Abstract
We have developed an assay specific for the PSA‐ACT (PSA‐α1‐antichymotrypsin) complex that effectively diminishes the problem of high assay background commonly reported by other investigators. The assay follows a two‐site ELISA format. Polyclonal anti‐PSA antibodies were coated on the microplate to capture the PSA complex from the serum, whereas the biotinylated anti‐ACT polyclonal antibodies and HRP‐conjugated streptavidin were used for detection. The high background ordinarily associated with this assay was greatly reduced when milk casein was added in addition to albumin for blocking and when the Super Block™ was also included in the diluents for sample dilution and dilution of enzyme conjugated detecting antibodies. The assay has a sensitivity of 0.05 ng/mL. The within‐run precision ranges from 4.2–7.2% and the between‐run precision falls between 5.8–8.5%. Cross reactions with ACT and free PSA (fPSA) are 0.0001% and 0.02%, respectively. The highest concentration of PSA‐ACT complex in the maternal sera was < 0.4 ng/mL by this assay, much less than reported in the literature. Using this improved assay, the sum of fPSA and PSA‐ACT concentrations were less than that of their corresponding total PSA (tPSA) most of the time. We believe that this improved assay should be used to replace the current tPSA assay for screening, monitoring, and managing patients with prostate cancer. J. Clin. Lab. Anal. 12:14–19, 1998. © 1998. Wiley‐Liss, Inc.
Keywords: PAGE, prostate cancer, PSA complexes, free PSA, total PSA, protease inhibitor, antichymotrypsin, protease activity, anti‐PSA antibody
References
- 1. Wu JT: Assay for prostate specific antigen (PSA): Current problems and possible solutions. J Clin Lab Anal 8: 51–62, 1994. [DOI] [PubMed] [Google Scholar]
- 2. Wu JT: Serum prostate specific antigen assay — An update. Western J Med (Epitomes‐Pathology) 162: 447–448, 1995. [PMC free article] [PubMed] [Google Scholar]
- 3. Catalona WJ, Smith DS, Ratliff T, et al.: Measurement of prostate‐specific antigen in serum as a screening test for prostate cancer. N Engl J Med 324: 1156–1161, 1991. [DOI] [PubMed] [Google Scholar]
- 4. Chan DW, Bruzek DJ, Oestlering JE, et al.: Prostate‐specific antigen as a marker for prostate cancer: A monoclonal and a polyclonal immunoassay compared. Clin Chem 3: 1916–1920, 1987. [PubMed] [Google Scholar]
- 5. Horton GL, Bahnson RR, Daft M, et al.: Differences in values obtained with 2 assays of prostate specific antigen. J Urol 139: 762, 1988. [DOI] [PubMed] [Google Scholar]
- 6. Graves HCB, Wehner N, Stamey T: Comparison of a polyclonal and monoclonal immunoassay for PSA: Need for an international antigen standard. J Urol 144: 1516–1522, 1990. [DOI] [PubMed] [Google Scholar]
- 7. Christensson A, Laurell CB, Lilja H: Enzymatic activity of prostate specific antigen and its reactions with extracellular serine proteinase inhibitors. Eur J Biochem 194: 755–763, 1990. [DOI] [PubMed] [Google Scholar]
- 8. Christensson A, Bjork T, Nilsson O, et al.: Serum prostate specific antigen complexed to al‐antichymotrypsin as an indicator of prostate cancer. J Urol 150: 100–105, 1993. [DOI] [PubMed] [Google Scholar]
- 9. Lilja H, Cockett ATK, Abrahamsson P‐A: Prostate specific antigen predominantly forms a complex with alphal‐antichymotrypsin in blood. Cancer Supplement 70: 230–234, 1992. [DOI] [PubMed] [Google Scholar]
- 10. Lilja H, Christensson A, Dahlen U, et al.: Prostate‐specific antigen in serum occurs predominantly in complex with α 1 ‐Antichymotrypsin. Clin Chem 37: 1618–1625, 1991. [PubMed] [Google Scholar]
- 11. Stenman UH, Leinonen J, Alfthan H, et al.: A complex between prostate‐specific antigen in serum 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. Can Res 51: 222–226, 1991. [PubMed] [Google Scholar]
- 12. Wu JT, and Stephenson RA: Microplate assays for free PSA, PSA‐ACT and total PSA: assay characteristics, selection of calibrators and in vitro stability studies. Proceeding for the 1st International Consultation on Prostate Cancer, 1997, p 159–168.
- 13. PSA‐ACT high background paper.
- 14. Wu JT, Zhang P, Wang T et al.: Evaluation of free PSA isoforms, PSA complex formation and specificity of anti‐PSA antibodies by HPLC and PAGE‐immunoblotting techniques. J Clin Lab Anal 9: 1–14, 1995. [DOI] [PubMed] [Google Scholar]
- 15. Wu JT, Liu GH, Zhang P: Effect of denaturation and curve fitting on the accuracy of microplate fPSA assay (submitted).