scholarly journals Dual-Label Immunoassay for Simultaneous Measurement of Prostate-specific Antigen (PSA)-α1-Antichymotrypsin Complex Together with Free or Total PSA

2003 ◽  
Vol 49 (1) ◽  
pp. 97-103 ◽  
Author(s):  
Lei Zhu ◽  
Jari Leinonen ◽  
Wan-Ming Zhang ◽  
Patrik Finne ◽  
Ulf-Håkan Stenman
Keyword(s):  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Simultaneous Measurement ◽  
Solid Phase ◽  
Specific Antigen ◽  
Cathepsin G ◽  
Serum Samples ◽  
Total Psa ◽  
Dual Label

Abstract Background: A major portion of prostate-specific antigen exists in circulation as a complex with α1-antichymotrypsin (PSA-ACT), whereas a minor part is free (fPSA). The proportion of PSA-ACT is increased in prostate cancer (PCa), but immunologic determination of PSA-ACT is hampered by a background produced by nonspecific adsorption of ACT to the solid phase. To reduce the nonspecific interference, we produced an antibody specific for complexed ACT and developed immunofluorometric assays (IFMAs) for simultaneous measurement of fPSA + PSA-ACT (fPSA/PSA-ACT) and PSA-ACT + total PSA (tPSA, PSA-ACT/tPSA). Methods: Monoclonal antibodies (MAbs) were produced by immunization with PSA-ACT. The dual-label time-resolved IFMAs for fPSA/PSA-ACT and PSA-ACT/tPSA used a capture MAb to tPSA, an Eu3+-labeled MAb to fPSA or complexed ACT, and an Sm3+-labeled MAb to complexed ACT or to tPSA as tracer antibodies. The clinical utility was evaluated using serum samples from individuals with or without PCa with PSA concentrations of 2.0–20.0 μg/L. Results: One MAb (1D10) showed low cross-reactivity with free ACT and cathepsin G-ACT. A sandwich assay for PSA-ACT with 1D10 as tracer had a detection limit of 0.05 μg/L, and with this assay, PSA-ACT was undetectable in female sera. The detection limit for fPSA was 0.004 μg/L. Determinations of the ratio of fPSA to PSA-ACT and the proportions of fPSA/tPSA and PSA-ACT/tPSA provided the same clinical specificity for PCa and provided significantly better clinical specificity than did tPSA. Conclusions: Background problems observed in earlier PSA-ACT assays are eliminated by the use of a MAb specific for complexed ACT as a tracer. The same clinical validity can be obtained by determination of fPSA or PSA-ACT together or in combination with tPSA.

Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum

1995 ◽  
Vol 41 (8) ◽  
pp. 1115-1120 ◽  
Author(s):  
K Mitrunen ◽  
K Pettersson ◽  
T Piironen ◽  
T Björk ◽  
H Lilja ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Simultaneous Measurement ◽  
Solid Phase ◽  
Characteristic Curve ◽  
Specific Antigen ◽  
Sample Volume ◽  
Free Psa ◽  
One Step ◽  
Total Psa ◽  
Dual Label

Abstract We developed a simple one-step dual-label immunoassay for simultaneous measurement of the free, noncomplexed form of prostate-specific antigen (PSA) and total PSA. The assay is based on time-resolved fluorescence and includes a stable fluorescent chelate of Eu to label a monoclonal antibody (mAb) that detects only free PSA, whereas a second mAb labeled with a fluorescent chelate of Tb provides equimolar detection of both free PSA and PSA complexed to alpha 1-antichymotrypsin. A third mAb on a solid phase captures the free and complexed forms of PSA in an equimolar fashion. The simultaneous measurement of the free-to-total PSA ratio (F/T) with the one-step dual assay is not sensitive to variations in the sample volume. The discrimination between benign prostatic hyperplasia and prostate cancer patients, i.e., the area under the receiver-operating characteristic curve, increased from 0.64 (total PSA assay) to 0.78 and 0.81 when the F/T ratio was measured with single and dual assays, respectively.

scholarly journals Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

2004 ◽  
Vol 50 (9) ◽  
pp. 1607-1617 ◽  
Author(s):  
Ville Väisänen ◽  
Susann Eriksson ◽  
Kaisa K Ivaska ◽  
Hans Lilja ◽  
Martti Nurmi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Solid Phase ◽  
Specific Antigen ◽  
Control Group ◽  
Serum Samples ◽  
Human Kallikrein 2 ◽  
Kallikrein 2 ◽  
Capture Antibody ◽  
Total Psa

Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA <1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

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.

scholarly journals Miniature Single-Particle Immunoassay for Prostate-specific Antigen in Serum Using Recombinant Fab Fragments

2000 ◽  
Vol 46 (11) ◽  
pp. 1755-1761 ◽  
Author(s):  
Harri Härmä ◽  
Piia Tarkkinen ◽  
Tero Soukka ◽  
Timo Lövgren
Keyword(s):  
Prostate Specific Antigen ◽  
Single Particle ◽  
Binding Capacity ◽  
Specific Antigen ◽  
Genetically Engineered ◽  
Covalent Attachment ◽  
Serum Samples ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Total Psa

Abstract Background: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay. Methods: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-μm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode. Results: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 μg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06 ± 0.03 and 1.03 ± 0.02, respectively (Sy|x = 0.084 and 0.057 μg/L), with intercepts of 0.013 ± 0.018 and 0.013 ± 0.017 μg/L (R >0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed. Conclusions: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.

scholarly journals Reference Reagents for Prostate-specific Antigen (PSA): Establishment of the First International Standards for Free PSA and PSA (90:10)

2000 ◽  
Vol 46 (9) ◽  
pp. 1310-1317 ◽  
Author(s):  
Brian Rafferty ◽  
Peter Rigsby ◽  
Matthew Rose ◽  
Thomas Stamey ◽  
Rose Gaines Das
Keyword(s):  
Confidence Interval ◽  
Prostate Specific Antigen ◽  
Recombinant Dna ◽  
Collaborative Study ◽  
Specific Antigen ◽  
International Standards ◽  
Serum Samples ◽  
International Standard ◽  
Free Psa ◽  
Total Psa

Abstract Background: Prostate-specific antigen (PSA) measurements in serum by immunoassay are widely used in the screening, diagnosis, and monitoring of patients with prostate cancer although the lack of common reference reagents has led in the past to wide differences in estimates. We report here the results of a WHO international collaborative study in which two preparations of PSA representative of the main immunoreactive components in serum, free PSA and PSA 90:10, and a preparation of recombinant DNA-derived PSA were assessed as potential standards for the calibration of diagnostic immunoassays for PSA. Methods: Coded vials of the candidate materials and serum preparations containing PSA in the clinically important range were provided to the 10 laboratories in the study, and participants were asked to perform PSA assays currently in use in their laboratories. Data from 89 immunoassays by 26 different method-laboratory combinations were contributed to the study and analyzed centrally at the National Institute for Biological Standards and Control. Results: Potency estimates of the preparations relative to the in-house calibrators were in good agreement with the target value of 1 μg of total PSA/vial, the preparation of free PSA giving 1.10 μg/vial (95% confidence interval, 0.99–1.21 μg/vial) and PSA 90:10, 1.11 μg/vial (95% confidence interval, 1.04–1.18 μg/vial). No immunoreactivity was detected in ampoules containing the recombinant material. Use of a common standard of PSA 90:10 significantly reduced the between-laboratory geometric coefficients of variation for serum samples included in the study and gave a much narrower range of potency estimates. Conclusions: The preparation of free PSA was established by WHO as the First International Standard for PSA (free) with an assigned content of 1 μg of total PSA per vial. In addition, the preparation of bound PSA was established as the First International Standard for PSA (90:10) with an assigned content of 1 μg of total PSA per vial.

scholarly journals Determination of Non-α1-Antichymotrypsin-complexed Prostate-specific Antigen as an Indirect Measurement of Free Prostate-specific Antigen: Analytical Performance and Diagnostic Accuracy

2003 ◽  
Vol 49 (6) ◽  
pp. 887-894 ◽  
Author(s):  
Sebastian Wesseling ◽  
Carsten Stephan ◽  
Axel Semjonow ◽  
Michael Lein ◽  
Brigitte Brux ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Sensitivity And Specificity ◽  
Specific Antigen ◽  
Roc Curves ◽  
Indirect Measurement ◽  
Free Psa ◽  
Specificity And Sensitivity ◽  
Diagnostic Sensitivity And Specificity ◽  
Total Psa

Abstract Background: A new assay measures prostate-specific antigen (PSA) not complexed to α1-antichymotrypsin (nACT-PSA) after removing PSA complexed to ACT by use of anti-ACT antibodies. We evaluated nACT-PSA and its ratio to total PSA (tPSA) as alternatives to free PSA (fPSA) and its ratio to tPSA in differentiating prostate cancer (PCa) and benign prostatic hyperplasia (BPH) in patients with tPSA of 2–20 μg/L. Methods: PSA in serum of 183 untreated patients with PCa and 132 patients with BPH was measured retrospectively on the chemiluminescence immunoassay analyzer LIAISON® (Byk-Sangtec Diagnostica) with the LIAISON tPSA and LIAISON fPSA assays. The nACT-PSA fraction was determined with a prototype assay measuring the residual PSA after precipitation of ACT-PSA with an ACT-precipitating reagent. Results:nACT-PSA was higher than fPSA in samples with fPSA concentrations <1 μg/L but lower in samples with >1 μg/L fPSA. The median ratios of fPSA/tPSA and of nACT-PSA/tPSA were significantly different between patients with BPH and PCa (19.4% vs 12.2% and 17.4% vs 13.0%, respectively). Within the tPSA ranges tested (2–20, 2–10, and 4–10 μg/L), areas under the ROC curves for the fPSA/tPSA ratios were significantly larger than those for nACT-PSA/tPSA. In the tPSA ranges <10 μg/L, the areas under the ROC curves for fPSA/tPSA were significantly larger than those for tPSA, whereas the areas for nACT-PSA/tPSA were not. At decision limits for 95% sensitivity and specificity, both ratios significantly increased specificity and sensitivity, respectively, compared with tPSA, but the fPSA/tPSA ratio showed higher values. Conclusions: nACT-PSA and its ratio to tPSA provide lower diagnostic sensitivity and specificity than fPSA/tPSA. The fPSA/tPSA ratio represents the state-of-the-art method for differentiating between PCa and BPH.

scholarly journals Development of immunoradiometric assay for quantitative determination of free prostate-specific antigen

2005 ◽  
Vol 24 (2) ◽  
pp. 129-134 ◽  
Author(s):  
Miroslava Jankovic ◽  
Maja Kosanovic ◽  
Ljiljana Hajdukovic-Dragojlovic ◽  
Snezana Golubovic
Keyword(s):  
Quantitative Determination ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Laboratory Diagnostics ◽  
Immunoradiometric Assay ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
One Step ◽  
Prostate Diseases

In this study we reported the development and analytical validation of new assay for quantitative determination of free prostate-specific antigen, fPSA. It is formulated as one step, two-site "sandwich" immunoradiometric assay. Specificity of this assay was achieved by using epitope-1-reactive anti-fPSA antibody as tracer antibody. Assay was calibrated against first international standard 96/668, and its detection limit was determined as 0.08 mg/L. Intra- and inter-assay coefficients of variation were 3.42-7.53% and 7.04-8.33%, respectively. Measured concentrations of serially diluted serum samples were close to the calculated concentrations, indicating good linearity with recovery percentage ranging from 98.7-107.4%. Analytical performance characteristics of fPSA assay speaks in favor of its use as a reliable tool in laboratory diagnostics relating to prostate diseases.

Determination of prostate-specific antigen in serum by immunoradiometric assay

1990 ◽  
Vol 36 (1) ◽  
pp. 53-58 ◽  
Author(s):  
G Lindstedt ◽  
A Jacobsson ◽  
P A Lundberg ◽  
H Hedelin ◽  
S Pettersson ◽  
...  
Keyword(s):  
Detection Limit ◽  
Los Angeles ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Reference Interval ◽  
Clinical Analysis ◽  
Detectable Effect ◽  
Serum Samples ◽  
Strict Adherence ◽  
Normal Reference

Abstract A better understanding is needed of the role of pre-analytical factors if prostate-specific antigen (PSA) is to be reliably used as a tumor marker. Reports on the analytical performance of TANDEM-R PSA (Hybritech, Inc., San Diego, CA) differ considerably with respect to detection limit and imprecision, differences that might be due (e.g.) to the use of different control matrices, to between-batch variations in reagent composition, or to nonrobustness of the assay. During nine months we determined PSA in 986 serum samples from 728 male urology patients and 54 samples from women (25 assay runs). As controls we used two serum pools with low PSA concentration and two widely used commercial controls. The within-assay CV for patients' samples was similar to that found with the commercial controls: 2.6% to 3.4% in the upper part of the normal reference interval. Precision was worse at lower concentrations (CVs 5-10% at about 0.5 micrograms/L). Imprecision tended to be higher at the end of runs. Assay drift for 100-tube runs was -4%. PSA was stable at -20 degrees C during six months. Neither the polyester polymer in SST tubes nor a hemolysate had any detectable effect on PSA values. Clinical analysis of the first 322 patients and all patients with PSA less than or equal to 0.20 micrograms/L highlighted the requirements for strict adherence to sampling instructions and to stringent quality control also at low analyte concentrations (analyte-free sera and sera with PSA concentrations 0.2-0.5 micrograms/L). Values with TANDEM-R PSA and IRMA-Count PSA (Diagnostic Products Corp., Los Angeles, CA) correlated well with no difference in detection limit or with samples from women. Within-assay precision was better with IRMA-Count PSA in the upper part of the normal reference interval and above. The designs of the two assays were compared in a format that is generally applicable for immunoassay kits (NORDKEM kit group, unpublished), and subjective impressions were recorded.

scholarly journals Analytical performance of the Tandem®-R free PSA immunoassay measuring free prostate-specific antigen

1997 ◽  
Vol 43 (7) ◽  
pp. 1203-1208 ◽  
Author(s):  
David L Woodrum ◽  
Chester M French ◽  
Timothy M Hill ◽  
Steven J Roman ◽  
Harold L Slatore ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Accurate Determination ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Analytical Performance ◽  
Minimum Detectable Concentration ◽  
Free Psa ◽  
Detectable Concentration ◽  
Total Psa

Abstract The analytical performance of the Tandem®-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA–α1-antichymotrypsin was determined to be <1%. The minimum-detectable concentration was <0.05 μg/L. The within-run and between-day CVs were ≤5% for samples with >0.3 μg/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.

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