Development of an immunofluorometric assay for human kallikrein 15 (KLK15) and identification of KLK15 in tissues and biological fluids.
BACKGROUND
Human kallikrein 15 (KLK15) may have some utility as a prostate, ovarian, and breast cancer biomarker, based on previous studies, which examined mRNA levels of KLK15. The aim of this study was to develop analytical technology for human kallikrein 15, including recombinant protein, specific antibodies, and a sensitive and specific ELISA immunoassay. The assay was then used to examine levels of KLK15 in tissues and biological fluids.


METHODS
We produced human, recombinant pro-KLK15 in HEK 293 cells. Recombinant KLK15 was purified with various chromatographic steps and used to immunize rabbits and mice for production of KLK15 polyclonal antibodies. We used these antibodies to develop a highly sensitive and specific KLK15 immunoassay and to study KLK15 expression in various tissues and biological fluids.


RESULTS
Large amounts of pure, recombinant KLK15 have been produced and characterized. KLK15 mouse and rabbit polyclonal antibodies have been employed for development of a KLK15 immunoassay. This assay has a lower detection limit of 0.05 microg/L, and no cross-reactivity with any of the other fourteen kallikreins. Using this assay, KLK15 was detected in prostate, colon, and thyroid tissues, as well as in breast milk and seminal plasma.


CONCLUSIONS
The KLK15 reagents developed here will allow for analysis of KLK15 protein expression levels in tissues and biological fluids, both normal and cancerous. This will expand upon previously characterized tissue KLK15 mRNA expression studies which suggested that KLK15 might be useful as a biomarker for breast, ovarian, and prostate cancer. KLK15 is another serine protease that is produced in prostate and other tissues and is secreted in seminal plasma and other fluids. Its physiological function needs to be further elucidated.
Shaw
Julie L V
JL
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5.
Grass
Linda
L
Sotiropoulou
Georgia
G
Diamandis
Eleftherios P
EP
D000595:Amino Acid Sequence; D000653:Amniotic Fluid / Q000737:chemistry; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D001826:Body Fluids / Q000737:chemistry*; D002138:Calibration; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015200:Fluoroimmunoassay / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007610:Kallikreins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D013058:Mass Spectrometry; D008895:Milk, Human / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D012661:Semen / Q000737:chemistry
D000906:Antibodies; D010455:Peptides; D011994:Recombinant Proteins; C501608:KLK15 protein, human; D007610:Kallikreins