ENZYME LINKED IMMUNOABSORBENT

ASSAY
Enzyme linked immunoabsorbent assay (ELISA) is a quantitative technique for the detection
of antigen, haptens and antibodies. Various enzyme linked to either antigen or antibody as a
label which can easily be detected by measuring the enzyme activity. This technique is
attracted considerable attention to the investigator interested in development of different
immunodiagnostic test for the detection of various diseases. This technique is comparable
with radio-immunoassay in determining in minute concentration of protein, hormones etc. in
pictogram range and those same times need no sophisticated equipment such as isotopes
counter and is easier to handle in a small lab as well.


ENZYME USED IN THE ELISA TECHNIQUE

Enzyme that have been frequently used include horseradish peroxidase, alkaline phosphatase,
lysozyme, Glucose 6 phosphate dehydrogenase, glucose oxidase, and B- galactosidase.
These enzyme are coupled to antigen or antibody by various cross linking agent. Virtually,
any enzyme can be use as long as it is soluble, stable and not present in the biological fluid.
In general horseradish peroxidase and alkaline phosphatase are used most commonly
in the localisation of antigen/antibodies. Peroxidase is actually the choicable enzyme
because:-

1. it has low molecular weight e.g. 4KD which is less than that of all other enzyme used in
immunocytochemicalproedures.
2. penetration of peroxidase in the interior of fixed cells is much more easily obtained.



CLASSIFICATION OF ELISA

Enzyme linked immunoabsorbent assay is classified into following categories :-

DIRECT ELISA :- this technique is used for the detection of antigen or immune complexes
in the sera.

INDIRECT ELISA :- this is used for the detection of antibodies in sera

SANDWICH ELISA:- this is used for the detection of antigen. This technique is commonly
used in the laboratory,
COMPITITIVE ELISA :- this system is commonly used for both identification and
quantitation of either antigen or antibody.

INHIBITION ELISA :- this assay system is used for determining the identify of specific
antigen or antibody.
i.e. IgG, IgM.

PRINCIPLE:-
In this type of assay, one of the reaction components is non specifically adsorbed or covalently bound
to the surface of a solid phase, such as that of a microtitre well, a magnetic particle, or a plastic bead.
This attachment facilitates separation of bound and free labelled reactants.
In the most common approach to using the ELISA techniques, an aliquot of sample or calibrator
containing the antigen to be quantified is added to and allowed to bind with a solid phase Ab-Ag-Ab-
enzymes.
Excess(unbound) antibody is then washed away and enzyme substrate is added. The enzyme
catalytically converts the substrate to products. The amount which is propotional to the quantity of
antigen in the sample.

Antibodies in a sample can also be quantified using an ELISA procedure in which antigen
instead of antibody is bound to a solid phase and the second reagent is an enzyme-labelled
antibody specific for the analyte antibody.


NOTE:
HRP/ALP :
Commonly used:-
- penitrating capacity more.
- low molecular weight, near abount 65 KD
- Easily handle for the reaction.































RADIO-IMMUNOASSAY
BREI F HI STROY:-
- It was developed in 1959 by solon and Rosalyn yellow for the estimation of insulin in human
serum.
- It is a technique for the estimation of several biological fluid in a very low concentration (
nanogram to pictogram)
- It is a highly sensitive and specific analytical tool.

PRI NCI PLE OF RI A :-
RIA combines the principles of radioactivity of isotopes and immunological reactions of antigen and
antibody.
- RIA is primarily based on the competition beteen the labelled and unlabelled antigen to bind
with antibody to form Ag-Ab complexes ( either labelled or unlabelled )
- The unlabelled Ag is the substance (say insulin) to be determined. The antibody to it is
produced by injecting the Ag to a goat or a rabbit.
- The specific Ab is then subjected to react with unlabelled Ag is then subjected to react with
ublabelled Ag in the prescence of excess amount of isotopically labelled (I
131
) antigen (Ag
+
)
with known radioactivity. There occurs a competition between the antigen (Ag
+
) and Ag bind
the antibody.
- Certainly, the labelled Ag
+
will have an upper hand due to its excess prescence.
- As the concentration of unlabelled antigen increases the amount of labelled Ag

Ab complex
decreases.
- The concentration of Ag-Ab is inversely related to the concentration of unlabelled Ag (i.e the
substance to be determined). This relation is almost linear.
The labelled Ag-Ab complex is separated by ppt. The radioactivity of I
131
present is Ag-Ab is
determined.

Ag
+
+ Ab Ag Ab Ag Ab.



APPLI CATI ON:-
1) hormone disorder
2) Cancer
3) Therapeutic monitoring of drugs.