CEDIA(TM), a new homogeneous immunoassay system

Abstract

Genetic engineering of β-galactosidase (EC 3.2.1.23) has led to the development of a new homogeneous assay system CEDIA(TM). The Z gene of the lac operon of Escherichia coli encodes a large enzymatically inactive polypeptide that spontaneously aggregates and folds to form active β-galactosidase. Using recombinant DNA techniques, we have been able to engineer β-galactosidase protein into a large polypeptide (an enzyme acceptor, EA) and a small polypeptide (an enzyme donor, ED). The EAs and EDs are both enzymatically inactive, but spontaneously associate to form enzymatically active tetramers. In the assay, hapten or analyte is attached to an ED, and an analyte-specific antibody is used to inhibit the spontaneous assembly of active enzyme. Analyte in a patient's serum competes with the analyte in the analyte-ED conjugate for antibody, modulating the amount of β-galactosidase formed. The signal generated by enzyme substrates is directly proportional to the analyte concentrations in the patient's serum. We describe quick (5-15 min) colorimetric tests for digoxin, requiring no serum pretreatments or predilutions and suitable for use with centrifugal and random-access analyzers.