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Technical Briefs |
Dept. of Chem., Cleveland
State Univ., Cleveland, OH 44115
1
Dept. of Pathol., MetroHealth Med. Center, Cleveland, OH 44109;
a author for
correspondence: fax 216-687-9298, e-mail y.xu{at}popmail.csuohio.edu
Serum prostate-specific antigen (PSA) has been recognized as a sensitive indicator of recurrent prostate cancer after radical prostatectomy (1)(2)(3)(4)(5). In the past 5 years, numerous PSA assays with improved limits of detection (6)(7)(8)(9)(10)(11)(12)(13)(14)(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®-E PSA assay (Hybritech, San Diego, CA). EIA coupled with electrochemical detection offers low limits of detection with high specificity (18)(19)(20)(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 +500 mV. The system had a custom-built injector with 1-µL sample loop. The carrier fluid (0.1 mol/L Tris, 1 mmol/L MgCl2, 65 mmol/L oxalic acid, and 0.2 g/L NaN3 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 µg/L) was prepared through serial dilutions of the PSA calibrators (2, 10, and 50 µg/L) with the zero diluent. The assay was carried out in duplicate as follows: (a) Pipette 100 µL of the zero diluent, calibrators, controls, and serum specimens, as well as 100 µL 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 °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 µL of 4 mmol/L p-aminophenyl phosphate (in 100 mmol/L Tris and 1 mmol/L MgCl2, pH 9.0) and incubate for 5 min at room temperature; (f) pipette 30 µL 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. 1
A). The calibration curve had a linear dynamic range from 0.02
to 1.0 µg/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 µg/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 µg/L, our assay lowered the LOD by
>37-fold.
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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® automated immunoassay
analyzer (Hybritech) was informative. We carried out a comparison study
with serum samples containing PSA ranging from 0.235 to 4.80 µg/L.
Because a lower LOD (0.008 µg/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 = 1.00).
In conclusion, the electrochemical EIA of PSA offers a much lower LOD (0.008 µg/L) and requires shorter enzyme reaction time (5 min) than those of the Tandem-E PSA assay (LOD 0.3 µg/L, enzyme reaction time 30 min). It has a potential for use in small-volume analysis (only 1 µL sample was injected for final detection), and its LOD may be further improved by increasing the enzyme reaction time.
Acknowledgments
This work was supported by a research grant from the American Association for Clinical Chemistry.
References
-fetoprotein in maternal serum by flow-injection amperometric detection of 4-aminophenol. Clin Chem 1990;36:1941-1944.
[Abstract]
The following articles in journals at HighWire Press have cited this article:
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  B. R. Grzeda, T. Le Bui, C. N. Warner, T. L. Pirucki, L. M. Dewey, M. Babich, and J. A. Maggiore Measurement of Prostate-specific Antigen by Use of a Novel Blood Collection and Analytical System Clin. Chem., August 1, 2002; 48(8): 1272 - 1278. [Abstract] [Full Text] [PDF]
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