Monitoring coupling of peptides to carrier proteins using biotinylated peptide

The amount of synthetic peptide coupled to a carrier protein is a critical parameter in production of antipeptide antibodies. In the method described, biotinylated peptide was included as a tracer to monitor the coupling of peptide to keyhole limpet hemocyanin or BSA. The extent of coupling was assessed by first fractionating free and coupled peptide by gel filtration, then slot blotting an aliquot of each fraction onto nitrocellulose. The biotinylated peptide was detected using an avidin-horseradish peroxidase conjugate. To estimate the mass of peptide incorporated, the fractions were analyzed using a competitive ELISA. The limit of detection of this assay was < 0.1 pmol and was linear to 2 pmol. The mass of biotinylated peptide in each column fraction was determined using this assay and the total mass of peptide coupled to the carrier was calculated. Using these techniques, biotin-labeling provided a sensitive and versatile means to assess the quality of peptide-carrier protein conjugates.