Mode of interaction between tumor-necrosis-factor-alpha and a monoclonal-antibody expressing a recurrent idiotype

Tumor Necrosis Factor alpha (TNFalpha) is an inflammatory cytokine which exists mainly as a 51kD complex built up of 3 identical, noncovalently-linked polypeptide subunits. We have raised monoclonal antibodies (mAb) against human TNFalpha (huTNFalpha). One of these mAb (mAb78, mouse IgG1k) was studied in detail. mAb78 expresses a recurrent idiotype typical of the BALB/c anti-huTNFalpha antibody response. HuTNFalpha bound to mAb78 with an affinity constant (K(obs)) of 3.2 x 10(10)M-1. The number of huTNFalpha-binding sites per mAb78 molecule was almost-equal-to 0.7. At concentrations higher than the K(obs) mAb78 neutralized huTNFalpha at a almost-equal-to 1.3 : 1 molar ratio. mAb78 precipitated huTNFalpha in a double immunodiffusion assay in agar. Gel-filtration experiments of mAb78-huTNFalpha mixtures, that had been set up in large antigen excess, detected complexes of 570 kD as the smallest ones formed under these conditions. We propose that these results are accommodated best by a model according to which cyclic complexes built up of 3 mAb78 and 2 huTNFalpha molecules are the smallest units formed upon interaction of the reagents. In view of this model we discuss how huTNFalpha and mAb78 can undergo a precipitin reaction.