A competitive enzyme immunoassay of human beta 2-microglobulin based on separation by filtration

A competitive enzyme immunoassay has been developed for the determination of beta 2-microglobulin in undiluted human serum. In the assay a beta 2-microglobulin-beta-galactosidase conjugate competes with beta 2-microglobulin from the sample for the binding to anti-beta 2-microglobulin antibodies bound to small size agarose particles (micro-Sepharose) via a double antibody. The conjugate is made by providing beta 2-microglobulin with 'reactive disulphide structures.' This is achieved by the heterobifunctional reagent SPDP and the subsequent linkage of the formed derivative to beta-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 beta 2-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.