Sensitive and specific immunodetection of human glandular kallikrein 2 in serum

Background: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunologica l cross-reactivity with prostate-specific antigen (PSA). Our objectives wer e to design an assay with improved analytical detection and functional sens itivity and nonsignificant cross-reactivity with PSA, and to characterize d ifferent immunoreactive forms of hK2. Methods: In the assay, critical PSA epitopes were blocked with four monoclo nal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured usin g a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA. Results: The analytical detection limit was <10 ng/L, and functional sensit ivity was 30 ng/L. Cross-reaction with PSA was <0.01%. Between-assay imprec ision was 3.1% for 1600 ng/L hK2 and 4.8% for 160 ng/L hK2; corresponding v alues for within-assay precision were 1.9% and 4.5%, respectively. Complexe s of hK2-alpha(1)-antichymotrypsin (ACT) were detected in vitro with -6% bi as compared with the free form of hK2. Gel filtration of patient samples sh owed that hK2 correlated in size mainly with free hK2; only 4-19% correspon ded to hK2 possibly complexed with ACT or protein C inhibitor. Conclusions: Our assay had extremely low cross-reactivity with PSA, provide d a very low detection limit, and allowed close to equimolar detection of t he free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum. (C) 2000 American Associa tion for Clinical Chemistry.