Immunoassays employ competitive binding equilibria between labelled and non-labelled antigen (or hapten), Ag* and Ag, respectively, for antibodies (Ab) developed to the antigen for the determination of the antigen:
Immunoassay techniques can be described as either heterogeneous or homogeneous. Heterogeneous techniques require the separation of the Ag-Ab and Ag*-Ab from the Ag* and Ag, whereas the homogeneous techniques avoid the separation step. The first immunoassay techniques employed antigens or haptens labelled with radioactive tracers. These techniques are by necessity heterogeneous because there is no way to distinguish between the radioactivity signal due to the Ag*-Ab from that of the Ag*. The use of alternative labels for immunoassays has enabled the development of homogeneous techniques in addition to the traditional heterogeneous techniques. Homogeneous fluoroimmunoassay (FIA) techniques in which fluorescent labels are used can employ differences in fluorescence intensity, spectral characterisitics, polarization, and/or lifetime between the Ag* and the Ag*-Ab.
$$Ag* + Ag + Ab \rightleftharpoons Ag* - Ab + Ag - Ab.$$
KeywordsHuman Serum Albumin Fluorescence Lifetime Immunoassay Technique Bromocresol Green Fluorescence Spectral
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