| 1. |
- Stenman, U H, et al.
(författare)
-
Summary report of the TD-3 workshop: characterization of 83 antibodies against prostate-specific antigen
- 1999
-
Ingår i: Tumor Biology. - : Springer Science and Business Media LLC. - 1423-0380 .- 1010-4283. ; 20:Suppl. 1, s. 1-12
-
Tidskriftsartikel (refereegranskat)abstract
- Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86-91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158-163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3-11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.
|
|
| 2. |
- Pettersson, K, et al.
(författare)
-
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
-
Ingår i: Clinical Chemistry. - 0009-9147. ; 41:10, s. 8-1480
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 3. |
- Piironen, T, et al.
(författare)
-
Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling
- 1998
-
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 7:2, s. 259-269
-
Tidskriftsartikel (refereegranskat)abstract
- Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence. Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-IV). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53-64, III (a and b) was localized to residues 80-91 (= kallikrein loop), and IV was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.
|
|
| 4. |
|
|
| 5. |
- Becker, Charlotte, et al.
(författare)
-
Clinical value of human glandular kallikrein 2 and free and total prostate-specific antigen in serum from a population of men with prostate-specific antigen levels 3.0 ng/mL or greater
- 2000
-
Ingår i: Urology. - 1527-9995. ; 55:5, s. 694-699
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: To investigate the clinical value of human glandular kallikrein 2 (hK2) compared with free (f) and total (t) prostate-specific antigen (PSA) in the early detection of prostate cancer (PCa). METHODS: In PCa screening conducted in 1995 to 1996 in Goteborg, Sweden, 5853 of 9811 randomly selected men (aged 50 to 66 years; median 61) accepted PSA testing; those with tPSA levels of 3. 0 ng/mL or greater were offered digital rectal examination, transrectal ultrasound, and sextant biopsies. Serum from 604 of 611 biopsied men (18% with positive digital rectal examinations, tPSA range 3.0 to 220 ng/mL, 144 men with PCa) was analyzed for hK2 (research assay) and tPSA and fPSA (Prostatus). Sera were stored at -20 degrees C for a maximum of 2 weeks for tPSA and fPSA and 3 years for hK2. RESULTS: hK2 levels and hK2 x tPSA/fPSA values were significantly elevated in men with PCa. Receiver operating characteristic data revealed that the area under the curve for hK2 x tPSA/fPSA was significantly greater than that for tPSA and greater, but not significantly greater, than that for percent fPSA. Also, the cancer-detecting sensitivity was significantly improved (P <0.05) using hK2 x tPSA/fPSA compared with tPSA and percent fPSA at specificity levels of 75% to 90%. At 75% specificity, a sensitivity of 74% was obtained compared with 64% or 54% using percent fPSA or tPSA; at 90% specificity, the corresponding sensitivity level was 55%, 41%, and 36%, respectively. CONCLUSIONS: Discrimination of men with and without PCa in a randomly selected population was improved by measuring hK2 in addition to tPSA and fPSA.
|
|
| 6. |
- Becker, Charlotte, et al.
(författare)
-
Discrimination of men with prostate cancer from those with benign disease by measurements of human glandular kallikrein 2 (HK2) in serum
- 2000
-
Ingår i: Journal of Urology. - 1527-3792. ; 163:1, s. 311-316
-
Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: To investigate the clinical value of measuring human glandular kallikrein 2 (hK2) compared with free and total prostate specific antigen (PSA-F and PSA-T) in serum from patients with prostate disease. MATERIALS AND METHODS: Serum from healthy controls, from men with benign prostate hyperplasia (BPH), clinically localized prostate cancer (PCa), and advanced PCa were analyzed for hK2 (using an in-house-research assay with detection limit of 0.05 ng./mL and <0.1% cross-reaction with PSA) and for PSA-F and PSA-T (using the Dual Prostatus assay from EG&G Wallac). RESULTS: HK2 concentrations were <0.05 ng./mL in 50/50 healthy volunteers but significantly higher (p <0.0001) and > or =0.05 ng./mL in 28/54 (52%) patients with BPH. In comparison to these men, the hK2 levels were significantly higher (p <0.0001, median 0.085 ng./mL) and > or =0.05 ng./mL in 100/136 (74%) men with clinically localized PCa. Compared with localized PCa, the hK2 levels were significantly higher (p <0.0001, median 0.57 ng./mL) and > or =0.05 ng./mL in 55/57 (96%) patients with advanced PCa. The median hK2 levels ranged from 1.3 to 1.6% of those of PSA-T in all three patient groups, whereas percent hK2/PSA-F and hK2 x PSA-T/PSA-F levels were significantly higher in cancer patients compared with those with BPH. In the discrimination of clinically localized PCa from BPH, hK2 x PSA-T/PSA-F gave the largest area under the receiver operating curve (AUC = 0.81) and significantly (p = 0.025) larger AUC than PSA-T alone (0.70). Further, at 95% sensitivity there was significant gain in specificity, and at specificity levels of 90 to 95% there was significant gain in sensitivity using the measurements of PSA-T+PSA-F+hK2 compared with analysis of PSA-T and/or percent free PSA. CONCLUSIONS: Discrimination of patients with benign prostate disease from those with prostate cancer was significantly enhanced using measurements of hK2 in addition to those of PSA-T and PSA-F. Percent hK2/PSA-F was higher in PCa than in BPH, a phenomena not yet understood.
|
|
| 7. |
- Becker, Charlotte, et al.
(författare)
-
Sensitive and specific immunodetection of human glandular kallikrein 2 in serum
- 2000
-
Ingår i: Clinical Chemistry. - 0009-9147. ; 46:2, s. 198-206
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2. METHODS: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA. RESULTS: The analytical detection limit was <10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was <0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4. 8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-alpha(1)-antichymotrypsin (ACT) were detected in vitro with -6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4-19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor. CONCLUSIONS: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.
|
|
| 8. |
- Björk, Thomas, et al.
(författare)
-
Rapid exponential elimination of free prostate-specific antigen contrasts the slow, capacity-limited elimination of PSA complexed to alpha 1-antichymotrypsin from serum
- 1998
-
Ingår i: Urology. - 1527-9995. ; 51:1, s. 57-62
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: To study the rates of elimination of total prostate-specific antigen (PSA-T), free PSA (PSA-F), and PSA complexed to alpha 1-antichymotrypsin (PSA-ACT) from blood after radical retropubic prostatectomy (RRP). METHODS: We obtained venous blood from 10 patients with prostate cancer who were undergoing RRP. We analyzed PSA-F and PSA-ACT and equimolar detection of both of these forms together (PSA-T) by using immunofluorometric assays. An attempt was made to fit the serum concentrations of PSA-F, PSA-ACT, and PSA-T for each patient to exponential curves by applying one- and two-compartment models for pharmacokinetic analysis. RESULTS: Manipulation of the prostate during RRP resulted in a 3- to 28-fold increase in PSA-F concentrations in serum. Removal of the prostate resulted in a rapid, biexponential elimination of PSA-F from serum, corresponding to a mean initial (alpha) half-life of 0.81 hours and a mean terminal (beta) half-life of 13.9 hours. Serum PSA-ACT concentrations decreased by 20% to 40% immediately after removal of the gland; the elimination after surgery was slow and nonexponential, corresponding to a mean rate of 0.8 ng/mL/day. The elimination of PSA-T reflects a combination of the elimination patterns for PSA-F and PSA-ACT. CONCLUSIONS: The main proportion of PSA-F is rapidly eliminated from serum, possibly by glomerular filtration. PSA-F released during surgery did not form complexes with ACT, as suggested by the lack of PSA-ACT elevation in serum. The size (approximately 90 kDa) and the extensive in vitro stability of the PSA-ACT complex prevents renal clearance. The nonexponential elimination of the PSA-ACT complex is evidence of a capacity-limited process (e.g., metabolic transformation).
|
|
| 9. |
|
|
| 10. |
|
|
