BACKGROUND OF THE INVENTION The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

In the following description, the abbreviation"PSA"means prostate specific antigen;"API"al-protease inhibitor; "PSA-API"complex between PSA and API;"ACT"al-antichymo- trypsin;"PSA-ACT"complex between PSA and ACT;"MAb" monoclonal antibody;"TBS"50 mmol/L Tris-HC1 buffer, pH 7.4 containing 150 mmol/L NaCl and 8 mmol/L sodium azide; "IFMA"immunofluorometric assay;"SDS-PAGE"sodium dodecyl sulfate polyacrylamide gel electrophoresis;''PCa'l prostate cancer;"BPH"benign prostatic hyperplasia;"ROC"receiver operating characteristic; and"AUC"area under the curve.

Prostate-specific antigen (PSA, also called hK3) is mainly produced by the prostatic epithelium and secreted into seminal fluid, where its concentration is 0.5-2.0 mg/mL (1, 2). It is a 30 kDa chymotrypsin-like serine protease (3, 4), which is a member of the human glandular kallikrein family (5,6). The enzyme activity of PSA is regulated by complex formation with protease inhibitors such as a2- macroglobulin and al-antichymotrypsin (ACT) (7,8).

Elevated PSA concentrations in serum are indicative of

prostate cancer (PCa), but false elevation of PSA in serum is often caused by benign prostatic hyperplasia (BPH) (9- 11), which limits the clinical utility of PSA for screening of PCa. It has been previously shown that PSA complexed with ACT (PSA-ACT) is the major immunoreactive form of PSA in serum whereas a minor fraction is free (12,13). The proportion of PSA-ACT to total PSA in serum is higher in PCa than in BPH, and a reduction in the number of false positive results caused by BPH can be achieved by measurement of the proportion of PSA-ACT or free PSA in serum (12,14,15). The dependency of the level of the PSA- ACT complex versus total PSA in PCa patient sera has also previously been shown (26).

In a serum with high PSA concentrations, a small fraction of PSA is bound to a1-protease inhibitor (API) (12) and purified PSA forms a complex with API (PSA-API) in vitro (16). We have now developed a quantitative time-resolved immunofluorometric assay (IFMA) for PSA-API and standardized it by using purified PSA-API formed in vitro.

This assay enabled us to determine the concentrations of PSA-API in serum and to evaluate its clinical utility for diagnosis of PCa.

OBJECT AND SUMMARY OF THE INVENTION The object of the present invention is to provide an improved diagnostic method for differentiating PCa patients from BPH patients or healthy male subjects without PCa, based on the use of the individual's body fluid level of total PSA, free PSA, PSA-ACT, and PSA-API.

According to the invention, either a stepwise ROC analysis, a logistic regression analysis combination or a similar statistical handling method of a) PSA-API or a quantity including PSA-API, and b) free PSA, PSA-ACT or total PSA, or any combination of free PSA, PSA-ACT and total PSA,

is used as marker distinguishing PCa patients from BPH and other non-PCa individuals.

According to another aspect this invention concerns a method for the purification of a PSA-API complex formed in vitro and useful as calibrator in an immunoassay, said method being characterized by the steps of subjecting said complex to hydrophobic interaction chromatograpy, immunoaffinity chromatography and anion exchange chromatography.

According to a further aspect, the invention concerns a method for stabilizing a PSA-API complex formed in vitro and useful as calibrator in an immunoassay, characterized by storing said complex in a buffer having a moderately acidic pH at a temperature below room temperature.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B show the separation of free PSA, PSA-API and PSA-ACT formed in vitro (Fig. 1A) and that occurring in serum (Fig. 1B) by anion exchange chromatography, on a Resource Q column. Fractions containing PSA immunoreactivity obtained by PSA immunoaffinity chromatography (Fig. 1A) or 0.5 mL serum diluted to 10 mL in buffer A (Fig. 1B) were subjected to anion exchange chromatography. Bound proteins were eluted with a linear gradient consisting of 60 mL buffer A and 60 mL buffer B.

Four-mL fractions were collected. Three peaks with PSA immunoreactivity were identified. Peak I was free PSA, II was PSA-API and III PSA-ACT. Note that a different scale is used for PSA-API in Fig. 1B.

Figure 2 shows the characterization of purified PSA-API and PSA-ACT formed in vitro by immunoblotting with a MAb to PSA (Panel A), and polyclonal antibodies to API (Panel B) and ACT (Panel C). The molecular mass markers used were: phosphorylase (97 kDa), bovine serum albumin (66 kDa),

ovalbumin (46 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (21 kDa) and lactalbumin (14 kDa). Lane M: molecular mass markers, lane 1: purified PSA, lane 2: purified API, lane 3: purified ACT, lane 4: peak I, lane 5: peak II, lane 6: peak III. Peaks I-III were those indicated in Fig. 1A.

Figures 3A and 3B show the stability of purified PSA-API during storage at different temperatures (Fig. 3A) and at different pH values (Fig. 3B). Purified PSA-API formed in vitro (50 Eg/L) diluted in TBS pH 7.4 containing 5% BSA was stored at 37,25 and 4°C (Fig. 3A). Figure 3B shows stability of PSA-API at pH values of 5.0,7.4 and 8.4 at 25 °C.

Aliquots were drawn at the time intervals indicated, frozen and stored at-20 °C until analysis. The aliquots were analysed by the IFMA for PSA-API.

Figures 4A and 4B show the fractionation of PSA-API formed in vitro (Fig. 4A) and that occurring in serum (Fig. 4B) by gel filtration on a Superdex-200 column. PSA-API was separated from other forms of PSA by anion exchange chromatography (Fig. 1A and 1B) and subjected to gel filtration. The fractions were analysed by IFMAs for total PSA, PSA-API and PSA-ACT.

Figure 5 shows the comparison of the concentrations of PSA- API in 14 serum samples measured by IFMA for PSA-API (X) with that determined by IFMA for total PSA in the PSA-API- containing serum fraction obtained fractionation of serum by anion exchange chromatography (Y). The correlation between the results was: Y = 1.3 X-2.1 and r = 0.97.

Figure 6 shows the concentration of PSA-API in serum from patients with prostate cancer (n = 82) and BPH (n = 66) as a function of total PSA. The concentrations of total PSA and PSA-API were determined by IFMA.

Figures 7A and 7B show the proportion of PSA-API as a

function of total PSA in sera with total PSA concentrations of 4-75 zg/L from patients with prostate cancer (n = 58) and BPH (n = 42). Total PSA and PSA-API were quantified by IFMA. The proportion of PSA-API was calculated by dividing PSA-API with total PSA (Fig. 7A). Fig. 7B shows the proportion of PSA-API after subtraction of the median background in female serum (0.1 zg/L) from the concentrations of PSA-API.

Figures 8A and 8B show receiver operating characteristic (ROC) curves for total PSA, the proportions of PSA-ACT, free PSA and PSA-API, and combined measurement of the proportion of PSA-API and that of PSA-ACT or free PSA in serum with total PSA concentrations of 4-20 ug/L. Fig. 8A shows PSA, PSA-API, PSA-ACT and PSA-API combined with PSA- ACT. Fig. 8B shows PSA, PSA-API, free PSA and PSA-API combined with free PSA.

Figures 9A and 9B show the proportion of PSA-API in sera with total PSA concentrations of 4-20 Rg/L in patients with prostate cancer (n = 43) and BPH (n = 36) as a function of the proportion of PSA-ACT (Fig. 9A) and that of free PSA (Fig. 9B). Free and total PSA, PSA-ACT and PSA-API in serum were quantified by IFMAs.

DETAILED DESCRIPTION OF THE INVENTION Logistic regression analysis is instrumental in providing the basis for various"risk analysis systems that can provide medical decision support". Other examples of such data handling systems are also: artificial neural networks (ANN), neuro fuzzy networks (NFN), multilayer perceptron (MLP), learning vector quantization (LVQ) [Freeman et a1.

In"Neural Networks: Algorithms, Applications and Programming Techniques by Addison-Wesley Publishing Company"1991, Zadeh Information and Control 1965; 8: 338- 353, Zadeh"IEEE Trans. on Systems, Man and Cybernetics" 1973; 3: 28-44, Gersho et al. In"Vector Quantization and

Signal Compression by Kluywer Academic Publishers, Boston, Dordrecht, London"1992, Hassoun"Fundamentals of Artificial Neural Networks by The MIT Press, Cambridge, Massachusetts, London"1995].

According to the invention, either a stepwise ROC analysis, a logistic regression analysis combination or a similar statistical handling method of a) PSA-API or a quantity including PSA-API and b) free PSA, PSA-ACT or total PSA, or any combination of free PSA, PSA-ACT and total PSA, is used to distinguish PCa patients from BPH and other non-PCa individuals.

According to a preferred embodiment, the stepwise analysis or the logistic regression analysis combination is preferably either c [ (PSA-API)/ (total PSA), (free PSA)/ (total PSA)] or c [ (PSA-API)/ (total PSA), (PSA- ACT)/ (total PSA)].

According to a preferred embodiment, the ratio (PSA- API)/ (total PSA) being less than 1.8 % is used as a primary test.

The method according to this invention is particularly useful for application on individuals having total PSA in the range 4 to 20 zg/L.

Combined measurement of the proportion of PSA-API and PSA- ACT or free PSA in sera with PSA levels of 4-20 zg/L improves the diagnostic accuracy for PCa, compared to known methods. As shown below, the area under the receiver operating characteristic curve is 0.86 for the combination of a proportion of PSA-API (< 1.8 %) with PSA-ACT or free PSA compared to 0.70 for total PSA, 0.81 for the proportion of PSA-ACT and 0.83 for the proportion of free PSA.

We recently showed that PSA slowly forms an SDS-stable complex with API in vitro (16). This is consistent with our

earlier finding showing that a small fraction of PSA in serum is bound to API (12). We have now developed a quantitative assay for PSA-API in serum, which shows no cross-reaction with free PSA or PSA-ACT (< 1%). By this assay we can show that a considerable proportion (1-12%) of the immunoreactive PSA in serum consists of PSA-API. This form of PSA is also measured by assays detecting total but not free PSA.

PSA-API in serum could be separated from free PSA and PSA- ACT by anion exchange chromatography and the chromatographic behavior of endogenous PSA-API in serum was indistinguishable from that of PSA-API formed in vitro.

Thus the PSA-API concentration in serum could also be determined"indirectly"by assay of total PSA in the PSA- API-containing fractions separated by anion exchange chromatography. The values obtained by this method correlated with those determined directly by the specific IFMA for PSA-API. However, measurement of PSA-API by the total PSA assay after chromatography overestimated the results by about 25% due to contamination of PSA-API with free PSA.

Determination of PSA-API in female serum devoid of PSA immunoreactivity indicated that there was a nonspecific assay background. This problem, which also concerns immunoassays for PSA-ACT (14,19), appears to be due to nonspecific binding of the huge excess of API (or other API-complexes) in serum to the solid phase. The effect was confirmed by testing the effect of pure API. The background in the PSA-ACT assays can be reduced by using heparin in the assay buffer (19). We found that preincubation with heparin in the microtitration wells was more effective, reducing the median background in female serum from 0.24 zg/L to 0.10 Rg/L.

The proportion of PSA-API in serum was significantly higher in BPH than in PCa and it was inversely related to the

concentration of total PSA. This behavior is opposite to that of PSA-ACT, the proportion of which increases with increasing PSA levels and is higher in PCa than in BPH (12).

The difference in the proportion of PSA-API between PCa and BPH is of potential clinical utility, but the proportion of PSA-API in serum alone did not improve the diagnostic accuracy of PSA for PCa. However, a fairly large number of prostate cancer patients had a very low proportion of PSA- API, i. e. <1.8%, which was below the lowest values in any BPH patient. When we used 1.8% as a cut-off, 11 (26%) of the cancers could be identified with only one (3%) falsely positive result among the BPH patients. Many of these patients could not be correctly diagnosed by using either free PSA or PSA-ACT. Thus the combination of a low proportion of PSA-API with the proportions of PSA-ACT or free PSA improved the validity for diagnosis of prostate cancer, as shown by an increase in the AUC of the ROC curves.

The standards for the PSA-API and PSA-ACT assays were prepared by adding purified PSA to a plasma fraction containing API and ACT but devoid of a2-macroglobulin. This resulted in formation of similar amounts of PSA complexed with ACT and API (about 25% each). The free PSA remaining was removed by hydrophobic interaction chromatography, after which PSA-ACT and PSA-API were separated by anion exchange chromatography. Although PSA gradually dissociates from PSA-API (16) less than 1% free PSA was detected by gel filtration of the PSA-API preparation. The higher proportion of free PSA and API (5-10%) observed in immunoblotting was apparently due to dissociation of PSA- API by SDS and heating of the sample before electrophoresis. During prolonged incubation at neutral or basic pH, PSA-API formed in vitro tended to dissociate. In serum, the PSA released forms a complexes with 2- macroglobulin (16). We therefore used an artificial buffer

matrix with a pH of 5 rather than female serum as the diluent for the calibrators. In this buffer, the stability of PSA-API was satisfactory, apparently due to low enzyme activity of PSA at this pH.

Because the molecular structure of API is different from that of ACT, it is possible that PSA epitopes exposed in the PSA-API complex differ from those in PSA-ACT. The antibodies used to measure total PSA react equally with free PSA and PSA-ACT (20). The fact that the concentration of total PSA did not change during dissociation of PSA-API indicates that the antibodies also react equally with PSA- API. Thus the assay for total PSA could be used to assign values to the PSA-API calibrators.

The results of this study indicate that the immunoreactive PSA in serum consists of three major molecular forms, i. e. the major form PSA-ACT comprising 60-95% (median 70-81%), free PSA 5-40% (median 11-29%) and PSA-API 1-12% (median 2- 3%). The sum of the concentrations were close to that measured by the assay for total PSA, suggesting that they together account for most, if not all, of the immunoreactive PSA in serum. In about 10% of the patients with PCa and 24% with BPH the proportion of PSA-API in serum was over 5%, thus it represents a notable part of PSA immunoreactivity measured by conventional PSA immunoassays.

In conclusion, we have developed a quantitative IFMA for PSA-API and showed that the proportion of PSA-API in serum is inversely correlated with the total PSA concentration and that it is higher in BPH than in PCa. Combined measurement of the proportion of PSA-API with that of PSA- ACT or free PSA in serum may improve the diagnostic accuracy for PCa as compared to using total PSA and the proportion of either PSA-ACT or free PSA in serum only.

The invention is disclosed more in deltail in the following experimental section.

Materials and methods Samples Serum samples were obtained from 66 patients with BPH, 82 with PCa and from 22 healthy females. The diagnosis was based on histological examination of tissue obtained by transurethral resection or biopsy. Serum samples were taken before initiation of therapy. Pooled EDTA plasma was prepared by mixing plasma samples from healthy females. All samples were stored at-20 °C until used.

Reagents Superdex-200 (60 x 1.6 cm) and Resource Q (6 mL) columns, Phenyl-Sepharose (high performance) and CNBr activated Sepharose 4B were obtained from Pharmacia Biotech (Uppsala, Sweden). PVDF membrane (Immobilon P) was from Millipore (Bedford, MA, USA). Heparin was from Leiras (Turku, Finland). The DELFIA assay buffer, washing and enhancement solutions used in IFMA (17) were from Wallac (Turku, Finland).

Proteins Intact PSA (isoenzyme B) was purified from seminal fluid as described (17). For standardization of IFMAs PSA-API and PSA-ACT were prepared in vitro (see below). Purified ACT was from Athens Research and Technology Inc. (Athens, Georgia, USA). API was purified from plasma as described (18). Protein molecular mass markers were from Pharmacia.

Bovine serum albumin (BSA) was from Sigma (St. Louis, MO) Antibodies A monoclonal antibody (MAb) to PSA (6C11) was produced by standard procedures and immobilized on CNBr-activated Sepharose 4B (2 mg/mL) according to the instructions of the

manufacturer. Anti-PSA MAbs H117 and H50 were from Abbott Diagnostics (Abbott Park, IL), and 5A10 was from Wallac.

MAbs H117 and H50 react equally with PSA and PSA-ACT, whereas MAb 5A10 reacts specifically with free PSA (19).

Polyclonal antibodies to ACT and API, peroxidase-conjugated swine anti-rabbit IgG and rabbit anti-mouse IgG antibodies were from Dakopatts (Glostrup, Denmark). The specificity of the polyclonal antibodies was tested by immunodiffusion (12). Polyclonal antibodies to API and ACT and anti-PSA MAb H50 were labeled with a Eu-chelate (12).

Preparation and purification of PSA-API and PSA-ACT formed in vi tro One hundred mL of pooled female EDTA-plasma was sequentially precipitated with ammonium sulfate at 50% and 80% saturation. The precipitate forming at 80% saturation was collected by centrifugation (15000 x g, 20 min, 4 °C), dissolved and dialysed against TBS with three changes.

Purified PSA (10 mg) was added to the dialysed solution at 37 °C. After 72 h, a saturated solution of ammonium sulfate was added to give 20% saturation. After 1 hour at 4 °C, the preparation was clarified by centrifugation (35000 x g, 20 min, 4 °C) and applied to a 10-ml Phenyl-Sepharose column. Unbound proteins in the flowthrough fractions were collected and applied to the PSA-immunoaffinity column (see below). Bound proteins were eluted with 1 mL/L of trifluoroacetatic acid, pH 2.0, and immediately neutralized by addition of 0.2 mL of 10-fold concentrated TBS to each 2 mL-fraction. After dialysis against buffer A, the eluted fractions were applied to the Resource Q column and eluted as described below.

Chromatographic methods Gel filtration was performed on a 1.6 x 60 cm Superdex-200 column using 50 mmol/L Tris-HCl buffer, pH 7.4 containing

0.15 mol/L sodium chloride (NaCl) and 8 mmol/L sodium azide (TBS). Flow rate was 15 ml/h and 1-mL fractions were collected. The column was roughly calibrated by measuring the absorbance at 280 nm in the fractions to identify IgG (150 kDa) and albumin (68 kDa) in serum.

Anion exchange chromatography was performed on a 6-mL Resource Q column equilibrated with 10 mmol/L Tris-HC1 buffer containing 8 mmol/L sodium azide, pH 8.4 (buffer A).

Serum samples of 0.5 mL were diluted to 10 mL (20-fold) in buffer A or protein fractions were dialysed against this buffer before chromatography. Bound proteins were eluted with a linear gradient composed of 60 mL of buffer A and 60 mL buffer B, which consisted of buffer A containing 300 mmol/L of NaCl.

Hydrophobic interaction chromatography was performed on a 10 mL Phenyl-Sepharose (HP) column equilibrated with 50 mmol/L Tris-HCl, pH 7.0 containing 0.8 mol/L ammonium sulfate and 8 mmol/L sodium azide. Flow rate was 1 mL/min and 4-mL fractions were collected. After sample application, the column was washed with 10 bed volumes of equilibration buffer. Bound proteins were eluted with a gradient composed of 30 mL water and 30 mL of 40% 2-propanol.

Immunoaffinity chromatography. Samples containing about 2 mg PSA were applied to a 15 mL PSA immunoaffinity column equilibrated with TBS, the column was then washed with TBS containing 1 mol/L NaCl and 2 mL/L Tween-20. After this, bound proteins were eluted with 1 mL/L trifluoroacetatic acid, pH 2.0.

Immunoassay IFMAs for free and total PSA. Total and free PSA were simultaneously determined by a dual-label IFMA (DELFIA Prostatus free/total PSA kit, Wallac) as described (20).

The assay was standardized against purified PSA (17).

IFMA for PSA-API and PSA-ACT. The solid phase antibody of the DELFIA Prostatus free/total PSA kit (Wallac) was used.

The coated microtitration wells were preincubated with heparin at a concentration of 50 IU/mL at 4 °C for 12 h to reduce the nonspecific background. Calibrators were prepared by dilution of PSA-API or PSA-ACT formed in vitro to concentrations of 0.1,0.5,2,10 and 50 ug/L in assay buffer, pH 5.0 containing 50 g/L bovine serum albumin. The PSA content in the complexes was determined by the IFMA for total PSA. For assay of serum, 25 uL of samples or calibrators in duplicates and 200 RL assay buffer were pipetted into the microtitration wells. For assay of chromatographic fractions a sample volume of 200 RL was used. Before this, 100 zL of assay buffer with a 10-fold concentration of BSA and bovine serum globulin was added to each 1-mL fraction. After incubation for 1 h at room temperature the wells were emptied, washed six times with wash solution with an automatic washer (DELFIA Platewash 1296-024, Wallac), and filled with 200 mL of assay buffer containing 100 ng of polyclonal antibody to either API or ACT labeled with Eu. After further incubation for 2 h the wells were emptied and washed six times with wash solution.

Enhancement solution (200 zL) was then added to the wells and after 5 min the fluorescence was measured with a 1234 DELFIA research fluorometer (Wallac).

Electrophoresis and immunoblotting Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed under reducing conditions in 2 mm thick and 10 x 10 cm 12.5% homogeneous polyacrylamide gels (21). Proteins were eletrophoretically transferred to an Immobilon P membrane (22). A monoclonal antibody (6C11) to PSA and polyclonal antibodies to API or ACT were used to probe the immunoreactivity of PSA and its inhibitor complexes.

Stability of PSA-API Purified PSA-API formed in vitro was dissolved in 50 mmol/L phosphate buffer containing 1 g/L bovine serum albumin and stored at either 4,25 or 37 °C. Alternatively, PSA-API was stored in buffers with different pH (5.0,7.4 and 8.5) at 25 °C. Aliquots were withdrawn at 0,24,72,120 and 168 h and analysed by IFMAs specific for total PSA and PSA-API.

Before assay, pH was adjusted to 7.4.

Statistical analysis The analytical detection limit was defined on the basis of the mean plus two standard deviations of the fluoroscence response of 10 aliquots of assay buffer only. Differences in the proportion of PSA-API between PCa and BPH patients or concentrations of PSA-API before and after heparin treatment were determined by the Wilcoxon rank sum test.

Differences in the proportion of PSA-API at different PSA concentrations were tested by the Kruskal-Wallis test. The correlation between the concentration of PSA-API determined by various methods was examined by linear regression analysis. The validity of the tests was analysed by receiver operating characteristic (ROC) curve analysis (23) using combinations of (PSA-API)/ (total PSA) and (PSA- ACT)/ (total PSA) or (PSA-API)/ (total PSA) and (free PSA)/ (total PSA).

Results Purification and characterization of PSA-API and PSA-ACT formed in vitro.

When PSA was incubated with the plasma protein fraction containing ACT and API at 37 °C for 72 h, about 25% was complexed with ACT and 24% with API (Table 1). When the mixture was applied to Phenyl-Sepharose (HP), the PSA complexes appeared in the flowthrough fraction whereas free

PSA was retained (not shown). The PSA complexes were applied to the PSA-immunoaffinity column, eluted with 1 mL/L of trifluoroacetatic acid, pH 2.0 and further fractionated by anion exchange chromatography. Three peaks (defined as I, II and III) with PSA immunoreactivity were observed (Fig. lA).

SDS-PAGE and immunoblotting showed that PSA in peak I contained a single 30 kDa band reacting only with the antibody to PSA (Fig. 2A), suggesting that it was free PSA.

PSA in peak II consisted of a major band of about 80 kDa, which reacted with antibodies to PSA and API (Fig. 2A and 2B). In addition, a faint band of about 45 kDa reacting with antibody to API and one of 30 kDa with antibody to PSA were observed. The intensity of these corresponded to 5-10% of the major band. PSA-API in Peak II contained about 14% of the added PSA (Table 1). Peak III contained a major 90 kDa band which reacted with antibodies to PSA and ACT (Fig.

2A and 2C). A faint band of 60-70 kDa reacted with antibody to ACT and one of 30 kDa reacted with antibody to PSA were also observed. PSA-ACT in peak III contained about 18% of the added PSA (Table 1). PSA-API did not react with the ACT-antibody and PSA-ACT did not react with the API- antibody (Fig. 2B and 2C).

When purified PSA-API was incubated at 4,25 and 37 °C for 168 h (Fig. 3A), the concentration of PSA-API decreased from 50 zg/L to 48 Rg/L (96%) at 4 °C, to 44; ig/L (88%) at 25 °C and to 36 g/L (72%) at 37 °C. When stored at pH 5.0 at 25 °C for 168 h (Fig. 3B), the concentration of PSA-API was virtually unchanged (49 Rg/L, 98%), it decreased to 44 Rg/L (88%) at pH 7. 4 and to 24 Rg/L (48%) at pH 8.5. The concentrations of total PSA did not change significantly under these conditions (not shown).

IFMAs for free and total PSA, PSA-ACT and PSA-API The analytical detection limit was 0.01 zg/L for the

Prostatus PSA free/total kit (20), and that for PSA-ACT was 0.16 Rg/L (8). In the range of 0-100 zg/L, the inter-and intra-assay coefficients of variation (CVs) were 3-5 and 5- 8% for the free and total PSA assays (20), and 4-9 and 8- 12% for the PSA-ACT assay, respectively.

The standard curve of the IFMA for PSA-API was linear over the range 0-50 zg/L. The analytical detection limit was 0.1 zg/L. The intra-and inter-assay CVs were 5-10% and 8-14%, respectively, determined by repeated measurement of three serum samples with PSA-API concentrations of 0.5, 2 and 11 zg/L ten times. Separation of PSA-API from PSA-ACT by anion exchange chromatography (see below) and analysis of the fractions showed that the cross-reaction between the IFMAs for PSA-ACT and PSA-API was less than 1%.

Comparison of PSA-API formed in vitro with that occurring in serum When purified PSA-API formed in vitro was separated by gel filtration, a major peak (> 99%) of about 80 kDa was detected by the IFMAs for total PSA and PSA-API (Fig. 4A).

A minor peak (< 1%) of 30 kDa was detected by the IFMA for free PSA, indicating that purified PSA-API contained less than 1% free PSA. When sera (n = 14) with PSA concentrations of 20-700 Rg/L were fractionated by anion exchange chromatography, the peak containing PSA-API was separated from the major peak containing PSA-ACT and several minor peaks detected by the IFMA for PSA but not by the PSA-ACT or PSA-API assays (Fig. 1B). Endogenous PSA-API in serum showed the same chromatographic behavior as that formed in vitro (Fig. 1A and B). The correlation between the concentrations of PSA-API determined by PSA-API IFMA (X) and that measured by an IFMA for total PSA (Y) in the PSA-API fractions separated by anion exchange chromatography was highly significant (Fig. 5, r = 0.97, Y = 1.3 X-2.1, p < 0.0001). The correlation was weaker in sera (n = 11) with low concentrations (< 2 ig/L) of PSA-API

(r = 0.42). When endogenous PSA-API in serum separated by anion exchange chromatography was further fractionated by gel filtration, a major peak (75%) of about 80 kDa was detected by the IFMAs for total PSA and PSA-API (Fig. 4B).

In addition, a minor peak 25%) of 30 kDa was detected by the IFMA for free PSA (Fig. 4B), indicating that PSA-API separated by chromatography from serum was partially contaminated by free PSA.

PSA-API in female serum When female sera (n = 22) devoid of PSA immunoreactivity were analysed by the IFMA for PSA-API, the apparent concentration of PSA-API ranged from 0.12 to 0.57 ug/L (median 0.24 ug/L). When purified API was diluted in assay buffer at concentrations of 0.5-2.0 mg/mL, which correspond to those occurring in normal serum, the apparent concentrations in the PSA-API IFMA were 0.15-0.46 rg/L (median 0.25 zg/L). Thus the concentrations of PSA-API measured in female serum or purified API preparation were probably due to a nonspecific background. This background was reduced by preincubation of the coated microtitration wells with heparin. This treatment reduced the apparent concentrations of PSA-API in female sera to 0.04-0.25 zg/L (median 0.1 pg/L, p < 0.0001).

PSA-API in serum of prostate cancer and BPH patients The concentrations of PSA-API in serum from patients with PCa and BPH increased with increasing concentrations of total PSA, but the proportion of PSA-API in relation to total PSA decreased both in PCa and BPH (Fig. 7A). The median proportion of PSA-API in serum was lower (1.5-2.3%) at high (> 20 zg/L) than at low (< 10 g/L) PSA concentrations (2.3-2.9%, Table 2). Because the nonspecific background affects the proportion of PSA-API more in sera with low PSA concentrations, we subtracted the median background observed in female serum (0.1 g/L) from the

concentration of PSA-API. After this, the proportion of PSA-API in serum was still inversely related to total PSA both in PCa and BPH (Fig 7B). However, because the concentrations of PSA-API in some male sera were below 0.1 g/L, the uncorrected results were used to compare the proportion of PSA-API in serum from PCa and BPH patients.

In sera with total PSA levels below 4 zg/L, the concentrations of PSA-API were mostly close to or below the detection limit of the assay, and they could therefore not be reliably measured. In 7 (10%) of the the serum samples from PCa and 10 (24%) from BPH patients, the proportion of PSA-API ranged from 5 to 12%.

Free and total PSA, PSA-ACT and PSA-API in serum of PCa and BPH patients The concentration of total PSA and the proportion of PSA- ACT were higher in PCa than that in BPH, whereas those of free PSA and PSA-API were lower (Table 3A). The differences were statistically significant in the whole material and in the PSA concentration range 4-20 zg/L. The sum of the median proportions of PSA-ACT and free PSA in serum was 95% of total PSA in PCa and 93% in BPH. The sum of PSA-ACT, PSA-API and free PSA was 97% both in PCa and BPH.

The area under the ROC curve (AUC) for the proportion of PSA-API in serum alone (AUC = 0.64) did not improve the separation between PCa and BPH as compared to total PSA (AUC = 0.70) whereas the proportion of PSA-ACT (AUC = 0.81) and free PSA (AUC = 0.83) did so (Fig 8). However, about one fourth (n = 11) of the PCa patients with a low proportion of PSA-ACT (Fig 9A) and/or a high proportion of free PSA (Fig 9B) had a low proportion of PSA-API (< 1.8%).

This could be used to reduce the number of false negative results obtained with PSA-ACT or free PSA. By using the proportion of PSA-API < 1.8% as a primary test for PCa, and the proportion of PSA-ACT or free PSA as secondary tests in those with PSA-API over 1.8%, the validity was improved

(AUC = 0.86) as compared to assay of total PSA and the proportion of PSA-ACT or free PSA in serum. (Fig. 8; Table 4).

It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the specialist in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

Table 1. Recovery of PSA-ACT and PSA-API formed in vitro during purification.

The concentrations of total and free PSA, PSA-ACT and PSA-API were determined by specific IFMAs.

Steps Volume Total PSA Free PSA PSA-ACT PSA-API (mL) mg (%) mg (%) mg (%) mg (%) 1) Purified PSA 5.0 10.0 (100) 10 (100) 0.0 (0) 0.0 (0) 2) Incubation of PSA with fractionated plasma at 37 °C for 72 h 50.0 9.9 (99) 5.0 (50) 2.5 (25) 2.4 (24) 3) Hydrophobic interaction chromatography 60.0 3.9 (39) 0.1 (1) 2.0 (20) 1.8 (18) 4) PSA-immunoaffinity chromatography 10.0 3.9 (39) 0.4 (4) 1.9 (19) 1.5 (15) 5) Anion exchange chromatography 32.0 3.6 (36) 0.4 (4) 1.8 (18) 1.4 (14) Table 2 Median proportions of PSA-API in serum from prostate cancer and BPH patients with various PSA concentrations.

- Patients No Total PSA Proportion of PSA-API Median (*) mean (lig/L) (%) (%) BPH 31 4.0-9.9 2.9 (2.3-3.3) 3.5 PCa 26 2.3 (1. 6-3.1) 2.7 BPH 5 10.0-19.9 2.6* 2.4 PCa 16 10.0-19.9 1.5 (1. 0-3.2) 2.0 BPH 6 20.0-73.6 2.3* 2.3 PCa 16 20.0-74.4 1.5 (0. 8-2.2) 1.6 *The 95% confidential interval for the median, can not be calculated if sample size < 7.

Table 3 Median proportions (with 95% confidential) of the various forms of PSA in serum from PCa and BPH patients with total PSA concentration in the range 4- 74 (A) and 4-20 llg/L (B).

Patients N o Total PSA (*) PSA-ACT (*) free PSA (*) PSA-API (*) (pg/L) (%) (%) (%) A. PSA range of 4-74 Rg/L BPH 42 7.0 (5. 9-9. 0) 69.7 (65.4-73. 3) 25.6 (20.7-28.6) 2.9 (2. 3-3.2) PCa 58 11.2 (9.2-14.2) 80.7 (78.6-83.9) 11.3 (10.7-13.1) 1.7 (1. 5-2.2) p value 0.001 < 0.001 < 0.001 0.005 B. PSA range of 4-19.9 pLg/L BPH 36 6.7 (5. 3-8.7) 69.7 (65.1-74. 0) 25.8 (18.6-29.2) 2.9 (2. 3-3.3) PCa 42 9.1 (8.0-10.7) 81.3 (77.0-85.7) 12.7 (10.8-16.4) 1.9 (1.5-2. 6) p value 0.002 < 0.001 < 0.001 0.04 Table 4 Clinical specificity and sensitivity for prostate cancer by assaying total PSA, the proportions of PSA-API, PSA-ACT and free PSA, combined measurement of the proportion of PSA-API with that of PSA-ACT or free PSA in serum with PSA concentration of 4-20 KLg/L from patients with prostate cancer (n = 43) and BPH (n = 36).

Specificity Sensitivity (%) (%) total PSA PSA-API PSA-ACT free PSA PSA-API & PSA-API & PSA-ACT free PSA 0 100 100 100 100 100 100 10 95 100 100 100 100 100 20 91 91 100 100 100 100 30 91 81 95 98 98 100 40 88 74 93 98 95 98 50 77 74 86 95 88 98 60 77 70 79 91 81 93 70 58 53 74 77 79 81 80 56 47 70 72 79 77 90 37 30 65 56 79 63 100 7 14 26 9 47 33

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