An ELISA for a multiphosphorylated peptide, alpha(s1)-casein(59-79)

Multiphosphorylated segments of proteins are predicted to be in or near epitopes. However, due to the very hydrophilic nature of multiphosphorylated peptides, epitope mapping by ELISA using conventional microtitre plates can produce false negatives due to poor antigen adsorption. We have developed a sensitive ELISA for a multiphosphorylated peptide alpha(s1)-casein(59-79) containing five phosphoseryl residues using Nunc-Immuno Maxisorp modules for antigen adsorption. The peptide alpha(s1)-casein(59-79) was detected in the ELISA at antigen coating concentrations of 1.0 microg/ml and above, using rabbit anti-casein antibodies at a dilution of 1/10,000 in Tris-buffered saline containing 0.05% (w/v) Tween 20 and 1.0% (v/v) normal goat serum. At an antigen coating concentration of 10 microg/ml, anti-casein antibodies bound to alpha(s1)-casein(59-79) and produced an absorbance of more than 100 times background. Using conventional polyvinyl chloride and polystyrene plates the peptide alpha(s1)-casein(59-79) could only be detected at very high antigen coating concentrations of 1-10 mg/ml. The addition of 0.05% (w/v) Tween 20 to the blocking, antibody diluting and wash buffers of the ELISA was shown to significantly reduce nonspecific binding of the primary antibody. Further, the inclusion of normal goat serum in the blocking and antibody diluting buffers resulted in a small but significant increase in absorbance. The ELISA developed in this study has been used successfully with a range of enzymatically derived and synthetic peptides containing one to five phosphorylated residues such that it should have general applicability to the study of antigenicity of multiphosphorylated peptides.