Page 355 - The Veterinary Laboratory and Field Manual 3rd Edition

324 Susan C. Cork, M. Faizal Abdul Careem and M. Sarjoon Abdul-Cader

or in a single test format. Thereafter testing • Add detecting antibody to antigen.
involves the following crucial steps. • Add enzyme labelled anti-species antibody.
• Add substrate.
1 Washing to remove unbound liquid phase
reagents. Indirect antibody capture (Figure 6.13b):
2 Addition of a buffer containing blocking
agent to fill in un-utilized binding site spaces. • Solid phase-antigen coated microtitre plate.
3 Addition of conjugate which is an antibody or • Add test antibody (serum).
antigen linked with enzyme. • Add enzyme labelled anti-species antibody.
4 Addition of chromogenic substrate which is • Add substrate.
specific for the enzyme used and will produce
a colour as a result of enzyme/substrate/ Points to remember.
chromogen interaction.
5 Stopping solution which stops the enzyme 1 Follow the kit test instructions carefully.
reaction after the optimal level of colour 2 pH is very important, so ensure that it is cor-
development is attained. rect.
3 Care should be taken when pipetting to
There are several ELISA procedures which will ensure accuracy and to avoid contamination
highlight reactants in different ways, some of of reagents between wells.
these are illustrated in Figure 6.13. ELISA tests 4 Use carefully washed glassware (several
can be developed which measure antigen (Figure changes of distilled water).
6.13a) or antibody (Figure 6.13b) in samples, 5 Ensure adequate washing between steps.
some kit tests currently available for the diag- 6 Mix reagents well, check shaker settings
nosis of viral diseases are outlined in Table 4.6. before use.
The following ELISA tests are those likely to be 7 Do not contaminate wells from droplets on
carried out in a regional laboratory. plastic covers.
8 Watch for uneven coating of plates (or other
format) and distribution of reagents in wells
The direct ELISA
through poor mixing or bubbles.
• Antigen capture: Solid phase-antibody coated
microtitre plate (or another format).
• Add enzyme labelled antigen. 6.4 Quality control and interpretation
• Antibody detection: Solid phase-antigen of results
coated microtitre plate (or another format).
• Add enzyme labelled antibody. As with any laboratory test, quality control
is very important. See Chapters 1 and 7 for
specific details about quality control in test sys-
The indirect ELISA:
tems. Most serological kit tests provide controls
Indirect antigen capture (that is, sandwich (positive and negative), a standard and often a
ELISA) (Figure 6.13a): reference sample. It is advisable to ensure that
the laboratory has its own supply of positive
• Solid phase-antibody coated microtitre plate (that is, hyperimmune) and negative sera for the
(or another format). tests that are performed most frequently. These
• Add test antigen material. can often be obtained commercially but known

Vet Lab.indb 324 26/03/2019 10:26