title: The reference range for complexed alpha 2-macroglobulin human plasma: development of a new enzyme linked in immunosorbent assay (ELISA) for quantitation of complexed alpha 2-macroglobulin.
abstract: Purified alpha 2-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 alpha 2-macroglobulin. Two monoclonal antibodies were selected, H11A11 (specific towards complexed alpha 2-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 alpha 2-macroglobulin in human plasma. The intra-assay coefficient of variation (CV) for measurement of complexed alpha 2-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 alpha 2-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 alpha 2-macroglobulin of 13.5-31.1 mg 1(-1) with a median value of 21.7 mg 1(-1). The concentration of total alpha 2-macroglobulin was measured by the same assay using the monoclonal antibodies 1CG4. For total alpha 2-macroglobulin we determined the reference range to be 1.12-3.54 g 1(-1) with a median value of 2.14 g 1(-1). Based on these results the reference range for complexed alpha 2-macroglobulin as a percentage of total alpha 2-macroglobulin was calculated to be 0.8-1.9% with a median value of 1.0%.
authors.affiliation: Department of Chemistry, Aarhus University, Denmark.
authors.forename: M H
authors.initials: MH
authors.lastname: Jespersen
authors.forename: J
authors.initials: J
authors.lastname: Jensen
authors.forename: L H
authors.initials: LH
authors.lastname: Rasmussen
authors.forename: E
authors.initials: E
authors.lastname: Ejlersen
authors.forename: J
authors.initials: J
authors.lastname: Møller-Petersen
authors.forename: H U
authors.initials: HU
authors.lastname: Sperling-Petersen
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D012016:Reference Values; D000511:alpha-Macroglobulins / Q000032:analysis* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000511:alpha-Macroglobulins; D010450:Endopeptidases