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Clinical Chemistry 45: 486-496, 1999;
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(Clinical Chemistry. 1999;45:486-496.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Epitope Analysis of a Prostate-specific Antigen (PSA) C-Terminal-specific Monoclonal Antibody and New Aspects for the Discrepancy between Equimolar and Skewed PSA Assays

Hiroshi Nagasaki1,a, Motoyuki Watanabe1, Naoki Komatsu1, Takashi Kaneko1, Jean Y. Dubé2, Tadahiro Kajita3, Yoshihiro Saitoh3 and Yohsuke Ohta3

1 Department of Medical Science, Cosmo Research Institute for Biomedical Research, 1134-2 Gongendo, Satte, Saitama 340-0193, Japan.

2 Laboratory of Hormonal Bioregulation, Centre de L'Université Lavel, Sainte-Foy, Quebec G1V 4G2, Canada.

3 Research & Development Department, International Reagents Corporation, Kobe 651-2241, Japan.
a Author for correspondence. Fax 81-480-42-3790; e-mail CSM01013@nifty.ne.jp or hiroshi_nagasaki{at}cosmo-oil.co.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum.

Methods: We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule.

Results: MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with {alpha}1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by {alpha}ACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was >0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA.

Conclusion: The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.© 1999 American Association for Clinical Chemistry


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate-specific antigen (PSA),1 a glycosylated protein that belongs to the kallikrein family (1), is produced in prostate tissues and secreted into seminal fluid (2)(3). PSA complexes with a serine protease inhibitor, {alpha}1-antichymotrypsin (ACT), in serum (4)(5)(6). PSA is an important marker for the detection of prostate cancer (CaP) (7). However, commercial immunoassays often give different values for the same patient samples, and the calibrators among immunoassays are not interchangeable (8)(9)(10). These differences may be attributed to heterogeneous reactivity of monoclonal antibodies (MAbs) with free PSA (F-PSA) and PSA-ACT as well as the abuse of nonstandardized calibrators. Some researchers have reported a difference of reactivity with F-PSA and PSA-ACT between the equimolar (Tandem-R) and skewed assays (IMx) (11)(12)(13). However, it is curious that the respective values determined by these assays are nearly equal for serum samples (14). A workshop on PSA standardization has recommended either the adoption of a molar ratio of 90% PSA-ACT complex and 10% F-PSA as an international standard or the adjustment of calibrators to the above standard (15). Clearly, it is important to reduce the discrepancy between PSA assays using the international standard and those using selected MAbs.

We determined the exact molecular weight of PSA for our calibrator by an electron spray ionization mass spectrometry coupled with liquid chromatography (ESI-LC/MS) (16). We also characterized the MAbs by BIAcore for the analysis of interactions such as those between antibodies and proteins (17)(18). In addition, we constructed 46 synthetic peptides that covered the whole primary amino acid sequence of PSA and analyzed their interactions with selected MAbs. We determined an epitope on PSA recognized by MAb PA313. It did not cross-react with human glandular kallikrein (hK2), which has high homology to PSA (78%) and is localized in the prostate (19)(20), and recognized the C-terminal PSA sequence.

Recently, several reports have shown the ratio between F-PSA and total PSA (T-PSA) values in serum (21)(22)(23)(24)(25)(26)(27). However, no reports have demonstrated the respective values of each form of PSA in serum.

In the present study, we used three novel assays to determine the material balance of PSA and to elucidate the discrepancy between equimolar and skewed assays on serum PSA values.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
PSA and PSA-ACT calibrators were obtained from Stanford University, Stanford, CA, as an international standard (15)(28). Our in-house calibrators were prepared using PSA from Scripps Laboratories with an absorptivity of 1.84 L · g-1 · cm-1 for PSA (cat. no. P0714) and 0.99 L · g-1 · cm-1 for PSA-ACT (cat. no. P0624) (15)(28)(29). To obtain PSA-ACT in terms of an equivalent mass weight of the free PSA, we multiplied the value for PSA-ACT by 0.32 to calculate the amount of complexed PSA (28). Renal/pancreatic glandular kallikrein (hK1; cat. no. 420313) was purchased from Calbiochem. 4-(2-Aminoethyl)-benzenesulfonyl fluoride (Pefabloc; cat. no. 1429 868) (30), trypsin (cat. no. 109 819), and chymotrypsin (cat. no. 103 306) were purchased from Boehringer Mannheim. Horseradish peroxidase (HRP; type XII) for labeling MAbs was purchased from Sigma. HRP-conjugated goat polyclonal anti-human ACT IgG (PP033) was purchased from Bindingsite. HRP-conjugated goat antimouse IgG (cat. no. 115-035-003) was purchased from Jackson Research Laboratories.

peptide synthesis
All synthetic peptides were obtained from Sawady Technology. They were purified by HPLC, and mass spectrometry was performed to confirm accuracy of synthesis. Forty-six peptides were designed and synthesized through the entire PSA molecule by every 10 amino acids, with 5-amino acid overlaps except for the C-terminal peptide. A cysteine residue was added to either end of the sequence for the thiol coupling method of the BIAcore analysis. The representative peptides were as follows: PSA3, CS11QPWQVLVAS20; PSA4, CV16LVASRGRAV25; PSA7, CV31HPQWVLTAA40; PSA8, CV36LTAAHCIRN45; PSA21, CR101LSEPAELTD110; PSA22, CA106ELTD-AVKVMD115; PSA37, CG181KSTCSGDSG190; PSA38, S186GDSGGPLVC195 (native cysteine); and PSA45, CK221VVHYRKWIK230. The sequences of the C-terminal PSA and hK2-derived peptides were as follows: PSA46A (PSA), CR226KWIKDTIVANP237; PSA46B (hK2), CR226KWIKDTIAANP237; PSA46C, CR226KWIKDTIVANS237; PSA46D, CR226KWIKDTIVADP237; PSA46F, CR226KWIKDTIVAN236; PSA46H, CR226KWIKDTIVANP237G; PSA46J, CRI233VANP237; and PSA46L, CR226KWIKDTLVANP237.

BIAcore ANALYSIS
MAb and peptide interaction.
A real-time biospecific interaction analysis was performed using BIAcore 1000 (Pharmacia Biosensor) with CM5 sensor chips. HBS buffer (10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 3.4 mmol/L EDTA) containing 0.5 mL/L BIAcore surfactant P20 was used for analysis. The measurements were based on changes in surface plasmon resonance signals from chips coated with carboxymethylated dextran to the covalently cross-linked protein (17). The changes in the signal were measured in real time, reflecting the changes in the refractive index caused by the increased protein concentration on the surface of the chip (18). Briefly, for immobilization of all molecules except MAbs, carboxy groups of the matrix were activated with 50 mmol/L N-hydroxysuccinimide and 200 mmol/L N-ethyl-N'-(3-diethyl aminopropyl)-carbodiimide for 10 min, and then 50 mg/L of PSA or PSA-ACT, or 2 g/L of a synthetic peptide in 10 mmol/L acetate buffer (pH 4.5) was injected onto the matrix for 10 min at a flow rate of 5 µL/min. The remaining activated groups were blocked with 1 mol/L ethanolamine (pH 8.5) for 20 min. The resonance units (RU) obtained after the immobilization were ~3000 RU for PSA, 5000 RU for PSA-ACT, and 1200 RU for synthetic peptides, respectively. For MAb immobilization, 10 mg/L of a MAb in 10 mmol/L acetate buffer (pH 4.5) was injected onto the matrix for 3 min at a flow rate of 5 µL/min. The obtained response was typically ~5000 RU for a MAb. Samples diluted with HBS buffer for analysis were injected over the immobilized protein or peptide for 3 min at a flow rate of 20 µL/min, and the surface of the immobilized matrix was regenerated by 10 µL of 10 mmol/L glycine-HCl (pH 2.0). The equilibrium dissociation constants (KD) were calculated from kinetic parameters.

MAb binding to PSA bound with Pefabloc.
PSA (20 mg/L) was incubated with and without 4 mmol/L Pefabloc in 10 mmol/L phosphate buffer (pH 7.0) for 12 h at 25 °C. The samples were concentrated using Centricon-3 centrifugal filtration units (Amicon), which exclude Pefabloc, at 1700g for 1 h at 4 °C. The recovered samples were diluted with HBS buffer and analyzed by BIAcore.

western blotting
PSA, hK2 from seminal fluid, and recombinant hK2 were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto nitrocellulose membranes and detected with the Amersham ECL system using two MAbs (PA313 and S42) for PSA and one MAb (9D5) specific for hK2 (20) as probes.

psa molecular weight determinations
ESI-LC/MS was performed using a Perkin-Elmer Sciex model API-300 solvent delivery system. The LC and MS conditions were described previously (31). Reconstruction was processed automatically with software (BioSpec Reconstruct Algorithm; Perkin-Elmer Sciex).

cross-competition assays among MAbs
Cross-competition assays among MAbs for PSA binding were performed using PSA-coated tubes and the peroxidase-conjugated Fab's (PA111, PA121, PA173, PA211, PA313, and S42) or the biotinylated MAbs (PA96, PA46, FPA503, and FPA811). The assay was performed on an automated instrument, ELSIA F-300 (International Reagents Corp.) according to the manufacturer's instructions with a slight modification. Briefly, 0.3 mL of PSA (1 mg/L) in 100 mmol/L phosphate buffer, pH 7.5, was coated on polystyrene tubes. A competitor MAb (10 mg/L) was incubated at 37 °C for 10 min. The tubes were washed with phosphate-buffered saline (PBS; 10 mmol/L phosphate buffer containing 150 mmol/L NaCl) and 0.2 mL/L Triton X-100, after which 0.3 mL of an HRP-labeled Fab' (~20 µg/L) in PBS containing 10 g/L bovine serum albumin (PBS-BSA) was added to the tubes and incubated at 37 °C for 10 min. For the biotinylated MAbs, after the above washing, 0.3 mL of a biotinylated MAb (~100 µg/L) in PBS-BSA was added to the tubes and incubated at 37 °C for 10 min. After incubation, the tubes were washed, and 0.3 mL of diluted streptavidin conjugated to HRP (1:5000; SA-5004; Vector Laboratories) in PBS-BSA was added to the tubes and incubated at 37 °C for 10 min. The tubes in both procedures were washed, and 0.3 mL of 0.1 mol/L potassium phosphate buffer, pH 7.0, containing 4 g/L of 3-(p-hydroxyphenyl)-propionic acid and 0.14 mL/L H2O2 was added to all tubes. After incubation at 37 °C for 1 min, the enzyme reaction was terminated with 1 mL of 100 mmol/L glycine-NaOH, pH 10.5. The fluorescence intensity was measured with the excitation wavelength at 340 nm and the emission wavelength at 410 nm.

commercial assays for psa and serum samples
Three commercial PSA assays were used for a comparative study: the Abbott IMx (Dainabot), an automated polyclonal/monoclonal enzyme immunoassay (EIA) (7)(14); the Hybritech Tandem-R (SRL), an RIA with dual different MAbs (4); and {gamma}-Sm (Chugai Pharmaceutical), a polyclonal/monoclonal EIA (27)(32)(33). The {gamma}-Sm assay has been reported as the first assay to detect the free form of PSA (27). The solid-phase MAb in the {gamma}-Sm assay (13A6) (32) was used in combination with the HRP-labeled MAbs for analyzing their reactivity with PSA and PSA-ACT. The IMx and Tandem-R assays were performed individually at commercial laboratories in Japan. Sera from patients with benign prostatic hyperplasia (BPH), CaP, or other prostate diseases and healthy subjects were collected and stored at -80 °C until use.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BIAcore ANALYSIS AND CROSS-COMPETITION ASSAYS FORMAbs
The affinity constants of MAbs to PSA and PSA-ACT were determined by BIAcore (Table 1 ). Of these, six MAbs (PA111, PA121, PA173, PA211, PA313, and S42) showed high affinities to PSA and PSA-ACT (~10-10 mol/L). Two MAbs (FPA503 and FPA811) also showed high affinities to PSA, but did not recognize PSA-ACT. To determine the epitopes on PSA recognized by the MAbs, competition analysis between the MAbs for the PSA binding was performed (Table 2 ). Ten MAbs were classified into seven groups (epitopes A to G). PA211 and PA46 recognized the same epitope (epitope E); FPA503 and FPA811 recognized epitope F; and PA313 and S42 recognized epitope G (Table 2 ).


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  		Table 1. Affinity constants of MAbs determined by BIAcore.


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  		Table 2. Cross-competition assays among MAbs.

reactivity of sandwich assays with psa-act in terms of free psa
The reactivity of sandwich assays with PSA and an equivalent mass weight of PSA-ACT is summarized in Table 3 . The combinations of 10 MAbs immobilized on tubes and 6 HRP-labeled Fab's were analyzed. Only the combination of PA313 for the labeled MAb and PA121 for the immobilized MAb (PA313/PA121) showed nearly equimolar reactivity with PSA and PSA-ACT among assays. However, the inverted combination using PA313 for the immobilized MAb and PA121 for the labeled MAb (PA121/PA313) showed less reactivity with PSA-ACT (76%) than with PSA. The combinations between immobilized 13A6 in the {gamma}-Sm assay and the other labeled MAbs showed trivial reactivity with PSA-ACT (<2%) as well as FPA503 and FPA811. In addition, the combination of the labeled PA211 and immobilized PA111 (PA211/PA111) reacted quite weakly with PSA-ACT (3.0%) despite the high affinities shown by both of these MAbs toward PSA-ACT as well as PSA in the BIAcore analysis (Table 1Up ).


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  		Table 3. Percentage of reactivity of sandwich assays with the PSA-ACT complex prepared by PSA mass weight [PSA-ACT].a,b

BIAcore ANALYSIS OF MAb BINDING TO SYNTHETIC PEPTIDES
The interactions between the 10 MAbs and the 46 synthetic peptides covering the whole molecule of PSA were analyzed for epitope mapping on PSA by BIAcore. Although we tried to immobilize peptides by a thiol-coupling method using the additional cysteine at the end of either the NH2 or COOH terminus as well as by the amine-coupling method following the manufacturer's instructions, the binding observed in both methods was not significant, and the affinities of the MAbs to some of the peptides were out of range in the BIAcore analysis (data not shown). Only MAb PA313 interacted substantially with the C-terminal peptide [PSA46A (CR226KWIKDTIVANP237)] of PSA (Fig. 1 A).



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  		Figure 1. Specific binding between PA313 and C-terminal synthetic peptides derived from PSA.

Each peptide was immobilized on a BIAcore CM5 sensor chip, and the binding of PA313 to the immobilized peptide was monitored by BIAcore 1000. (A), interactions between immobilized PSA46A on the CM5 sensor chip and PA313 as analyte were measured in the following reactions: with 5 mg/L PA313 [PSA46A-PA313 (5 mg/L)]; 10 mg/L PA313 [PSA46A-PA313 (10 mg/L)]; and 5 mg/L PA313 with 1 mmol/L PSA46A as competitor [PSA46A-(PA313 (5 mg/L) + PSA46A (1 mmol/L))]. Interactions between PSA46L and PA313 were measured in the following reactions: with 5 mg/L PA313 [PSA46L-PA313 (5 mg/L)]; 10 mg/L PA313 [PSA46L-PA313 (10 mg/L)]; and 5 mg/L PA313 with 1 mmol/L PSA46L as competitor [PSA46L-(PA313 (5 mg/L) + PSA46L (1 mmol/L))]. (B), interactions between a synthetic peptide on the CM5 chip and PA313. Each peptide [ PSA46A (46A), PSA46B (46B), PSA46C (46C), PSA46D (46D), PSA46F (46F), PSA46H (46H), PSA46J (46J), or PSA45 (45)] was immobilized on the CM5 sensor chip and interacted with PA313 (10 mg/L).

The affinity of PA313 binding to peptide PSA46A was reduced ~100-fold relative to its affinity for intact PSA (Table 1Up and Fig. 1BUp ). Its binding to PSA46A was specifically inhibited by the competition of PSA46A (1 mmol/L; Fig. 1AUp ). PA313 did not interact with either PSA3 or PSA4, which has the LVA sequence (data not shown). It also did not interact with PSA7 and PSA8, which have the TLV sequence, or with PSA21 and PSA22, which have the DTLEA sequence in reverse order, respectively (data not shown). In addition, it did not interact with PSA45 (CK221VVHYRKWIK230). PA313 did not interact with hK1 in either BIAcore or EIA analyses (data not shown). Because hK1 has the D232TI234 sequence (34), we assumed that the C-terminal sequence (V234ANP237) would be important for PA313 binding to PSA. Consequently, we synthesized the C-terminal peptides of PSA to investigate interactions with PA313. PA313 interacted with PSA46L (CR226KWIKDTLVANP237), which has an amino acid change from I233 to L233. The affinity of PA313 binding to PSA46L was similar to that of PSA46A, although the response of PA313 to PSA46L was reduced by about threefold relative to PLA46A (Fig. 1BUp ). PA313 did not interact with PSA46B (CR226KWIKDTIAANP237), which has the hK2-derived C-terminal sequence (Fig. 1BUp ). It also did not interact with either PSA46H, which has an additional glycine next to the C-terminal proline in the PSA sequence, or PSA46F, which lacks the C-terminal proline (Fig. 1BUp ). In addition, it did not interact with other peptides that have a mutation in the PSA sequence [PSA46C (P237S), PSA46D (N236D)], or with a short peptide [PSA46J (CRI233VANP237); Fig. 1BUp ]. Although S42 shared the epitope on PSA with PA313, it did not show the interaction to any C-terminal peptides (data not shown).

MAb BINDING TO PSA BOUND WITH PEFABLOC
To analyze reactivity of the MAbs specific for the free form of PSA, we added Pefabloc, a serine protease inhibitor in the sulfonyl fluoride family (30), to the PSA solutions to determine whether it could block the binding of the MAbs to PSA. When PSA was bound with Pefabloc before interacting with the MAbs, the binding of FPA503 or FPA811 to PSA was almost completely blocked in the BIAcore analysis (Fig. 2 ). In contrast, the binding of PA313 to PSA showed a nearly equivalent response (Fig. 2 ) and the same affinity for PSA blocked by Pefabloc as for PSA (data not shown). Both MAbs (FPA503 or FPA811) could not bind with either of the two synthetic peptides [PSA37 (CG181KSTCSGDSG190) and PSA38 (S186GDSGGPLVC195)] in the BIAcore analysis (data not shown). The serine-189 residue (S189) would be blocked by serine protease inhibitors in the highly conserved trypsin-like motif (GDSGG) of the serine protease family (34). Both MAbs also did not interact with other serine proteases such as hK1, trypsin, and chymotrypsin, which also have the conserved motif (GDSGG), in the BIAcore and EIA analyses (data not shown).



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  		Figure 2. Specific inhibition by a serine protease inhibitor (Pefabloc) against a MAb binding to PSA.

Each MAb was immobilized on a BIAcore CM5 sensor chip, and the binding the immobilized MAb to the 20-fold diluted PSA samples with and without pretreatment with 4 mmol/L Pefabloc (initial concentration) was monitored by BIAcore 1000. Interactions between FPA503 and PSA (FPA503-PSA), FPA503 and PSA treated with Pefabloc [FPA503-(PSA + Pefabloc)], FPA811 and PSA (FPA811-PSA), FPA811 and PSA treated with Pefabloc [FPA811-(PSA + Pefabloc)], PA313 and PSA (PA313-PSA), and PA313 and PSA treated with Pefabloc [PA313-(PSA + Pefabloc)] were measured.

western blotting
The reactivity of the two MAbs (PA313 and S42) with PSA or with hK2, detected by Western blotting, is shown in Fig. 3 , A and B. Both MAbs did not cross-react with hK2 from seminal fluid (180 ng) and glutathione S-transferase-hK2 fusion proteins (up to 2 µg). MAb 9D5, which is specific for hK2 (20), was used as control and detected low concentrations of hK2 (1 ng) without showing cross-reactivity with PSA (up to 5 µg; Fig. 3C ).



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  		Figure 3. Western blot analysis of PSA and hK2.

PSA and hK2 were electrophoresed on an sodium dodecyl sulfate-polyacrylamide gel and blotted to a nitrocellulose membrane. Membranes were probed with 50 µg/L HRP-labeled PA313 (A) or 500 µg/L HRP-labeled S42 (B). (C), hK2-specific MAb 9D5 (1.0 mg/L) was used as the primary antibody, and the immune complexes were detected by 10 000-fold diluted HRP-labeled goat antimouse IgG for hK2. (A and B), lane 1, PSA (40 ng); lane 2, PSA (180 ng); lane 3, hK2 purified from seminal fluid (180 ng); lane 4, recombinant GST-hK2 (1.0 µg); lane 5, recombinant GST-hK2 (2.0 µg). (C), lane 1, PSA (5.0 µg); lane 2, PSA (1.0 µg); lane 3, hK2 purified from seminal fluid (1.0 ng); lane 4, hK2 purified from seminal fluid (5.0 ng); lane 5, hK2 purified from seminal fluid (25.0 ng). Values to the left of each gel, molecular weight markers; k, x 103.

comparison of antigenic reactivity and correlation among assays
The antigenic reactivity of the PA313/PA121 and commercial assays was compared using the international standard and our in-house calibrators. The analysis of PSA by ESI-LC/MS is illustrated in Fig. 4 . The molecular weight of the main peak in the ionized PSA fragments was 1778.0 with 16 H+ ions (Fig. 4A ) and calculated as Mr 28 432 (16). The obtained spectrum was similar to that of Bélanger et al. (31) and had two additional peaks at Mr 28 449 and 28 304 on the statistical deconvolution (Fig. 4B ). The calibrators for the PSA and PSA-ACT assays were prepared according to the preparation of the international standard (see Materials and Methods) (15)(28)(29). Differences of reactivity with the PSA and PSA-ACT preparations were observed among assays (Table 4 ). The assay using the PA313/PA121 combination showed equimolar reactivity with both the international standard and our in-house calibrators. The IMx showed ~40% lower reactivity with the PSA-ACT than with the PSA preparations regardless of the origins. The Tandem-R showed nearly equimolar but slightly stronger reactivity with PSA-ACT than with PSA. The correlation coefficients were almost equal to 1.0 between assays (Figs. 5 and 6). The slopes ranged from 0.948 to 1.006 and from 0.912 to 0.983 when the manufacturers' or the Stanford 90:10 calibrators were used, respectively (Figs. 5 and 6 ). These observations raise the possibility that a skewed assay might correlate well with an equimolar assay when the proportion of F-PSA to T-PSA (F/T) in serum is low.



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  		Figure 4. ESI-LC/MS analysis for PSA.

(A), PSA (70 pmol) dissolved in 10 µL of H2O was injected, and the molecular weights of the ionized PSA fragments were analyzed by MS. (B), the peaks in A were reconstructed statistically from m/z to molecular scales.


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  		Table 4. Reactivity of PSAs and PSA-ACTs prepared by PSA mass weight in three different assays.1



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  		Figure 5. Correlation of PSA values measured by three different assays in sera from 29 individuals, using the manufacturers' calibrators.

(A), PA313/PA121 vs Tandem-R; (B), PA313/PA121 vs IMx; (C), Tandem-R vs IMx.



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  		Figure 6. Correlation of PSA values measured by three different assays in sera from 29 individuals, using the Stanford 90:10 calibrators.

(A), PA313/PA121 vs Tandem-R; (B), PA313/PA121 vs IMx; (C), Tandem-R vs IMx.

quantification of psa in serum samples
To clarify the above possibility, we reassessed the above 29 samples (22 CaP patients, 2 BPH patients, and 5 healthy subjects), using the F-PSA assay. The determined values using the PA313/FPA503 combination did not correlate well with those using PA313/PA121 (Fig. 7 ). The observed F/T ratios varied from 0% to 30% and were <10% except for five samples (Fig. 7 ). Two samples (M12 and K61) showed higher F/T ratios (>15%; Table 5 and Fig. 7 ) (21). This result supports our hypothesis that samples with higher F/T ratios would emphasize discrepancies in the liner regression analysis between an equimolar and a skewed assay and illustrate the difference of the respective values determined by them. Additional serum samples from 21 patients with CaP, 31 patients with BPH, and 25 patients with other prostate diseases were then screened using both the PA313/PA121 and the PA313/FPA503 MAb combinations to find samples with higher F/T ratios (>15%). Of these, four samples that showed higher F/T ratios (>15%) were assayed again in detail. The respective values of each form of PSA and PSA values determined by the equimolar and skewed assays are summarized in Table 5 . The values measured by either of the equimolar assays were nearly equal to the sum of the values obtained using PA313/FPA503 and {alpha}ACT/PA313 (the HRP-labeled polyclonal antibody against ACT and the immobilized PA313). In contrast, the values measured by the IMx assay were higher than the equimolar assays, especially sample 2K71 (F/T = 79%), which gave a value at least 1.5-fold higher in the IMx assay than in the equimolar assays. However, these values were in agreement with the calculated values (F-PSA/0.55 + C-PSA), where C-PSA is the complexed form of PSA.



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  		Figure 7. Correlation of PSA values measured by the total (PA313/PA121) and free (PA313/FPA503) PSA assays.

The dashed line indicates the 10% F/T ratio.


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  		Table 5. PSA in sera determined by different assays, µg/L.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
On the basis not only of the cross-competition assays but also of the reactivity of the MAbs in sandwich assays with PSA-ACT, we classified our MAbs into seven groups (epitopes A to G). Our results were similar to those in the previous report by Nilsson et al. (35). Because each MAb in epitope F showed different reactivity with the combination of the labeled MAbs, e.g., FPA503 could detect PSA with labeled PA111 (epitope A), PA173 (epitope D), PA211 (epitope E), PA313, and S42 (epitope G), but not with PA121 (epitope C), we divided the F-specific epitopes into three subgroups. FPA811 and 13A6, the solid-phase MAb in the {gamma}-Sm assay (32), have also been classified in the same manner.

The PA313/PA121 assay showed equimolar reactivity with PSA and PSA-ACT. However, the PA121/PA313 assay showed 76% reactivity with PSA-ACT. This observation could be demonstrated by the reaction steps in the assay. Our assay is a two-step sandwich assay: the immobilized MAb first captures PSA or PSA-ACT, and then the labeled Fab'-HRP reacts with the captured antigens. Possible conformational changes between the solid-phase MAb and the captured PSA or PSA-ACT induce different reactivities despite the same combination of MAbs used in the assay. This phenomenon is explained indirectly by the reactivity results of both the PA211/PA111 and PA111/PA211 assays in EIA. That is, both of these MAbs (PA211 and PA111) can recognize PSA-ACT with high affinities by the BIAcore analysis; in contrast, neither the PA211/PA111 combination nor the PA111/PA211 combination recognizes PSA-ACT in our sandwich assay. It must be noted that these two MAbs are classified into the different epitope groups on PSA.

To develop PSA assays, we must consider cross-reactivity with related antigens to reduce false positives and/or false negatives, in this case such as hK2 and other kallikreins. Of the 10 MAbs tested, PA313 bound to PSA46A and PSA46L, whereas it could not bind with PSA46B, which is derived from hK2, or with other C-terminal peptides (PSA46C, D, F, H, and J, and PSA45). The difference between PSA and hK2 in the C-terminal sequence (residues 221–237) is only one amino acid at residue 234, from V234 in PSA to A234 in hK2. The difference between PSA46A and PSA46B is two additional methyl groups in V234 (PSA46A) to A234 (PSA46B) and that between PSA46A and PSA46D is a change from an amide in N236 (PSA46A) to a carboxylic group in D236 (PSA46D). In addition, PA313 could not interact with a longer C-terminal peptide with a mutation at A235 [PSAC (C210ALPERPSLYTKVVHYRKWIKDTIVENP237); data not shown]. Therefore, V234, A235, N236, and P237 with the C-terminal carboxylic acid in the PSA conformation would be a prerequisite for the binding of the PA313 to PSA. The homology between human PSA (R226KWIKDTIVANP237) and trypsin (V213KWIKNTIAANS224) in the C-terminal sequence is highly conserved (sequence homology, 67%; sequence similarity, 83%) (31)(34)(36)(37). The C-terminal structure of PSA (R226KWIKDTIVAN236) calculated by computer analysis is predicted to be an {alpha}-helix (data not shown) from the crystal structure of human trypsin (37). We checked the interaction between PA313 and PSA46J (CRI233VANP237) instead of the CIVANP peptide by BIAcore because the CI233VANP237 and CT232IVANP237 peptides did not dissolve in the immobilizing buffer. In view of the computer analysis, we assume that PSA 46J might not hold the same conformation of the IVANP in the {alpha}-helix of PSA46A (data not shown). The structure of the PA313-PSA interaction has not been analyzed in detail yet, but it is clear that the C-terminal {alpha}-helix and P237 with carboxylic acid is crucial for the binding between the PA313 and PSA. It is one of the representative examples in other interactions that the PDZ domain can tightly bind the S/TXV with the C-terminal carboxylic acid (38)(39). These results indicate that the interaction between the PA313 and PSA is quite specific and that the cross-reaction between the PA313 and other kallikreins is negligible.

It is also important to clarify the epitopes on PSA recognized by the MAbs that can interact only with PSA and not with PSA-ACT because the molecular weight of ACT is approximately twice as high that of PSA (28) and it might interfere the binding between a MAb and PSA. In fact, both of the PA211/PA111 and PA111/PA211 assays recognize PSA-ACT only faintly. The epitopes of the F-PSA-specific MAbs are still ambiguous, but we could obtain important information. A synthetic serine protease inhibitor (Pefabloc; Mr 239.5) abolished the interaction between the F-PSA-specific MAbs (FPA503 or FPA811) and PSA in the BIAcore analyses. Its function is thought to be similar to that of phenylmethylsulfonyl fluoride (30). PSA forms a complex with ACT at S189 in the active pocket (34), and this serine residue could be blocked by it. These two MAbs could probably recognize the tertiary but not the primary structure of the vicinal domain of S189 without cross-reaction with other kallikreins and serine proteases.

We next compared our assay with both the equimolar (Tandem-R) and skewed assays (IMx). The IMx assay has already shown ~50% lower responses to the PSA-ACT than to the F-PSA calibrators (12)(13)(15). In our study, the IMx assay showed ~40% lower reactivity with the PSA-ACT than with the F-PSA calibrators, and its assay calibrators in the kit showed ~55% responses to the highly purified F-PSA calibrators (data not shown). In contrast, both the correlation coefficients and slopes between equimolar and IMx assays on serum samples were nearly 1.0 with either the manufacturers' or the Stanford 90:10 calibrators. The result of the regression analysis between the Tandem-R and IMx in this study is consistent with the previous report (14). It seems contradictory that the reactivity of the equimolar and IMx assays with PSA and PSA-ACT is clearly different but the values they measure are nearly equal. These data imply that the IMx assay could not detect the PSA-ACT concentration in sera precisely, but it might yield correct values of serum PSA when F/T ratios in samples are low by the use of approximately twofold-diluted F-PSA for the calibrators instead of the correct concentration of the PSA calibrators. In the randomly selected 29 samples, we had only 2 samples that showed higher F/T ratios (>15%) (21). These results suggest that the low concentrations of PSA in serum samples do not cause discrepancies derived from the assay methods, which means that the serum values measured by either an equimolar or a skewed assay could be correlated well in the regression analysis when F/T ratios are low (26). Thus, we hypothesized that, if the F/T ratios in sera are remarkably high, the serum values measured by the IMx assay should be higher than those measured by the PA313/PA121 or Tandem-R assay.

Petersson et al. (40) have shown the epitope map of PSA and developed an assay for C-PSA, but the C-PSA was measured as protein concentration and not as PSA mass weight. Recently, researchers have also studied the F-PSA-specific assays and discussed the F/T ratios (21)(22)(23)(24)(25)(26)(27). Before this study, however, none of the assays could have illustrated the material balance of PSA in serum. Therefore, we tried to determine the respective values of each form of PSA in serum by the three different assays. The T-PSA concentration measured by the equimolar assays is nearly equal to the sum obtained by the PA313/FPA503 and {alpha}ACT/PA313 (F-PSA + C-PSA), whereas the T-PSA concentration measured by the IMx assay is higher than F-PSA + C-PSA. However, T-PSA measured by the IMx is nearly consistent with the values by calculation (F-PSA/0.55 + C-PSA). This coefficient (0.55) could be induced from the reactivity of the IMx assay between PSA and PSA-ACT as well as that between the manufacturer's calibrator and the highly purified F-PSA calibrators. Semjonow et al. (26) have reported that the slope in the regression analysis between the Tandem-R (x) and IMx (y) assays is larger than 1.0 for samples with higher F/T ratios. It is consistent with the idea that the discrepancy of the serum PSA values determined by the equimolar and skewed assay is dependent on the F/T ratios in sera.

Additional study of PSA assays will provide valuable insights into the diagnosis of other diseases such as breast tumors (41)(42).


   Acknowledgments
 
We thank Dr. T. A. Stamey, Department of Urology, Stanford University, Stanford, CA, for providing PSA calibrators and Dr. O. Nilsson, CanAg Diagnostics, Gothenburg, Sweden, for providing MAbs to do the BIAcore analysis. We thank Drs. T. Arai and S. Tominaga for helpful discussions and Dr. J. Inagawa for suggestions concerning the BIAcore analysis. We thank Dr. B. Whittaker for analysis of the enzyme reactivity of PSA, and S. Yamazaki and K. Matsuura for the ESI-LC/MS analysis. We also thank Dr. J. Kajihara and K. Shibata, Development and Research Laboratories, JCR Pharmaceuticals, Kobe, Japan, for providing the purified PSA from urine. We thank A. Kanou for the computer analysis and J. Iwase for technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: PSA, prostate-specific antigen; ACT, {alpha}1-antichymotrypsin; CaP, prostate cancer; MAb, monoclonal antibody; F-PSA, free prostate-specific antigen; T-PSA, total prostate-specific antigen; ESI-LC/MS, electron spray ionization mass spectrometry coupled with liquid chromatography; hK2, human glandular kallikrein; hK1, human pancreatic/renal glandular kallikrein; HRP, horseradish peroxidase; HBS, 10 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 3.4 mmol/L EDTA; RU, resonance unit(s); PBS, phosphate-buffered saline; BSA, bovine serum albumin; EIA, enzyme immunoassay; F/T; ratio of free to total prostate-specific antigen; BPH, benign prostatic hyperplasia; and C-PSA, complexed prostate-specific antigen.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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