TUMOR-NECROSIS-FACTOR (TNF)-ALPHA QUANTIFICATION BY ELISA AND BIOASSAY - EFFECTS OF TNF-ALPHA-SOLUBLE TNF-RECEPTOR (P55) COMPLEX DISSOCIATION DURING ASSAY INCUBATIONS

It has been previously reported that different quantitative results ca n be obtained when TNF alpha is measured in biological fluids by bioas say and immunoassay. This is thought to be related to the presence of antigenic forms of TNF alpha that cannot be detected by bioassay, such as complexes with soluble receptors (sTNF-R) or TNF alpha monomers. I n this work we have observed discrepancies between antigenic and bioac tive TNF alpha even when we used a sandwich-ELISA, unable to detect TN F alpha monomers, based on antibodies that bind epitopes overlapping w ith the soluble-receptor binding site of TNF alpha. Moreover, we found that antigenic TNF alpha levels in the presence of p55-sTNF-R (sTNF-R 1) measured by different immunoassays were variable, depending on the immunoreagents and incubation time. To investigate whether TNF alpha-s oluble receptor complex dissociation occurring during assay incubation s contributes to the variability of results, we studied the kinetics o f TNF alpha-soluble receptor interactions and examined the effect of c omplex dissociation using different analytical systems. TNF alpha asso ciation (k(on)) and dissociation (k(off)) rate constants with sTNF-R1, measured by real-time biospecific interaction analysis, were 5.01 x 1 0(5) s(-1) M(-1) and 2.8 x 10(-4) s(-1), corresponding to an equilibri um 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 TNF alph a-sTNF-R1 bound:free ratio, in different analytical systems, markedly affects TNF alpha quantification.