Synthesis of mono- 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 monofunctional peptide-dextran conjugates. The immunodetection of such carrier immobilized peptides on ELISA plates was compared to that of peptides adsorbed directly to immunoplates. The main features observed with peptide-dextran conjugates were as follows: only small amounts of peptide (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 carrier immobilized peptide to be calculated; an estimated 15-17 peptides 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 the carrier apart from the peptide ligand. It can be assumed that some peptide ligands provide the forces for an interaction with the plate surface whereas other 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 conjugates was demonstrated by investigating the titer and specificity of a polyclonal anti-peptide serum developed against human gastrointestinal glutathione peroxidase. The introduction of biotin as a second ligand of the dextran conjugate permitted its capture on streptavidin coated plates. This synthesis of bifunctional peptide-biotin-dextran conjugates opens up additional possibilities for applications.
Böcher
M
M
Department of Enzymology, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany. boecher@gbf-braunschweig.de
Böldicke
T
T
Kiess
M
M
Bilitewski
U
U
D000327:Adsorption; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000941:Antigens / Q000032:analysis*; D003911:Dextrans / Q000032:analysis / Q000138:chemical synthesis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis*
D000596:Amino Acids; D000941:Antigens; D003911:Dextrans; D010455:Peptides