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Licensed Unlicensed Requires Authentication Published by De Gruyter June 26, 2006

A sensitive proximity ligation assay for active PSA

  • Lei Zhu , Hannu Koistinen , Ping Wu , Ale Närvänen , Edith Schallmeiner , Simon Fredriksson , Ulf Landegren and Ulf-Håkan Stenman
From the journal Biological Chemistry

Abstract

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 μg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.

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References

Armitage, T.G., Cooper, E.H., Newling, D.W., Robinson, M.R., and Appleyard, I. (1988). The value of the measurement of serum prostate specific antigen in patients with benign prostatic hyperplasia and untreated prostate cancer. Br. J. Urol.62, 584–589.10.1111/j.1464-410X.1988.tb04431.xSearch in Google Scholar

Catalona, W.J., Bartsch, G., Rittenhouse, H.G., Evans, C.L., Linton, H.J., Amirkhan, A., Horninger, W., Klocker, H., and Mikolajczyk, S.D. (2003). Serum pro prostate specific antigen improves cancer detection compared to free and complexed prostate specific antigen in men with prostate specific antigen 2 to 4 ng/ml. J. Urol.170, 2181–2185.10.1097/01.ju.0000095460.12999.43Search in Google Scholar

Finne, P., Zhang, W.M., Auvinen, A., Leinonen, J., Maattanen, L., Rannikko, S., Tammela, T.L., and Stenman, U.H. (2000). Use of the complex between prostate specific antigen and α1-protease inhibitor for screening prostate cancer. J. Urol.164, 1956–1960.10.1016/S0022-5347(05)66927-3Search in Google Scholar

Fredriksson, S., Gullberg, M., Jarvius, J., Olsson, C., Pietras, K., Gustafsdottir, S.M., Ostman, A., and Landegren, U. (2002). Protein detection using proximity-dependent DNA ligation assays. Nat. Biotechnol.20, 473–477.10.1038/nbt0502-473Search in Google Scholar

Gullberg, M., Gustafsdottir, S.M., Schallmeiner, E., Jarvius, J., Bjarnegard, M., Betsholtz, C., Landegren, U., and Fredriksson, S. (2004). Cytokine detection by antibody-based proximity ligation. Proc. Natl. Acad. Sci. USA101, 8420–8424.10.1073/pnas.0400552101Search in Google Scholar

Kumar, A., Mikolajczyk, S.D., Hill, T.M., Millar, L.S., and Saedi, M.S. (2000). Different proportions of various prostate-specific antigen (PSA) and human kallikrein 2 (hK2) forms are present in noninduced and androgen-induced LNCaP cells. Prostate44, 248–254.10.1002/1097-0045(20000801)44:3<248::AID-PROS10>3.0.CO;2-DSearch in Google Scholar

Leinonen, J., Zhang, W.M., Paus, E., and Stenman, U.H. (1999). Reactivity of 77 antibodies to prostate-specific antigen with isoenzymes and complexes of prostate-specific antigen. Tumour Biol.20 (Suppl. 1), 28–34.10.1159/000056527Search in Google Scholar

Leinonen, J., Wu, P., and Stenman, U.H. (2002). Epitope mapping of antibodies against prostate-specific antigen with use of peptide libraries. Clin. Chem.48, 2208–2216.10.1093/clinchem/48.12.2208Search in Google Scholar

Lilja, H., Christensson, A., Dahlen, U., Matikainen, M.T., Nilsson, O., Pettersson, K., and Lovgren, T. (1991). Prostate-specific antigen in serum occurs predominantly in complex with α1-antichymotrypsin. Clin. Chem.37, 1618–1625.10.1093/clinchem/37.9.1618Search in Google Scholar

Michel, S., Collomb-Clerc, E., Geourjon, C., Charrier, J.P., Passagot, J., Courty, Y., Deleage, G., and Jolivet-Reynaud, C. (2005). Selective recognition of enzymatically active prostate-specific antigen (PSA) by anti-PSA monoclonal antibodies. J. Mol. Recognit.18, 225–235.10.1002/jmr.729Search in Google Scholar

Mikolajczyk, S.D., Grauer, L.S., Millar, L.S., Hill, T.M., Kumar, A., Rittenhouse, H.G., Wolfert, R.L., and Saedi, M.S. (1997). A precursor form of PSA (pPSA) is a component of the free PSA in prostate cancer serum. Urology50, 710–714.10.1016/S0090-4295(97)00449-4Search in Google Scholar

Mikolajczyk, S.D., Millar, L.S., Wang, T.J., Rittenhouse, H.G., Marks, L.S., Song, W., Wheeler, T.M., and Slawin, K.M. (2000). A precursor form of prostate-specific antigen is more highly elevated in prostate cancer compared with benign transition zone prostate tissue. Cancer Res.60, 756–759.Search in Google Scholar

Mikolajczyk, S.D., Marker, K.M., Millar, L.S., Kumar, A., Saedi, M.S., Payne, J.K., Evans, C.L., Gasior, C.L., Linton, H.J., Carpenter, P., and Rittenhouse, H.G. (2001). A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer. Cancer Res.61, 6958–6963.Search in Google Scholar

Niemela, P., Lovgren, J., Karp, M., Lilja, H., and Pettersson, K. (2002). Sensitive and specific enzymatic assay for the determination of precursor forms of prostate-specific antigen after an activation step. Clin. Chem.48, 1257–1264.Search in Google Scholar

Schedlich, L.J., Bennetts, B.H., and Morris, B.J. (1987). Primary structure of a human glandular kallikrein gene. DNA6, 429–437.10.1089/dna.1987.6.429Search in Google Scholar

Stamey, T.A., Yang, N., Hay, A.R., McNeal, J.E., Freiha, F.S., and Redwine, E. (1987). Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N. Engl. J. Med.317, 909–916.10.1056/NEJM198710083171501Search in Google Scholar

Stenman, U.H., Leinonen, J., Alfthan, H., Rannikko, S., Tuhkanen, K., and Alfthan, O. (1991). A complex between prostate-specific antigen and α1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res.51, 222–226.Search in Google Scholar

Stenman, U.H., Paus, E., Allard, W.J., Andersson, I., Andres, C., Barnett, T.R., Becker, C., Belenky, A., Bellanger, L., Pellegrino, C.M., et al. (1999). Summary report of the TD-3 workshop: characterization of 83 antibodies against prostate-specific antigen. Tumour Biol.20 (Suppl. 1), 1–12.10.1159/000056523Search in Google Scholar

Vaisanen, V., Lovgren, J., Hellman, J., Piironen, T., Lilja, H., and Pettersson, K. (1999). Characterization and processing of prostate specific antigen (hK3) and human glandular kallikrein (hK2) secreted by LNCaP cells. Prostate Cancer Prostatic Dis.2, 91–97.10.1038/sj.pcan.4500289Search in Google Scholar

Wu, P., Leinonen, J., Koivunen, E., Lankinen, H., and Stenman, U.H. (2000). Identification of novel prostate-specific antigen-binding peptides modulating its enzyme activity. Eur. J. Biochem.267, 6212–6220.10.1046/j.1432-1327.2000.01696.xSearch in Google Scholar

Wu, P., Zhu, L., Stenman, U.H., and Leinonen, J. (2004). Immunopeptidometric assay for enzymatically active prostate-specific antigen. Clin. Chem.50, 125–129.10.1373/clinchem.2003.026146Search in Google Scholar

Zhang, W.M., Leinonen, J., Kalkkinen, N., Dowell, B., and Stenman, U.H. (1995). Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid. Clin. Chem.41, 1567–1573.10.1093/clinchem/41.11.1567Search in Google Scholar

Zhang, W.M., Finne, P., Leinonen, J., Salo, J., and Stenman, U.H. (2000). Determination of prostate-specific antigen complexed to α2-macroglobulin in serum increases the specificity of free to total PSA for prostate cancer. Urology56, 267–272.10.1016/S0090-4295(00)00609-9Search in Google Scholar

Published Online: 2006-06-26
Published in Print: 2006-06-01

©2006 by Walter de Gruyter Berlin New York

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