Analysis of prostate specific antigen and alpha 1-antichymotrypsin interaction using antipeptide monoclonal antibodies

Purpose: The synthetic peptides E30D and D10P that correspond to prostate s pecific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclona l antibodies (mAbs). Materials and Methods: Antipeptide mAb characteristics were studied using b iosensor technology and enzyme-linked immunosorbent assay, and analyzing th e mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PS A enzymatic activity. Epitope mapping of these mAbs was performed using ove rlapping peptide synthesis on nitrocellulose membrane. Results: Anti-E30D mAbs bound PSA coated on the solid phase only, whereas a nti-D10P mAbs recognized PSA in detection as well as in capture. However, t hese mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of P SA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was invo lved in the interaction site of PSA with ACT. Furthermore, we were unable t o inhibit the enzymatic activity of PSA as well as PSA-ACT complex formatio n. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA . Conclusions: The linear anti-D10P mAb epitope is located outside of the PSA -ACT binding site. However, these mAbs may be of value for evaluating the p resence of different molecular PSA forms in sera.