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, an 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 micrograms/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 culture supernatants.

Human Vascular Smooth Muscle Cells Express Interleukin-1β–converting Enzyme (ICE), but Inhibit Processing of the Interleukin-1β Precursor by ICE

“…Rabbit polyclonal antisera directed against IL-1 ␣ (L1), IL-1 ␤ (L4), or ICE ( ␣ -ICE P20 ) were generated by immunization with recombinant human mature IL-1 ␣ , mature IL-1 ␤ , or the ICE 20-kD subunit (ICE P20 ), respectively. The anti-IL-1 ␤ 251-269 antibody (Fib 3) was raised as described elsewhere (30).…”

Properties of Multinucleated Giant Cells in a Newin Vitro Model for Human Granuloma Formation

“…The antibodies used in this study were as follows: anti-CD14 (HB44, American Type Culture Collection), anti-CD4 (Leu 3a, Becton Dickinson), anti-gp 140 kd (Ber-Mac3 18 ), anti-CD8 (Leu 2a, Becton Dickinson), anti-CD19 (MD-37, Dako), anti-IL1 (Fib-3 19 ), anti-IL-6 (B-E8, Biosource International), and anti-TNF (TNFE 20 ). Histochemical staining for NADPHdiaphorase was performed according to Vincent and Kimura.…”

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A

“…Immunocytochemical staining of intracellular TNF and IL-1/3 was performed using murine monoclonal antibodies against rh TNF (TNF-E, kindly provided by Dr. G.R. Adolf, Ernst Boehringer Institute, Vienna, Austria [33]) and against peptide 251-269 of human IL-lfl (FIB-3) [34,35], and the alkaline phosphatase anti-alkaline phosphatase method as described by Cordell et al [36] followed by substrate reaction with New Fuchsin [37]. The percentage of TNFor IL-1/3-positive cells was calculated based on counting 200 PBMo on each slide.…”