MONOCLONAL-ANTIBODY BASED ENZYME-LINKED AND CHEMILUMINESCENT ASSAYS FOR THE HUMAN INTERLEUKIN-1 RECEPTOR ANTAGONIST - APPLICATION TO MEASURE HIL-1RA LEVELS IN MONOCYTE CULTURES AND SYNOVIAL-FLUIDS

Interleukin-1 receptor antagonist (IL-1ra) has the potential to counte ract at least part of the biological effects of interleukin-1. The out come of an inflammatory reaction may therefore be determined by the ba lance between IL-1 and IL-1ra, rather than by IL-1 alone. We have deve loped an immunoassay to address this issue as well as to assess the ef fects of anti-inflammatory agents on the expression of IL-1 and IL-1ra in vitro or in body fluids. Recombinant human IL-1ra was expressed in an E. coil system, purified to homogeneity, and used to derive monocl onal antibodies in mice as well as polyclonal antibodies in rabbits. A sandwich ELISA was constructed with F(ab'>(2) fragments of a high aff inity monoclonal antibody and the rabbit serum as a source of secondar y antibody. The assay required no sample treatment to avoid interferen ce by rheumatoid factor. The measuring range was 0.020-2 ng/ml. By lab elling a second monoclonal antibody with an acridinium ester, a chemil uminescence assay with a wider measuring range (0.050-15 ng/ml) was ge nerated. In accord with published data, we found that IL-1ra was secre ted by human monocytes stimulated with LPS, Zymosan, IL-1 alpha, or hu man IgG. After an induction phase of ca. 4 hours and depending on the stimulus, IL-1ra accumulated linearly for periods up to 96 h. IL-1ra l evels in synovial fluids of 19 patients suffering from various inflamm atory joint diseases were compared with the cytokine levels of IL-1 be ta, IL-6, IL-8, and TNF-alpha. Highest positive correlations were foun d with IL-8 and IL-1 beta. In normal blood donors IL-1ra serum levels were 150-800 pg/ml (Median: 387 pg/ml). Owing to its sensitivity and l arge measuring range the newly developed assays appear to be suitable for measuring IL-1ra in cell cultures as well as in biological fluids.