David J. Litman

Enzyme channeling immunoassay: A new homogeneous enzyme immunoassay technique☆

Enzyme immunoassays have been widely used for the determination of specific substances in human serum (1). The sample, antibody, and an enzyme-antigen conjugate are combined and the amount of enzyme activity associated with the immune complex is determined. In heterogeneous enzyme immunoassays (ELISA) the free and bound enzyme are physically separated prior to measurement of the enzyme activity (2). In homogeneous enzyme immunoassays the activities of the free and bound enzyme are different and total activity of the solution is determined without a prior separation step (3). The serum concentration of numerous drugs are monitored clinically in this way. The small size of most drugs permits intimate interaction of the antibody and enzyme within the complex and provides mechanisms for modification of enzyme activity. By contrast, it is more difficult to modulate the activity of enzyme conjugates of large antigens such as proteins.

One-step enzyme immunochromatographic assay for theophylline

The convenience of the previously described enzyme immunochromatography method for visually quantifying theophylline in whole blood has been improved with the development of a one-step protocol. The capillary migration and color generation in the two-step enzyme immunochromatographic assay have been combined into a single step. Ascorbic acid is used as a signal inhibitor to delay enzymatic color product formation until the inhibitor itself is consumed. The concept of internal delay reaction is presented and the mechanism of ascorbates action as an inhibitor to temporarily delay color generation is described. The internal delay reaction has been applied to a practical one-step quantitative visual enzyme immunochromatographic assay for theophylline in whole blood.