[New biomarkers and application of multivariate models for detection of prostate cancer]. / Neue Biomarker und Anwendung multivariater Modelle zur Detektion des Prostatakarzinoms.

The specificity of PSA has been enhanced by using molecular forms of PSA and free PSA (fPSA) such as percent free PSA (%fPSA), proPSA, intact PSA or BPHA and / or new serum markers. Most of these promising new serum markers like EPCA2 or ANXA3 still lack confirmation of the outstanding initial results or show only marginally enhanced specificity at high sensitivity levels. PCA3, TMPRSS2-ERG, and other analytes in urine collected after digital rectal examination with application of mild digital pressure have the potential to preferentially detect aggressive PCa and to decrease the number of unnecessary repeat biopsies. The combination of these new urinary markers with new and established serum markers seems to be most promising to further increase specificity of tPSA. Multivariate models, e. g., artificial neural networks (ANN) or logistic regression (LR) based nomograms have recently been performed by incorporating these new markers in several studies. There is generally an advantage to include the new markers and clinical data as additional parameters to PSA and %fPSA within ANN and LR models. Results of these studies and also unexpected pitfalls are discussed in this review.

Human kallikrein 2 (hK2), but not prostate-specific antigen (PSA), rapidly complexes with protease inhibitor 6 (PI-6) released from prostate carcinoma cells.

Human kallikrein 2 (hK2) is a secreted, trypsin-like protease that shares 80% amino acid sequence identity with prostate-specific antigen (PSA). hK2 has been shown to be a serum marker for prostate cancer and may also play a role in cancer progression and metastasis. We have previously identified a novel complex between human kallikrein 2 (hK2) and protease inhibitor 6 (PI-6) in prostate cancer tissue. PI-6 is an intracellular serine protease inhibitor with both antitrypsin and antichymotrypsin activity. In the current study we have shown that PI-6 forms a rapid in vitro complex with hK2 but does not complex with PSA. Recombinant mammalian cells expressing both hK2 and PI-6 showed hK2-PI-6 complex in the spent media only after cell death and lysis. Similarly, LNCaP cells expressing endogenous hK2 and PI-6 showed extracellular hK2-PI-6 complex formation concurrently with cell death. Immunostaining of prostate cancer tissues with PI-6 monoclonal antibodies showed a marked preferential staining pattern in cancerous epithelial cells compared with noncancerous tissue. These results indicate that the hK2-PI-6 complex may be a naturally occurring marker of tissue damage and necrosis associated with neoplasia. Both hK2 and PI-6 were shed into the lumen of prostate cancer glands as granular material that appeared to be cellular necrotic debris. The differential staining pattern of PI6 in tissues suggests a complex regulation of PI-6 expression that may play a role in other aspects of neoplastic progression.

A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa) but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia, because both PCa and benign prostatic hyperplasia release PSA into the serum. We have identified previously a truncated form of precursor PSA (pPSA) in prostate tumor extracts consisting of PSA with a serine-arginine pro leader peptide ([-2]pPSA) instead of the normally expressed 7 amino acid pro leader peptide. In the current study we developed monoclonal antibodies to detect [-2]pPSA and other isoforms of pPSA for Western blot analysis. PSA was immunoaffinity purified from 100 to 200 ml of serum from each of five men with biopsy-proven cancer and three biopsy-negative men, all with total PSA levels in the diagnostically relevant range near 10 ng/ml. The truncated [-2]pPSA was estimated to range from 25 to 95% of the free PSA in the five PCa samples but only 6-19% of the free PSA in the biopsy-negative men. Immunohistochemical studies showed positive staining for [-2]pPSA in PCa epithelium and that [-2]pPSA was enriched in cancer cell secretions. In vitro activation studies showed that human kallikrein 2 and trypsin readily activated full-length pPSA but were unable to activate [-2]pPSA to mature PSA. Thus, [-2]pPSA, once formed, is a stable but inactive isoform of PSA. Truncated [-2]pPSA may represent an important new diagnostic marker for the early detection of PCa.

Development of a dual monoclonal antibody immunoassay for total human kallikrein 2.

BACKGROUND: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-alpha(1)-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. METHODS: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. RESULTS: The minimum detectable concentration in the thK2 assay was 0.008 microg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2-serum protease complexes. The median serum concentration of thK2 in control samples (0.013 microg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 microg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at -70 degrees C. CONCLUSIONS: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.

Seminal plasma contains "BPSA," a molecular form of prostate-specific antigen that is associated with benign prostatic hyperplasia.

BACKGROUND: We previously reported that levels of BPSA, a modified form of prostate-specific antigen (PSA), are significantly elevated in prostate transition-zone tissue exhibiting nodular hyperplastic changes associated with the presence of benign prostatic hyperplasia (BPH). BPSA was purified and found to contain a characteristic clip between Lys182 and Ser183. We now describe the identification of BPSA in seminal plasma. METHODS: PSA was purified from seminal plasma by immunoaffinity chromatography. The purified PSA was further resolved by hydrophobic interaction chromatography, and the individual PSA forms were analyzed by gel electrophoresis and N-terminal amino-acid sequencing. RESULTS: BPSA comprised about 8% of the PSA in pooled seminal plasma, and was identical to BPSA purified from prostate tissues. BPSA was cleanly resolved from all active and inactive forms of PSA. Other inactive forms of PSA in seminal plasma consisted largely of PSA clipped at Lys145, though about 30% of the inactive seminal plasma PSA was intact, mature PSA. CONCLUSIONS: BPSA represents a distinct form of inactive PSA in the seminal plasma that may represent a specific marker for the biochemical changes associated with nodular development in the prostate transition zone found in patients with BPH.

Benign prostatic hyperplasia-associated prostate-specific antigen (BPSA) shows unique immunoreactivity with anti-PSA monoclonal antibodies.

We previously identified a modified molecular form of prostate-specific antigen that is significantly elevated in the nodular transition zone tissue of prostates with benign prostatic hyperplasia. This prostate-specific antigen form, designated BPSA, is inactive and contains clipped polypeptide bonds at amino-acid residues Lys145-146 and Lys182-183. BPSA is not elevated in prostate cancer tissues and may therefore be a prostate-specific antigen marker to better discriminate benign prostatic hyperplasia from early prostate cancer. In this work we characterize the immunoreactivity of BPSA in competition assays with prostate-specific antigen using anti-prostate-specific antigen mAb recognizing six different epitopes on the prostate-specific antigen molecule. One mAb showed > 50% loss of immunoreactivtiy with BPSA compared with prostate-specific antigen, while the binding of two mAbs was largely unaffected and three mAbs had intermediate reactivity. BPSA purified from prostate tissue and seminal plasma, as well as BPSA generated in vitro by mild trypsin-treatment were found to have a similar pattern of reactivity to the six mAbs. However, other forms of inactive seminal plasma prostate-specific antigen, either intact or clipped at Lys145 only, had immunoreactivity similar to total prostate-specific antigen. These results demonstrate that BPSA has unique immunological properties from other forms of prostate-specific antigen, which should allow the development of BPSA-specific mAbs for the study of benign prostatic hyperplasia. Measurement of BPSA levels in the serum may help discriminate benign prostatic hyperplasia from early prostate cancer.

A precursor form of prostate-specific antigen is more highly elevated in prostate cancer compared with benign transition zone prostate tissue.

Prostate-specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but in the critical diagnostic range of 4-10 ng/ml it has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). PSA in serum is comprised of a variety of both "free" and "complexed" forms that have been used to improve the specificity of PSA for prostate cancer detection. We previously reported that pro PSA (pPSA), the zymogen or precursor form of PSA, is a component of free PSA in the serum of PCa patients. In the current study, we examined prostate tissues to understand the origin and specificity of pPSA. PSA was immuno-affinity purified from matched sets of prostate tissues including peripheral zone cancer (PZ-C); peripheral zone noncancer; and benign tissue from the transition zone (TZ), the primary site of BPH within the prostate. We found that pPSA is differentially elevated in PZ-C, but is largely undetectable in TZ. N-terminal sequencing revealed that the pPSA was comprised primarily of [-2]pPSA and minor levels of [-4]pPSA, containing pro leader peptides of 2 and 4 amino acids, respectively. The median value of pPSA was 3% in PZ-C and 0% (undetectable) in TZ (P < 0.0026). No pPSA was detected in 13 of 18 transition zone specimens (72%), but only 2 of the 18 matched cancer specimens (11%) contained no measurable pPSA. These results demonstrate that pPSA is more highly correlated with prostate cancer than with BPH. The pPSA in serum may represent a more cancer-specific form of PSA that could help distinguish prostate cancer from BPH.

"BPSA," a specific molecular form of free prostate-specific antigen, is found predominantly in the transition zone of patients with nodular benign prostatic hyperplasia.

OBJECTIVES: The biologic mechanism for the increased proportion of noncomplexed ("free") prostate-specific antigen (PSA) found in the serum of patients with benign prostate disease is unknown. We recently reported that most of the PSA found in benign, hyperplastic, and cancerous prostatic tissue is in the free form. To determine whether specific molecular forms of free PSA are associated differentially with normal, hyperplastic, or cancerous prostatic tissue, we have further characterized the free PSA in each type of prostatic tissue. METHODS: PSA was purified by immunoaffinity chromatography from matched prostatic tissue samples of peripheral zone cancer (PZ-C), PZ noncancer (PZ-N), and transition zone (TZ) tissue from 10 large-volume (greater than 50 g) and 8 small-volume (less than 25 g) radical prostatectomy specimens. Eight TZ specimens obtained during transurethral resection of the prostate for benign prostatic hyperplasia (BPH) were also analyzed. The different molecular forms of PSA were further resolved by high-performance hydrophobic interaction chromatography. Clipped forms of PSA were identified by N-terminal amino acid sequencing. RESULTS: More than 99% of the PSA in prostatic tissues was in the free, noncomplexed form. Specimens from the prostate TZ were found to contain elevated levels of an altered form of PSA, which we designated BPSA. Purified BPSA contained a distinctive cleavage at lysine 182. The median percent BPSA (%BPSA) was 11.4 in the TZ of specimens with nodular BPH compared with a %BPSA of 4.1 in the TZ of specimens without nodular BPH (P <0.0014). The median %BPSA levels of the PZ-N and PZ-C tissues ranged from 3.2 to 4.9 and were not significantly different from one another or from the %BPSA level of TZ tissues without nodular BPH. CONCLUSIONS: We have identified a specific molecular form of clipped free PSA, called BPSA, that is increased within the prostatic TZ of patients exhibiting nodular BPH. Higher levels of percent free PSA in serum have been found to correlate strongly with prostate volume, which in turn is closely associated with the progressive enlargement of nodular BPH tissue within the TZ of the prostate. Thus, it is possible that a proportion of the serum percent free PSA found in patients with BPH may be composed of BPSA released into the serum.

Use of human glandular kallikrein 2 for the detection of prostate cancer: preliminary analysis.

OBJECTIVES: Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of a multigene family of serine proteases that share approximately 80% sequence homology. Both are expressed in the prostate epithelium, are under androgen regulation, are present in serum and seminal fluid, and can form complexes with endogenous protease inhibitors (eg, alpha2-macroglobulin and alpha1-antichymotrypsin). Differences in immunohistochemistry and substrate specificity suggest hK2 may provide unique information for early detection and characterization of prostate cancer. METHODS: Nine hundred thirty-seven archived serum samples from men treated at two academic institutions were studied. All men underwent biopsy, had a histologically confirmed diagnosis of cancer or noncancer, and a total PSA level greater than 2 ng/mL. Samples were tested in Hybritech's Tandem-R PSA and Tandem-R free PSA (fPSA) assays and a research prototype assay for total hK2 (thK2). RESULTS: The thK2/fPSA ratio provided additional specificity for cancer detection over PSA and the percentage of fPSA (%fPSA). A model for cancer detection using %fPSA and the thK2/fPSA ratio when PSA is 2 to 4 ng/mL is proposed that would identify as many as 40% of the cancers and would require biopsy in only 16.5% of the men in this PSA range. CONCLUSIONS: In this study, %fPSA and thK2/fPSA provided unique information for prostate cancer detection and increased the specificity of cancer detection.

Identification of a novel complex between human kallikrein 2 and protease inhibitor-6 in prostate cancer tissue.

Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation.

Use of percentage of free prostate-specific antigen to identify men at high risk of prostate cancer when PSA levels are 2.51 to 4 ng/mL and digital rectal examination is not suspicious for prostate cancer: an alternative model.

OBJECTIVES: Currently, many clinicians do not recommend prostate biopsy for men with digital rectal examination (DRE) results that are not suspicious for cancer and prostate-specific antigen (PSA) values between 2.51 and 4 ng/mL. We propose a new model for the detection of prostate cancer using the percentage of free PSA (%FPSA) in the limited range of PSA values between 2.51 and 4 ng/mL that maximizes clinical specificity (ie, minimizes false-positive results). This model identifies higher risk patients in this relatively low-risk population. METHODS: Three hundred sixty-eight archived serum samples from men evaluated and treated at two academic institutions were reviewed. All men had a histologic diagnosis, findings not suspicious for cancer on DRE, and PSA levels between 2.51 and 4 ng/mL. Samples were tested in Hybritech's Tandem-R PSA and Tandem-R free PSA (FPSA) assays in the same laboratory at each institution. RESULTS: Various models for cancer detection using %FPSA when PSA is 2.51 to 4 ng/mL and DRE is not suspicious for cancer are proposed. These models recommend biopsy for only 10% to 36% of the men in this population and would identify as many as 30% to 54% of the detectable cancers. There is evidence that the cancers that would be detected are the most aggressive cancers in this population. CONCLUSIONS: Our models identified men with a higher risk of prostate cancer in a relatively low-risk population that currently does not routinely undergo biopsy. This may allow for a more cost-effective way to increase cancer detection when PSA values are between 2.51 and 4 ng/mL and DRE is not suspicious for cancer. This model has the potential to detect a greater number of clinically important and potentially curable cancers than would be detected with current practice.

Highly sensitive automated chemiluminometric assay for measuring free human glandular kallikrein-2.

BACKGROUND: Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to alpha1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results. METHODS: We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS(R) analyzer. RESULTS: The detection limit of the assay was 1.5 ng/L, the "functional sensitivity" (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations. CONCLUSION: The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.

Serum and semen prostate specific antigen concentrations are different in young spinal cord injured men compared to normal controls.

PURPOSE: Recent investigations have indicated that factors within the seminal plasma may contribute to the condition of low sperm motility in men with spinal cord injury. To determine whether the prostate gland functions normally in these men we chose prostate specific antigen (PSA) as a marker of prostatic function, and compared serum and semen concentrations in spinal cord injured and healthy noninjured men. MATERIALS AND METHODS: The study included 21 spinal cord injured men (mean age 33.3+/-1.2 years) and 22 noninjured normal men (mean age 30.3+/-1.5 years). Blood was obtained from subjects following at least 24 hours of abstinence from ejaculation and serum PSA was determined by modified enzyme immunoassay. Antegrade ejaculates from all subjects were frozen to -80 C, exactly 15 minutes after collection. Seminal plasma PSA was determined using Hybritech Tandem MP assay. RESULTS: Mean serum PSA concentration was 1.20+/-0.19 ng./ml. in spinal cord injured and 0.69+/-0.07 ng./ml. in noninjured men (p<0.02). Mean seminal plasma PSA concentration was 0.59+/-0.11 mg./ml. in spinal cord injured and 1.29+/-0.15 mg./ml. in noninjured men (p<0.001). CONCLUSIONS: Our findings of elevated serum and decreased seminal plasma PSA concentrations indicate that prostatic secretory dysfunction is present in men with spinal cord injury.

Human glandular kallikrein 2 expression in prostate adenocarcinoma and lymph node metastases.

OBJECTIVES: To describe the expression of a potential new tumor marker, human glandular kallikrein 2 (hK2), in primary adenocarcinoma and lymph node metastases that may be useful as an adjunct to prostate-specific antigen (PSA) in the diagnosis and monitoring of prostate cancer. METHODS: We evaluated 151 radical prostatectomy specimens removed at Mayo Clinic with node-positive adenocarcinoma to compare cytoplasmic expression of hK2, pro-hK2, and PSA in benign tissue, prostate adenocarcinoma, and lymph node metastases. Monoclonal antibodies for mature hK2 (hK2-G586), pro-hK2 (pro-hK2-G464), and PSA (PSA-773) were used. A polyclonal antibody for PSA was also used. Immunoreactivity in each case was tested to determine whether cancer recurrence could be predicted. RESULTS: Intense epithelial cytoplasmic immunoreactivity was observed in every case for hK2-G586, pro-hK2-G464, PSA-773, and polyclonal PSA (100% of cases, respectively). The intensity and extent of hK2 expression was greater in lymph node metastases than in primary cancer; furthermore, the expression in primary cancer was greater than in benign epithelium. Pro-hK2 was expressed in a greater percentage of cells in primary cancer than in benign tissue; furthermore, pro-hK2 was expressed to a greater extent in primary cancer than in lymph node metastases. In marked contrast to mature hK2, monoclonal PSA immunoreactivity was expressed to a higher extent in primary cancer than in lymph node metastases. Polyclonal PSA showed an incremental increase in expression from benign tissue to primary cancer and a further increase in expression in lymph node metastases. CONCLUSIONS: hK2 was expressed in every cancer, and the expression incrementally increased from benign epithelium to primary cancer and lymph node metastases. Pro-hK2 was expressed to the greatest extent in primary cancer. Monoclonal PSA displayed inverse immunoreactivity compared with hK2. Polyclonal PSA showed incremental increases, suggesting that both hK2 and PSA were being detected. Tissue expression of hK2 appears to be regulated independently of PSA in benign epithelium, adenocarcinoma, and lymph node metastases.

Polyclonal and monoclonal antibodies to prostate-specific antigen can cross-react with human kallikrein 2 and human kallikrein 1.

OBJECTIVES: The human tissue kallikrein family contains three closely related proteases: human kallikrein 1 (hK1), human kallikrein 2 (hK2), and prostate-specific antigen (PSA). The structural homology between these three proteins suggests potential cross-reactivity interference when different immunologic techniques are used. This study evaluated PSA and hK2 monoclonal antibody (mAb) and polyclonal antibody (pAb) reactivities to hK1, hK2, and PSA. METHODS: mAbs and pAbs to hK2 and PSA were evaluated using Western blot analysis on hK1, hK2, PSA, and seminal plasma. RESULTS: pAbs to PSA and hK2 recognized all three human kallikreins, as well as fragments of hK2 and PSA. An mAb with minimal (less than 0.4%) cross-reactivity between PSA and hK2 and a cross-reactive mAb were found. mAbs specific to PSA or hK2 did not cross-react with the less homologous hK1 protein. A PSA mAb raised specifically to PSA fragments recognized both PSA and hK2 but did not cross-react with hK1. pAbs to hK1 cross-reacted slightly with PSA and not at all with hK2. CONCLUSIONS: Both pAbs and mAbs to hK2 and PSA may exhibit immunocross-reactivity. pAbs to PSA or hK2 react with all three human tissue kallikreins. The potential for cross-reactivity should be considered in any clinical or research procedures that use hK1, hK2, and PSA antibodies.

Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay.

The prostate-specific antigen (PSA) immunoassay is an important tool for the detection and monitoring of prostate cancer. PSA exists in serum mainly as complexes with serine protease inhibitors including alpha1 antichymotrypsin (ACT) and alpha2 macroglobulin (MG). PSA-MG complex is not detected by the existing PSA immunoassays since MG (720 kDa) sequesters PSA and masks the antibody binding sites. Existing immunoassays for quantitation of total serum PSA measure PSA-ACT (CPSA) and free PSA (FPSA), which comprise the major and minor components of total PSA, respectively. Monoclonal antibodies (MAb) specific for CPSA alone were generated using a unique immunogen prepared by blocking the major antigenic determinants on FPSA and ACT. The blocked immunogen greatly enhanced the frequency of hybridomas reactive against the CPSA complex. CPSA prototype immunoassays were established using anti-CPSA (PX1G359) or anti-ACT (AC1A212) MAb as tracer MAb and anti-PSA (PSA399) MAb as capture MAb. The complex-selective MAbs demonstrated minimal cross-reactivity with Cathepsin-G (CG) ACT (CG-ACT), ACT or FPSA. CG-ACT complex interfered with the accuracy of initial prototype assays specific for CPSA measurement and caused over-recovery (1 to 3 ng/mL, with 40 to 180 ng/mL range of CG-ACT in serum) of apparent CPSA values. Addition of 0.4% NP-40 and 0.1% 0.088 micron microparticles in clinical specimens eliminated this interference. Specimens from 39 prostate cancer (PCa) patients and 44 benign prostatic hyperplasia (BPH) patients were analyzed with the PX1G359 CPSA assay. In this study, the area under the curve (AUC) values for ROC analysis of total PSA (CPSA+FPSA), FPSA to total PSA ratio (f/t), and FPSA to CPSA ratio (f/c) were 0.357, 0.634, and 0.624, respectively. In a second study using AC1A212 CPSA assay, where specimens from 16 PCa patients and 48 BPH patients were tested, the AUC values for total PSA, f/t and f/c ratios were 0.62, 0.785, and 0.732, respectively. Using the CPSA assay with minimal interference our studies are consistent with previous CPSA data showing that the f/t PSA ratio remains superior to the f/c PSA ratio in differentiating PCa and BPH diseases. Complex PSA by itself or as ratio with free or total PSA does not provide any advantage over the established method of FPSA to total PSA ratio.

Western blotting analysis of antibodies to prostate-specific antigen: cross-reactivity with human kallikrein-2.

Immunoassays for prostate-specific antigen (PSA) using monoclonal or polyclonal antibodies have clinical applications, such as monitoring and early detection of prostate cancer. However, PSA shares 80% sequence homology with human glandular kallikrein (hK2). In the present study, we have used SDS-PAGE and Western blotting of a recombinant form of hK2 to determine the degree of cross-reactivity in a panel of 83 antibodies submitted to the ISOBM TD-3 PSA Workshop. From this panel, 24 of the 83 antibodies showed cross-reactivity with hK2. The majority of these antibodies showed binding to conformationally independent or linear epitopes. Fourteen of these antibodies appear to map to the same epitope group, indicating the existence of a dominant and linear immunogenic domain shared by PSA and hK2.

Western blotting analysis of antibodies to prostate-specific antigen: specificities for prostate-specific antigen and prostate-specific antigen fragments.

Approximately 40% of the prostate-specific antigen (PSA) purified from seminal fluid comprises cleaved or fragmented forms of PSA. These fragments are observed by reduced SDS-PAGE and have been identified in preparations of purified seminal plasma. The comparative analysis of 53 antibodies, submitted to an international PSA Workshop, with different PSA variants was studied using SDS-PAGE and Western blotting. Six different patterns of reactivity were observed which may reflect different epitopes recognized by this panel of antibodies. Additional antibody studies to nonreduced intact PSA suggest the epitopes are conformation-dependent.