Production and Characterization of Novel Anti-Prostate-specific Antigen (PSA) Monoclonal Antibodies That Do Not Detect Internally Cleaved Lys145-Lys146 Inactive PSA

Abstract Background: The nature of free, uncomplexed prostate-specific antigen (PSA) in the circulation is still unknown. In this study, we developed novel anti-PSA antibodies using PSA produced by a metastasized cancer cell line, LNCaP, as an immunogen. Methods: Hybridoma cell lines were screened with different methods that aimed at finding antibodies specific for the forms of free PSA produced by LNCaP cell line. Obtained antibodies were further studied for their characteristics related to previously characterized monoclonal antibodies. Results: Numerous anti-PSA antibodies were obtained, of which four represented unique epitopes previously unrecognized by us. One free-PSA-specific antibody was bound to PSA on two distinct epitopes, and one antibody was bound to the carboxyl-terminal peptide of PSA. Two antibodies were found to bind to the peptide sequence adjacent to the internal cleavage site Lys145-Lys146. These antibodies failed to recognize internally cleaved PSA at Lys145-Lys146. We could not find anti-proPSA antibodies despite the fact that LNCaP PSA contained more than one-half of the zymogen form of PSA. Conclusions: We report, for the first time, novel anti-PSA antibodies that do not recognize internally cleaved PSA at Lys145-Lys146 and thus are specific for intact, unclipped PSA.

Download Full-text

Characterization of Monoclonal Antibodies for Prostate-specific Antigen and Development of Highly Sensitive Free Prostate-specific Antigen Assays

Clinical Chemistry ◽

10.1093/clinchem/45.3.347 ◽

1999 ◽

Vol 45 (3) ◽

pp. 347-354 ◽

Cited By ~ 24

Author(s):

Margot H Black ◽

C Linda Grass ◽

Jari Leinonen ◽

Ulf-Håkan Stenman ◽

Eleftherios P Diamandis

Keyword(s):

Monoclonal Antibodies ◽

Prostate Specific Antigen ◽

Specific Antigen ◽

Cross Reactivity ◽

Free Form ◽

Time Resolved ◽

Free Psa ◽

Glandular Kallikrein ◽

Highly Sensitive

Abstract Background: The recent elucidation of the importance of serological free prostate-specific antigen (PSA) in the diagnosis of prostate cancer has created a demand for immunoassays specific for free PSA. Methods: We developed and characterized 11 monoclonal antibodies with high affinities for PSA (Kavalues from 1.1 × 108 to 1.8 × 1010L/mol), only 3 of which cross-react with human glandular kallikrein (hK2). Using these antibodies and PSA antibodies developed by others, in conjunction with time-resolved fluorometry, we developed ultrasensitive sandwich immunoassays specific for the free form of PSA. Results: The analytical detection limit of these immunoassays is 0.001 μg/L. To our knowledge, this is the most sensitive free PSA assay reported to date. The free PSA immunoassays exhibit <1% cross-reactivity with PSA-α1-antichymotrypsin, show no cross-reactivity with hK2, and correlate well with established free PSA kits. The 11 antibodies developed by our group, in conjunction with 4 commercially available antibodies, were used to generate a putative epitope map of the PSA molecule. Conclusion: The highly sensitive free PSA immunoassays may be used for measuring PSA subfractions in female serum, an application currently impossible with other reported free PSA immunoassays.

Download Full-text

Characterization of Novel Monoclonal Antibodies for Prostate-Specific Antigen (PSA) with Potency to Recognize PSA Bound to α2-Macroglobulin

Clinical Chemistry ◽

10.1373/clinchem.2004.039636 ◽

2005 ◽

Vol 51 (1) ◽

pp. 84-92 ◽

Cited By ~ 11

Author(s):

Yvonne Baumgart ◽

Andreas Otto ◽

Angelika Schäfer ◽

Elke Usbeck ◽

Christiane Cott ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Phage Display ◽

Prostate Specific Antigen ◽

Specific Antigen ◽

Molecular Forms ◽

Phage Display Technology ◽

Free Psa ◽

Α2 Macroglobulin ◽

Display Technology

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.

Download Full-text

Characterization of Monoclonal Antibodies Raised Against the Prostatic Cancer Cell Line PC-82

The Journal of Urology ◽

10.1016/s0022-5347(17)43200-9 ◽

1987 ◽

Vol 138 (2) ◽

pp. 458-458

Author(s):

M.P.W. Gallee ◽

C.C.J. van Vroonhoven ◽

H.A.G.M. van der Korput ◽

T.H. van der Kwast ◽

F.J.W. ten Kate ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Cell Line ◽

Cancer Cell ◽

Prostatic Cancer ◽

Cancer Cell Line

Download Full-text

Clathrin Gene Expression Is Androgen Regulated in the Prostate*

Endocrinology ◽

10.1210/endo.139.4.5926 ◽

1998 ◽

Vol 139 (4) ◽

pp. 2111-2119 ◽

Cited By ~ 6

Author(s):

James L. Prescott ◽

Donald J. Tindall

Keyword(s):

Gene Expression ◽

Cell Line ◽

Prostate Specific Antigen ◽

Heavy Chain ◽

Messenger Rna ◽

Specific Antigen ◽

Cancer Cell Line ◽

Secretory Proteins ◽

Clathrin Heavy Chain ◽

Glandular Kallikrein

Abstract Androgens are required for the development and function of the prostate. In a normal human prostate, androgens control the synthesis of proteins such as prostate-specific antigen and human glandular kallikrein. The prostate secretes these proteins as well as a number of other compounds to form the prostatic fluid. Using differential display PCR to detect novel androgen-regulated genes, clathrin heavy chain expression was identified as potentially being up-regulated by androgens in the prostate cancer cell line LNCaP. We report here that the clathrin heavy chain and light chain genes are regulated by androgens. Clathrin heavy chain messenger RNA was up-regulated by androgens in a concentration- and time-specific manner in the LNCaP cell line. Translation of clathrin heavy chain messenger RNA was stimulated by androgens. Steady state levels of clathrin light chains a and b were up-regulated in the presence of androgen in LNCaP cells. Clathrin gene expression was examined in normal rat prostates, and similar results were found. Clathrin heavy chain protein levels in the rat prostate are also affected by the androgen status of the animal. We hypothesize that clathrin may be involved in the exocytosis of androgen-regulated secretory proteins such as prostate-specific antigen and human glandular kallikrein.

Download Full-text