Characterization of novel monoclonal antibodies for prostate-specific antigen (PSA) with potency to recognize PSA bound to α2- macroglobulin

Abstract

Background: Different molecular forms of prostate-specific antigen (PSA) have been used to differentiate between benign prostatic hyperplasia and prostate cancer. Detecting PSA bound to endogenous inhibitors such as α1-antichymotrypsin (ACT) and α2-macroglobulin (α2M) is often difficult because of epitope masking or sensitivity problems. Here we report the characterization of four novel mouse monoclonal antibodies (mabs) obtained by immunization with PSA- α2M complexes. Their ability to detect free PSA and PSA-inhibitor complexes was shown, and their epitopes were analyzed by phage display technology. Methods: The properties of the mabs were studied by competition and sandwich assays and by Western blotting. Epitope mapping was performed by screening of a phage display peptide library. Results: All four mabs recognized free PSA, PSA-ACT, and PSA-α2M complexes, but to various degrees. With different combinations of mabs in competition experiments, antibodies were identified that enhance binding of other mabs to PSA, forming the molecular basis of a very sensitive assay for the detection of PSA and PSA-ACT complexes. Mabs with highest reactivity for PSA- α2M were selected to establish an immunoassay for that complex. Western blot analysis revealed that all mabs recognized conformational epitopes of PSA. These findings were supported by phage display results demonstrating mimotopes in the PSA molecule. Conclusion: The results presented here could aid in the further development of clinically relevant assays for PSA and PSA-α2M complexes. © 2005 American Association for Clinical Chemistry.