Synthetic peptides reactive with anti-human milk fat globule membrane monoclonal antibodies

Mammary mucins are increased in amounts in breast cancer patient sera, and most anti-breast cancer antibodies react with such mucins. One such mucin is found in human milk fat globule membrane and consists predominantly of O-linked sugars and a protein core. Partial complementary DNA clones for the protein core have recently been obtained. The nucleotide sequence is of interest as it contains a 60-base pair repeat, giving rise to a repeated 20-amino acid sequence (PDTRPAPGSTAPPAHGVTSA). Peptides with various lengths were synthesized using this sequence and the adjacent 4 amino acids (PDTR). Three anti-human milk fat globule membrane antibodies produced in our laboratory (BC1, BC2, and BC3) were tested to determine their reactivity with these synthetic peptides. Using three different assays (direct enzyme-linked immunosorbent assay test on peptides, direct enzyme-linked immunosorbent assay test on bovine serum albumin-conjugated peptides, and an inhibition test with the peptides in liquid, rather than solid phase), it was shown that APDTR was the minimum amino acid sequence required to form a reactive epitope with all 3 antibodies, although individual differences in the reactivities of the antibodies were noted. The addition of alanine (A) converted a nonreactive PDTR peptide to a reactive one, and the deletion of arginine (R) did the reverse; thus APDTR is the smallest peptide which reacts with these anti-human milk fat globule membrane antibodies.