Functional and Molecular Characterization of a Monoclonal Antibody against the 165–186 Peptide of Human 1β

Abstract

A synthetic peptide of human recombinant interleukin 1.beta. (hrIL-1.beta.) 165-186, which exhibits biological activity in the human fibroblast assay, was used as an immunizing antigen to obtain a murine monoclonal antibody (MoAb) termed FIB 1. This MoAb, and IgG1, reacts specifically with hrIL-1.beta., but not with hrIL-1.alpha., as measured in enzyme-linked immunosorbent assays (ELISA). The MoAb FIB 1 detects the characteristic 17 kDa IL-1 protein in Western blots. Binding to the antigen is specific, as deduced also from the close correlation of ELISA immunoreactivity with IL-1 biological activity. The anti-IL-1.beta. 165-186 Ab specifically neutralizes the biological activity of hrIL-1.beta. and native IL-1, as measured by the IL-1 induced proliferation of murine thymocytes and human fibroblasts and the IL-1-dependent IL-2 production by murine T cells (EL4-6.1). Fifty per cent of hrIL-1.beta. activity (25 U/ml, or 0.25 ng/ml) has neutralized by less than 30 .mu.g/ml of MoAb. Furthermore, FIB 1 recognizes intracellular IL-1 in lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The anti-IL-1.beta. 165-186 Ab does not react with the shorter IL-1.beta. fragment 161-173 in solid-phase ELISA, therefore the binding region seems to be localized in the amino acid sequence VALGLKEKNLYLS. A sandwich-ELISA, using a polyclonal sheep anti-IL-1.beta. 251-269 Ab as the capture antibody and an anti-IL-1.beta. 165-186 MoAb as the detecting probe, allowed the determination of IL-1.beta. from crude supernatants.