Determinations of total cytokine concentration in biological fluids by immunoassays face two major problems: the biochemical heterogeneity of the analyte and the interference of cytokine-binding proteins. We developed an ultrasensitive enzyme immunoassay for interleukin-6 (IL-6), using monoclonal antibodies and acetylcholinesterase as the tracer enzyme. The antibodies recognized recombinant and glycosylated forms of IL-6 equally. The antibodies measured dimeric recombinant IL-6, yet we could not detect IL-6 oligomers in plasma samples. We investigated the potential interference of soluble IL-6 receptor (sIL-6R), which is present at high concentrations in plasma samples (1 to 2 nmol/L). Heat treatment of the sample obviated the sIL-6R interference. Using calibrators in a plasma matrix, we demonstrated by fractionation, dilution, and recovery experiments that the immunoassay accurately measured total IL-6 in both normal and pathological serum and plasma samples.
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