PAP Prostatic Acid Phosphatase
PAP Prostatic Acid Phosphatase
PREPARATION FOR ASSAY
1. Bring all reagents and samples to room temperature (20oC - 25oC) and shake 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.
2. Dispense 25 ul of references, controls or serum samples into the appropriate wells.
3. Dispense 100 ul of enzyme conjugate into wells. Incubate for 30 minutes at room temperature.
4. Remove incubation mixture and rinse the wells 5 times with running water.
5. Dispense 100 ul of Solution A and Solution B into each Well and incubate for 10 minutes at room temperature.
6. Stop reaction by adding 50 ul of 1 N H2SO4 to each well and read absorbance at 450 nm with ELISA reader against Blank well (only solution A and solution B)
CALCULATION OF RESULTS
1. Plot the concentration (X) of each reference standards against its absorbance (Y) on a full logarithmic graph paper.
2. Obtain the PAP 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 PAP
No. (ng/ml) 450nm (ng/ml)
A1 0 0
A2 1 0.043
A3 3 0.139
A4 5 0.24
A5 15 0.88
A6 30 1.686
A7 PATIENT A 0.602 11.4
1. Gorman, L. and Statland, B.E. Clinical usefulness of alkaline phosphatase Isoenzyme determinations. Clin. Biochem., 10:171, 1977.
2. Kutscher, W. and Wolbergs H.. Prostatic Phosphatase I. Z. Physiol Chem. 236:237, 1935.
3. Kutscher, W. and Warner, A. Prostatic Phosphatase II. Z. Physiol. Chem. 239:109, 1936.
4. Gutman, A. B. and Gutman, E. B. An AAcid A Phosphate Occuring in the Serum of Patients with Metastasizing Carcinoma of the Prostate Gland. J. Clin Invest 17:473-478, 1938.
5. Yam, Y. T. Clinical Significance of the Human Acid Phosphatases. A Review. Am. J. Med. 56:604, 1974.
6. Bernstein, L.H. Rudolph, R.A., Pinto, M.M., Viner, N. and Zuckerman, H. Medically Significant Concentrations of Prostate-Specific Antigen in Serum Assessed. Clin. Chem. 36/3, 515-518, 1990.
7. Nguyen, L., Chapdelaine, A., and Chevvalier. S.. Prostatic Acid Phosphatase in Serum of Patients with Prostatic Cancer is a Specific Phosphotyrosine Acid Phosphatase. Clin. Chem. 36/8, 1450-1455, 1990.
8. Ames, B.N. Assay of Inorganic Phosphate. Total Phosphate and Phosphatases. Methods Enzu,p; 8:115-118, 1966.
9. Fishman, W.H. and Lerner, F. A. A method of Estimating Serum Acid Phosphatase of Prostatic Origin. J. Biol. Chem. 200:89, 1953.
10. Chu,T.M. et. Al. Immunochemical detection of Serum Prostatic Acid Phosphatase. Methodology and Clinical Evaluation. Invest. Urology 15:319, 1978.
11. Fu, J., Hsu B.R.S., Lu, W. MagiwelTM Enzyme Immunoassay for Prostate Acid Phosphatase (PAP) using Microtiter wells as solid phase. Clin.Chem . 34/6: 1297, 1988.
12. Bruce, A.W. Mahan, D.E., Sullivan, L.D. and Goldenberg, L. The Significance of Prostatic Acid Phosphatase in Adenocarcinoma of the Prostate. J. Urol. 125:357, 1981.