Comparison of Peptide-Coating Conditions in Solid Phase Plate Assays for Detection of Anti-Peptide Antibodies

Abstract

Mice were immunized with 14 free (i.e. not conjugated to any carrier) synthetic peptides representing the entire human hemoglobin α-chain. Antibodies against each peptide were determined using solid phase radioimmunoassay, both with free peptides and peptides coupled to a protein carrier as the coating antigen. It has been demonstrated that large improvements in the ability to detect anti-peptide antibodies were achieved in some cases by precoating the assay wells with free peptides and in other cases by precoating with peptide-protein conjugates. Sodium carbonate buffer, pH 9.6, had a favorable effect on the coating of two of the free peptides when compared with phosphate-buffered saline, pH 7.2. The assay with the plates coated with optimum peptide form (free peptide or peptide-protein conjugate) was superior in the detection of antibody binding to 9 of the peptides when compared with the assay using chemically activated plates. The results suggest that the appropriate form (conjugated or free) and conditions for immobilizing small peptides to plastic supports are not universal but will have to be determined for each test peptide.