European Journal of Immunology vol:23 issue:8 pages:2026-9
Mice pretreated with monoclonal anti-interleukin-6 (IL-6) antibody and then challenged with lipopolysaccharide (LPS), paradoxically develop higher levels of circulating biological IL-6 activity, as measured by the hybridoma growth promotion assay, than mice similarly challenged but not pretreated with antibody. Here we provide evidence that this increased biological activity was entirely accounted for by the presence of increased amounts of IL-6 protein, which could be isolated by immunoaffinity chromatography and subsequently visualized after gel electrophoresis. Chromatography on a protein G matrix and a sandwich ELISA allowed to demonstrate that all IL-6 present in the serum was in the form of antigen-antibody complexes. Serum samples of antibody-treated animals which contained the highest biological activity typically contained near equimolar concentrations of IL-6 and antibody. In vitro neutralization tests with pure antibody and IL-6 demonstrated that, with both antibodies tested, more than 1000-fold molar excess of antibody is needed for neutralization in the hybridoma growth assay. It is concluded that increased biological activity in serum of the anti-IL-6 antibody-treated mice is due to sequestration of the endogenous IL-6 in the form of antigen-antibody complexes which, due to the lack of sufficient antibody excess, produce nearly full activity in the hybridoma growth assay.