Biotechnology and Bioinformatics (1+1)

4.2.12. Immunological techniques

4.2.12.1. Enzyme immunoassays

The interaction of an antibody with an antigen forms the basis of all immunochemical techniques. Immunoassays are both qualitative and quantitative. A labelled antibody/ /antigen is used to visualize the immune reaction. The Enzyme-Linked Immunosorbent Assay (ELISA), also known as the Enzyme Immuno Assay (EIA), has become a widely-used serological technique.

There are two basic methods.

i) Direct ELISA

ii) Indirect ELISA

i) Direct ELISA

• Coat the ELISA plate wells with antigen and incubate (4°C) it overnight followed by washing.
• Block the uncoated sites with milk powder or BSA, wash to remove excess of blocking agent.
• Add antibody which were raised against the antigen and conjugated with enzyme followed by washing to remove unbound antibody.
• Add substrate and read the colour developed under spectrophotometer.

ii) Indirect ELISA

• Coat the ELISA plate wells with antigen and incubate (4° C) it overnight followed by washing.
• Block the uncoated sites with milk powder or BSA, wash to remove excess of blocking agent.
• Add primary antibody which were raised against the antigen followed by washing to remove unbound antibody.
• Add secondary antibody conjugated with enzyme followed by washing to remove unbound antibody.
• Add substrate and read the colour developed under spectrophotometer.

A microtitre plate with numerous shallow wells is used in both procedures. There are three enzymes gene rally used for colour development with the second antibody. The earliest used was alkaline phosphatase and the commonest now is horseradish peroxidase. The third is β-galactosidase, from E. coli, which is active at the higher pH values preferred for antigen absorption.

The substrates generally employed for alkaline phosphatase and β-galactosidase are p-nitrophenylphosphate and o-nitrophenlbeta-D-galactopyranoside, respectively, with colour development being detected at 405 and 420 nm. In the case of horseradish peroxidase, o-phenylenediamine (OPD) or tetramethyl benzedine (TMB) are most common.

A more sensitive ELISA detection system may be obtained using fluorogenic substrates for alkaline phosphatase or betagalactosidase. Most ELISA are read in a spectrophotometer adapted for microtitre plates.

Crawford et al. (1999) developed an Enzyme-linked Immunosorbent assay for detection of antibodies to Channel Catfish Virus (CCV) in Channel catfish.

Sharif (1999) developed and standardized an ELISA for diagnosis of Aeromonas hydrophila infection in fish.