante estudios serolgicos, al observarse que cuando en un conejo se inyectaban
anticuerpos antisalmonella de otro conejo del mismo alotipo, producan anticuerpos que reaccionaban con el
anticuerpo inyectado, incluso aunque los dos conejos fueran genticamente idnticos. Estos anticuerpos
anti-idiotipo, en la mayora de los casos, estn dirigidos contra la estructura exclusiva de la porcin fijadora de
antgeno y por tanto solo reconocen a inmunoglobulinas de la misma especificidad, sin embargo en algunos casos,
los anticuerpos anti-idiotipo pueden estar dirigidos contra zonas de la regin hipervariable distintas de la porcin
fijadora del antgeno y en este caso podrn unirse a inmunoglobulinas de varias especificidades distintas
regulando la respuesta inmune frente a varios antgenos.
sta teora est relacionada con la regulacin de la respuesta inmune y sugiere que existe una autoregulacin por
medio de la estimulacin de la produccin de clulas complementarias anti-idiotpicas por antgenos receptores
-idiotipos-. Estas clulas o sus productos disminuiran la produccin del idiotipo original. Los genes de las clases
II y III, especialmente los localizados en la regin HLA-D/DR -genes de respuesta inmmune (Ir)-,
proporcionaran una gran individualidad y especificidad a la respuesta inmune y determinan las interacciones
entre linfocitos y clulas del Sistema Fagocitario Mononuclear para la presentacin de antgenos y la proliferacin
de linfocitos.
La idea de las redes idiotpicas, y su posible implicacin en la regulacin del sistema inmune se debe a Niels
Jerne (quien la propuso en 1973, y que obtuvo por ello el premio Nobel en 1984).
Como sabemos, durante el desarrollo del sistema inmune se establece la tolerancia a los auto-antgenos,
esencialmente porque se eliminan los clones de linfocitos autoreactivos (que reconocen molculas propias).
Consideremos las inmunoglobulinas como auto-antgenos. En las primeras fases de vida, se induce la tolerancia
hacia las porciones constantes (Fc) porque globalmente existen en grandes concentraciones, pero no se induce
tolerancia frente a los idiotipos, (residentes en la parte variable de Fab) porque cada uno de ellos est presente en
muy pequeas cantidades: esta es la razn por la que las inmunoglobulinas son inmunognicas en el mismo
individuo.
Teora_de_la_red_idiotpica
Consideremos un antgeno que entra a un individuo, y fijmonos en uno de los pptidos que resultan de su
procesamiento. Frente a dicho pptido el sistema inmune monta una respuesta humoral a base de anticuerpos
(llammosles Ac#1). Pues bien, por las razones del prrafo anterior, dicho Ac#1 provocar a su vez la produccin
de otros anticuerpos (Ac#2), que reconocen los idiotopos del primero: a estos segundos anticuerpos se les
denomina anticuerpos anti-idiotpicos. A su vez, estos Ac#2 podran inducir una tercera "oleada" de anticuerpos
(Ac#3, anti-anti-idiotpicos), etc. De esta forma, se ira formando una red (red idiotpica) que se autorregula. El
mismo principio se puede extender a los receptores clonotpicos (TCR) de los linfocitos T.
Aunque estas ideas son muy atractivas, el papel real de la red idiotpica en el control normal del sistema inmune
an no est aclarado del todo, estando sujeto a debates.
Un corolario (confirmado) de la teora de la red idiotpica es que algunos de los anticuerpos anti-idiotpicos
reconocern al paratopo del Ac#1, y por lo tanto, son como la imagen interna que tiene el organismo del epitopo
del antgeno exgeno. Esta imagen interna podra servir para seguir activando al sistema inmune aun cuando
hubiera desaparecido el antgeno exgeno que desancaden la respuesta, asegurando suficiente expansin clonal y
clulas de memoria.
Hay algunas evidencias experimentales de que la red idiotpica acta fisiolgicamente: Ya se estn ensayando
varias aplicaciones clnicas de estos hallazgos, principalmente el diseo de vacunas ms seguras a base de
anti-idiotipos que sean la imagen interna de determinado antgeno. Esto puede ser interesante sobre todo cuando
se desconoce el antgeno exgeno real o cuando ste es carbohidrato o glucoprotena, y por lo tanto no se puede
recurrir a la clonacin de genes.
Ejemplos de vacunas basadas en anti-idiotipos que se han usado con xito experimentalmente en animales de
laboratorio:
vacunas frente a virus (de Newcastle, Sendai, reovirus, virus de la rabia, hepatitis B, citomegalovirus)
vacunas frente a bacterias (Listeria monocytogenes, Escherichia coli, Streptococcus pneumoniae)
vacunas frente a parsitos (Schystosoma mansoni, Trypanosoma rhodiense).
Se han hecho intentos de vacunas anti-idiotpicas frente al VIH (virus del sida), a base de anti-Id hacia anticuerpos
anti-CD4. Aunque el anti-Id es capaz de neutralizar al virus tanto in vitro como in vivo, la incertidumbre sobre su
capacidad de provocar respuestas celulares ha hecho que no se empleen clnicamente.
Una lnea interesante que se est explorando en ratones es el uso de ciertos anti-Id como vacunas neonatales
capaces de superar el efecto inhibidor de las IgG maternas:
Los ratones neonatales no pueden vacunarse frente a E. coli K-13 por el efecto supresor de las IgG
maternas recibidas pasivamente. Se ha obtenido IgG1 monoclonal anti-idiotpica frente a una IgM
monoclonal que se saba era capaz de conferir inmunidad pasiva. Pues bien, la vacunacin de ratones
neonatos con esa IgM o con IgG1 les capacitaba para resistir una infeccin letal de E. coli. En estas
condiciones el anti-Id (imagen interna) era eficaz, mientras que el antgeno real no lo era. Adems, si
inyectamos ese anti-Id a las hembras que acaban de parir, confieren inmunidad pasiva (a travs de la
leche) a las cras.
Referencias
1. Curso de Inmunologa General - Universidad de Granada
Referencias
THE GENERATIVE GRAMMAR OF

THE IMMUNE SYSTEM
Nobel lecture, 8 December 1984

by
NIELS K. JERNE
Chteau de Bellevue, F-30210 Castillon-du-Gard, France

Grammar is a science that is more than 2000 years old, whereas immunology
has become a respectable part of biology only during the past hundred years.
Though both sciences still face exasperating problems, this lecture attempts to
establish an analogy between linguistics and immunology, between the descriptions of language and of the immune system. Let me first recall some of the
essential elements of the immune system, with which I shall be concerned. In
1890, von Behring and Kitasato (12) were the first to discover antibody
molecules in the blood serum of immunized animals, and to demonstrate that
these antibodies could neutralize diphtheria toxin and tetanus toxin. They also
demonstrated the specificity of antibodies: tetanus antitoxin cannot neutralize
diphtheria toxin, and vice versa. During the first 30 years, or more, after these
discoveries, most immunologists believed that all cells of our body are capable
of producing antibodies, and it took until the 1950s before it became clear, and
until 1960 before it was demonstrated (13), that only the white blood cells
named lymphocytes can produce antibodies. The total number of lymphocytes
represent a little more than 1% of the body weight of an animal. Thus, it would
not be wrong to say that our immune system is an organ consisting of about l0 12
lymphocytes
(human) immune system = 1012 lymphocytes
or in a mouse that is 3000 times smaller than WC
(mouse) immune system = 3x108 lymphocytes
This brief description of the immune system disregards the fact that lymphocytes interact with most other cells in the body, which in my definition do not
belong to the immune system sensu strictu.
Let me draw attention to the fact that this number of lymphocytes in the
immune system is at least one order of magnitude larger than the number of
neurons in the nervous system. Also, we should note that lymphocytes travel
among most other cells of our body, that they circulate in blood and lymph,
and occur in large concentrations in the spleen, lymph nodes, appendix,
thymus and bone marrow. Strangely enough, however, they seem to be
excluded from the brain. The 1960s was a very fruitful decade of immunological
discoveries, of which I shall name a few: In the beginning of the decade, the
primary structure of antibody molecules was clarified (14, 15); then followed
the demonstration that the dictum of Burnet (2,16) was correct, namely that all
212
Physiology or Medicine 1984
antibody molecules synthesized by one given lymphocyte are identical; and

finally, towards the end of that decade, lymphocytes were shown to fall into two
classes, called T cells and B cells, existing in the body in almost equal numbers
(17, 18, 5). Only B lymphocytes, or B cells, however, can produce and secrete
antibody molecules.
Schematically, I could picture this as in Fig. 1.
Fig. 1.
What I should like you to retain from this picture is both what we know as well
as what we do not know at present. Thus, B lymphocytes are known to carry
so-called receptor molecules on their surface (about 105 identical receptors per
B cell), and when such a resting B cell is properly stimulated to divide and to
mature, its descendants will end up excreting about 2000 antibody molecules
per second, all of which are identical, and similar or identical to the receptors
that the resting B cell originally displayed. This clonal nature of antibody
formation was clearly demonstrated in the early 1970s (19, 20). The normal
antibody response of an animal to a foreign antigen involves a large number of
different clones, however, and is characterized by the polyclonal production,
usually of several hundreds of different antibody molecules (21). T lymphocytes are also known to carry receptor molecules on their surface, but firstly
these molecules are yet not well known because they have been discovered only
during the past two years and secondly, the T cells do not excrete such
molecules: these T cell receptors are antigen-recognizing molecules, but they
do not contribute to the population of freely circulating antibody molecules
which are purely B cell products. Furthermore, there are at least two different
types of T cells, one of which is called T helper cells (T H) (22) because they
The Generative Grammar of the Immune System
213
help B cells to become stimulated (or, in their absence, they deny B cells to
receive a proper stimulus); the other type is called T killer cells (T K ) (23)
because they are capable of killing other cells which they consider undesirable
(such as virus-infected cells, or cells transplanted from another individual);
moreover, as suppressor cells, they may prevent B cells from being stimulated
(6).
Thus, in this picture, B cells have the single-minded desire to express their
antibody language, but are subordinate to the T cells, that can either enhance
or suppress this capacity.
Before looking at grammar, I must briefly describe the structure of an
antibody molecule. No matter what you try to investigate in biology turns out
to become increasingly complex thus also the structure of antibody molecules. The basic element of all antibody molecules has been shown to be a Yshaped protein structure (26) of about 150,000 daltons molecular weight. This
is a three-dimensional structure, like all molecules and cells that biology has to
deal with. Three-dimensionality tends to perplex our mind which is most at
ease with one-dimensional, linear sequences, but I shall try to make a rough
two-dimensional sketch of an antibody in Fig. 2.
Fig. 2.
214
We can make some important cuts through this molecule. The vertical cut
shows symmetry. It divides the antibody molecule in two identical halves. The
two other cuts separate a so-called constant part (c) from a variable part
(v). By constant part, we mean that molecules of different antibody specificity
have this part in common (such as, for example, diphtheria antitoxin and
tetanus antitoxin). By variable part, we mean that this is the region of the
antibody molecule that determines its specificity. The two variable parts are
identical; that is to say, that the molecule, with regard to specificity, is divalent.
The difficulty we face is not to transform this sketchy two-dimensional picture
into a one-dimensional primary structure, but into a three-dimensional tertiary
structure. The primary structure (14, 15) has been clarified: the half molecule
is made up of a light polypeptide chain of about 214 amino acid residues, and a
heavy polypeptide chain of a little more than 400 amino acid residues, as in Fig.
3.
Fig. 3.
It turned out that antibody molecules of different specificity have identical

amino acid sequences in their carboxyl-ended regions, but that they vary with
respect to the amino acid sequences in the amino-ended regions of both heavy
and light chains (24). It became obvious immediately that the great diversity of
antibody molecules, the great number of different molecules which antibodies
can recognize, or in other words, the great repertoire of antibody specificities,

must result from an enormous number of varieties in the variable regions with
respect to amino acid sequences. This insight does not solve our problems,
however. It is like saying that the great variety of words or sentences in a
language results from the enormous number of varieties with respect to the
sequences of letters or of phonemes.
Interpretation in immunology remained practically as it had traditionally
been, namely that the variable region of an antibody molecule forms a threedimensional combining site, and that specificity simply means that
this combining site is complementary in shape to part of the threedimensional profile of an antigen molecule. Antigen is the word that was given,
and is still in use, for molecules that can induce the immune system to produce
specific antibodies which can recognize these antigens. Traditionally, the antibody combining site was conceived of as a cleft which recognizes a protuberance on the outer shape of an antigenic molecule, and all antibodies were
named after the antigens that they recognized, such as diphtheria antitoxin,
antisheep red blood cell antibodies, anti-TNP, etc. (25). I shall now try to give
you an impression of the size of this system of antigens and specific antibody
molecules. Let us first consider macromolecules of molecular weights exceeding
10,000 daltons; they may be polysaccharides, proteins, lipoproteins, nucleic
acids, viruses, bacteria in fact any such molecule or particle existing in the
world is an antigen to which the immune system can make specific antibodies.
Moreover, molecules such as nitrophenol, or arsonate, or any organic or
inorganic molecules you care to mention are antigenic when attached to a socalled carrier molecule, for example to a protein: the immune system will then
produce antibodies that specifically recognize these molecules, even if they
have been synthesized in a chemical laboratory without ever before having
existed in the world (1). How is this possible? For example, the immune system
of a mouse possesses no more than about 108 B lymphocytes, which would be
the maximal available repertoire of variable regions on its antibody molecules.
We realize that recognition need not be perfect, and that the same combining site might recognize, with more or less precision, a number of similar
antigens.
I shall now turn to some remarkable discoveries, made during the past 25
years, showing that the variable regions of antibody molecules are themselves
antigenic and invoke the production of anti-antibodies. Kunkel (27) showed
that monoclonal myeloma antibodies, when injected into another animal,
induce specific antibodies which recognize the particular myeloma antibodies
used, but which do not recognize any other myeloma antibodies isolated from
other myeloma patients. This work was extended by others, but mainly by
Jacques Oudin and his colleagues in Paris, who showed that ordinary antibody
molecules that arise in an immunized animal are antigenic and invoke the
formation of specific anti-antibodies (28, 29, 30, 31). In other words, the
variable region of an antibody molecule constitutes not only its combining
site, but also presents an antigenic profile (named its idiotype) against which
anti-idiotypic antibodies can be induced in other animals. Moreover, it turned
216
out that this antigenic, idiotypic profile of the variable region of a given
antibody molecule is not a single site, but consists of several distinct sites
against which a variety of different anti-idiotypic antibody molecules can be
made. These individual sites are now named idiotopes, implying that the
idiotype of one antibody molecule can be described as a set of different
immunogenic idiotopes. And finally, it has been shown that the immune system
of a single animal, after producing specific antibodies to an antigen, continues
to produce antibodies to the idiotopes of the antibodies which it has itself made.
The latter anti-idiotopic antibodies likewise display new idiotypic profiles, and
the immune system turns out to represent a network of idiotypic interactions
(7, 8, 9, 10, 11).
I shall now show, in Fig. 4, a preliminary picture of how we might vaguely
try to imagine the shape of the variable region of an antibody molecule.
Fig. 4.
This picture is an historical compromise: we uphold our antigen-centered
tradition (25) by retaining the notion of a combining site which enables the
antibody molecule to recognize the antigenic molecule that induced its production, and we simply add a number of idiotopes on the same variable region,
which are capable of inducing the production of other antibody molecules that
have combining sites recognizing these idiotopes.
We are now getting into trouble, however, when trying to interpret our
experimental results. As I said earlier, a resting B lymphocyte displays on its
surface about 100,000 identical receptor molecules which are representations of
the type of antibody molecules this B cell and its progeny will produce if
stimulated by an antigen.
217
Fig. 5.
Let us return to this picture for a moment, as in Fig. 5,
and let us make an enlargement, in Fig. 6, of a small part of the surface of this B
cell, focusing on one receptor molecule only, making the usual cuts between
constant and variable regions:
Fig. 6.
218
As you see, I have retained the traditional distinctions between the antigenrecognizing combining site, and a number of antigenid idiotopes. I have also
added an imaginary profile of an antigenic molecule, part of which is recognized by the combining site of the B cell receptor. This is the basic picture of
the selective theories of antibody formation, as most clearly formulated by
Burnet (16, 2). The antigen selects the lymphocytes by which it is recognized, and stimulates these cells to proliferate, to mature, and to secrete
antibodies with fitting combining sites. Clearly, T cell control is also involved,
as well as growth factors, maturation factor, etc., but this picture remains the
basic idea of antibody induction. Fig. 7 shows one of these antibody molecules,
which recognizes part of the surface profile (epitope) of the antigen.
Fig. 7.
Now imagine, however, that this antibody molecule (Abl), displaying both its
combining site as well as its antigenic idiotopes, is itself used as antigen. It is
then possible to envisage two situations, a and .
Fig. 8 shows, schematically, a freely circulating Abl molecule which recognizes,
and sticks to, a receptor on a B cell. The figure envisages the stimulation of two
different B cells by Abl molecules now acting as antigens. In the case, the
combining site of the receptor on a B cell recognizes an idiotope of Abl, and the
cell may be stimulated to produce the corresponding anti-idiotopic antibodies
(Ab2). In the case, however, it is the combining site of the Abl molecule
which recognizes an idiotope of the receptor on a B cell which may thus be
stimulated to produce antibodies which possess idiotopes that have a shape
that is similar to the epitope displayed by the original antigen. Experiments
have shown both these situations actually to occur. For example, if the original
antigen is insulin, and Abl is an anti-insulin antibody, then some of the antiidiotypic antibodies (Ab2) of the type show a similarity to insulin and have
219
Fig. 8.
been shown, by Sege and Peterson, to function like insulin (32). Similar results
in other systems have been obtained by Cazenave and Roland, by Strosberg, by
Urbain, and their colleagues, and by others (33, 10, 39, 35).
The point I wish to make, however, is to consider whether the two situations
and are fundamentally different, or not. Is there a difference between
saying that Abl recognizes Ab2 or that Ab2 recognizes Abl? Can we, at this
three-dimensional, molecular level, distinguish between recognizing and
being recognized? If not, it becomes meaningless to distinguish between
idiotopes and combining sites, and we could merely say that the variable region
of an antibody molecule displays several equivalent combining sites, or a set of
idiotopes, and that every antibody molecule is multi-specific. I do not have to
belabour this point which has been made repeatedly (36, 37, 38, 25). Instead, I
should now like to introduce some numerology into this discussion. How large
is the number of different antibodies that the immune system of one single
animal (be it a human or a mouse) can make? This number, during the past
few decades, has been estimated, on more or less slender evidence, to exceed
ten millions, and this enormous diversity has been designated as the repertoire of the B lymphocytes. Such a repertoire has been characterized by
Coutinho as complete (39). Completeness means that the immune system
can respond, by the formation of specific antibodies, to any molecule existing in
the world, including, as I said earlier, to molecules that the system has never
220
before encountered. Immunologists sometimes use words they have borrowed

from linguistics, such as immune response. Looking at languages, we find
that all of them make do with a vocabulary of roughly a hundred thousand
words, or less. These vocabulary sizes are a hundred-fold smaller than the
estimates of the size of the antibody repertoire available to our immune system.
But if we consider that the variable region that characterizes an antibody
molecule is made up of two polypeptides, each about 100 amino acid residues
long, and that its three-dimensional structure displays a set of several combining sites, we may find a more reasonable analogy between language and the
immune system, namely by regarding the variable region of a given antibody
molecule not as a word but as a sentence or a phrase. The immense repertoire of
the immune system then becomes not a vocabulary of words, but a lexicon of
sentences which is capable of responding to any sentence expressed by the
multitude of antigens which the immune system may encounter.
At this point, I shall make a quotation from Noam Chomsky (3) concerning
linguistics: The central fact to which any significant linguistic theory must
address itself is this: a mature speaker can produce a new sentence of his
language on the appropriate occasion, and other speakers can understand it
immediately, though it is equally new to them Grammar is a device that
specifies the infinite set of well-formed sentences and assigns to each of these
one or more structural descriptions. Perhaps we should call such a device a
generative grammar which should, ideally, contain a central syntactic component , a phonological component and a semantic component. That is the
end of my quotation. For the size of the set of possible sentences in a language,
Chomsky uses the word open-endedness, and I now think that openended is the best description also of the completeness of the antibody
repertoire. As for the components of a generative grammar that Chomsky
mentions, we could with some imagination equate these with various features
of protein structures. Every amino acid sequence is a polypeptide chain, but
not every sequence will produce a well-folded stable protein molecule with
acceptable shapes, hydrophobicity, electrostatics, etc. Some grammatical rules
would seem to be required. It is harder, however, to find an analogy to
semantics: does the immune system distinguish between meaningful and meaningless antigens? Perhaps the distinction between self and non-self is a
valid example. It would seem, at first sight, that the immune response to a
sentence presented by an invading protein molecule is merely to select, from
amongst its enormous preformed antibody repertoire, a suitable mirror image
of part of this antigenic sentence. As you will know, Leonardo da Vinci wrote
his private journal in the mirror image of ordinary handwriting. It is difficult,
without considerable practice, to write and read mirror handscript. Let me
show you an example in Fig. 9.
221
Fig. 9.
On the following two figures, I shall use the device of showing ordinary letters
in black, and of using greyly marked zones to indicate the mirror images of
these letters.
Fig. 10.
Fig. 10 shows an antigenic sentence, part of which is mirrored by Abl. The
anti-idiotopic Ab2 mirrors part of Abl, but bears no relation to the original
antigen. Fig. 11 is a little more complex. Here, the original antigen is insulin,
and the letter-sequence OF INSULIN DE represents its active site, which is
mirrored by Abl. Of the two anti-idiotopic antibodies shown, Ab2a and Ab2,
the latter mirrors this mirror image, and thus displays the active site of insulin
(32).
Fig. II.
I should perhaps again emphasize that the sentences representing antibodies

possess partial mirror images of an antigenic sentence. These antibodies are not
echoes of the invading antigen, but were already available to the animal in its
repertoire of B cells before the antigen arrived. This is the important insight
that followed the introduction into immunology of the selective theories in the
1950s. Also, I must emphasize another important quantitative aspect of the
situation facing the immune system. It has been estimated that one human
individual produces about 10,000 different proteins, such as enzymes, hormones, cell surface proteins, etc. At the same time, we estimate that the
immune system maintains a repertoire exceeding ten million different proteins,
namely antibody molecules. This is a thousand-fold more than all other body
proteins taken together. Man and mice, normally, have about ten milligrams of
antibodies in a milliliter of their blood. Thus, a normal human possesses
between about 50 to 100 grams of freely circulating antibodies, called immunoglobulins. If we divide this figure by l07 different specificities, we are still left
with an average of 5 to 10 micrograms of every specificity in the available
13
repertoire, representing an average of about 310 monoclonal antibodies of
every specificity. For mice that are 3000 times smaller, we would have to divide
these figures by 3000, which would still leave a mouse with an average of 2 to 3
nanograms of antibodies of every of l07 specificities. That even such nanogram
quantities of monoclonal anti-idiotypic antibodies, when introduced into mice,
produce remarkable effects has been shown by Rajewsky and his colleagues
(40, 41, 42).
223
I should therefore like to conclude that in its dynamic state our immune
system is mainly self-centered, generating anti-idiotypic antibodies to its own
antibodies, which constitute the overwhelming majority of antigens present in
the body. The system also somehow maintains a precarious equilibrium with
the other normal selfconstituents of our body, while reacting vigorously to
invasions into our body of foreign particles, proteins, viruses, or bacteria, which
incidentally disturb the dynamic harmony of the system.
The inheritable deep structure of the immune system is now known:
certain chromosomes of all vertebrate animals contain DNA segments which
encode the variable regions of antibody polypeptides. Furthermore, experiments in recent years have demonstrated the generative capacities of this
innate system. In proliferating B lymphocytes, these DNA segments are the
targets for somatic mutations, which result in the formation of antibody variable regions which differ, in amino acid sequences, from those encoded by the
stem cell from which these B cells have arisen (43, 44, 45, 46, 47). The
experiments showed that it was still possible, however, to identify the original
stem cell genes that must have undergone these mutations. Expressed in
linguistic terms, such investigations belong to the etymology of the immune
system.
As immunologists, we should like to know the semantics of the inheritable
gene structures. What is the meaning of the basic lexicon, or what are the
specificities of the antibodies, B cell receptors and T cell receptors as encoded
in the genes of our germ cells? It is known that B cells recognize the language of
the T cell receptors. I have said so little about the latter because T cell
receptorology is still in too early a stage of development. An immune system of
enormous complexity is present in all vertebrate animals. When we place a
population of lymphocytes from such an animal in appropriate tissue culture
fluid, and when we add an antigen, the lymphocytes will produce specific
antibody molecules, in the absence of any nerve cells (48). I find it astonishing
that the immune system embodies a degree of complexity which suggests some
more or less superficial though striking analogies with human language, and
that this cognitive system has evolved and functions without assistance of the
brain.
It seems a miracle that young children easily learn the language of any
environment into which they were born. The generative approach to grammar,
pioneered by Chomsky (4), argues that this is only explicable if certain deep,
universal features of this competence are innate characteristics of the human
brain. Biologically speaking, this hypothesis of an inheritable capability to
learn any language means that it must somehow be encoded in the DNA of our
chromosomes. Should this hypothesis one day be verified, then linguistics
would become a branch of biology.
224
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Press (1959)
3. Chomsky, N., Current Issues in Linguistic Theory. Janua Linguarum, Series minor. Mouton,
The Hague (1964)
4. Chomsky, N., Language and Mind. Harcourt Brace Jovanovich, New York (1972)
5. Greaves, M.F., Owen, J.J.T., and Raff, M.C., T and B Lymphocytes. American Elsevier, New
York (1974)
6. Golub, E.S., The Cellular Basis of the Immune Response. Sinauer Associates, Massachusetts
(1977)
7. Immunoglobulin Idiotypes. Eds. Janeway, C., Sercarz, E.E., and Wigzell, H., Academic Press,
New York (1981)
8. Idiotypes: Antigens on the Inside. Ed. Westen-Schnurr, I.. Editiones Roche, Base1 (1982)
9. Immune Networks. Eds. Bona, C.A., and Khler, H., Annals N.Y. Acad. Sci. 4 1 8 (1983)
10. Idiotypy in Biology and Medicine. Eds. Khler, H., Urbain, J., and Cazenave. P.-A., Academic Press, New York (1984)
11. The Biology of Idiotypes. Eds. Greene, M.J., and Nisonoff, A., Plenum Press, New York (1984)
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14. Porter, R.R., Biochem. J. 73, 119 (1959)
15. Edelman. G.M., J. Amer. Chem. Soc. RI, 3155 (1959)
16. Burnet, F.M., Aust. J. Sci. 20, 67 (1957)
17. Miller, J.F.A.P., and Mitchell, G.F., Proc. Natl. Acad. Sci. USA 59, 296 (1968)
18. Raff, M.C., Immunology 19, 637 (1970)
19. Bosma, M., and Weiler, E.,J. Immunol. 104, 203 (1970)
20. Lefkovits, I., Eur. J. Immunol. 2, 360 (1972)
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Fluids. Ed. Peeters, H., p. 559, Pergamon Press, Oxford (1980)
22. Claman, H.N., Chaperon, E.A., and Triplett, R.F., Proc. S O C. Exp. Biol. Med. 1 2 2 , 1 1 6 7
(1966)
23. Govaerts, A., J. Immunol. 85, 516 (1960)
24. Hilschmann, N., and Craig, L.C., Proc. Natl. Acad. Sci. USA 53, 1403 (1965)
25. Coutinho, A., Forni, L., Holmberg, D., Ivars, F., and Vaz, N., Immunol. Rev., ed. Mller, G.,
79, 151 (1984)
26. Valentine, R.C., and Green, N.M., J. Mol. Biol. 27, 615 (1967)
27. Slater, R.J., Ward, S.M., and Kunkel, H.G., J. Exp. Med. 101. 85 (1955)
28. Oudin, J., and .Michel, M., C.R. Acad. Sci. Paris 257, 805 (1963)
29. Oudin, J., and Michel, M., J. Exp. Med. 130, 595, 619 (1969)
30. Kunkel, H.G., Mannik, M., and Williams, R.C., Science 140, 1218 (1963)
31. Gell, P.G.H., and Kelus, A.S., Nature 201, 687, (1964)
32. Sege K. and Peterson P.A., Proc. Natl. Acad. Sci. USA 75, 2443 (1978)
33. Jerne, N.K., Roland, J., and Cazenave, P.-A., EMBOJ. I, 243 ( 1 9 8 2 )
34. Strosberg, A.D., Couraud, P.-O., and Schreiber, A., Immunol. Today 2, 75 (1981)
35. Guillet, ,J.G., Kaveri, S.V., Durieu, O., Delavier, C., Hoebeke, J., and Strosberg, A.D., Proc.
Natl. Acad. Sci. USA, 82, 1781 (1985)
36. Richards, F.F., and Konigsberg, W.H., Immunochemistry 10, 545 (1973)
37. Varga, J.M., Konigsberg, W.H., and Richards, F.F., Proc. Natl. Acad. Sci. USA 7 0 , 3 2 6 9
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38. Jerne, N.K., Immunol. Rev., ed. Mller, G., 79, 5 (1984)
39. Coutinho, A.. Ann. Immunol. (Inst. Pasteur) 1 3 1 D , 235 (1980)
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41. Rajewsky, K., and Takemori, T., Ann. Rev. Immunol. 1, 569 (1983)
42. Mller, C.E., and Rajewsky, K., J. Exp. Med. 159, 758 (1984)
43. McKean, D.M., Hppi, K., Bell, M., Staudt, L., Gerhard, W., and Weigert, M., Proc. Natl.
Acad. Sci. USA 81, 3180 (1984)
44. Rdikoff, S., Pawlita, M., Pumphrey, J., and Heller, M., Proc. Natl. Acad. Sci. USA 81, 2162
(1984)
45. Bothwell, A.L.M., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewsky, K., and Baltimore,
D., Cell 24, 625 (1981)
46. Sablitzky, F., Wildner, G., and Rajewsky, K., EMBOJ. 4, 345 (1985)
47. Sims, J., Rabbitts, T.H., Estess, P., Slaughter, C., Tucker, P.W., and Capra, J.D., Science 2 1 6 ,
309 ( 1982)
48. Mishell, R.I., and Dutton, R.W., J. Exp. Med. 126, 423 (1967)
FROM THE STRUCTURE OF ANTIBODIES TO

THE DIVERSIFICATION OF THE IMMUNE
RESPONSE
Nobel lecture, 8 December, 1984
by
CSAR MILSTEIN
Medical Research Council Laboratory of Molecular Biology, Hills Road,

Cambridge, U.K.
Cuando se acerca elfin, escribi Cartaphilus, ya no quedan imgenes de1

recuerdo; slo quedan palabras. Palabras, palabras desplazadas y mutiladas,
palabras de otros, fu la pobre limosna que le dejaron las horas y los
siglos.
J. L. Borges
When an animal is infected, either naturally or by experimental injection, with
a bacterium, virus, or other foreign body, the animal recognises this as an
invader and acts in such a way as to remove or destroy it. There are millions of
different chemical structures that the animal has never seen and yet which it is
able to recognise in a specific manner. How is this achieved? Scientists have
been fascinated by this question for most of this century, and we continue to be
fascinated by the intricacies and complexities that still need to be clarified.
Even so, looking back over the years since I myself became involved in this
problem, progress in the understanding of the process has been phenomenal.
Suffice it to remind our younger colleagues that 20 years ago we were still
trying to demonstrate that each antibody differed in its primary amino acid
sequence.
What attracted me to immunology was that the whole thing seemed to
revolve around a very simple experiment: take two different antibody molecules and compare their primary sequences. The secret of antibody diversity
would emerge from that. Fortunately at the time I was sufficiently ignorant of
the subject not to realise how naive I was being.
Back in 1962, when I had by accident become the supervisor of Roberto Celis
in Argentina, it occurred to me that antibody diversity might arise from the
joining by disulphide bridges of a variety of small polypeptides in combinatorial patterns. I dont know whether anybody else had the same idea at that time,
but of-all the prevailing theories about antibody diversity that I am aware of,
this is one that was widest of the mark. I hold it to my credit that I never put it
into print. But it was of great value to me as it provided an intellectual
justification to work on disulphide bonds of antibodies. By the time I joined the
Laboratory of Molecular Biology in 1963, the model of two heavy and two light
248
From the Structure of Antibodies
249
Light
Fig. 1. Antibodies are made of two or more pairs of heavy and light chains joined by disulphide
bonds. Each chain has two regions. The variable region differs in structure from one antibody to
another and contains the combining site. The antibody combining site is located at the tips of a Yshaped three-dimensional structure. The constant region is invariant within a given class or
subclass, and is responsible for effector functions (complement binding, attachment to and transport across membranes etc). The number and position of the interchain disulphide bonds is
characteristic for the different classes and subclasses. In this figure, the structure depicted is the
mouse myeloma protein MOPC 21 which was the subject of much research in our laboratory.
chains joined by disulphide bonds (Fig. 1) had been established (l), and I was
eager to accept Dr. Sangers proposal that I should engage in studies of
antibody combining sites.
The nature of antibody diversity
At first I looked for differences in fingerprints of digests of iodinated antibodies
directed against different antigens. The pattern that emerged from those studies implied that purified antibodies were too complex and differed only in a
subtle quantitative way from the totally unfractionated immunoglobulin. I
never published those results, which only led me to the conviction that the
protein chemistry of antibodies at that level was too difficult to tackle, and that
a different approach was needed.
The study of the amino acid sequence around the disulphide bonds of the
immunoglobulins was my own short-cut to the understanding of antibody
diversity. I soon recognised the existence of what appeared to be a variable
250
Physiology or Medicine I984
disulphide bridge and a common disulphide bridge (2,3), but the full meaning
of that observation only became obvious when Hilschmann and Craig described the variable and constant halves of antibody light chains (4). The
variable half contained one disulphide bond, and the constant half the other.
This was followed, in later studies with Pink, Frangione, Svasti and others, by
the observation of the repeating pattern of similar S-S loops as a distinctive
common architectural feature of the different classes and subclasses of immunoglobulin chains. What distinguished them from each other was the diversity
of interchain S-S bonds (5).
The period between 1965 and 1970 was full of excitement, both at the
experimental and theoretical level. How were these variable and constant
regions going to be explained? It was now not only a problem of millions of
antibody structures, but that in addition those millions of structures were part
of a polypeptide which otherwise had an invariant primary sequence encoded
by only one or very few genes. How to solve the puzzle? Dreyer and Bennett (6)
suggested that there were thousands of genes in the germline and that the
paradox was easy to solve if we postulated a completely unprecedented scheme.
This became known as the two genes-one polypeptide hypothesis. At the
time we did not like that, and proposed a mechanism of hyper-mutation
operating on selected segments of a gene (7). There were other ideas at the time
to generate antibody diversity. One of them, widely discussed in a Cold Spring
Harbor Symposium in 1967, was based on a mechanism of somatic cross-over
between gene-pairs (8). It was very exciting for me when soon after the
symposium I could show that in the human kappa chains at least three genes
must be involved (9). The predicted thousands of V-regions could be grouped
into a small number of families or subgroups. The fact that these families were
encoded by non-allelic V-genes (10) - coupled to the genetics of the C-region,
which indicated a single Mendelian C-gene - provided the experimental
evidence that convinced me and many others that the two genes-one polypeptide hypothesis was inescapable.
After that, there was a period of consolidation and extension of the results.
The concept of V-gene families or subgroups became firmly established, as was
the existence of hypervariable residues within the variable segment (9,ll).
Crystallographic data showed that such hypervariable residues were near to
each other, justifying the idea that they were part of the antibody combining
site. This was directly shown with crystals of myeloma protein-antigen complexes (12). The work with myelomas was not only totally vindicated, but also
generally accepted. The idea of separate pools of V- and C-genes that were
under continuous expansion and contraction was the last element added to the
picture. By 1970 we became convinced that the section of the genome involved
in the coding of immunoglobulin chains undergoes an expansion-contraction
evolution: that the number of individual genes coding for basic sequences is not
large, and that it varies in different species and even within species at different
stages of its own history. The task of providing for the endless variety of
individual chains is left to somatic processes (13).
251
Light chain mRNA and the signal for secretion

I now began to feel a bit restless. It seemed that protein chemistry alone was
not going to get us much further. Furthermore, there was a lot of excitement in
the laboratory with the new methods for sequencing RNA being developed by
Sanger and his group. Perhaps even more important, one of my closest friends
at the laboratory, George Brownlee, was beginning to feel that the time was
ripe to attack molecules more complicated than 5S or 6S RNA. So we joined
forces in an attempt to isolate immunoglobulin mRNA. This was a difficult
problem and when Georges new research student, Tim Harrison, joined us we
decided to move from solid tumours (14) to cell lines in culture which were
kindly provided by colleagues from the Salk Institute (15). The first important
breakthrough in the field was a paper reporting in vitro synthesis of immunoglobulin light chains (16). We immediately set to work to follow up that approach,
and to our delight ran into the unexpected observation of the existence of a
biosynthetic precursor of light chains. Further experiments led us to propose
that the extra N-terminal sequence was a signal for vectorial transport across
membranes during protein synthesis. That was the first evidence which indicated that the signal for secretion was an N-terminal segment, rapidly cleaved
during protein synthesis (17,18).
However, our major concern remained the sequence of the messenger RNA
for the light chains. In those days there was no DNA sequencing, only mRNA
sequencing via elaborate fingerprints of radioactive mRNA. Every radioactive
messenger preparation on which we could do sequence analysis involved the
32
at levels of 50 mCi. So there we
labelling of cells with inorganic P- hosphate
p
were, dressed up in our new-style laboratory coats (namely heavy lead aprons),
behind a thick plastic screen, labelling cells and then frantically working up our
messenger purification procedures and performing fingerprinting experiments,
before the inexorable radioactive decay. Although we didnt go very far in our
sequencing, we could isolate oligonucleotides that corresponded to the protein
sequences (19). Among these were oligonucleotides spanning the V- and Cregions, demonstrating that the protein chain was made from a single messenger RNA and that, therefore, integration of the V- and C-genes did not
take place during or after protein synthesis (20). At this stage the radioactive
approach was stopped and we tested alternative methods for the sequencing of
mRNA, using synthetic primers and cDNA synthesis. This approach went on
in the background while our main efforts were moving in a different direction.
Eventually however, it paid off (21). I will come back to that later, because it
forms part of my story.
Spontaneous somatic mutants of a myeloma protein

The introduction of tissue culture methods to our laboratory had a major
impact on the direction of our research. With my new research student, D. S.
Secher, and soon after with R. G. H. Cotton, we decided to embark on an
analysis of the rate and nature of somatic mutation of myeloma cells in culture.
We were hoping that we might reveal a high rate of mutation of the hypervaria-
252
Fig. 2. Protocol used for the screening of the isoelectric focusing pattern of the immunoglobulin
secreted by 7,000 clones of P3 myeloma cells. Mutants were detected and their primary defect
analysed by amino acid and mRNA sequence analysis. The results are described in Table 1 (taken
from Ref. 23).
ble segments. (The protocol is described in Fig. 2.) A continuous culture was
grown for a minimum of three months to allow mutants to accumulate, and
individual cells were taken and grown as colonies. These were incubated with
labelled amino acids and the radioactive immunoglobulin analysed to detect
mutants with altered electrophoretic properties. Our first structural mutant
appeared after a few thousand clones (22), and the final analysis of 7,000
individual clones gave us a pool of mutants which are described in Table 1. We
were believed that this elaborate experiment provided the first evidence at the
protein and nucleic acid levels of the existence of somatic mutations of mammalian cells (23). Furthermore, the rate at which these mutations occurred suggested an important role in the generation of diversity (24). But the mutations
were not in the variable region, and we were forced to conclude that in the cells
253
IF3
IF4
we were studying, there was no evidence for a hypermutable segment. So that

in a sense we were back to square one.
Hybrid myelomas
While this work was going on, Cotton was preparing another type of experiment which turned out to be more important than we anticipated (25). This
involved the fusion of two myeloma cells in culture (Fig. 3). That fusion
demonstrated that the phenomenon of allelic exclusion was not dominant. On
the contrary, fusion of two myeloma cells gave rise to a hybrid co-dominantly
expressing the antibody chains of both parents. In addition, we proved that the
expression of V- and C-regions was cis, probably because the V- and Csegments were already integrated at the DNA level by a translocation event in
the precursors of plasma cells. This was in contrast to the assembly of heavy
and light chains, which combined with each other to give rise to hybrid
molecules.
Armed with these results, I went to Base1 to give a seminar, and the
important consequence was that Georges Khler came to Cambridge. He
joined in our main research project of looking at somatic mutants in immunoglobulin-producing cells, and in the other minor project concerning the phenotypic expression of somatic cell hybrids prepared between myelomas and
myeloma mutants. It became increasingly clear that we could not go on looking
for mutants by the procedure we had employed before, and the only way ahead
was to use a culture of a myeloma cell line capable of expressing an antibody.
Mutants from that cell could then be made based on the antibody activity.
Although at that time there had been reports in the literature of myeloma cells
254
Fig. 3. Co-dominant cis expression of antibody genes in hybrids of myeloma cells. The diagram
describes data taken from Ref. 25.
capable of fulfilling that role, none proved suitable in our hands. The myeloma
cell line P3 (MOPC 21) would have been ideal from a chemical point of view,
because at the time sequencing the protein was a major undertaking and
we knew how to deal with MOPC 21. But we were unable to find a suitable
antigenic binding activity to this myeloma protein. We failed, but others who
were pursuing similar types of experiments succeeded. Scharff and his coworkers were the first to demonstrate that one can isolate somatic mutants of a
variable region in that way (26).
255
Fig. 4. The first successful hybridoma was prepared from cells from a mouse immunized with sheep
red blood cells (SRBC) (56). These were fused to a myeloma cell line producing the IgG protein
MOPC 21 (Fig. 1) growing in tissue culture and made resistant to azaguanine. Hybrids
selected by growth in HAT medium (57).
were
And yet in a funny way our lack of success led to our breakthrough; because,
since we could not get a cell line off the shelf doing what we wanted, we were
forced to construct it. And the little experiment being done in the background
concerning hybridization between myeloma cells developed into a method for
256
the production of hybridomas. Thus, as illustrated in Fig. 4, instead of hybridizing two myelomas, we hybridized a myeloma and an antibody producing
cell. The resultant hybrid was an immortal cell capable of expressing the
Fig. 5. Most generally used protocol for the derivation of hybridomas (taken from Ref. 58)
257
antibody activity of the parental antibody-producing cell the immortality

being acquired from the myeloma.
So finally, we were able to obtain a continuously-growing cell-line expressing
a specific antibody and use it to search for mutants of the hypervariable region.
This was undertaken by my research student, Deborah Wilde. While she got
more and more discouraged by her lack of success in what she called looking
for a needle in a haystack, it dawned on me that it was up to us to demonstrate
that the exploitation of our newly-acquired ability to produce monoclonal
antibodies la carte was of more importance than our original purpose.
After our early success we ran into technical difficulties and could not get our
Table 2. Selected list of monoclonal antibodies derived in our laboratory
258
fusion experiments to work for quite some time. Then Giovanni Galfr, who
had recently joined us, got us out of the deadlock when he discovered that one
of our stock solutions had become contaminated with a toxic substance. After
this an improved reliable protocol was developed (Fig. 5) and quick progress
made towards the first practical applications of the technology. For several
years I shelved the antibody diversity problem to demonstrate the practical
importance of monoclonal antibodies in other areas of basic research and in
clinical diagnosis (Table 2). W e were able to show that the hybrid myelomas
were capable of being used for the production of standard reagents such as antihistocompatibility antigens (27) and anti-Ig-allotypes (28). The procedure was
ideally suited to the study of cell surface and tumour antigens and to providing
reagents for cell fractionation (29-3 1). Monoclonal antibodies produced in
this way were suitable for radioimmunoassays and for neuropharmacology
(32), as blood group reagents (33) and for large scale purification of natural
products (34). W e also extended the hybrid myeloma technology to a second
species-the rat (35) and to the production of bi-specific immunoglobulins
(hybrid-hybridomas) (36).
Genetic origin of antibody diversity
In the period 1970- 1975, a considerable effort was being made to measure the
number of germline genes coding for the variable regions of immunoglobulin
chains. Our own contributions started when we persuaded Terry Rabbitts to
join us. After considerable effort and a lot more radioactivity we obtained
results indicating that the number of germ line genes was not much higher than
would be predicted from our understanding of subgroups, and this view was
shared and reinforced by parallel work being conducted by others (37,38). By
1976 this view was gaining general support (39). But then the impact of the
recombinant DNA revolution began to be very strongly felt. Within a few
years, and largely through the work of Tonegawa, Leder, Rabbitts, Hood,
Baltimore, and others, a coherent picture of the arrangement and rearrange-
259
ment of immunoglobulin genes and their involvement in the generation of

diversity began to emerge (40).
The precursors of the antibody producing cells do not express an immunoglobulin, but during their differentiation into pre-B cells and B cells, they
express first the heavy chain and then the light chain (Fig. 6). The first
antibody produced is membrane bound and functions as a receptor
molecule, which receives antigenic signals. Triggered cells divide and differentiate to antibody producing cells and memory cells. These events at the cellular
level are correlated with changes in the DNA structure (Fig. 7). The germline
DNA contains the V- and C-genes on different DNA fragments, as predicted.
But in addition, there are further fragmentations, and only some of them are
shown in the figure. Light and heavy chains can only be transcribed and
translated when certain fragments (one of the V and J in light chains, V, D and
J in heavy chains) are integrated by a deletion mechanism. During this process
of integration enormous diversity is generated.
To theorize about the genetic origin of antibody diversity was a must
among molecular immunologists for quite a number of years. How do those
theories contrast with the reality of today? The hard experimental facts made
possible by the methodological advances in molecular biology show that, while
none of them was right, most of them contained at least a grain of truth. There
were two major currents of opinion. One consisted of germline theories whereby all the diversity was inherited as genes present in the germline. The other
included somatic diversification theories, whereby somatic processes were responsible for the generation of diversity, starting from a small number of
germline genes. As it turns out, the genetic mechanisms responsible for the
Fig. 7. Genetic arrangement of immunoglobulin genes in the germline. During differentiation into
pre B cells and B cells large deletions of DNA lead to the integration of fragments (rearranged
genes). Further proliferation leads to somatic mutation of the integrated gene and this is of major
importance in the maturation of the response.
260
Table 3. Mechanisms that generate antibody diversity

I. GERMLINE: multiple V-gene segments
2. COMBINATORIAL: a) Different combinations of V-(D)-J
b) Different combinations Of V H a n d VL
3. JUNCTIONAL: variation at at V-J, V-D, and D-J boundaries
4. SOMATIC POINT MUTATION: nucleotide substitutions throughout the V region
generation of diversity include a little bit of everything (Table 3). There are
between 50 and 300 gene fragments in the germline encoding the light or the
heavy chains. The number varies from species to species. So there is a considerable germline contribution. Recombination and gene conversion arc probably
important genetic events in the evolution and maintenance of that germline
gene pool. We still do not know whether these events are significant as somatic
generators of diversity (41). As shown in Fig. 7, the V-region is encoded by V,
D and J segments (heavy chain) and V and J segments (light chain). Their
combinatorial integration into a single gene, although an important component
of the generation of diversity, is not the critical mechanism predicted by the
mini gene hypothesis (42). Al so important is the diversity generated during the
joining process, and this contains an element of the errors and aberrations
during repair predicted by other theories (7,43). And then there are the somatic
point mutations for which a mechanism remains to be elucidated. It may
involve error-prone repair enzymes (7), genetic hot-spots (24), appropriate
selection either by antigen (44) or by other network elements (45), or quite
possibly by a mixture of all or some of these. The instructional theories were
largely forgotten as soon as the chemical diversity of antibodies was established
(46). Yet they also may contain a grain of truth. It has recently been proposed
that peptide segments of the antigen which appear to be mobile are better
immunogens, presumably because they adapt their structure to a predefined
antibody structure (47,48). It is also possible that to some extent the antibody
combining site itself has a certain degree of mobility, which has a limited
capacity to accommodate its own structure to that of the antigen. Of course
dynamic adaptation has a price to pay in terms of affinity. Adaptability should
not be confused with the generation of specificity. As I discuss below, an
improved fit of binding to the ligand is the result of somatic mutation and
antigenic selection.
Molecular analysis of an immune response using monoclonal antibodies and mRNA
sequencing
Let us return to an animal that is being immunized with a certain substance.
The immune system recognizes the substance as foreign, and the B cells are
triggered to produce antibody (Fig. 8). The different antibodies are secreted
and mixed in the serum. The individual antibody molecules are extremely
similar and once mixed cannot be separated from each other. For this reason,
and until the advent of the hybridoma technology, it was impossible to study
the diversity of the antibody response to a given immunogen. The derivation of
immortal cell hybrids solved this problem, because it affords individual anti-
261
Fig. 8. The dissection of the immune response by the hybridoma techniquc. When an animal is
injected with an immunogen the animal responds by producing an enormous diversity ofantibody
structures directed against different antigens, different determinants of a single antigen, and even
different antibody structures directed against the same determinant. Once these are produced they
are released into the circulation and it is next to impossible to separate all the individual
components present in the serum. But each antibody is made by individual cells. The immortalization of specific antibody-producing cells by somatic cell fusion followed by cloning of the appropriate hybrid derivative allows permanent production of each of the antibodies in separate culture
vessels. The cells can be injected into animals to develop myeloma-like tumours. The serum of the
tumour-bearing animals contains large amounts of monoclonal antibody.
262
bodies separately produced, in culture vessels and as mouse myelomas. This

permits dissection of the individual components of the antigen. Monoclonal
antibodies prepared against hitherto undefined cellular components can themselves be used to identify the chemical nature of those components, to probe for
their function, and later for use as reagents for diagnostic and therapeutic
purposes. These are the fundamental properties behind the most important of
the general applications of monoclonal antibodies. When we started to explore
these applications, and until some years ago, it was possible to some extent to
summarize the main results obtained (49). In recent years their application to
basic research, clinical biochemistry, medical therapy, and in industry has
been so widespread that I do not intend even to attempt to discuss it any
further here.
Fig. 9. Derivation of monoclonal antibodies at the onset and during the maturation of the response
to oxazolone.
263
Fig. IO. Avidity of monoclonal antibodies 7 and 14 days after immunization. Haptenated phage
inhibition (HPI) per g of anti-phlOx immunoglobulin from supernatants of IgG-secreting hybridomas. Those on the left were from day 7 and those on the right from day I4 fusions. Black circles
represent oxazolone idiotype-positive IgG and open circles represent idiotype-negative IgG (taken
from Ref. 50).
Different antibodies recognize different antigenic determinants of the immunogen, and the recognition of each determinant is complex in itself (Fig. 8).
It has been known for a long time that even the simplest antigenic determinants
are recognized by an unknown variety of antibody molecules. Monoclonal
antibodies can be made pure and used to answer the old questions of how
complex the collection of antibody molecules produced by the animal as a
response to a particular antigen is, and how the individual molecules differ
from each other. This brings me back to sequencing messenger RNA.
While in the late 70s the excitement about monoclonal antibodies and DNA
recombinant methods was simmering, Pamela Hamlyn was quietly adapting
Sangers fast DNA sequencing methods to the sequencing of light chain
mRNA. Her eventual success (21) added to our capacity to derive cell lines
secreting monoclonal antibodies to a predefined antigen, and to our ability to
264
Physiology o-r Medicine 1984
sequence quickly the messenger RNA of the antibody molecule they produce.
So, instead of asking the question What is the nature of antibody diversity?,
we were now in a position to ask the question How do antibodies diversify
during an immune response? In other words, how, in real life in the animal,
are all those genetic events capable of producing antibody diversity actually
operate in response to an antigenic stimulus?
In collaboration with Matti Kaartinen, Gillian Griffiths and Claudia Berek,
we have been conducting a study of the response to the hapten phenyl oxazolone (50,51). The essence of the experiment is described in Fig. 9. The hapten
conjugated to chicken serum albumin as carrier is injected into mice, and 7
days and 14 days later animals are sacrificed, hybridomas are prepared and a
number of random clones isolated in each case. Other animals are left for a
couple of months, and hybridomas of the secondary response arc prepared.
Hybridomas prepared 7 days and 14 days after primary immunization are
compared in Fig. 10. Each point on the figure represents the avidity of each one
of 32 monoclonal antibodies. The mixture of antibodies at each stage, as a first
approximation, represents a cross-section of the complexity of a typical antiserum. The average titres of the antibodies at both stages are not very different,
although the day 14 average is slightly higher. This is as expected. The
antibody titre of an antiserum, as well as its average avidity, increases during
the course of an immunization. It is what we refer to as the maturation of the
response. What distinguishes the results of the day 7 and day 14 is that while
the day 7 results cluster around the average, the scatter at day 14 is much
wider.
Since each monoclonal antibody was the product of an immortal hybridoma,
we could go one step further and study the total amino acid sequence of each
one of these monoclonal antibodies. Better still, we could study the sequence of
Fig. 1 1 . mRNA sequencing strategy. Synthetic oligonucleotide primers designed to pair with
defined bases within segments of mRNAs were used to initiate reverse transcription. Using
dideoxynucleotides, specific stops in the cDNA can be generated and the nucleotide sequence
determined by gel methods (taken from Ref. 59).
265
the mRNA coding for each amino acid sequence. This not only provided more
information, but was also technically simpler. To do so, RNA was prepared
from the hybridoma cells and direct sequencing done on the impure messenger
preparations, as shown in Fig. 11. In this way, sequences of antibodies at
different stages of the immune response could be compared.
What we have learned from this is that the majority of antioxazolone
antibodies at day 7 express a single set of germline V-genes taken from the total
pool of over 100 for each of the two chains (Fig. 12). This pair of germline genes
(which we refer to as VH-Ox1 and Vk-Ox1) are at this stage expressed in their
unmutated form. The few differences between them arise by junctional diversity - that is the variations introduced during integration of the DNA fragments
V, D and J which make up the variable region of the antibodies. At day 14 the
same germ line genes VH-Ox1 and Vk-Ox1 still seem to dominate the response.
Fig. 12. Diagrammatic comparison of the mRNA sequences from anti-phOx-secreting hybridomas
derived at different stages after immunization with Ox-CSA. Only sequences closely related to the
prototype are shown. The variable region sequences of each hybridoma have been compared with
the sequences of V H -Ox1 and V h -Ox1 respectively. Unbroken horizontal lines denote identical
sequences, broken lines represent extensive sequence differences. A black circle indicates that these
changes predict an amino acid difference at this position. Complementarity determining regions
(CDR-1, -2, -3) have been marked as have the D and J regions. Where different J segments are
observed these are represented accordingly. Dissociation constants determined by fluorescence
quenching (Kd in moles/litre) are shown on the right (taken from Ref. 51).
266
However, in sharp contrast to day 7, the day 14 antibodies express a small

number of point mutations which are responsible for a significant increase in
affinity for the same hapten. In other words, as the response matures, new
somatic mutants appear in a seemingly endless variety.
The antibodies obtained during the secondary response, expressing the
germline gene combination characteristic of the primary response, show a
further small increase in point mutations (Fig. 12). However, the most important feature of the secondary response is a shift towards other germline genes
(see Table 4).
It appears therefore that the development and maturation of the immune

response to oxazolone - which we take as a model system - proceeds basically
in three stages. In the first the majority of the antibody reflects a very restricted
choice from a vast repertoire of germline gene combinations, self-selected for
their capacity to bind the antigen. In the second stage, cells expressing these
combinations proliferate, and during this proliferation mutants arise which
improve the affinity of the antibody for the antigen. In the third stage, as the
first type of germline gene combinations and their mutants reach a certain limit
of dissociation constants, new germline gene combinations and somatic mutants arc selected for further improvements. Of course the three stages are not
absolutely separate and all three processes overlap to a certain extent. In many
ways, the system behaves as a Darwinian system, where adaptation is an
improvement in antigen binding. It remains to be seen to what extent other
regulatory constraints are critical to the process.
From monoclonal antibodies to antibody engineering
The immortalization of antibody-producing cells not only allows the permanent supply of an antibody of a constant chemical structure but, more important, affords all the advantages that can be derived from the techniques of cell
culture and somatic cell genetics. The most obvious is cell cloning, and this has
been at the root of the explosion in the use of this technology. And yet the
derivation of cell lines producing specific antibodies cannot go beyond the
immortalization of what already exists. We select hybrids producing monoclonal antibodies of desired properties, but if the immunized animal does not
make it, there is no way of immortalizing it. Fortunately we can go further.
Hybridomas are established cell lines and are therefore capable of other in
vitro manipulations using somatic cell genetic and molecular engineering
techniques. We are at the beginning of a new era of immunochemistry, namely
the production of antibody based molecules. The derivation of hybrid hybri-
267
domas is one example of the utilization of such methods for the biosynthesis of
bi-specific antibodies (36). Another example is the derivation of class switch
mutant antibodies (52).
Some years ago, I discussed the eventual use of recombinant DNA techniques to make more drastic changes (53). R ecent developments have shown
the feasibility and potential of the approach. Antibody genes have been put into
suitable vectors, propagated, modified and re-introduced into myeloma cells
which will then secrete recombinant antibodies possessing novel properties.
For instance, in my laboratory Neuberger has developed a cell line which
secretes a mouse-human antibody molecule with a mouse anti-nitrophenacetyl
variable region and a human epsilon heavy chain constant region (54). In
another example, the Fc portion of the mouse antibody was replaced by
staphylococcal nuclease (55). A novel antibody was thus made which contains
an antigen specific Fab portion joined to an enzymatic effector function replacing the normal Fc portion.
More elaborate modifications will be made possible by the fast-developing
techniques of site-directed mutagenesis. These will allow well-planned specific
modifications of antibody combining sites. In this way we will be able to test
the contribution of individual point mutations to the generation of high affinity
antibody during the process of the maturation of the response. This brings us
back to the problems of the diversity of molecular recognition and the maturation of the immune response.
Exciting as these prospects are, they still require the basic starting genes
taken from a hybridoma line. With them, we can introduce changes at the
amino acid sequence level but with the exception of simple changes, the
ultimate folding pattern and their effect on protein-ligand interaction cannot
yet be reliably predicted. This will remain so for the time being. Total construction of antibody molecules to suit specific needs depends on a much better
understanding of protein folding.
While selection is the strategy of the antibody response of an animal, the
immunochemistry of the future will revert to an instructional approach where
the antigen will tell us what antibody structure we should construct. Although
this is not science fiction, we need to overcome the theoretical problems
involved in the translation of one-dimensional reality into a valid three-dimensional prediction. Although the way ahead is full of pitfalls and difficulties, this
is indeed an exhilarating prospect. There is no danger of a shortage of forthcoming excitement in the subject. Yet, as always, the highlights of tomorrow
are the unpredictabilities of today.
Acknowledgements
The hybridoma technology was a by-product of basic research. Its success in
practical applications is to a large extent the result of unexpected and unpredictable properties of the method. It thus represents another clear-cut example
of the enormous practical impact of an investment in research which might not
have been considered commercially worthwhile, or of immediate medical relevance. It resulted from esoteric speculations, for curiositys sake, only motivat-
268
ed by a desire to understand nature. It is to the credit of the Medical Research

Council in Britain to have fully appreciated the importance of basic research to
advances in medicine. We are delighted to belong to the small, lucky group of
those who are at the window-dressing end of the justification for the wisdom of
that policy.
I learned what research was all about as a research student of Stoppani in
Argentina, and then with Sanger in the Department of Biochemistry at Cambridge. I owe an enormous debt to the atmosphere of the Laboratory of
Molecular Biology, where all the work I have described here was done, mostly
under the Chairmanship of Max Perutz, and within the Division of Protein and
Nucleic Acid Chemistry, of which Fred Sanger was the Head. From them, I
always received an unspoken message which in my imagination I translated as
Do good experiments, and dont worry about the rest.
During my lecture I have tried to acknowledge those of my collaborators
whose contributions were critical at specific stages of the work. In addition, so
far unmentioned, is John Jarvis, my personal assistant for well over 20 years only months less than my involvement with immunology. Since this prize
mentions the discovery of the principles of the hybridoma technology, I would
like to acknowledge specifically the importance of the contributions of Dick
Cotton and David Secher, with whom preliminary work leading to that discovcry was made, and my tissue culture assistant, Shirley Howe, who was directly
involved not only in the preliminary work but also in some of the specific
experiments conducted with Georges Khler. I would also like to acknowledge
other collaborators who were concerned in our own contributions to the demonstration of the practical potential of the hybridoma technology in a variety of
fields, particularly G. Galfr, A. F. Williams and A. C. Cuello, and the
technical assistance of Mr. B. W. Wright. The list of acknowledgements is
certainly much longer, but I wouldnt like to end it without recording my
indebtedness to my secretaries, Margaret Dowding, and Judith Firth. Their
handling of the press in the immediate period after the announcement of this
prize ranks high among my memories of that exciting moment.
269
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INDICADORES QUMICOS CIDO-BASE I

La aplicacin de la ley de accin de masas a este equilibrio, nos da que:

[In ][H ] , de lo que

Indicadores qumicos cido-base naturales.

Mientras que para las antoxantidinas, sera:

La mayora de los ptalos de las flores

Prcticas de indicadores qumicos cido-base naturales.

2. Con extracto de lombarda.

En medio alcohlico, debido a la diferente tensin superficial, la gota de indicador se desparrama y es

Desde rosa a violeta casi incoloro

4. Con extracto de fresas en etanol absoluto.

5. Con tornasol (extracto de lquenes de los gneros Rocella, Variolaria y Lecanora).

En medio cido resulta incoloro, mientras

INDICADORES QUMICOS CIDO-BASE II

Indicadores cido-base sintticos

Uso de los indicadores cido-base sintticos.

Prctica con indicadores sintticos.

Obsrvese la aparicin del color verde en la fig 38

CESAR MILSTEIN, PREMIO NOBEL DE MEDICINA 1984

nace en Baha Blanca, Argentina, el 8 de agosto de 1927;

en 1944 completa sus estudios de Bachiller en Baha Blanca;

En esta apretada sntesis no encuentra su lugar aquello que caracteriza a Milstein

Milstein, Csar. Curriculum Vitae. Manuscrito. 1989.

A partir de ese momento, el Milstein hombre se identifica con el Milstein

la adhesin de sus colegas, al coincidir con problemas hondamente sentidos por la

mutaciones en un cultivo de clulas de mieloma, un tumor cuyas clulas poseen la

Se abre la posibilidad de producir anticuerpos a voluntad, y en cantidades

Pero todava no es un conjunto monoclonal. Esto es as, puesto que un agente

Finalmente haban obtenido una lnea de clulas que produca un anticuerpo

convencionales, y que luego fue utilizada en gran escala por la industria

Cuando Ehrlich habla de esta manera, no se refiere nicamente a la especificidad

Canguilhem, George (1986), pp. 70-71.

Bachelard, Gaston, (1953), Le matrialisme rationnel, PUF. Paris.

THE GENERATIVE GRAMMAR OF

Nobel lecture, 8 December 1984

Chteau de Bellevue, F-30210 Castillon-du-Gard, France

Physiology or Medicine 1984

antibody molecules synthesized by one given lymphocyte are identical; and

The Generative Grammar of the Immune System

Physiology or Medicine 1984

It turned out that antibody molecules of different specificity have identical

can recognize, or in other words, the great repertoire of antibody specificities,

Physiology or Medicine 1984

The Generative Grammar of the Immune System

Physiology or Medicine 1984

The Generative Grammar of the Immune System

Physiology or Medicine 1984

before encountered. Immunologists sometimes use words they have borrowed

The Generative Grammar of the Immune System

Physiology or Medicine 1984

I should perhaps again emphasize that the sentences representing antibodies

The Generative Grammar of the Immune System

Physiology or Medicine 1984

FROM THE STRUCTURE OF ANTIBODIES TO

Medical Research Council Laboratory of Molecular Biology, Hills Road,

Cuando se acerca elfin, escribi Cartaphilus, ya no quedan imgenes de1

From the Structure of Antibodies

Physiology or Medicine I984

From the Structure of Antibodies

Light chain mRNA and the signal for secretion

Spontaneous somatic mutants of a myeloma protein

Physiology or Medicine 1984

From the Structure of Antibodies