Fluorimetric Measurements in Enzyme Immunoassays
The sensitivity of enzyme immunoassays (EIAs) depends on the specificity and avidity of the immunochemical reagents, the specific activity of the enzyme label, and the detection limit of the enzyme product. When the antibody and enzyme systems have been optimized, the ability to detect the enzyme product controls assay sensitivity. When applicable, one of the simplest and most sensitive quantitative methods is fluorimetry. Colorimetry is limited to the parts per billion (10 -3µg/mL) level whereas fluorimetry achieves parts per trillion (10 6µg/mL) and, with the use of laser excitation, even sub-parts per trillion detection limits (Bradley and Zare, 1976, and Imasaka and Zare, 1979). This advantage is because fluorimetry measures a signal increase above relatively low background luminescence while colorimetry measures a small decrease due to absorbance from a large signal of transmitted light. Using fluorimetric microscopy, Rotman (1961) was able to detect single molecules of the enzyme β-galactosidase. Under more practical circumstances, the detection limits of fluorimetry are frequently limited by background fluorescence such as that arising from serum or other biological samples.
KeywordsHigh Performance Liquid Chromatography Testosterone Oestradiol NADH Gelatin
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