A competitive enzyme immunoassay of human β2-microglobulin based on separation by filtration

Abstract

A competitive enzyme immunoassay has been developed for the determination of β2-microglobulin in undiluted human serum. In the assay a β2-microglobulin-β-galactosidase conjugate competes with β2-microglobulin from the sample for the binding to anti-β2-microglobulin antibodies bound to small size agarose particles (micro-Sepharose) via a double antibody.The conjugate is made by providing β2-microglobulin with ‘reactive disulphide structures’. This is achieved by the heterobifunctional reagent SPDP and the subsequent linkage of the formed derivative to β-galactosidase by a thioldisulphide exchange reaction. This procedure gives conjugates with high immunoreactivity and enzyme activity.The assay is fast and simple. The reagents are incubated together for 60 min in the wells of a Millititer plate and separation is performed by filtration. Bound enzyme-labelled β2-microglobulin is measured by incubation with substrate for 15 min. Both incubations are performed at room temperature without agitation. Due to the high capacity of the micro-Sepharose no sample predilution is needed. The use of the Millititer filtration system gives a rapid and efficient separation and the advantages of access to equipment for dispensing and reading adapted to the microtitre format.