Immunoassay for the discrimination of free prostate-specific antigen (fPSA) forms with internal cleavages at Lys(145) or Lys(146) from fPSA without internal cleavages at Lys(145) or Lys(146).

Total levels of circulating prostate-specific antigen (tPSA) are strongly associated with prostate cancer (PCa) risk and outcome but benign prostate disease is the most frequent cause of a moderately elevated PSA level. Free PSA (fPSA) forms are independently associated with PCa risk and contribute modest diagnostic enhancements above and beyond tPSA alone. We developed an immunoassay for fPSA subfractions containing internal cleavages at Lys(145) or Lys(146) (fPSA-N). The assay was based on blocking intact single-chain fPSA (fPSA-I) with antibody 4D4 which does not detect PSA containing internal cleavages at Lys(145) or Lys(146). We also measured fPSA-N in blood from healthy volunteers and in anti-coagulated plasma from 76 men with or without evidence of PCa at biopsy. The analytical and functional detection limits of this assay were 0.016 ng/mL and 0.10 ng/mL, respectively. The median recovery of male fPSA-N from female plasma was 95.0%. All 12 female samples (average age 28 years) had fPSA-N concentrations at or below the analytical detection limit. The median fPSA-N concentration (0.050 ng/mL) in 9 healthy male volunteers (age<40 years) was below the functional detection limit, 0.420 ng/mL in 27 patients with benign prostate conditions and 0.239 ng/mL in 49 patients with PCa. Deming regression analysis of the patient samples showed that the measured fPSA-N concentrations were generally 23% lower than the previously calculated (fPSA minus fPSA-I) concentrations, likely due to differences in the antibody combinations used. In conclusion, we have developed a sensitive, specific and direct immunoassay for fPSA-N which can be used to study the clinical relevance of this PSA isoform.

Intact and internally cleaved free prostate-specific antigen in patients with prostate cancer with different pathologic stages and grades.

OBJECTIVES: To measure the concentration levels of free prostate-specific antigen (PSA) isoforms in patients with prostate cancer selected for curative treatment using radical prostatectomy and to study the association between the isoforms and the pathologic cancer stage and grade. METHODS: Preoperative plasma samples were obtained from 309 consecutive patients scheduled to undergo radical prostatectomy at Turku University Hospital. The pathologic TNM stage, Gleason score, and World Health Organization grade of the tumors were recorded. The total, free, and intact PSA (tPSA, fPSA, and fPSA-I, respectively) concentrations of the archived samples were measured with in-house immunoassays, and the nicked PSA (fPSA-N) concentrations (fPSA minus fPSA-I) and ratios of different PSA forms were calculated. These were compared with the prostate cancer stage, Gleason score, and World Health Organization grade. RESULTS: The median fPSA-I and fPSA-N concentrations in the patients with prostate cancer was 0.42 and 0.28 ng/mL, constituting an average of 60% and 40% of fPSA, respectively. The nicked/total PSA and free/total PSA ratios had the strongest negative correlations with a higher pathologic stage, Gleason score, and World Health Organization grade (Spearman rho -0.205 to -0.262, P < .05). Within a patient subgroup with tPSA <10 ng/mL, fPSA-N as a single marker had a negative correlation with a higher Gleason score (rho -0.160, P = .021). CONCLUSIONS: Lower proportions of fPSA-I and fPSA-N to total PSA were associated with a more advanced cancer stage and grade. A long-term follow-up study and a comparison with currently used clinical methods are needed to evaluate the usefulness of the analytes as prognostic markers for cancer aggressiveness in individual patients.

Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin.

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions.

Association of free-prostate specific antigen subfractions and human glandular kallikrein 2 with volume of benign and malignant prostatic tissue.

BACKGROUND: We investigated the association of different subfractions of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), such as total PSA (tPSA), complexed PSA (cPSA), free PSA (fPSA), "single-chain Intact fPSA" (fPSA-I), "multi-chain nicked fPSA" (fPSA-N), and total hK2 with volumes of total prostate gland, transition zone (tz), and prostate cancer (PCa) tissue in patients with benign and malignant prostatic disease. METHODS: Serum samples were collected from men with negative biopsy (n=164) and PCa (n=252). Total and fPSA were measured using a commercially immunoassay. We measured hK2 and fPSA-I by previously reported in-house research assays specific for hK2 and single-chain, non-cleaved fPSA, respectively. Levels of fPSA-N (=fPSA-fPSA-I) and cPSA (=tPSA-fPSA) were calculated. Total prostate and tz volume were measured using transrectal ultrasound (TRUS); PCa volume was calculated using a computer assisted volumetric program. Association with tz and cancer volumes (CaVols) was performed by linear regression analysis. RESULTS: All PSA subfractions and hK2 were associated with tz volume in multivariable linear regression analysis. Only hK2, fPSA, and fPSA-N were significantly associated with CaVol in multivariable analysis, fPSA-I seemed to be cancer related. CONCLUSIONS: The multi-chain fPSA-N subfractions of fPSA may be a valuable predictor of both benign prostate hyperplasia (BPH) and CaVol that is likely to be more useful in predicting tz volumes than CaVols. fPSA-I may provide information on cancer without being influenced by the presence of BPH.

Characterization of monoclonal antibodies against prostate specific antigen produced by genetic immunization.

Prostate specific antigen (PSA) is the most important marker for prostate cancer. Antibodies against minor variants of PSA may be useful in the development of novel diagnostic tests for prostate cancer, but it has been difficult to produce such antibodies by protein immunization. In this study, we have compared the characteristics of monoclonal antibodies (MAbs) obtained by genetic immunization with those obtained by protein immunization. The whole coding region of PSA-cDNA was cloned in a mammalian expression vector pCDNA-3. Six mice were immunized four times by intra-muscular (i.m.) injection of the PSA-pCDNA3 plasmid. The MAbs produced were characterized with respect to subclass, epitope specificity, binding to various molecular forms of PSA and affinity. After intra-muscular injection of DNA, anti-PSA antibodies were detected in the serum of all mice, but the antibody titers were markedly lower than after protein immunization. After fusion of the spleen cells from the mice, five hybridomas producing MAbs to PSA were obtained. The MAbs were of IgG1 and IgG2a isotype and they all recognized equally different forms of free PSA, namely enzymatically active, nicked and proPSA. Epitope mapping showed that these MAbs reacted with the same antigenic regions as those obtained by protein immunization. Thus, genetic immunization leads to production of anti PSA MAbs with similar characteristics to those obtained by immunizing with PSA protein. As applied in the present study, it is less efficient than protein immunization, but it is a useful technique when the antigen is not available in the quantities needed for immunization.

Sensitive and specific enzymatic assay for the determination of precursor forms of prostate-specific antigen after an activation step.

BACKGROUND: The proteome of the serine protease prostate-specific antigen (PSA) and its enzymatic properties have been clarified only recently. We have developed a specific and sensitive method for the measurement of active PSA and used it to measure proPSA in blood. METHODS: We used the synthetic peptide KGISSQY, which possesses a PSA-specific cleavage site, as substrate. To ascertain the specificity of the assay, we used an anti-PSA monoclonal antibody that captures known forms of PSA. An activation step enabled us to measure proPSA by converting it to mature, active PSA. RESULTS: The detection limit of the optimized assay was 0.5 microg/L. In blood samples from patients, the activation step substantially increased the concentration of active PSA, thus showing the presence of proPSA in the samples. ProPSA was 0-79% (median, 45%) of the amount of free PSA in 15 samples with total PSA concentrations of 5.3-423 microg/L. In samples obtained from three benign prostatic hyperplasia (BPH) patients after transurethal resection of the prostate, no significant increase in activity was detected after the activation step, thus showing that proPSA was not a portion of free PSA in plasma of BPH patients. CONCLUSIONS: Proforms of PSA are a considerable fraction of free PSA in the blood of patients with increased total PSA. The approach described can be used to study the diagnostic value of proPSA and active PSA in patients with BPH and prostate cancer.