Bilirubin Oxidase Label for an Enzyme-Linked Affinity Assay with O₂ as Substrate in a Neutral pH NaCl Solution

Laccase, a copper enzyme catalyzing the four-electron reduction of O(2) to water, has been shown by others to be a useful label in enzyme-linked immunoassays, in which the substrate is ambient O(2) instead of an added chemical, such as hydrogen peroxide, or a phosphate ester of a phenol. Laccase-catalyzed O(2) reduction is, however, inhibited by halides, which complex the enzyme's copper ions. Replacement of laccase by bilirubin oxidase, a copper enzyme retaining its maximal activity at high chloride concentrations and at pH 7.2, allows enzyme-amplified affinity assays with O(2) as the substrate in neutral-pH chloride solutions, exemplified here by the assay of DNA, the duplexes of which are unstable at low ionic strength but are stable in strong NaCl solutions.