1.        Bring all reagents and samples to room temperature (20oC - 25oC) and mix gently before beginning the test. Have all reagents and samples ready before the start of the assay. Once
the test is begun it must be performed without any interruption to get the most reliable and consistent results.
2.        Use new disposable tips for each specimen.


1.        Secure the desired number of coated wells in the holder. Mark data sheet with sample identification.
2.        Dispense 25 ul of references, controls or serum samples into the appropriate wells.
3.        Dispense 100 ul of enzyme conjugate into wells.
4.        Incubate for 30 minutes at room temperature.
5.        Remove incubation mixture and rinse the wells 5 times with running water.
6.        Dispense 100 ul of solution A and solution B into each well.
7.        Incubate for 10 minutes at room temperature.
8.        Stop reaction by adding 50 ul of 1 N H2SO4 to each well and read
9.        O.D. at 450 nm with a micro-well reader.


1.        Plot the concentration (X) of each reference standards against its absorbance (Y) on a full logarithmic graph paper.
2.        Obtain the PSA value of patient by reference to the standard curve as follows: (These data are for demonstration purpose only and must not be used in place of data generated for each assay).

Well Description Absorbance PSA
No. (ng/ml) 450 nm (ng/ml)
A1 0 0
B1 0
A2 1.5 0.038
B2 0.042
A3 5 0.144
B3 0.142
A4 15 0.414
B4 0.457
A5 30 0.821
B5 0.888
A6 60 1.616
B6 1.784
A7 PATIENT A 0.397 14.5
B7 0.4

1.        Wang, M.c. L.A. Valenzuela, G.P. Murphy, and T.M. Chu. Purification of a Human Prostate Specific Antigen. Invest Urol. 17:159. 1979
2.        Frankel. A.E., R.V. Rouse, M.C. Wang, T.M. Chu and L.A. Herzenberg. Monoclonal Antibodies to a Human Prostate Antigen. Cancer Res. 42:3714, 1982.
3.        Papsider, L.D., M.C. Wang, L.A. Valenzuela, G.P. Murphy, G.P. and T.M. Chu. A Prostate Antigen in Sera Cancer Patients. Cancer Res. 40:2428. 1980.
4.        Ponts, J.E., T.M. Chu, N.Slack, J.Karr, G.P. Murphy. Serum Prostatic Antigen Measurement in Localized Prostatic Cancer: Correlation with Clinical Course. J. Urol. 128:1216-1218, 1982.
5.        Killian, C.S., N. Yang, L.J. Emrich, F.P. Vargas, M. Kuriqyama, M.C. Wang, N.H. Slack, L.D. Murphy and T.M. Chu. Prognostic Importance of Prostate-Specific Antigen for Monitoring Patients with Stages B2 to D1 Prostate Cancer. Cancer Res. 45:886. 1985.
6.        Kuriyam, M., M.C. Wang, L.D. Papsidero, C.S. Killian. T. Shimano, L. Valenzuela, T. Nishiura, G. P. Murphy, and T.M. Chu. Quantitation of Prostate-Specific Antigen in Serum by a Sensitive Enzyme Immunoassay: Cancer Res. 40:4658, 1980.
7.        Kuriyama, M., M.C. Wang, C.L. Lee, C.S. Killian, L.D. Papsidero, H. Inaji, R.M. Loor, M.R. Lin, G.P. Murphy and T.M. Chu. Multiple Marker Evaluation in Human Prostate Cancer with the Use of Tissue-Specific Antigens. J. Nat. Canc. 68:99, 1982.
8.        Schifman, R.B., F.R. Ahmann, A. Elvick, K. Coulis and K. Brawer. Analytical and Physiological Characteristic Prostate-Specific Antigen and Prostatic Acid Phosphatase in Serum Compared. Clin. Chem. 33/11, 2086-2088, 1987.