SYNTHESIS OF MONOFUNCTIONAL AND BIFUNCTIONAL PEPTIDE DEXTRAN CONJUGATES FOR THE IMMOBILIZATION OF PEPTIDE ANTIGENS ON ELISA PLATES - PROPERTIES AND APPLICATION

Dextran has been used as a carrier molecule for the synthesis of monof unctional peptide-dextran conjugates. The immunodetection of such carr ier immobilized peptides on ELISA plates was compared to that of pepti des adsorbed directly to immunoplates. The main features observed with peptide-dextran conjugates were as follows: only small amounts of pep tide (1-2 mg) were necessary for coupling via alpha- or epsilon-amino groups to NaIO4-activated dextran (4 mg); the coupling yield was up to 68%; an amino acid analysis of the conjugate enabled the amount of ca rrier immobilized peptide to be calculated; an estimated 15-17 peptide s were bound per dextran molecule (MW 73,500); using a carbohydrate as carrier reduces the possibility of non-specific interactions because no hydrophobic or ionic sites and no protein-like epitopes exist on th e carrier apart from the peptide ligand. It can be assumed that some p eptide ligands provide the forces for an interaction with the plate su rface whereas others remain free for the interaction with the antibody . Thus, the detection with monoclonal anti-peptide antibodies allowed peptide-dextran conjugates to be used at coating concentrations of 1-3 nM peptide, corresponding to 0.6-2.6 ng peptide-dextran per well. In contrast, concentrations of 150-500 nM were required for coating with peptides. The applicability of monofunctional peptide-dextran conjugat es was demonstrated by investigating the titer and specificity of a po lyclonal anti-peptide serum developed against human gastrointestinal g lutathione peroxidase. The introduction of biotin as a second ligand o f the dextran conjugate permitted its capture on streptavidin coated p lates. This synthesis of bifunctional peptide-biotin-dextran conjugate s opens up additional possibilities for applications. (C) 1997 Elsevie r Science B.V.