Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10

Radio iodinated recombinant human IL-10 was prepared and validated for the measurement of natural human anti-IL-10 antibodies. Iodination of IL-10 was accomplished by means of the chloramine-T method. The crude tracer was purified by size chromatography as homo-dimeric IL-10 with a specific activity of 75 cpm/pg. Validation of the tracer confirmed preserved antibody epitopes and receptor binding ability. A robust Radio Immuno Assay (RIA) was developed and validated to detect natural human anti-IL-10 antibodies based on the formation of 125I-labeled IL-10–IgG complexes in solution and separation of the complexes by chromatography on mini-columns. The RIA was applied to 3360 plasma samples derived from normal Danish blood donors. Generally, IL-10 did not bind to plasma factors other than natural anti-IL-10 IgG antibodies. The prevalence of donors high positive for antibodies against IL-10 was 0.5%. These levels were apparently associated with IL-10 deficiency, since the antibodies blocked IL-10-induced STAT3 phosphorylation in normal blood leukocytes. This methodology will be used to explore the clinical significance of natural anti-IL-10 antibodies with respect to inflammatory disorders.