Standardization of Two Immunoassays for Human Glandular Kallikrein 2

Abstract

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.

Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

“…Multicenter studies have shown that different total hK2 assays used to measure the exact same patient sample give significantly different results, leading to low correlation coefficients (57,58 ). Even small differences in the molecular structure (nicking, prosequences, or changes in amino acid composition) can affect the way hK2 is recognized by the capture or tracer antibodies (58 ). Therefore, the reaction patterns of different hK2 antibodies should be well characterized and care should be taken when comparing results obtained with different hK2 assays.…”

Standardization as a Private Enterprise

“…Although standards are available for most clinically important analytes, reference methods for immunoassays are available only for haptens (1 ). The study of Haese et al (2 ) in this issue aims at standardization of assays for human kallikrein 2 (hK2); it highlights many of the problems associated with standardization of sensitive immunoassays and with the use of recombinant proteins as standards.…”

“…On the tissue level, PSA expression decreases with increasing tumor grade (and aggressiveness), whereas hK2 expression is constant or less affected (3 ). Several studies indicate that this is reflected in an increased hK2/PSA ratio in serum, but this has not been observed in all studies (2 ). The somewhat conflicting results may be attributable to differences between the methods used or to insufficient analytical sensitivity of the assays.…”

“…Haese et al (2 ) have taken a pragmatic approach to the selection of a standard, i.e., they selected the one giving the best agreement when used to measure hK2 in serum by two different assays. This standard is not wild-type hK2, but a variant engineered to improve stability.…”

“…Some of the hK2 standards have been calibrated in mass units on the basis of amino acid composition and molecular weight, but the standard finally selected was calibrated by immunoassay using a PSA assay assumed to measure PSA and hK2 equally. The authors point out that this standard needs to be characterized by amino acid analysis and mass spectrometry (2 ). Because amino acid analysis reflects the molar content of protein, it would be advisable to start using substance concentrations (mol/L) for hK2 as has been done with the recently issued WHO standards for chorionic gonadotropin (7 ).…”