Fluorogenic Substrate Labeled Separation-Free Enzyme Mediated Immunoassays for Haptens and Macromolecules

Abstract

Fluorogenic substrate labeled separation-free enzyme mediated immunoassay, hereafter abbreviated as FSIA (Boguslaski et al., 1980; Burd, 1981) (it is also known as Substrate-labeled Fluorescent Immunoassay) uses a modified fluorogenic enzyme substrate as a label to form a stable covalent substrate-analyte ligand conjugate. This is in contrast to most enzyme mediated immunoassays which use an enzyme rather than an enzyme substrate as the label (Ngo and Lenhoff, 1981 and 1982). There are two critical prerequisites that must be satisfied before a fluorogenic substrate can be used as a label in FSIA development: (1) the modified fluorogenic substrate after covalent conjugation to the analyte ligand must retain its function as an enzyme substrate and (2) the substrate-analyte ligand conjugate upon binding to an antibody specific to the ligand must not be able to serve as an enzyme substrate. By definition, a fluorogenic enzyme substrate should not fluoresce at the wavelength used to monitor the assay; however, its enzymatic product should exhibit strong fluorescence at the appropriate monitoring wavelengths. The substrate-analyte ligand conjugate in FSIA serves as a modified enzyme substrate capable of binding to the active-site of an enzyme and being transformed into a detectable product.