Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications

Background: The human kallikrein gene family has contributed the best prost atic biomarkers currently available, including prostate-specific antigen (P SA) and human glandular kallikrein 2 (hK2). Recently, new members of the hu man kallikrein gene family have been identified. One new member is the KLK6 gene, encoding for human kallikrein 6 (hK6), which is also known as zyme/p rotease M/neurosin. In this paper, we describe development of antibodies an d a sensitive immunofluorometric procedure for hK6 protein. Methods: Recombinant hK6 protein was used as immunogen to develop polyclona l antibodies in rabbits and mice. These antibodies were used to develop a s andwich-type time-resolved immunofluorometric procedure for hK6. Results: The newly developed hK6 immunofluorometric assay has a detection l imit of 0.5 mu g/L and upper concentration range of 200 mu g/L. The assay i s highly specific (no detectable crossreactivity from PSA and hK2) and was used to quantify hK6 protein in various biologic fluids. Highest concentrat ions of hK6 were found in milk of lactating women, cerebral spinal fluid, n ipple aspirate fluid, and breast cyst fluid. hK6 was also detected in male and female serum, in the majority of seminal plasmas and in a small fractio n of amniotic fluids and breast tumor cytosols. hK6 was not detectable in u rine. Chromatographic studies indicated that hK6 is present in these biolog ic fluids in its free, 30-kDa form. Conclusions: This is the first reported sensitive immunofluorometric proced ure for quantifying hK6 protein. hK6 is a secreted proteolytic enzyme that is found at high levels in cerebrospinal fluid and all breast secretions. T his assay will facilitate further studies to examine the possible applicati on of hK6 in diagnostics, including cancer and neurodegenerative disorders. Copyright (C) 2000 The Canadian Society of Clinical Chemists.