Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications.
BACKGROUND
The human kallikrein gene family has contributed the best prostatic biomarkers currently available, including prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). Recently, new members of the human 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/protease M/neurosin. In this paper, we describe development of antibodies and a sensitive immunofluorometric procedure for hK6 protein.


METHODS
Recombinant hK6 protein was used as immunogen to develop polyclonal antibodies in rabbits and mice. These antibodies were used to develop a sandwich-type time-resolved immunofluorometric procedure for hK6.


RESULTS
The newly developed hK6 immunofluorometric assay has a detection limit of 0.5 microg/L and upper concentration range of 200 microg/L. The assay is highly specific (no detectable cross-reactivity from PSA and hK2) and was used to quantify hK6 protein in various biologic fluids. Highest concentrations of hK6 were found in milk of lactating women, cerebral spinal fluid, nipple 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 fraction of amniotic fluids and breast tumor cytosols. hK6 was not detectable in urine. Chromatographic studies indicated that hK6 is present in these biologic fluids in its free, 30-kDa form.


CONCLUSIONS
This is the first reported sensitive immunofluorometric procedure for quantifying hK6 protein. hK6 is a secreted proteolytic enzyme that is found at high levels in cerebrospinal fluid and all breast secretions. This assay will facilitate further studies to examine the possible application of hK6 in diagnostics, including cancer and neurodegenerative disorders.
Diamandis
E P
EP
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada. ediamandis@mtsinai.on.ca
Yousef
G M
GM
Soosaipillai
A R
AR
Grass
L
L
Porter
A
A
Little
S
S
Sotiropoulou
G
G
D000653:Amniotic Fluid / Q000201:enzymology; D000818:Animals; D000906:Antibodies; D015415:Biomarkers; D001826:Body Fluids / Q000201:enzymology; D001943:Breast Neoplasms / Q000201:enzymology; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005348:Fibrocystic Breast Disease / Q000201:enzymology; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D007610:Kallikreins; D008297:Male; D008895:Milk, Human / Q000201:enzymology; D005810:Multigene Family; D009363:Neoplasm Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism*; D014018:Tissue Distribution
D000906:Antibodies; D015415:Biomarkers; D009363:Neoplasm Proteins; C103551:KLK6 protein, human; D007610:Kallikreins; D012697:Serine Endopeptidases