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Standardization of Two Immunoassays for Human Glandular Kallikrein 2

Departments of
¹ Urology and
² Pathology, The James Buchanan Brady Urological Institute, The Johns Hopkins University Medical Institutions, Baltimore, MD 21287.
³ Hybritech Incorporated, a subsidiary of Beckman Coulter Inc, San Diego, CA 92822.

⁴ Department of Biotechnology, University of Turku, 20520 Turku, Finland.

⁵ Department of Clinical Chemistry, Lund University, 20502 Malmo, Sweden.

^aAddress correspondence to this author at: The Johns Hopkins University School of Medicine, James Buchanan Brady Urological Institute, 600 N. Wolfe St., Baltimore, MD 21287. Fax 410-614-8096; e-mail alexanderhaese{at}gmx.de.

Background: Measurement of human kallikrein 2 (hK2) has improved early detection and staging of prostate cancer. However, reported concentrations of hK2 among currently used assays have not been standardized in any way. We compared two hK2 assays and five different recombinant hK2 variants (rhK2) and suggest a common calibrator as an important step and putative reference substance in hK2 assay standardization.

Methods: We measured 146 sera by two hK2 assays, using assay-specific calibrators to assess the difference between the two assays. Serial dilutions of five rhK2 preparations were measured repeatedly, with one preparation assigned as calibrator and the others as unknowns to define which variant provided the closest match between the two assays. This rhK2 variant was used to recalibrate both assays. We measured hK2 concentrations in the same 146 patients to evaluate the change in the difference.

Results: Use of assay-specific calibrators for comparison of the two assays yielded a Deming regression equation of: y = 0.789 (95% confidence interval, 0.674–0.922)x + 0.014 (0.004–0.025) µg/L; R² = 0.667. Analysis of five rhK2 variants revealed that the enterokinase (ek)-rhK2 form provided the best match between both assays. Using the ek-rhK2 as a common calibrator, we observed a change in the slope of the regression curve to: y = 1.106 (0.872–1.340)x + 0.006 (-0.002 to 0.016) µg/L; R² = 0.648, suggesting an increase in the mean estimate of agreement between the two assays.

Conclusion: Calibration with a common calibrator substantially increased agreement between the assays. The ek-rhK2 variant provided the best performance of all tested rhK2 variants and should undergo mass spectrometry and amino acid analysis for exact mass determination and value assignment to evaluate its potential as a reference material for immunoassays for hK2.

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