AGGLUTINATION REACTIONS Antibody must be able to bridge the gap
between cells in such a way that one molecule
Agglutination is the visible aggregation of particles
caused by combination with specific antibody.
Reaction takes place on the surface of the
particle, antigen must be exposed and able
to bind with antibody
A two-step process, involving sensitization
or initial binding followed by lattice
formation, or formation of large aggregates.
RBCs, Bacterial cells, inert carriers (latex
particles) multiple antigenic or
determinant sites
Gruber and Durham (1896) first to report the
ability of antibody to clump cells, based on
observations of agglutination of bacterial cells
by serum.
Widal and Sicard detection of antibodies
occurring in typhoid fever, brucellosis and
tularemia.
Sensitization - antigenantibody combination
through single antigenic determinants on the
particle surface and is followed by the law of
mass action and is rapid and reversible.
The affinity and avidity of an individual
antibody determine how much antibody
remains attached.
The class of immunoglobulin (IgM is more
efficient than IgG)
If epitopes are sparse or if they are
obscured by other surface molecules, they
are less likely to interact with antibody
Lattice Formation - sum of interactions between
antibody and multiple antigenic determinants
on a particle, is dependent on environmental
conditions and the relative concentrations of
antigen and antibody
Bordet - governed by physicochemical factors such
as the milieus ionic strength, pH, and
temperature
can bind to a site on each of two different cells
Enhancement of Lattice Formation
LISS decreasing the buffers ionic strength
Albumin 5 to 30% helps to neutralize the
surface charge and allows red cells to
approach each other more closely
Increase viscosity using enzymes, agitating
centrifuging, altering temperature or the Ph
PEG and Dextran reduce the water
hydration around cells and allow them to
come into closer proximity for antibody to
join together
Bromelin, Papain, Trypsin and Ficin
reduces the surface charge on the RBCs
through cleaving of chemical groups and
decreasing hydration.
Ficin cleaves sialoglycoproteins from the
RBCs surface and may change the external
configuration of the membrane to reveal
more antigenic determinant sites.
IgGs reacts best at 30 to 370C, IgM reacts
best between 4 to 270C
pH optimal 6.5 7.5
TYPES OF AGGLUTINATION REACTIONS:
1. DIRECT AGGLUTINATION
2. PASSIVE AGGLUTINATION
3. REVERSE PASSIVE AGGLUTINATION
4. AGGLUTINATION INHIBITION
5. COAGGLUTINATION
Direct Agglutination - occurs when antigens are
found naturally on a particle.
Patient serum is diluted into a series of tubes
or wells on a slide and reacted with bacterial
antigens specific for the suspected disease.
Used in diagnosis of diseases for which the
bacterial agents are extremely difficult to
cultivate.
 Fourfold increase in antibody titer over time
when paired dilutions of serum samples are
tested with any of these antigens
Hemagglutination - agglutination reaction
involves red blood cells
Examples: ABO typing
Passive/Indirect Agglutination - employs particles
that are coated with antigens not normally found
on their surfaces.
Carrier particles:
RBCs possibility of cross-reactivity
(heterophile antibody)
Latex inexepensive, relatively
stable, not subject to cross-reactivity
Gelatin
Silicates
Use to detect:
Rheumatoid Factor
Antinuclear antibody
ASO
Abs to Trichinella spiralis
Abs to Treponema pallidum
Abs to CMV, Rubella, Varicella-zoster,
and HIV-1/2
There is always a risk of non-specific
agglutination caused by the presence of
other IgM antibodies
Reverse Passive Agglutination - antibody rather
than antigen is attached to a carrier particle.
The antibody must still be reactive and is
joined in such a manner that the active sites
are facing outward.
Adsorption may be spontaneous, or it may
require some of the same manipulation as
is used for antigen attachment.
Agglutination Inhibition - reactions are based on
competition between particulate and soluble
antigens for limited antibody-combining sites, and
a lack of agglutination is an indicator of a positive
reaction.
Haptens that are complexed to proteins; the
haptenprotein conjugate is then attached
to a carrier particle.
The patient sample is first reacted with a
limited amount of reagent antibody that is
specific for the hapten being tested.
Indicator particles that contain the same
hapten one wishes to measure in the patient
are then added.
If the patient sample has no free hapten, the
reagent antibody is able to combine with the
carrier particles and produce a visible
agglutination. agglutination is a negative
reaction, indicating that the patient did not
have sufficient hapten to inhibit the
secondary reaction
Either antigen or antibody can be attached
to the particles.
The sensitivity of the reaction is governed by
the avidity of the antibody itself. It can be a
highly sensitive assay capable of detecting
small quantities of antigen.
Hemagglutination Inhibition reactions are
the same principle, except RBCs are the
indicator particles.
Coagglutination - systems using bacteria as the
inert particles to which antibody is attached.
Staphylococcus aureus is most frequently
used, because it has a protein on its outer
surface, called protein A, which naturally
adsorbs the fragment crystallizable (FC)
portion of antibody molecules.
These particles exhibit greater stability than
latex particles and are more refractory to
changes in ionic strength.
Antiglobulin-Mediated Agglutination

1. DIRECT ANTI-GLOBULIN TEST

2. INDIRECT ANTI-GLOBULIN TEST

AHG (Coombs Test) - technique that detects non-

agglutinating antibody by means of coupling with a
second antibody.
Antibody will react with the FC portion of the
human antibody attached to red blood cells.
Agglutination
Takes place because the antihuman globulin
is able to bridge the distance between cells
that IgG alone cannot do.

DIRECT ANTI-GLOBULIN TEST (DAT)

- Used to demonstrate in-vivo attachment of
antibody or complement to an individuals
red blood cells.

Serves as an indicator of autoimmune

haemolytic anemia, HDN, Sensitization of
RBCs caused by the presence of drugs, or a
transfusion reaction.
RBCs are washed to remove any antibody
that is not specifically attached and then
cells are tested directly with antibody to IgG
or complement
A positive test indicates that an immune
reaction is taking place in that individual.

INDIRECT ANTI-GLOBULIN TEST (IAT)

- Used to determine the presence of a
particular antibody in a patient or it can be
used to type patient red blood cells for
specific blood group antigens.

All reactions are run at 37C to detect

clinically significant antibodies.
Used to check for the presence of clinically
significant alloantibodies in patient serum.