Immunofluorescence / Immunoperoxidase

Mechanism: The antigen is grown in a test system (usually cell culture on cover slips) and the test sera are simply applied to the surface of the infected cells. After washing to remove any non-reacting antibody the surface is treated with a labeled anti-globulin to "develop" the image of the attached antibody. In the immunofluorescence test the labeling is by way of a chemical which fluoresces under ultra-violet light (as viewed with a U-V microscope). In the immunoperoxidase test an anti-globulin is labeled with a peroxidase enzyme which produces a colour reaction (visible with a conventional microscope) after the addition of a substrate which is modified by the enzyme.

Reagents: As soon as the antigen in question can be grown well in an in-vitro culture system the infected cells can be used to test sera for antibodies. This lends these methods to the rapid development of serological test systems when new pathogens are discovered. The only other reagent required for immunofluorescence is fluorescein-labeled anti-globulin. The immunoperoxidase requires the substrate, in addition to enzyme-labeled anti-globulin.

Methods: These tests are usually carried out by growing viruses on tissue culture on glass cover slips. The infected cells may be fixed and stored until required. The serum and other reagents may be applied to cover-slips in the wells of a plate or in Petri dishes. After carrying out the reactions and washes the cover slips are mounted on glass slides for microscopic examination.\par \par Assays: Not currently used in routine serological screening in poultry. Quite commonly applied as a research tool when a new pathogen is identified (e.g. Chick Anaemia Virus).

Comments: These tests tend to be used more widely as research tools rather than as routine tests, partly because the results are read by microscopic examination. This requires considerable experience and can be somewhat subjective. However the same basic mechanism has been developed into the Elisa tests which are widely used (see below).