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Clinical Chemistry 49: 97-103, 2003; 10.1373/49.1.97
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(Clinical Chemistry. 2003;49:97-103.)
© 2003 American Association for Clinical Chemistry, Inc.

Dual-Label Immunoassay for Simultaneous Measurement of Prostate-specific Antigen (PSA)-{alpha}1-Antichymotrypsin Complex Together with Free or Total PSA

Lei Zhu1, Jari Leinonen1, Wan-Ming Zhang1, Patrik Finne1 and Ulf-Håkan Stenman1a

1 Department of Clinical Chemistry, Helsinki University Central Hospital, Biomedicum, PB 700, FIN-00029 Helsinki, Finland.

aAuthor for correspondence. Fax 358-9-47171737; e-mail ulf-hakan.stenman{at}hus.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: A major portion of prostate-specific antigen exists in circulation as a complex with {alpha}1-antichymotrypsin (PSA-ACT), whereas a minor part is free (fPSA). The proportion of PSA-ACT is increased in prostate cancer (PCa), but immunologic determination of PSA-ACT is hampered by a background produced by nonspecific adsorption of ACT to the solid phase. To reduce the nonspecific interference, we produced an antibody specific for complexed ACT and developed immunofluorometric assays (IFMAs) for simultaneous measurement of fPSA + PSA-ACT (fPSA/PSA-ACT) and PSA-ACT + total PSA (tPSA, PSA-ACT/tPSA).

Methods: Monoclonal antibodies (MAbs) were produced by immunization with PSA-ACT. The dual-label time-resolved IFMAs for fPSA/PSA-ACT and PSA-ACT/tPSA used a capture MAb to tPSA, an Eu3+-labeled MAb to fPSA or complexed ACT, and an Sm3+-labeled MAb to complexed ACT or to tPSA as tracer antibodies. The clinical utility was evaluated using serum samples from individuals with or without PCa with PSA concentrations of 2.0–20.0 µg/L.

Results: One MAb (1D10) showed low cross-reactivity with free ACT and cathepsin G-ACT. A sandwich assay for PSA-ACT with 1D10 as tracer had a detection limit of 0.05 µg/L, and with this assay, PSA-ACT was undetectable in female sera. The detection limit for fPSA was 0.004 µg/L. Determinations of the ratio of fPSA to PSA-ACT and the proportions of fPSA/tPSA and PSA-ACT/tPSA provided the same clinical specificity for PCa and provided significantly better clinical specificity than did tPSA.

Conclusions: Background problems observed in earlier PSA-ACT assays are eliminated by the use of a MAb specific for complexed ACT as a tracer. The same clinical validity can be obtained by determination of fPSA or PSA-ACT together or in combination with tPSA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate-specific antigen (PSA)1 is a 30-kDa serine protease secreted by the epithelial cells of the prostate (1)(2). It is a reliable marker for monitoring of prostate cancer (PCa) and is also widely used for early detection and screening of PCa (3)(4)(5)(6). However, the use of PSA for screening is hampered by the fact that increased PSA concentrations are also found in many patients with benign prostatic hyperplasia (BPH) and prostatitis (3)(7)(8).

In plasma, PSA occurs predominantly as a complex with the serine protease inhibitor {alpha}1-antichymotrypsin (ACT), whereas a smaller portion exists in the free form (fPSA) (4)(9). PSA also occurs in complex with other serine protease inhibitors in plasma, including {alpha}2-macroglobulin and {alpha}1-protease inhibitor (API) (4)(10). The proportion of PSA-ACT is higher, whereas those of fPSA and PSA-API are lower in the serum of PCa patients than in those with BPH (4)(11)(12). Numerous studies have shown that the clinical accuracy of the PSA determination can be improved by measuring the proportion of either fPSA or PSA-ACT in relation to total PSA (tPSA) (4)(13)(14). However, accurate measurement of PSA-ACT is complicated by a nonspecific background caused by other ACT complexes and uncomplexed ACT that bind nonspecifically to the solid phase (15)(16)(17). Furthermore, for metrologic reasons, measurement of the ratio of PSA-ACT to PSA, which occur at similar concentrations, is more demanding than measuring the ratio of fPSA to tPSA. This is because the relative difference in the proportions of PSA-ACT between cancers and controls are smaller (6% based on mean values 82% and 77%, respectively) than those of fPSA/tPSA (30% based on the corresponding mean values of 18% and 23%, respectively). Therefore, to obtain the same reproducibility for measurement of the PSA-ACT/tPSA ratio, it is necessary to have a fivefold better precision than for fPSA/tPSA. This can be demonstrated by calculation of the impact of imprecision on the overlap in the proportions in samples from cancer patients and controls. Assuming random distribution, ~60% of the samples will have results within the mean ± 1 SD (and 97% within the mean ± 2 SD). Thus, at a CV of 5%, the range of values obtained for PSA-ACT/tPSA in cancer cases will be 0.78–0.87 and that in control cases 0.73–0.81, when the true ratios are 0.82 and 0.77, respectively. Thus, ~20% of the results (one-half of 40%) in each group will overlap because of assay imprecision. With the same CVs, the corresponding measured values for fPSA/tPSA will be 0.17–0.19 and 0.22–0.24, respectively, i.e., there is no overlap.

The variation of the ratio can be reduced by measuring the two components simultaneously with a double-label assay. With this approach, the effect of pipetting errors is eliminated because it is the same for both analytes. When measured with an assay based on this principle, the proportion of PSA-ACT was found to provide substantial improvement in cancer specificity. However, the high background of this assay and a moderate analytical sensitivity limited its use for samples with low PSA concentrations (15).

To eliminate the limitations of earlier assays for PSA-ACT, we have developed dual-label time-resolved immunofluorometric assays (IFMAs) for simultaneous determination of the serum concentrations of PSA-ACT together with fPSA or tPSA, using a novel monoclonal antibody (MAb) specific to complexed ACT in combination with antibodies to fPSA and tPSA. The diagnostic performance of this method was evaluated with serum samples from individuals with and without PCa.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients
Serum samples were collected from 35 patients with PCa and from a reference group (n = 90) without evidence of cancer in sextant biopsies. Of these, 25 had BPH, 5 had other prostatic diseases, and 60 had normal histology. Samples from 132 healthy females were also studied. Serum samples were frozen once and stored for 1 month to 2 years at -80 °C until analyzed. All patient samples were taken before initiation of therapy. The diagnoses were based on histologic analysis of tissue obtained by biopsy or at surgery.

reagents
Eu3+ and Sm3+ chelates, plates coated with MAb H117 for tPSA, Sm3+-labeled MAb H50 for tPSA, and Eu3+-labeled MAb 5A10 for fPSA were from Perkin-Elmer Wallac. Polyclonal antibodies to ACT and to cathepsin G were purchased from Dako. Anti-PSA MAbs 5E4, 9C5, and 5C7 were developed in our laboratory by standard techniques. These MAbs have been shown to recognize distinct epitopes on PSA (18). ACT was from Athens Research. Cathepsin G was from Sigma. Purified PSA, PSA-API, and PSA-ACT were prepared as described previously (19)(20). The cathepsin G-ACT complex was prepared by incubating purified cathepsin G with ACT at 37 °C for 30 min. Complex formation was verified by measuring the loss of enzymatic activity of cathepsin G with the chromogenic substrate S-2586 (Kabi) and by monitoring the response in the cathepsin G-ACT IFMA (see below). A MAb (1D10) prepared against PSA-ACT reacting with complexed ACT was used as a tracer in the assay for PSA-ACT.

labeling of antigens and antibodies
Antibodies, PSA, and PSA-ACT were labeled with either Eu3+ or Sm3+ chelates of N1-(p-isothiocyanatobenzoyl)-diethyleneamine-N1,N2,N3,N3-tetraacetic acid (DTTA; Wallac) as described previously (15).

development of MABS to PSA-ACT
BALB/c mice were immunized with 10–30 µg of purified PSA-ACT by intraperitoneal injection with Freund’s complete adjuvant. A booster dose of 10 µg was administered after 4 weeks, with additional boosters of 100 and 150 µg administered 1 and 2 days after the first booster dose, respectively. After the final booster, the splenic lymphoid cells of the mice were fused with the mouse myeloma cell P3x63-Ag8.653 (American Type Culture Collection). The fused cells were harvested in HAT medium supplemented with interleukin-6 for 4 weeks, after which the cells were harvested in HT medium.

Antibody production was assayed by IFMA using microtiter wells coated with rabbit anti-mouse immunoglobulin. Briefly, 25 µL of hybridoma supernatant and 200 µL of IFMA assay buffer (15) were incubated for 1 h in the well. After the wells were washed twice with wash solution in an automatic washer (DELFIA Platewash 1296-024), 50 ng of Eu3+-labeled PSA-ACT or PSA in 200 µL of assay buffer was added. After additional incubation for 30 min, the wells were washed four times, and 200 µL of enhancement solution (Wallac) was added to the wells. After 5 min, the fluorescence was measured with a Wallac 1420 Victor2 V fluorometer. MAbs were purified from culture fluid by affinity chromatography on a protein G column (Amersham Pharmacia Biotech AB).

The reactivities of the ACT antibodies with PSA-ACT and PSA-cathepsin G was determined with a sandwich assay. Cathepsin G-ACT (2.5 ng) or PSA-ACT (0.25 ng) were captured by polyclonal anti-cathepsin G antibody or anti-PSA MAb H117, respectively. After the wells were washed, 50 ng of each Eu3+-labeled anti-ACT MAb (1E2, 1D10, or 8E2) was added and the bound fluorescence was measured by time-resolved fluorometry.

time-resolved IFMAS for FPSA and the PSA-ACT complex
Calibrators.
Sera with high PSA concentrations were pooled and used as a calibrator (15). The concentrations of fPSA, PSA-ACT, and PSA-API were estimated by fractionating a serum sample with high PSA concentration (960 µg/L) by anion-exchange chromatography on a Resource Q column (Pharmacia Biotech AB). The tPSA concentration in the fractions was measured with the Prostatus PSA Free/Total Kit. Calibrators were prepared by diluting with pooled female serum. The concentration range of the calibrators was 0.8–120 µg/L calculated on the basis of total PSA.

Assay procedure.
In the dual-label assays for fPSA and PSA-ACT, calibrators or serum samples (25 µL) were added to wells coated with MAb H117, followed by 100 µL of assay buffer (Wallac). After incubation for 1 h at room temperature with shaking, the wells were washed twice, and 200 µL of assay buffer containing 50 ng of Eu3+-labeled antibody (5A10) was added to each well. After incubation for 1 h at room temperature, the wells were washed twice, and 200 µL of assay buffer containing 100 ng of Sm3+-labeled antibody (1D10) was added. After further incubation for 2 h and washing four times, enhancement solution (200 µL) was added to the wells. After the plate was shaken for 5 min at room temperature, the fluorescence was measured. The dual-label assays for fPSA plus tPSA and PSA-ACT plus tPSA were performed according to the Prostatus PSA Free/Total assay protocol (16) except that dilutions of the serum pool were used as calibrators. Eu3+-labeled (1D10) and Sm3+-labeled (H50) antibodies were used in the double-label assay of PSA-ACT plus tPSA. Briefly, calibrators or samples and assay buffer were added into the wells coated with capture MAb. After incubation for 1 h with shaking, the plate was washed and Eu3+- and Sm3+-labeled tracer MAbs in assay buffer were added together and allowed to incubate with shaking for 2 h. The plate was washed again, and enhancement solution was added and allowed to incubate for 5 min before fluorescence was measured. The earlier described double-label assay for PSA-ACT plus tPSA based on a polyclonal ACT antibody (15) was used for comparison.

Statistical analysis.
Pearson parametric correlation coefficients were used to describe the correlation between different assays. Differences in tPSA and the ratio of fPSA to PSA-ACT between various patients were calculated with the Mann–Whitney U-test. The validity of the diagnostic tests was analyzed by ROC curve analysis. Differences in specificity at various sensitivities were compared by the McNemar test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
development of MABS and IFMAS
We identified three MAbs (1D10, 8D2, and 1E2) that reacted more strongly with PSA-ACT than with free ACT, but not with fPSA or PSA-API. These MAbs bound to PSA-ACT in sandwich assays using as capture tPSA antibody MAb 5E4, 9C5, or 5C7. The cross-reactivity of Eu3+-labeled MAbs 1D10, 8D2, and 1E2 with native ACT captured by a polyclonal anti-ACT antibody was estimated to be 2.5%, 5%, and 25%, respectively, compared with that with PSA-ACT. The reactivities of these MAbs were further studied with cathepsin G-ACT captured on the solid phase by a polyclonal anti-cathepsin G antibody. Each MAb recognized the captured cathepsin G-ACT complex, but MAb 1D10 showed the lowest reactivity with cathepsin G-ACT (Fig. 1 ). The binding of MAbs 8D2 and 1E2 to cathepsin G-ACT was 2- to 2.3-fold that of 1D10. Because MAb 1D10 showed the lowest cross-reactivity with free ACT and cathepsin G-ACT, it was selected as a tracer in the PSA-ACT assay.



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  		Figure 1. Reactivity of anti-ACT MAbs with cathepsin G-ACT (CatG-ACT; {blacksquare}) and PSA-ACT () complexes.

calibration curves
In the serum pool used as a calibrator, the proportions of PSA-ACT, fPSA, and PSA-API determined in fractions separated by ion-exchange chromatography (12) were 88.5%, 10%, and 1.5%, respectively. These results were used to assign values to the calibrators. Fig. 2 shows calibration curves for the double-label assays for fPSA plus PSA-ACT and for PSA-ACT plus tPSA. The higher signal in the Eu3+-based compared with the Sm3+-based assays is explained by the stronger fluorescence of Eu3+.



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  		Figure 2. Calibration curves for the double-label assays for fPSA/PSA-ACT (A) and PSA-ACT/tPSA (B).

precision
Between-assay imprecision was studied by analyzing two serum pools with fPSA values of 1.01 and 1.67 µg/L and PSA-ACT values of 4.16 and 11.5 µg/L, respectively, in six successive analyses. Within-assay imprecision was measured on 12 replicates of the same samples. The intraassay and interassay CVs were ~0.4% and 6.1% for all assays. The imprecision for the ratio PSA-ACT/PSA measured by the dual-label assay was clearly better (<1%) than for the other assays (Table 1 ).


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  		Table 1. Precision and reproducibility of double-label assays.

detection limit and nonspecific background
The detection limit, defined as the concentration corresponding to the fluorescence signal of the zero calibrator + 2 SD (calculated from 28 replicates), was 0.05 µg/L for PSA-ACT and 0.004 µg/L for fPSA. In the dual-label assay for PSA-ACT and tPSA, the detection limits were 0.03 µg/L for PSA-ACT and 0.02 µg/L for tPSA, respectively.

With the PSA-ACT assays incorporating a polyclonal ACT antibody, the apparent mean value of PSA-ACT in 84 female sera was 0.12 µg/L (95% confidence interval, 0.10–0.15 µg/L), whereas the corresponding value for the assay using MAb 1D10 as a tracer was 0.02 µg/L (95% confidence interval, 0.01–0.04 µg/L), which is below the detection limit of the assay (none of 84 values exceeded the detection limit).

linearity and analytical recovery
Two serum samples containing 4.28 and 5.51 µg/L fPSA and 32.7 and 6.62 µg/L PSA-ACT, respectively, were diluted 2- to 64-fold with zero calibrator. The results showed good dilution linearity (data not shown). Three sera containing 4.3–18.3 µg/L PSA were diluted in a male serum containing 0.84 µg/L PSA and in a female serum with undetectable PSA, respectively. The mean recovery of fPSA was 93.3% (range, 80.6–106.5%), and that of PSA-ACT was 99% (97.5–102.8%).

correlation between assays
For fPSA, the correlation between the double-label assays for fPSA/PSA-ACT and fPSA/tPSA was y =1.15x - 0.13 (r = 0.98). The correlation between PSA-ACT concentrations measured by the fPSA/PSA-ACT and PSA-ACT/tPSA assays was: y = 1.03x + 0.15 (r = 0.99). The calculated concentrations of tPSA based on the sum of fPSA and PSA-ACT obtained with the fPSA/PSA-ACT assay and those measured by the fPSA/tPSA and PSA-ACT/tPSA assays also correlated well (y = 1.07x - 0.27; r = 0.99 and y = 1.07x - 0.29; r = 0.99). Bland–Altman plots did not reveal any systematic difference between the assays (data not shown).

differentiation between PCA and bph
To estimate the clinical validity of the assays, we analyzed sera from patients with and without PCa with PSA concentrations in the range 2.0–20.0 µg/L (Table 2 ). The ratio fPSA to PSA-ACT was significantly lower in samples from patients with PCa than in those with benign conditions (P = 0.0015).


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  		Table 2. Patient characteristics and distribution of the various marker values in PCa samples and controls.

The ability of the tests to differentiate between cancers and controls was calculated by ROC analysis. In all ranges of tPSA, the areas under the curves (AUC) for the ratio of fPSA to PSA-ACT were higher than those for either tPSA or PSA-ACT, and the difference was highly significant (P <0.001) in the range 2–20 µg/L (Fig. 3 ). There were no significant differences in AUC values between the proportions of fPSA/tPSA, PSA-ACT/tPSA, and fPSA/PSA-ACT (Table 3 ). Even the AUC values calculated on the basis of the sums of fPSA and PSA-ACT or the differences between tPSA and fPSA/PSA-ACT were quite similar to those measured directly. The AUC values for PSA-ACT alone or the calculated complexed PSA values (tPSA minus fPSA) were not significantly better than those for tPSA.



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  		Figure 3. ROC curves for individual analytes and ratios based on 35 PCa and 90 control samples with PSA concentrations of 2.0–20.0 µg/L.


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  		Table 3. AUC values of the different forms of PSA and their ratios in various tPSA ranges.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The first study on PSA-ACT in patients with prostatic disease showed that it is the major form of PSA immunoreactivity in serum and that the proportion of PSA-ACT is higher in PCa than in benign prostatic disease (4). PSA-ACT alone improved the discrimination between PCa and benign prostatic disease compared with tPSA, but the proportion of PSA-ACT provided the best discrimination. These results have been confirmed in several studies (11)(15)(21), but no improvement has been observed in other studies using different assays (22). This may be explained by differences in the extent to which various assays are affected by the background caused by nonspecific binding to the solid phase of a >10 000-fold excess of uncomplexed ACT and variable concentrations of ACT-protease complexes (23). The nonspecific background may be reduced by adding proteins and other substances (e.g., heparin) that reduce nonspecific binding (16). Another approach is to use a tracer antibody that does not recognize free ACT and ACT-protease complexes other than PSA-ACT, but previous attempts to use this approach have not been very successful (24). In the present study, we developed MAbs with improved specificity by careful selection of antibodies obtained by immunization with PSA-ACT. One antibody showed very low reactivity with free ACT, and it reacted less with the cathepsin-G-ACT complex than with PSA-ACT. As a result, the apparent PSA-ACT immunoreactivity in female sera was below the detection limit of the assay. This was achieved without additional proteins or heparin in the assay buffer. Thus, the antibody specificity is suitable for measurement of PSA-ACT in males.

Another problem for measurement of the proportion of PSA-ACT is assay imprecision when calculating the ratio of two concentrations of similar magnitude. We controlled imprecision caused by pipetting errors by use of a double-label assay design, which is possible with time-resolved immunofluorometry. We have used the same approach before with an assay that included a polyclonal tracer antibody to ACT (15). Although that assay was affected by a rather high nonspecific background, we achieved a substantial improvement in cancer specificity compared with that of tPSA. Thus, in the concentration range 4–20 µg/L, the AUC increased from 0.64 to 0.78, an improvement similar to that obtained in the present study. However, in the concentration range 2–4 µg/L, the assay did not improve cancer specificity, apparently because of limited assay sensitivity (0.16 µg/L) and a higher nonspecific background (15). With the monoclonal ACT antibody, the detection limit was 0.05 µg/L and the apparent concentrations in female sera were below the detection limit. The CVs were <4%; thus, the assay was useful at PSA values down to 2 µg/L. The ratio of PSA-ACT to tPSA also provided the same discrimination between cancers and controls as fPSA/tPSA in this range, which is becoming increasingly important because of the trend to lower the cutoff for tPSA as an indication to perform biopsy.

An extremely low CV (<1%) was obtained when we measured the PSA-ACT/tPSA ratio with a double-label assay. In the double-label assays for PSA-ACT/tPSA and fPSA/tPSA, the tracer antibodies were added together. However, in the double-label assay for fPSA and PSA-ACT, there was interference between the Sm3+-labeled MAb to complexed ACT and the Eu3+-labeled MAb to fPSA. If the two MAbs were added simultaneously or the Sm3+-labeled MAb to complexed ACT was added first and the fPSA antibody 1 h later, there was 10-fold increase in the background of the fPSA assay. This problem was eliminated when the Eu3+-labeled MAb to fPSA was added first and incubated for 1 h before addition of the Sm3+-labeled MAb to complexed ACT. The complicated double-label assay for fPSA/PSA-ACT probably explains why the precision of the ratio was not better than those of the individual components.

All the calculated ratios, i.e., fPSA/PSA-ACT, PSA-ACT/tPSA, and fPSA/tPSA, provided the same improvement in cancer specificity. This was the case in the whole material and in limited concentration ranges. This finding confirms that the assays and the ratio perform as theoretically expected.

An alternative approach for measurement of the proportion of fPSA or PSA-ACT in serum is to measure the two complexed forms of PSA, i.e., PSA-ACT and PSA-API, together in an assay based on blocking of the reactivity of fPSA with an antibody and measuring the remaining PSA immunoreactivity. The results of the first clinical study with the assay for the so-called complexed PSA suggested that it was as good as the proportion of fPSA (25), but this has not been confirmed in later studies (26)(27). Because complexed PSA comprises PSA-ACT, which increases, and PSA-API, which decreases in PCa, the PSA-ACT/tPSA ratio should theoretically provide the best discrimination between PCa and benign prostatic diseases of the indices studied. However, the contribution of PSA-API to total PSA is small, i.e., the median proportions being 0.9% in PCa and 1.6% in benign disease (28). Thus, the theoretical advantage of PSA-ACT is small and could probably be demonstrated only in a larger study.

In conclusion, we have developed an assay for PSA-ACT that avoids the background problems associated with earlier assays and combined this method with double-label assays for free or total PSA. These assays provided the same discrimination between PCa and BPH as a double-label assay for free and total PSA.


   Footnotes
 
1 Nonstandard abbreviations: PSA, prostate-specific antigen; PCa, prostate cancer; BPH, benign prostatic hyperplasia; ACT, {alpha}1-antichymotrypsin; tPSA and fPSA, total and free prostate-specific antigen; API, {alpha}1-protease inhibitor; IFMA, immunofluorometric assay; MAb, monoclonal antibody; and AUC, area under the curve.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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The following articles in journals at HighWire Press have cited this article:


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