AN ELISA FOR A MULTIPHOSPHORYLATED PEPTIDE, ALPHA(S1)-CASEIN(59-79)

Multiphosphorylated segments of proteins are predicted to be in or nea r epitopes. However, due to the very hydrophilic nature of multiphosph orylated peptides, epitope mapping by ELISA using conventional microti tre 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 N unc-Immuno Maxisorp modules for antigen adsorption. The peptide alpha( s1)-casein(59-79) was detected in the ELISA at antigen coating concent rations of 1.0 mu g/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 co ncentration of 10 mu g/ml, anti-casein antibodies bound to alpha(s1)-c asein(59-79) and produced an absorbance of more than 100 times backgro und. Using conventional polyvinyl chloride and polystyrene plates the peptide alpha(s1)-casein(59-79) could only be detected at very high an tigen 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 pri mary antibody. Further, the inclusion of normal goat serum in the bloc king and antibody diluting buffers resulted in a small but significant increase in absorbance.The ELISA developed in this study has been use d successfully with a range of enzymatically derived and synthetic pep tides containing one to five phosphorylated residues such that it shou ld have general applicability to the study of antigenicity of multipho sphorylated peptides. (C) 1998 Elsevier Science B.V. All rights reserv ed.