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Electrochemical enzyme immunoassay for serum prostate-specific antigen at low concentrations.

Serum prostate-specific antigen (PSA) has been recognized as a sensitive indicator of recurrent prostate cancer after radical prostatectomy [1-5]. In the past 5 years, numerous PSA assays with improved limits of detection [6-15] have been developed by both clinical researchers and diagnostic assay manufacturers. The rationale behind the development of more sensitive PSA assays (e.g., lower limits of detection) is that the relapse of prostate cancer or the tumor-doubling time after radical prostatectomy can be detected much earlier if patients are monitored with more sensitive assays [16, 17].

Here we report a rapid enzyme immunoassay (EIA) for serum PSA at low concentrations by flow-injection electrochemical detection, which is based on the modification of the Tandem[R]-E PSA assay (Hybritech, San Diego, CA). EIA coupled with electrochemical detection offers low limits of detection with high specificity [18-21]. Electrochemical EIA is usually based on the conversion of an electroinactive substrate to an electroactive product by the enzyme label. In this work, the enzyme was alkaline phosphatase (EC 3.1.3.1), which converted p-aminophenyl phosphate to p-aminophenol [22]. The concentration of p-aminophenol was then determined amperometrically in the flow-injection system.

We had illustrated the flow-injection electrochemical detection system used for this work elsewhere [21]. Basically, it was a BAS chromatograph (Bioanalytical Systems, West Lafayette, IN) without the separation column. The thin-layer electrochemical cell had dual glassy carbon working electrodes (in the parallel mode), a Ag/AgCl (3 mol/L NaCl) reference electrode, and a stainless-steel auxiliary electrode. The detection voltage between the working and reference electrode was set at 1500 mV. The system had a custom-built injector with 1-[micro]L sample loop. The carrier fluid (0.1 mol/L Tris, 1 mmol/L Mg[Cl.sub.2], 65 mmol/L oxalic acid, and 0.2 g/L Na[N.sub.3] at pH 3.2) was pumped at 0.2 mL/min.

In our assay procedure, Tandem-E PSA assay kit was used. A set of low-concentration PSA calibrators (0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 [micro]g/L) was prepared through serial dilutions of the PSA calibrators (2, 10, and 50 [micro]g/L) with the zero diluent. The assay was carried out in duplicate as follows: (a) Pipette 100 [micro]L of the zero diluent, calibrators, controls, and serum specimens, as well as 100 [micro]L of anti-PSA antibody-alkaline phosphatase conjugate, into each appropriately labeled test tube; (b) add one anti-PSA antibody-coated bead to each tube and vortex-mix the tubes; (c) shake the test tube rack gently in the water bath of the shaking incubator at room temperature (~23 [degrees]C) for 2 h; (d) aspirate the liquid from the tubes and wash the beads with the wash solution (4 x 1 mL); (e) place each bead into a fresh test tube containing 200 [micro]L of 4 mmol/L p-aminophenyl phosphate (in 100 mmol/L Tris and 1 mmol/L Mg[Cl.sub.2], pH 9.0) and incubate for 5 min at room temperature; (f) pipette 30 [micro]L of 0.5 mol/L oxalic acid into each tube to stop the enzyme reaction (oxalic acid lowers the pH from 9.0 to 3.2) and vortex-mix; and (g) draw the solution from each tube with a syringe and inject the sample into the flow-injection system, where the enzyme product, p-aminophenol, is detected by an amperometric detector.

With our electrochemical EIA procedure, a six-point calibration curve for serum PSA was constructed with oxidative currents of p-aminophenol vs PSA concentrations (Fig. 1A). The calibration curve had a linear dynamic range from 0.02 to 1.0 [micro]g/L with a correlation coefficient of 1.00 and CVs <5.5% over its range. The limit of detection (LOD) of the method was calculated to be 0.008 [micro]g/L, which was defined as the mean signal (n = 19) of the zero diluent + 2 SD. Compared with the Tandem-E PSA assay, which has a LOD of 0.3 [micro]g/L, our assay lowered the LOD by >37-fold.

[FIGURE 1 OMITTED]

For serum sample analysis, two PSA calibrators and two PSA controls (which were prepared by serial dilutions of the high-concentration PSA controls provided in the assay kit with the zero diluent) were used. Because there is no internationally accepted PSA reference standard available, we could not study the accuracy of our assay procedure. However, comparing the results of our assay with the Tandem-E PSA assay performed on the Photon ERA[R] automated immunoassay analyzer (Hybritech) was informative. We carried out a comparison study with serum samples containing PSA ranging from 0.235 to 4.80 [micro]g/L. Because a lower LOD (0.008 [micro]g/L) was obtained by our assay procedure, a 10-fold dilution of sample with the zero diluent was performed before the analysis. The results of our electrochemical EIA were plotted against the results of the Tandem-E PSA assay by a factor of 0.1 (Fig. 1B). On 16 patients' samples, a good correlation (r = 0.999) was obtained between these two assays, and no bias was observed (m 5 1.00).

In conclusion, the electrochemical EIA of PSA offers a much lower LOD (0.008 [micro]g/L) and requires shorter enzyme reaction time (5 min) than those of the Tandem-E PSA assay (LOD 0.3 [micro]g/L, enzyme reaction time 30 min). It has a potential for use in small-volume analysis (only 1 [micro]L sample was injected for final detection), and its LOD may be further improved by increasing the enzyme reaction time.

This work was supported by a research grant from the American Association for Clinical Chemistry.

References

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[4.] Stamey TA, Kabalin JN, McNeal JE, et al. Prostate specific antigen in the diagnosis and treatment of adenocarcinoma of the prostate. II. Radical prostatectomy treated patients. J Urol 1989;141:1088-90.

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[14.] Yu H, Diamandis EP, Prestigiacomo AF, Stamey TA. Ultrasensitive assay of prostate-specific antigen used for early detection of prostate cancer relapse and estimation of tumor-doubling time after radical prostatectomy. Clin Chem 1995;41:430-4.

[15.] Ferguson RA, Yu H, Kalyvas M, Zammit S, Diamandis EP. Ultrasensitive detection of prostate-specific antigen by a time-resolved immunofluorometric assay and the Immulite[R] immunochemiluminescent third-generation assay: potential applications in prostate and breast cancers. Clin Chem 1996;42:675-84.

[16.] Diamandis EP, Yu H, Melegos DN. Ultrasensitive prostate-specific antigen assays and their clinical application. Clin Chem 1996;42:853-7.

[17.] Vessella RL, Noteboom J, Lange PH, Rittenhouse H, Liu R, Kusnierz E. Ultrasensitive PSA immunoassays. How low do we need to go? J Urol 1993;149:484A.

[18.] Xu Y, Halsall HB, Heineman WR. Solid-phase electrochemical enzyme immunoassay with attomole detection limit by flow injection analysis. J Pharm Biomed Anal 1989;7:1301-11.

[19.] Xu Y, Halsall HB, Heineman WR. Heterogeneous enzyme immunoassay of a-fetoprotein in maternal serum by flow-injection amperometric detection of 4-aminophenol. Clin Chem 1990;36:1941-4.

[20.] Gil EP, Tang HT, Halsall HB, Heineman WR, Misiego AS. Competitive heterogeneous enzyme immunoassay for theophylline by flow-injection analysis with electrochemical detection of p-aminophenol. Clin Chem 1990; 36:662-5.

[21.] Yu Z, Xu Y, Ip MPC. An ultrasensitive electrochemical enzyme immunoassay for thyroid stimulating hormone in human serum. J Pharm Biomed Anal 1994;12:787-93.

[22.] Thompson RQ, Barone GC III, Halsall HB, Heineman WR. Comparison of methods for following alkaline phosphatase catalysis: spectrophotometric versus amperometric detection. Anal Biochem 1991;192:90-5.

Sung-Fang Chen, Yan Xu, * and Michael Po-Chee Ip (1)

(Dept. of Chem., Cleveland State Univ., Cleveland, OH 44115 and (1) Dept. of Pathol., MetroHealth Med. Center, Cleveland, OH 44109; *author for correspondence: fax 216-687-9298, e-mail y.xu@popmail.csuohio.edu)
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Title Annotation:Technical Briefs
Author:Chen, Sung-Fang; Xu, Yan; Ip, Michael Po-Chee
Publication:Clinical Chemistry
Date:Aug 1, 1997
Words:1580
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