Human kallikrein 10 ELISA development and validation in breast cancer sera.

BACKGROUND: Human kallikrein 10 (hK10) is a putative secreted serine protease belonging to the same gene family as prostate specific antigen (hK3; PSA). There is significant interest in measuring hK10 which may act as a tumor suppressor in some cancers. We have developed an ELISA for hK10 and determined its analytical and clinical performance in normal and breast cancer sera. METHODS: The assay used a previously described pair of monoclonal anti-hK10 antibodies and recombinant hK10 protein. Serum hK10 was detected quantitatively in a 2-step sandwich ELISA with colorimetric detection. The assay was analytically validated and used to determine serum levels of hK10 in a set of breast cancer, benign breast disease and normal samples. RESULTS: The assay covered a linear range of 0.2 to 15 ng/mL and had a detection limit of 0.08 ng/mL. The within-run and between-run imprecision was <9%. The average spike and dilution linearity recoveries were 96 and 103% respectively. Mean hK10 concentration in normal female sera was 0.79+/-0.26 ng/mL. Concentrations were not age related and were not significantly different from benign fibrocystic disease or breast cancer. However, in a subset of breast cancer patients with both early and late stage disease, serum hK10 levels were elevated, at >1.55 ng/mL, above all normal female and benign disease samples. CONCLUSIONS: We report in detail the analytical performance of a colorimetric hK10 ELISA validated in human serum and report for the first time the hK10 serum concentration in benign and breast cancer samples.

Expression system for high levels of GAG lyase gene expression and study of the hepA upstream region in Flavobacterium heparinum.

A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.