Induction and detection of anti-peptide antibody specificity is critically affected by the mode of hapten presentation.

C-Terminal cholecystokinin (CCK)-peptides of increasing chain lengths were all linked at their N-termini to the single surface-exposed cysteine residue 107 of yeast iso-1-cytochrome c by the maleimide/thiol reaction. The resulting CCK/cytochrome 1:1 conjugates with the haptenic peptides in the identical protein environment were used to immunize outbred guinea pigs in order to assess the critical size of CCK peptides required for the expression of a CCK-specific epitope and the induction of antibodies not crossreacting with the homologous gastrin sequence. By using standard ELISA techniques with polystyrene-adsorbed antigen to evaluate the specificity of the antisera, none of the conjugates were found to induce anti-CCK antisera not crossreacting with gastrin. However, when the biotinyl-CCK-antigen was immobilized by polystyrene-adsorbed avidin, i.e. via a procedure which assures maximum accessibility of the bound antigen, we were able to demonstrate that with CCK-12 and particularly CCK-13, linked through their N-termini to the carrier, the critical length for the expression and recognition of a CCK-specific epitope was reached. The related polyclonal antisera did not crossreact with the homologous gastrin in the modified ELISA.

Alkylation of histidine with maleimido-compounds.

Introduction of the maleimide function via a spacer into histidine-containing peptides was found to produce ring closure by nucleophilic addition of the Nim-imino function of the histidine side-chain to the activated double bond of the maleimide. As an intramolecular cyclization reaction it proceeds at remarkably higher rates than the bimolecular alkylation of histidine derivatives with N-ethyl-maleimide. Correspondingly, in the case of the histidine-peptides examined only mixtures of the cyclic isomeric compounds were isolated and structurally characterized by 1H-NMR analysis. As expected, prevention of this reaction in histidine-containing maleoyl-peptides can be achieved by Nim-protection of the imidazole group. However, upon removal of this protection, the reaction takes place again, thus remarkably hampering the usefulness of the maleimide/thiol addition principle in conjugate chemistry for peptides. On the other hand this reaction could represent an interesting new approach for the design of cyclic peptidomimetic analogs.

Enzyme immunoassay with captured hapten. A sensitive gastrin assay with biotinyl-gastrin derivatives.

Two N-terminal biotinyl-gastrin derivatives were synthesized to investigate the effect of the length and chemical properties of the biotin-spacer on both the capture of the hapten by streptavidin or avidin adsorbed on polystyrene, and the antigenicity of the captured peptide. The observed full retainment of antibody binding capacity of the biotinyl-gastrins upon their immobilization, allowed to develop a sandwich-type ELISA with a sensitivity of one order of magnitude better than the standard ELISA with polystyrene-adsorbed gastrin. This hapten capture system reduces desorption particularly pronounced for low mass peptides, and avoids possible modifications or suppression of epitopes by the adsorption process with concomitant reduction of antibody binding affinity of the antigen. This new type of assay procedure may also represent a useful tool particularly for epitope mapping with relatively low mass synthetic protein fragments.