Specialized methods

These include radiolabelling, flow cytometry and enzyme-linked immunosorbent assays (ELISAs) used for the estimation of the number of antigen sites on red cells and when more sensitive antiglobulin techniques are required. The essence of these approaches is the use of a labelled antiglobulin: for radiometric assays, 125I is usually used to 'tag' the antiglobulin molecules; in flow cytometry, fluorescent anti-IgG is used (or antibodies can be directly coupled with a fluorescent dye); in the case of ELISA, enzymes are attached covalently (i.e. conjugated) to the antiglobulin.

The execution of these assays requires antibody-coated cells or particles to be incubated with the labelled antiglobulin reagent. After incubation, excess unbound antiglobulin is washed away and bound, labelled antiglobulin is then measured. For radio-metric assays, this is achieved by simply counting radioactivity in a gamma counter. For flow cytometry, a cytofluorimeter is used to measure fluorescence of each cell. For ELISA tests, the bound enzyme conjugate is detected through the enzyme's ability to modify, and usually effect a colour change, in its substrate (Figure 14.6). The colour intensity of the modified substrate can then be measured with a spectrophotometer.

Figure 14.6 The enzyme-linked immunosorbent assay (ELISA) for cell-bound antibodies. Enzyme substrate is represented by open and closed stars, for colourless and coloured derivatives respectively.

Figure 14.6 The enzyme-linked immunosorbent assay (ELISA) for cell-bound antibodies. Enzyme substrate is represented by open and closed stars, for colourless and coloured derivatives respectively.

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