Date of Award
Doctor of Philosophy (PhD)
Biomedical Sciences - Microbiology & Immunology
George L. Wright, Jr.
Ann E. Campbell
A murine monoclonal antibody (MAb), 7E11-C5, recognizes a prostate organ-specific antigen. Under reducing conditions, the 7E11-C5 antigen exhibited major 100-Kd and minor 70-Kd bands, suggesting that the antigen comprises two peptide chains. The antigen was detected in pooled normal human seminal plasma, xenograft tissue extract of a prostate adenocarcinoma cell line, LNCaP, and tissue extracts of benign prostatic hyperplasia (BPH) and prostate carcinomas, but was not identified in any of the non-prostate tissue extracts tested. Antibodies of the three major prostate-associated antigens (prostatic acid phosphatase (PAP), prostate specific antigen (PSA) and prostate secretory protein (PSP)) were unable to block the binding of MAb 7E11-C5 to its target antigen. Western blot analysis demonstrated that there was no similarity between the 7E11-C5 antigen and these three antigens. The 7E11-C5 antigen was purified 400-fold from LNCaP xenograft tissue extract by immunoaffinity chromatography with an estimated antigen recovery of 64%. High pressure liquid chromatography (HPLC) molecular exclusion column was used to further purify the antigen and results showed that the molecular weight of the antigen, under native condition, was about 500-Kd. Biochemical characterization showed that the antigen was sensitive to both periodate oxidation and protease treatment, indicating that the antigen was a glycoprotein; both carbohydrate and peptide were required for the antigenicity. The antigen was found to be sensitive to pH 2.0, while remaining unaffected after treatment with NaOH (pH 11.0). Glycosidase treatment and lectin competitive binding study suggested that the carbohydrates involved in the antigenic determinant were galactose and N-acetyl-D-galactosamine. The antigen was not demonstrable in thin-layer chromatography (TLC) immunoblot of glycolipid extracts of LNCaP tissue. Western blot analysis of 48 serum samples from age-matched normal males, BPH patients and prostate cancer patients (stages A, B, C and D) failed to clearly show the presence of the 7E11-C5 antigen. Likewise, the antigen was not detected in these sera using a competitive binding inhibition assay. These results suggest that the antigen is either not shed into the circulation or that the antigen is either masked by serum inhibitors or disassociated into fragments not recognized by the MAb.
"Purification and Characterization of the Prostate Cancer-Associated Antigen: 7E11-C5"
(1991). Doctor of Philosophy (PhD), Dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/a6tb-nx91