Development of a dual monoclonal antibody immunoassay for total human kallikrein 2

Background: Human kallikrein 2 (hK2) shares 80% sequence identity with pros tate-specific antigen (PSA). Because both hK2 and hK2-alpha (1)-antichymotr ypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. Methods: We developed monoclonal antibodies (mAbs) with high specificity fo r hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was asses sed. Serum samples (n = 1035) from healthy volunteers, patients with increa sed PSA, and men who had undergone radical prostatectomy were assayed for t hK2. We also measured thK2 in samples before and after storage under common laboratory conditions. Results: The minimum detectable concentration in the thK2 assay was 0.008 m ug/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and t hree different hK2-serum protease complexes. The median serum concentration of thK2 in control samples (0.013 mug/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 mu g/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at -70 degreesC. Conclusions: The thK2 assay recognizes all forms of hK2 that have been foun d in bodily fluids to date. (C) 2001 American Association for Clinical Chem istry