A novel mechanism for cytokine regulation: Screening, selection, and characterization of anticytokine monoclonal and polyclonal autoantibodies

We have recently described an immunoregulatory mechanism involving release of neutralizing autoantibodies (Aab) to cytokines during bacterial infectio ns. Intraperitoneal inoculation of Haemophilus influenzae type b (Hib) into Sprague-Dawley rats resulted in high levels of inflammatory mediators earl y after infection. Increased titers of cytokine Aab were observed, with a p eak at day 7, We cloned Aab-producing B cells. Screening of the clones with five different cytokines resulted in detection of Aab-producing clones rea ctive with each cytokine. After repeated subcloning, monoclonal Aab (mAab) were selected and characterized for their specificity, isotypes, and affini ties. To elucidate regulatory importance, mAab to interferon-gamma (IFN-gam ma) and tumor necrosis factor-alpha (TNF-alpha) dose-dependently inhibited IFN-gamma-induced MHC expression by peritoneal macrophages and TNF-alpha-in duced thymocyte proliferation, respectively. Fab fragments exhibited bindin g and neutralizing effects, confirming specificities, Cross-reactivity with other rat cytokines was excluded. Pools of clones containing several mAab to each cytokine were obtained and served as polyclonal Aab, The relative a ffinity of the Aab was determined and found to be of high index. The charac terized Aab were tested in methodologic assays for cytokine detection, reve aling that some Aab were useful in a cell release capturing (CRC) ELISA.