The following points highlight the two important immunological techniques in antigens and antibody. The techniques are: 1. Radio-Immuno Assay (RIA) 2. Enzyme-Linked Immunosorbent Assay (ELISA).
Technique # 1. Radio-Immuno Assay (RIA):
One of the most sensitive techniques for detecting antigen or antibody is Radio-immuno assay (RIA).
History:
The technique was first introduced by two endocrinologists, S. A. Berson and Rosalyn Yalow, in 1960. It was first used to determine the levels of insulin-anti-insulin complexes in diabetics. After a lot of controversy, the technique was proved as an effective one for quantification of hormones, serum proteins, drugs and vitamins even at concentrations of 0.001 μg or less.
In 1977, the significance of the technique of RIA was acknowledged by the award of a Novel Prize to Yalow, which was after the death of Berson.
1. Radio-immuno assays for antigen:
i. Principle:
RIA is a highly sensitive method of estimation of antigens or haptens. The principle of RIA is a competition between a radioactive antigen or a hapten (a ligand) and a non-radioactive counterpart of the same antigen or hapten for binding to the sites of the cognate antibody molecules. The antigen is generally labelled with a gamma- emitting isotope as for e.g.125I.
ii. Types:
RIA has been widely used in clinical diagnostic laboratories due to its high sensitivity.
Technique of RIA is of two types:
(i) Direct RIA and
(ii) Indirect RIA.
(i) Direct RIA:
When antigens or primary antibodies are directly labelled with a radionuclide, it forms the basis of Direct RIA. This technique utilizes radiolabelled antibody or its ligand (antigen). Antibody is incubated with ligand and unbound reactants are removed from the system (phase separation).
It may utilize precipitation of bound reactants (quantitative precipitin reaction), particulate antigens (such as bacteria), the immobilization of the nonradioactive reactant onto a solid matrix (such as plastic), and so on (Fig. 9.1A).
(ii) Indirect RIA:
Anti-immunoglobulin antibody (secondary antibody) is radio-labelled and used in the indirect RIA.
This technique uses radiolabeled secondary antibody (anti-immunoglobulin) to detect the binding of a primary antibody (Fig. 9.1B).
Methods of quantification by RIA:
(i) The labelled antigen is mixed with antibody at a concentration that just saturates the antigen-binding sites of the antibody molecules and then increasing amounts of un-labelled antigen of unknown concentration are added.
(ii) The antibody does not distinguish labelled from un-labelled antigen for which two kinds of antigen compete for available binding sites on the antibody.
(iii) Gradually, the concentrations of un-labelled antigen is increased and more labelled antigen will be displaced from the binding sites.
(iv) Now, the decrease in the amount of radio-labelled antigen bound to specific antibody in the presence of test sample is measured in order to determine the amount of antigen present in the test sample.
Explanation of RIA by detection of hepatitis B-virus:
(i) A solid-phase Radio-immuno assay (RIA) is used to detect hepatitis B virus in blood samples:
Microtiter wells are coated with a constant amount of antibody specific for HBsAg, the surface antigen on hepatitis B virions.
A serum sample and [125I]HBsAg are then added. After incubation the supernatant is removed and the amount of radioactivity bound to the antibody is determined.
If the sample is infected, the amount of label bound will be less than in controls with uninfected serum (Fig. 9.2).
(ii) A standard curve can be obtained by adding the increasing concentrations of un-labelled HBs Ag to a fixed quantity of [125I] HBs Ag and specific antibody (Fig. 9.3).
From the plot of the percentage of labelled antigen bound versus the concentration of un-labelled antigen, the concentrating of HBsAg in unknown serum samples can be determined from the linear portion of the curve.
Reaction of RIA:
In RIA, the essential components of the analytical system are the antigen (Ag) to be determined, a fixed amount of labelled antigen (Ag*) and a fixed limited amount of antibody (Ab).
The reaction is:
Technical requirements for Radio-immuno assay:
(i) Antigen is required in the preparation of all the main reagents (standard, label and antiserum).
(ii) Antigen purification can be done in case of those hormones which are labile or present in minute amounts in tissues and plasma (Fig. 9.4).
Technique # 2. Enzyme-Linked Immunosorbent Assay (ELISA):
The most important of the immuno-enzyme assays are the Enzyme-linked immunosorbent assays, commonly called ELISA. It is a type of solid-phase enzyme immunobinding assay. In ELISA antigen is linked to a solid phase anchored antibody in such a way that retains both immunological and enzymatic activity. The solid phase may be of polystyrene/polyvinylchloride.
So ELISA can be called as a qualitative or quantitative assay for antibodies i.e., an assay for quantitating either antibody or antigen by use of an enzyme-linked antibody and a substrate that forms a coloured reaction product.
Principle:
The basic principle of ELISA depends on the presence of enzyme. An enzyme conjugated to an antibody reacts with a colourless substrate to generate a coloured reaction product. ELISA is similar to RIA in principle, depends on an enzyme but avoiding the involvement of radioactive label.
Enzyme involved in the technique:
A number of enzymes are usually used for ELISA, such as, alkaline phosphatase, horseradish peroxidase and β-galactosidase (Table 9.1).
Besides enzymes, some auto-antibodies are also detected by enzyme-linked immunosorbent assay (ELISA) (Table 9.2).
Requirements:
(i) Antigen coated microtiter well
(ii) Primary antibody (Ab1)
(iii) Secondary antibody (Ab2) remain conjugated with enzyme.
(iv) Specific enzyme
(v) Specific substrate
(vi) Suspected blood sample (serum).
Types:
A number of variations of ELISA have been developed. Each type of ELISA can be used qualitatively to detect the presence of antigen/antibody.
I. Indirect ELISA
II. Sandwich ELISA
III. Competitive ELISA
I. Indirect ELISA:
Antibody can be detected or quantitated with an indirect ELISA.
Steps involved:
1. Serum or some other sample contains primary antibody (Ab1) is added to an antigen- coated microtiter well and allowed to react with the bound antigen.
2. After reactions, free Ab1 is washed away and antigen bound antibodies are present within the microtiter well.
3. Addition of enzyme conjugated secondary antibody (Ab2) to the microtiter well which will bind with the primary antibody.
4. After sometime, a wash is done to remove excess Ab2 from the set up.
5. Now, specific substrate for specific antibody is added and as a result, a coloured reaction product is formed.
6. The coloured reaction product is measured by specialized spectro-photometric plate reader (Fig. 9.5).
II. Sandwich ELISA:
Antibody can be detected or quantitated by a sandwich ELISA.
Steps involved:
1. The antibody (Ab1) is placed (immobilized) on a microtiter well.
2. A sample contains antigen is added and allowed to react with the bound antibody (Ab1).
3. A wash is taken to remove excess free antibody from the well.
4. After that, a second antibody (Ab2) specifically bound with enzyme is added which binds with different epitope present on the bound antigen.
5. A second wash is needed to remove free second antibody.
6. Again a specific substrate is added and the coloured reaction product is measured (Fig. 9.6).
III. Competitive ELISA:
Another variation for measuring amounts of antigen is Competitive ELISA.
Steps involved:
1. In this technique antibody is first incubated in solution with a sample containing antigen.
2. The antigen-antibody mixture which is thus formed, is then added to an antigen-coated microtiter well.
3. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well.
4. An enzyme conjugated secondary antibody (Ab2) specific for the iso-type of the primary antibody is added.
5. Now, primary antibody is used to quantitate the amount of primary antibody bound to the well.
6. Colour changes are being monitored. More the colour is observed less will be the analyte in the test specimen, i.e., in the competitive assay, the higher will be the concentration of antigen in the original sample, indicates the lower absorbance (Fig. 9.7).
