Enzyme-Linked Immuno-Sorbent Assay (ELISA)

Mechanism: The basic mechanism involved in these test utilizes absorption of antigen to a solid substrate which is placed in contact with a dilution of serum. The reaction which detects and quantifies the binding of antibody uses an antibody labeled with an enzyme followed by the addition of an appropriate substrate on which the enzyme can act to produce a colour reaction. Two distinct test mechanisms are used which we will designate "standard" and "competitive". These are described in detail under "Methods" below.

Reagents: Antigens may be produced using any of the standard techniques. They may be purified by precipitation and ultra-centrifugation or by column chromatography before being absorbed onto the surface. An alternative method which allows the use of relatively impure antigens is to bind specific antibody to the surface and allow it to absorb the antigen from the preparation. Since the latter approach usually adds an extra stage to the test this is not the preferred approach for commercial test kits. In most cases the antigen is delivered pre-absorbed onto plates, though they need to be carefully dried and packed to maintain their stability at refrigerator temperature for a reasonable period. The labeled anti-globulin used in the standard test is included in the test kits as is the appropriate substrates and stop-solutions for use with it. All of these reagents may be purchased separately from a number of sources however their titration to a standard level of activity for a specific purpose is rarely worthwhile for a commercial laboratory carrying out routine serology. Finally, and most importantly, standard negative sera and positive sera of known potency are required. These are even more important than those used in other serological tests since it is common practice to express the results of the test sera by comparing them with those of the control sera (serum-to-positive or serum-to-negative ratios). The objective of this is to remove some of the variation due to operator, environment and plate effects.

Methods:A. Standard. The antigen is absorbed on to a suitable physical surface. Usually this will be the well walls of a polystyrene micro-titre plate, although other surfaces have also been used (Rivetz et alii, 1985). A test-serum dilution (usually between 1:100 and 1:500) is made in a special diluent designed to inhibit non-specific binding of globulins to the physical surface but allowing their binding to the absorbed antigen. A range of tests can be carried out using a single serum dilution (Snyder et alii, 1984). The diluted serum is removed and the surface washed before adding an chicken anti-globulin labeled with an enzyme (usually a phosphatase). The anti-globulin will bind to any globulin attached to the antigen. Once again the excess reagent is removed and the surface washed before adding the enzyme substrate. Any attached enzyme-labeled conjugate causes a chemical change in the substrate which results in a colour change. The enzyme is inactivated after a fixed incubation time to stop the reaction, before reading the results using a colourimeter (Figure 2.7).

B. Competitive. The first part of this mechanism is very similar to the standard test in that antigen is absorbed on the solid surface and a serum dilution is applied. Serum dilutions tend to be much lower however (typically 1:2). The difference lies in the use of a standard anti-antigen conjugate to bind to any antigen sites which have not been bound to by the serum antibody as opposed to the anti-globulin in the standard test which binds to attached antibody. The addition of substrate, stop solution and reading is for the standard test. The interpretation is the opposite of the standard test in that a lack of colour development is indicative of a positive reaction.\par Statistical methods have been developed for the conversion of serum-to-positive ratios into serum titres (Chalquest, R.R., 1988, Briggs et alii, 1986)

Comments: An Elisa for Infectious Bursal Disease (IBD) has been found to be up to 25000 times more sensitive than DID/AGP (Marquardt et alii, 1980). Since 2 attachment sites are not required for the globulin to attach to the antigen this test can detect "incomplete" antibody in the same way as the Coombes test.

Figure 2.7. Micro-titre plate with a standard Elisa test seen from below. The first 2 wells at top right are the negative controls. The following 2 are positive controls. The remaining sera are field test sera. Usually at least 2 wells with a known laboratory positive control are included on each plate.

Assays: A large numbers of different assays based on Elisa technology have been developed for the detection and quantification of antibody response of poultry. A somewhat smaller number of assays are available commercially. See Table 3 for a list of assays and references.

Note: No endorsement is intended by citing specific manufacturers in the above list. For an up-to-date list of ucts please contact the manufacturer direct: