Detection of heterogeneity of Cu, Zn-superoxide dismutase with monoclonal antibodies and the establishment of a highly sensitive fluorescence sandwich enzyme-linked immunosorbent assay

Abstract

Murine monoclonal antibodies directed against a native form of Cu, Zn-superoxide dismutase (SOD) were produced by immunizing SOD purified from human erythrocytes. The monoclonal antibodies able to bind SOD were further screened for their ability to absorb SOD activity using anti-mouse IgG conjugated iron beads as solid supports in magnetic separation. This new screening method revealed the heterogeneity of native SOD in the reactivity with the antibodies. One monoclonal antibody successfully absorbed the entire activity of SOD detected by an inhibition assay of cypridina luciferin analog (MCLA)-dependent chemiluminescence induced by superoxide anion production, while other absorbed only a part of the SOD activity. The evidence that all of the latter antibodies failed to react with recombinant artificial SOD free of charge isomers suggested correlation of the heterogeneity with the presence of charge isomeric forms. The former antibody was further used to establish a fluorescence sandwich enzyme immunoassay, and this assay provided a very sensitive detection limit as low as 100 pg/ml.