Enhanced Chemiluminescence Immunoassay

Abstract

Chemiluminescence results from reactions with a very high energy yield, which produce a potentially fluorescent product molecule; reaction energy passed to the product may result in an excited state and subsequent production of a single photon of light. The light yield is usually low, but can approach one photon per molecule in bioluminescent reactions catalyzed by dedicated enzymes. A wide variety of immunoassay systems that use chemiluminescence or bioluminescence have been developed with the aim of detecting low concentrations of biologically active molecules. Even when emission efficiencies are <1%, chemiluminescence is a sensitive label detection method compared with isotopic methods in which very large numbers of molecules must be present for each detected disintegration (e.g., about 1 × 10⁷ atoms of ¹²⁵I give 1 count/s). The production of light against a low background permits detection of small numbers of reacting molecules by measuring total light output. Luminescent emissions can be measured over a range of at least six orders of magnitude by all but the simplest luminometers. This is in marked contrast to fluorescent or spectrophotometric detection of reaction products, where sensitivity and instrument linear range are limited by the stability of light sources and wavelength selection. For example, a good spectrophotometer may achieve a linear range slightly greater than three orders of magnitude.