A solid-phase immunosorbent assay to determine the proteinase binding capacity of α2-macroglobulin using 125I-trypsin as indicator proteinase

Abstract

Hyperimmune sera against human α 2macroglobulin were raised in rabbits following immunization with ‘s’ α 2-macroglobulin ∗∗ ∗∗ Nomenclature according to Barrett et al. [18]. over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for ‘s’ and ‘f’ α 2-macroglobulin (plasma α 2-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4·10 −6 as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, ‘s’ α 2-macroglobulin and both ‘f’ α 2-macroglobulins (27 and 82% saturation of ‘s’ α 2-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact α 2-macroglobulin subunit ( M r 182 000) with 125I-labeled trypsin binding capacity of immunoimmobilized α 2-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between ‘s’ α 2-macroglobulin and ‘f’ α 2-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol α 2-macroglobulin was observed. Saturation of solid phase immunoimmobilized ‘s’ α 2-macroglobulin with trypsin could be accomplished by incubation with a 100–200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol α 2-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized α 2-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to α 2-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of α 2-macroglobulin immunoadsorbed from synovial fluids ( n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive α 2-macroglobulin of 0–53% when compared to the trypsin-binding capacity of normal plasma α 2-macroglobulin.