The
kallikrein family is a group of 15
serine protease genes clustered on chromosome 19q13.4. Human
kallikrein (hK) gene 5 (KLK5) is a member of this family and encodes for a secreted
serine protease (hK5). KLK5 was shown to be differentially expressed at the
mRNA level in breast and
ovarian cancer. Until now, detection of hK5
protein in either
biological fluids or tissues has not been described due to lack of suitable
reagents and methods. The aim of this study was to develop immunological
reagents and a sensitive and specific fluorometric immunoassay (ELISA) for hK5, to examine the presence of hK5 in human tissues and
biological fluids, and to study the possible clinical utility of hK5 as a
biomarker for endocrine-related
malignancies. Recombinant hK5
protein was produced and purified using a Pichia pastoris yeast expression system. The
protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK5
antibodies. A sandwich-type microplate immunoassay (ELISA) was developed using these
antibodies, coupled with a time-resolved fluorometric detection technique. The ELISA assay was then used to measure hK5 in various
biological fluids,
tissue extracts, and serum samples from normal individuals and patients with various
malignancies. The hK5 ELISA immunoassay has a lower detection limit of 0.1 micro g/liter, is specific for hK5, and has no cross-reactivity with other homologous
kallikreins. The dynamic range is 0.1-25 micro g/liter, and within-run and between-run coefficients of variation within this range are <10%. hK5 is found in many tissues, with the highest expression levels seen in the skin, breast, salivary gland, and esophagus. hK5 is present at relatively high levels in milk of lactating women. Whereas the levels of hK5 are almost undetectable in serum of normal individuals (male and female) and patients with diverse
malignancies, higher concentrations were found in a proportion of patients with ovarian (69%) and breast (49%)
cancer. High levels were also detected in
ascites fluid from metastatic
ovarian cancer patients and in
ovarian cancer tissue extracts. In conclusion, we report development of the first immunofluorometric assay for hK5 and describe the distribution of hK5 in
biological fluids and
tissue extracts. Our preliminary data indicate that hK5 is a potential
biomarker in patients with ovarian and
breast cancer.