Alkitab University – Collage of Medical Techniques

Department of Medical Analysis

ELISA - RIA

Written by:

Muhammed Raheem Hussein

Sandy Adel Ishaaq
Falah Faridoon Ahmed

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 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
0obtained.

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.

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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.

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RADIO-IMMUNOASSAY

Radioimmunoassay is a heterogeneous assay that employs radiolabeled drug

(typically 125I). There are two different methods of RIA that are commonly
employed for drug detection in biological matrices, double-antibody RIA and
coated-tube RIA. With double-antibody RIA, a second antibody is added to
facilitate precipitation of the bound primary antibody. Once the
primary/secondary antibody–antigen complex precipitates, the unbound labeled
drug can be easily removed. With coated-tube RIA, the primary antibody is
coated on the inside of each tube. The unbound labeled drug can be easily
removed by pouring off the supernatant. The samples from each RIA method are
analyzed in a gamma counter to determine the counts per minute, which is
inversely proportional to the amount of drug present in the original specimen.
Radioimmunoassay’s are both sensitive and specific but require special handling
and disposal of radioactive waste. Additionally, shelf life is limited to
approximately two months due to radioactive decay.

BREIF HISTROY:-
- 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.

PRINCIPLE OF RIA :-
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

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 isotopically labelled (I131) 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.

APPLICATION:-
1) hormone disorder
2) Cancer
3) Therapeutic monitoring of drugs.

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