Tumor Necrosis Factor (TNF) α quantification by ELISA and bioassay: effects of TNFα-soluble TNF receptor (p55) complex dissociation during assay incubations

It has been previously reported that different quantitative results can be obtained when TNFα is measured in biological fluids by bioassay and immunoassay. This is thought to be related to the presence of antigenic forms of TNFα that cannot be detected by bioassay, such as complexes with soluble receptors (sTNF-R) or TNFα monomers. In this work we have observed discrepancies between antigenic and bioactive TNFα even when we used a sandwich-ELISA, unable to detect TNFα monomers, based on antibodies that bind epitopes overlapping with the soluble-receptor binding site of TNFα. Moreover, we found that antigenic TNFα levels in the presence of p55-sTNF-R (sTNF-R1) measured by different immunoassays were variable, depending on the immunoreagents and incubation time. To investigate whether TNFα-soluble receptor complex dissociation occurring during assay incubations contributes to the variability of results, we studied the kinetics of TNFα-soluble receptor interactions and examined the effect of complex dissociation using different analytical systems. TNFα association (k_on) and dissociation (k_off) rate constants with sTNF-R1, measured by real-time biospecific interaction analysis, were 5.01 × 10⁵ s^-1 M^-1 and 2.8 × 10^-4 s^-1, corresponding to an equilibrium constant (K_d) of 0.59 nM and to a half life for these complexes of 38 min. Complex dissociation and differential changes in the TNFα-sTNF-R1 bound:free ratio, in different analytical systems, markedly affects TNFα quantification.