Page 283 - The Veterinary Laboratory and Field Manual 3rd Edition

252 Susan C. Cork and Roy Halliwell

Virus haemagglutination (HA) test ELISa
In 1941, Hirst observed that agglutination of
chicken embryo erythrocytes occurred when Enzyme-linked immunosorbent assay (ELISA),
they were mixed with amniotic and allantoic also known as an enzyme immunoassay (EIA), is
fluids, which contained high levels of influenza a biochemical technique used to detect the pres-
virus. Subsequent research has shown that many ence of an antibody or an antigen in a sample.
viruses will agglutinate chicken and some mam- The ELISA is widely used as a diagnostic tool in
malian erythrocytes. Haemagglutination tests veterinary and human medicine. ELISAs can be
are available to test for the presence of a number developed to detect either antigen or antibody.
of viruses (for example, influenza A and some In simple terms, for a direct antibody ELISA, a
paramyxoviruses) and can also be used to show known amount of antigen is affixed to a surface,
the quantity of virus present in a given fluid. and then serum is added over the surface so that
The minimum quantity of virus that will pro- any antibody present can bind to the antigen.
duce haemaggluntination can be quite accurately A specific anti-species antibody linked to an
titrated by the HA test. In the performance of enzyme is then added, and in the final step a
the HA titration, doubling dilutions of the virus- substrate is added so that the enzyme can con-
containing material are made in saline. After a vert the substrate to some detectable signal,
suspension of washed erythrocytes is added, most commonly a colour change in a chemical
the tubes are shaken and then allowed to stand substrate (see Figures 6.13a and b).
at room temperature until sedimentation of Performing an antigen ELISA involves at
the cells occurs and the results can be read. If least one antibody with specificity for a par-
agglutination is present, a granular mat, often ticular antigen of interest. The sample with an
with curling at the edges, will be observed in the unknown amount of antigen is immobilized on
bottom of the tube. Absence of agglutination is a solid support (usually a polystyrene microtitre
shown by the cells settling to the very bottom of plate via adsorption to the surface or via capture
the tube as a rather compact round ‘button’ (see by another antibody specific to the same antigen
Figure 6.7a). as in a ‘sandwich’ ELISA). After the antigen is
immobilized, the detection antibody is added,
forming a complex with the antigen. The detec-
Haemagglutination-inhibition (HI) test tion antibody can be covalently linked to an
enzyme or can itself be detected by a secondary
The discovery that certain viruses would agglu- antibody that is linked to an enzyme. Between
tinate erythrocytes was promptly followed by each step, the microtitre plate (or other sur-
the observation that antibodies to the virus face) is typically washed with a mild detergent
could inhibit the reaction. HI tests have been solution to remove any proteins or antibodies
a convenient method of detecting the presence that are not specifically bound. After the final
of specific antibody in the serum of infected, or wash step, the plate is developed by adding an
convalescent, individuals. Furthermore, by using enzymatic substrate to produce a visible colour
serial dilutions of the serum, the comparative change, which can be measured using a spectro-
amount of antibody can be determined. The viral photometer to determine the quantity of antigen
antigen used in this test must be titrated accu- in the sample (see also Chapter 6).
rately by using the HA test previously described.
More details are provided in Chapter 6.

Vet Lab.indb 252 26/03/2019 10:25