Comparison of reactivity of monoclonal antibody (3F2) to trimeric tumor necrosis factor (TNF-alpha) with that to monomeric TNF-alpha.

Biologically active TNF-alpha is a trimer. Our newly developed monoclonal antibody against recombinant human TNF-alpha (3F2) bound to TNF-alpha immobilized on a solid phase precoated with the same monoclonal antibody, 3F2. We therefore examined the possibility that 3F2 preferentially recognized trimeric TNF-alpha, and obtained the following results. (i) Treatment of TNF-alpha with 1% NP40 enhanced the dissociation from the trimer to the monomer. The treatment also reduced the reactivity of 3F2 to TNF-alpha immobilized on a solid phase precoated with either 3F2 or a rabbit anti-TNF-alpha antibody (PT50). (ii) When trimeric and monomeric TNF-alpha, obtained by gel filtration of NP40-treated TNF-alpha, were assayed with either PT50 (solid phase)/3F2 (fluid phase) or PT50/PT50, PT50/3F2 showed a higher response to trimeric TNF-alpha than to monomeric TNF-alpha, while PT50/PT50 showed the opposite result. (iii) The values for two fractions containing only trimeric TNF-alpha obtained on ELISA with PT50/3F2 fitted the standard curve for ELISA with PT50/3F2 which was made by using unseparated TNF-alpha. Taken together, these findings suggested that 3F2 preferentially recognizes trimeric TNF-alpha.