Detection of antibodies using labeled antiimmunoglobulin reagents

The most widely used tests are those which use a second antibody, labeled in some way, to detect the binding of primary antibody to antigen. The availability of excellent, highly specific reagents from many commercial sources has made these techniques simple to apply in any laboratory. 'Second antibodies' are available which recognize imunoglobu-lins from humans and from most laboratory and veterinary animals. The second antibody can be labeled with radioactivity (usually 125I), a fluorescent or luminescent dye, or coupled covalently to enzyme molecules. Each type of assay has its own advantages and disadvantages.

In quantitative assays, the antigen is usually coated on to plastic tubes or, more frequently, 96-well microplates to provide a solid phase on which to capture antibody. Sensitive detectors measure enzyme activity colorimetrically or detect chemiluminescence or fluorescence in this format. Flexible plates can be cut up for counting radioactivity. The enzyme-linked immunosorbent assay (ELISA) is the most widely used technique. Bound antibody is usually detected using alkaline phosphatase, horseradish peroxidase or 3-galactosidase-conjugated second antibody. All of these enzymes cleave suitable substrates to yield highly colored products allowing detection of antibody in the ng ml-1 range.

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