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.
Moroder
L
L
Max-Planck-Institut für Biochemie, Martinsried/München, Germany.
Papini
A
A
Siglmüller
G
G
Köcher
K
K
Dörrer
E
E
Schneider
C H
CH
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D002766:Cholecystokinin / Q000737:chemistry / Q000276:immunology*; D003429:Cross Reactions; D003574:Cytochrome c Group / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D005755:Gastrins / Q000737:chemistry / Q000276:immunology; D006168:Guinea Pigs; D006241:Haptens / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D014611:Vaccination
D003574:Cytochrome c Group; D000939:Epitopes; D005755:Gastrins; D006241:Haptens; D010446:Peptide Fragments; C050803:cholecystokinin 10 C-terminal fragment; D002766:Cholecystokinin