Anti-ricin-A-chain monoclonal antibodies (mAbs) were raised in order to study epitopes of the A-chain involved in the initiation of its transmembrane passage. Five nonoverlapping epitopes were selected by cluster formation using a cross-inhibition assay of mAbs from 172 specific hybridomas. For each cluster, representative high affinity mAbs were selected. These mAbs were disulfide-linked to F(ab')2 fragments of a mAb directed against the CD5 antigen. The cytotoxicity of ricin A-chain bound by affinity through different epitopes to these hybrid mAbs was explored on the CD5-positive CEM cells. Without any enhancer, the hybrids displayed cytotoxicity varying in IC50 within a range of 1-10; in the presence of the enhancer NH4Cl, this variation of activity was maintained ranging from 1 to 20; with the enhancer monensin, the variation of cytotoxicity of hybrids extended over a 1-80-fold range. These differences of cytotoxicity were not correlated with mAb properties such as the inhibition of A-chain enzymatic activity, the A-chain binding affinity, the capacity for releasing A-chain at low pH, or the capacity of hybrids for delivering A-chain on the cell surface. This lack of correlation strongly suggests that the different presentations of A-chain epitopes to the cell membrane may be the essential reason for cytotoxicity variations.

Ammonium Chloride↗

PubMedDate not supplied

[Artificial H-2 dependent carrier determinants of the insulin molecule].

The known tertiary structure of insulin allows the study of carrierdeterminants of insulin: their nature, their distance to antigenic determinants and their H-2 dependance. We examined the influence of "foreign " amino acids of insulins from two different species and the influence of chemically coupled new amino acids at the B-chain of insulin in congenic resistant mice. Three strains were immunized with bovine insulin and three derivatives: LeuB-insulin, LysB-insulin and GluB-insulin. H-2d mice were high responders to insulin and all derivatives, while H-2k mice showed no detectable antibodies. The H-2b strain had an intermediate antibody response to bovine insulin. LeuB-insulin, LysB-insulin and GluB-insulin produced similar titers as unmodified insulin in H-2d mice. In H-2b mice, GluB-insulin produced significantly more antibodies than did all other insulins tested with antibody specificities directed against A8-A10. Therefore Glu at position B0 acts as a carrierdeterminant and A8-A10 as antigenic determinant at the dose utilised in H-2b mice. The distance between both determinants is about 15-20 A, allowing therefore the cooperation of T and B cell.

Animals↗