Multiple forms of prostate-specific antigen in serum: differences in immunorecognition by monoclonal and polyclonal assays

1993 ◽  
Vol 39 (12) ◽  
pp. 2483-2491 ◽  
Author(s):  
A M Zhou ◽  
P C Tewari ◽  
B I Bluestein ◽  
G W Caldwell ◽  
F L Larsen
Keyword(s):  
Prostate Specific Antigen ◽  
Molecular Sieve ◽  
Specific Antigen ◽  
Prostatic Hyperplasia ◽  
Multiple Forms ◽  
Early Detection Of Disease ◽  
Peak Integration ◽  
Free Psa ◽  
Predominant Form

Abstract Prostate-specific antigen (PSA) in serum is primarily complexed with alpha 1-antichymotrypsin (alpha 1-ACT). However, 12-15% of prostate cancer (PCa) patients present with the predominant form being uncomplexed (free) PSA (Lilja et al., Clin Chem 1991;37:1618-24). We report that commercial immunoassays demonstrate variations in reactivity, especially to the uncomplexed form. We fractionated and analyzed commercial controls, PSA complexes prepared in vitro, and sera from patients with PCa or benign prostatic hyperplasia, using molecular sieve chromatography and Hybritech Tandem-R, Abbott IMx, and Ciba Corning ACS PSA assays. Peak integration of PCa samples demonstrated ACS:Tandem-R ratios of 1-1.3 for PSA/alpha 1-ACT complex. In contrast, ratios of uncomplexed peaks ranged from 2 to 4, suggesting a greater reactivity of the uncomplexed form in the ACS PSA assay. Discrepancies between assays, when PSA was measured in unfractionated sera, correlated directly with the percentage of the uncomplexed form. In controls, fractionation revealed the presence of uncomplexed PSA only, with ratios of ACS:Tandem-R and IMx:Tandem-R of 3:1 and 1.8:1, respectively. Immunoblots of PCa sera detected uncomplexed PSA (approximately 30 kDa) and PSA complexes of approximately 95 kDa (PSA/alpha 1-ACT) and > 200 kDa, indicative of alpha 2-macroglobulin. Maximal recognition of all forms of PSA may be important for early detection of disease progression.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

scholarly journals The Combination of Human Glandular Kallikrein and Free Prostate-specific Antigen (PSA) Enhances Discrimination Between Prostate Cancer and Benign Prostatic Hyperplasia in Patients with Moderately Increased Total PSA

1999 ◽  
Vol 45 (11) ◽  
pp. 1960-1966 ◽  
Author(s):  
Angeliki Magklara ◽  
Andreas Scorilas ◽  
William J Catalona ◽  
Eleftherios P Diamandis
Keyword(s):  
Prostate Cancer ◽  
Benign Prostatic Hyperplasia ◽  
Prostate Specific Antigen ◽  
Prostatic Carcinoma ◽  
Specific Antigen ◽  
Area Under The Curve ◽  
Prostatic Hyperplasia ◽  
Free Psa ◽  
Glandular Kallikrein ◽  
Total Psa

Abstract Background: Prostate-specific antigen (PSA) is the most reliable tumor marker available and is widely used for the diagnosis and management of prostate cancer. Unfortunately, PSA cannot distinguish efficiently between benign and malignant disease of the prostate, especially within the range of 4–10 μg/L. Among the refinements developed to enhance PSA specificity is the free/total PSA ratio, which is useful in discriminating between the two diseases within the diagnostic “gray zone”. Recent data indicate that human glandular kallikrein (hK2), a protein with high homology to PSA, may be an additional serum marker for the diagnosis and monitoring of prostate cancer. Methods: We analyzed 206 serum samples (all before treatment was initiated) from men with histologically confirmed benign prostatic hyperplasia (n = 100) or prostatic carcinoma (n = 106) with total PSA in the range of 2.5–10 μg/L. Total and free PSA and hK2 were measured with noncompetitive immunological procedures. Statistical analysis was performed to investigate the potential utility of the various markers or their combinations in discriminating between benign prostatic hyperplasia and prostatic carcinoma. Results: hK2 concentrations were not statistically different between the two groups of patients. There was a strong positive correlation between hK2 and free PSA in the whole patient population. hK2/free PSA ratio (area under the curve = 0.69) was stronger predictor of prostate cancer than the free/total PSA ratio (area under the curve = 0.64). At 95% specificity, the hK2/free PSA ratio identified 30% of patients with total PSA between 2.5–10 μg/L who had cancer. At 95% specificity, the hK2/free PSA ratio identified 25% of patients with total PSA between 2.5 and 4.5 μg/L who had cancer. Conclusions: Our data suggest that hK2 in combination with free and total PSA can enhance the biochemical detection of prostate cancer in patients with moderately increased total PSA concentrations. More specifically, the hK2/free PSA ratio appears to be valuable in identifying a subset of patients with total PSA between 2.5 and 4.5 μg/L who have high probability of cancer and who should be considered for biopsy.

scholarly journals PREPARASI PEREAKSI KIT IMMUNORADIOMETRlCASSAY FREE PROSTATE SPECIFIC ANTIGEN UNTUK DETEKSI KANKER PROSTAT

2013 ◽  
Vol 15 (2) ◽  
pp. 14-24
Author(s):  
Puji Widayati ◽  
Gina Mondrida ◽  
Sri Setiyowati ◽  
Agus Ariyanto ◽  
V. Yulianti Susilo ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Prostate Specific Antigen ◽  
Prostate Gland ◽  
Specific Antigen ◽  
Prostatic Hyperplasia ◽  
Free Condition ◽  
Free Psa ◽  
Prostate Enlargement ◽  
Assay Design ◽  
Benign Prostate

Prostate Specific Antigen (PSA) is a glycoprotein with a molecular weight of approximately 34,000 daltons serine protease secreted exclusively by prostatic epithelial cells that lining acini and prostate gland. Increased of PSA levels can be caused by prostate cancer or benign prostate enlargement (benign prostatic hyperplasia, BPH). PSA in the blood was found in the free condition (free PSA) and most of the bound protein (complexed-PSA, c-PSA). Measuring levels of PSA was found in the blood can be done by several methods such as by immunoradiometricassay (IRMA) methods or ELISA methods. IRMA method is one of immunoassay techniques using radionuclides ,/' 125 oJ I as a tracer, so the sample in small 13 quantity can be detected The purpose of this study was obtained PSA reagent kit that includes 1251labeled PSA as a tracer, PSA coated tube and PSA standard that requirements of the kit, then it can be optimized assay design, that eventually PSA reagent kit can be used for early detection of prostate cancer. It has been done labeling of Mab PSA using 125 1with reaction time was 90 seconds, amount of PSA MAb was 75 ugram and the activity of Na_ 125I was 1000 flCi. Preaparation of PSA coated tube using 0.05 M Na2C03 solution, at pH: 9.6 with volume was 250 ml., standard PSA with 0.025 Mphosphate buffer at pH 7.4 containing 5% BSA and 0.1% NaN3, and resulting at 1,25% and 14,12% respectively of NSB and BIT that requirement of the kit.Keywords: Prostate cancer, PSA, IRMA,NSB, Maximum Binding

Free and complexed prostate-specific antigen (PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-alpha 1-antichymotrypsin complex

1995 ◽  
Vol 41 (10) ◽  
pp. 1480-1488 ◽  
Author(s):  
K Pettersson ◽  
T Piironen ◽  
M Seppälä ◽  
L Liukkonen ◽  
A Christensson ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Solid Phase ◽  
Specific Antigen ◽  
Close Correlation ◽  
Cathepsin G ◽  
Free Psa ◽  
Total Psa ◽  
The Stability ◽  
Storage Buffer

Abstract Generation of 15 monoclonal antibodies (MAbs) allowed construction of epitope maps and specific two-site immunofluorometric assays for free prostate-specific antigen (PSA) and PSA complexed with alpha 1-antichymotrypsin (ACT). Close correlation of PSA concentrations obtained with the use of different assays of free PSA suggested extensive similarity in immunodetection of free PSA in serum. Assays of the PSA-ACT complex overestimated the concentration of PSA-ACT in serum because of nonspecific adsorbance of ACT or cathepsin G-complexed ACT to the solid phase. This interference was substantially decreased in the presence of heparin. In studying the stability of purified PSA and PSA-ACT complexes formed in vitro, we found that the free PSA was stable during storage for 4 weeks at 35 degrees C, whereas PSA-ACT complexes largely dissociated in these conditions. The instability of PSA-ACT complexes was counteracted by storage at low temperatures, by adjusting the pH of the storage buffer between 6.8 and 7.4, and through addition of 100-1000-fold molar excess of native ACT. The ease of calibration and the accuracy of free PSA assays in comparison with assays of the PSA-ACT complex suggest that measurements of free to total PSA most accurately reflect the inverse of the proportion of PSA complexed to ACT in serum.

scholarly journals Measurement of the Complex between Prostate-specific Antigen and α1-Protease Inhibitor in Serum

1999 ◽  
Vol 45 (6) ◽  
pp. 814-821 ◽  
Author(s):  
Wan-Ming Zhang ◽  
Patrik Finne ◽  
Jari Leinonen ◽  
Satu Vesalainen ◽  
Stig Nordling ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Protease Inhibitor ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Serum Samples ◽  
Free Psa ◽  
Total Psa ◽  
Healthy Females

Abstract Background: Prostate-specific antigen (PSA) occurs in serum both free and in complex with protease inhibitors. The complex with α1-antichymotrypsin (ACT) is the major form in serum, and the proportion of PSA-ACT is higher in prostate cancer (PCa) than in benign prostatic hyperplasia (BPH). PSA also forms a complex with α1-protease inhibitor (API) in vitro, and the PSA-ACT complex has been detected in serum from patients with prostate cancer. The aim of the present study was to develop a quantitative method for the determination of PSA-API and to determine the serum concentrations in patients with PCa and BPH. Methods: The assay for PSA-API utilizes a monoclonal antibody to PSA as capture and a polyclonal antibody to API labeled with a Eu-chelate as a tracer. For calibrators, PSA-API formed in vitro was used. Serum samples were obtained before treatment from 82 patients with PCa, from 66 patients with BPH, and from 22 healthy females. Results: The concentrations of PSA-API are proportional to the concentrations of total PSA. PSA-API comprises 1.0–7.9% (median, 2.4%) of total immunoreactive PSA in PCa and 1.3–12.2% (median, 3.6%) in BPH patients with serum PSA concentrations >4 μg/L. In patients with 4–20 μg/L total PSA, the proportion of PSA-API serum is significantly higher in BPH (median, 4.1%) than in PCa (median, 3.2%; P = 0.02). Conclusions: The proportion of PSA-API in serum is lower in patients with PCa than in those with BPH. These results suggest that PSA-API is a potential adjunct to total and free PSA in the diagnosis of prostate cancer.

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