Enzyme Immunoassays - Antigen Antibody Reactions

Enzyme Immunoassays

Enzyme immunoassays (EIAs) can be used for detection of either antigens or antibodies in serum and other body fluids of the patient. In EIA techniques, antigen or antibody labeled with enzymes are used. Alkaline phosphatase, horseradish peroxi-dase, and galactosidase are the enzymes used in the EIA tests.

The commonly used EIAs are enzyme-linked immunosorbent assays (ELISAs). The ELISA technique was first conceptualized and developed by Peter Perlmann and Eva Engvall at Stockholm University, Sweden.

These assays involve the use of an immunosorbent specific to either the antigen or antibody. Following the antigen–antibody reaction, chromogenic substrate specific to the enzyme (o-phenyl-diamine dihydrochloride for peroxidase, p-nitrophenyl phosphate for alkaline phosphatase, etc.) is added. The reaction is detected by reading the optical density. Usually, a standard curve based on known concentrations of antigen or antibody is prepared from which the unknown quantities are calculated. There are different types of ELISAs available for the detection and quantitation of either the antigen or antibodies in serum and other body fluids. These include: (a) indirect ELISA, (b) sandwich ELISA, (c) competi-tive ELISA, and (d) ELISPOT assay.

◗ Indirect ELISA

The indirect ELISA is used for the quantitative estimation of antibodies in the serum and other body fluids. In this method, specimens are added to microtiter plate wells coated with antigen to which specific antibodies are to be detected. After a period of incubation, the wells are washed. If antibody was present in the sample, antigen–antibody complex would have been formed and will not get washed away. 

On the other hand, if the specific antibody was not present in the specimen, there would not be any complex formation. Next, an anti-isotype antibody conjugated with an enzyme is added and incubated. After another washing step, a substrate for the enzyme is added. If there was complex formation in the initial step, the second-ary anti-isotype antibody would have bound to the primary antibody, and there would be a chromogenic reaction between the enzyme and substrate. By measuring the optical density val-ues of the wells, after a stop solution has been added to arrest the chromogenic reaction, one can determine the amount of antigen–antibody complex formed in the first step (Fig. 14-14).

◗ Sandwich ELISA

The sandwich ELISA is used for the detection of antigen. In this test, the known antibody is coated and immobilized onto the wells of microtiter plates. The test sample containing the suspected antigen is added to the wells and is allowed to react with the antibodies in the wells. After the step of washing the well, a second enzyme-conjugated antibody specific for a dif-ferent epitope of the antigen is added and allowed to incubate. After removing any free secondary antibody by rewashing, the specific substrate is added, and the ensuing chromogenic reac-tion is measured. The chromogenic reaction is then compared with a standard curve to determine the exact amount of the antigen present in the test sample. In a positive test, an enzyme acts on the substrate to produce a color, and its intensity can be measured by spectrophotometer or ELISA reader. The change of color can also be observed by the naked eye (Fig. 14-15).

◗ Competitive ELISA

Competitive ELISA is another technique used for the esti-mation of antibodies present in a specimen, such as serum. Principle of the test is that two specific antibodies, one conjugated with enzyme and the other present in test serum (if serum is positive for antibodies), are used. Competition occurs between the two antibodies for the same antigen. Appearance of color indicates a negative test (absence of anti-bodies), while the absence of color indicates a positive test (presence of antibodies).

In this test, the microtiter wells are coated with HIV anti-gen. The sera to be tested are added to these wells and incu-bated at 37°C and then washed. If antibodies are present in the test serum, antigen–antibody reaction occurs. The antigen– antibody reaction is detected by adding enzyme-labeled-specific HIV antibodies. In a positive test, no antigen is left for these antibodies to act. Hence, the antibodies remain free and are washed away during the process of washing. When substrate is added, no enzyme is available to act on it. Therefore, positive result indicates no color reaction. In a negative test, in which no antibodies are present in the serum, antigen in the coated wells is available to combine with enzyme-conjugated antibodies and the enzyme acts on the substrate to produce color.

◗ ELISPOT Assay

ELISPOT assay is a modification of ELISA. It allows the quanti-tative determination of number of cells in a population that are producing antibodies specific for a given antigen or an antigen for which one has a specific antibody. These tests have found application widely in the measurement of cytokines.