The reference range for complexed α2-macroglobulin human plasma: Development of a new enzyme linked in immunosorbent assay (ELISA) for quantitation of complexed α2-macroglobulin

Abstract

Jespersen MH, Jensen J, Rasmussen LH, Ejlersen E, M0ller-Petersen J, Sperlitig-Petersen HU. The reference range for complexed a2-macroglobulin in human plasma: development of a new enzyme-linked immunosorbent assay (ELISA) for quantitation of complexed a2-macroglobulin. Scand J Clin Lab Invest 1993; 53: 639-648.Purified α2-macroglobulin was complexed by reaction with methylamine and used to raise monoclonal murine antibodies. A four-step enzyme linked immunosorbent assay (ELISA) was developed to determine the antibody-specificity of the produced monoclonal murine antibodies towards human native and complexed α2-macroglobulin.Two monoclonal antibodies were selected, HI 1 All (specific towards complexed a2-macroglobulin) and 1CG4 (recognizes both forms of the molecule), and purified by affinity chromatography on protein G. The purified antibodies were used to develop a fast three-step ELISA for exact quantitation of complexed and total a2-macroglobulin in human plasma. The intra-assay coefficient of variation (CV) for measurement of complexed a2-macroglobulin is 2.2-9.9%, whereas the inter-assay CV was determined to be 3.7-10.5% and the recovery of the assay is 93-108%. The assay for total a2-macroglobulin has an intra-assay CV of 3.0-15.5%, an interassay CV of 5.1-21.2% and a recovery of 91-116%.Citrated plasma samples from 139 healthy blood donors were examined, resulting in a reference range for complexed a2-macroglobulin of 13.5 31.1 mgl−1 “with a median value of 21.7mgl”. The concentration of total a2-macroglobulin was measured by the same assay using the monoclonal antibodies 1CG4. For total a2-macroglobulin we determined the reference range to be 1.12-3.54g 1_1 with a median value of 2.14g 1_1. Based on these results the reference range for complexed a2-macroglobulin as a percentage of total a2-macroglobulin was calculated to be 0.8-1.9% with a median value of 1.0%.