Preparation Of Anti-hK5Antibodies And Establishment Of Sandwich ELISA Method

ObjectiveThe human kallikrein gene family has recently been discovered as a new cancerbiomarker which consists of15genes. Human kallikrein5(hK5, encoded by the KLK5gene) is a new member of the kallikrein gene family, which has been deeply researched.Results of previous studies have suggested that hK5abnormally express in varied cancers,and their difference expression is relative to the formation, development and metastasis ofcancers. In this study, we used recombination protein to prepare polyclonal antibodies(pAb) and monoclonal antibodies (mAb) against hK5. We aim to establish double antibodysandwich ELISA method to quantitative hK5in all kinds of biologic fluids. We thencalculated the diagnostic sensitivity and specificity of this test by detecting hK5concentration in serum of patients and healthy adult.Methods1．Human recombinant hK5expression plasmid was constructed and expressed inE.coli BL27. The recombination protein was expressed by inducing prokaryotic expressionvector with IPTG and further purified by Ni-NTA Spin Kit. The purified material was thenseparated by SDS-PAGE and stained with Coomassie Blue to detect its purity and itsmolecular mass. An anti-his antibody was used to monitor hK5production by Western blotanalysis.The concentration of the hK5was determined by the bicinchoninic acid method,which uses BSA as calibrator.2．The purified recombinant hK5protein was used as immunogen to developantibodies. We have generated anti-hK5polyclonal antibodies by immunizing rabbits withpurified hK5protein. We have also prepared anti-hK5monoclonal antibodies anddesignated hK5mAb by using hybridoma technology. The antiserum and ascites werefurther purified with Protein A IgG purification Kit. The specificity of hK5antibodies wascharacterized by Western blot and immunohistochemistry (IHC). 3．To quantitatively examine hK5in serum samples, we established double antibodysandwich ELISA method using mouse hK5mAb as capture and rabbit anti-hK5pAb astracer antibody. We have detected hK5concentration in many kinds of clinical serumsamples to ensure that our sandwich ELISA measurement with high sensitivity andspecificity.Results1．The prokaryotic expression vector induced by IPTG to express the recombinationprotein at28℃and the8thhour, the product was purified and analyzed. The resultsshowed that a new anticipated28kD protein band appeare on Western blot.2． Polyclonal antibodies were obtained by immunizing rabbits with purifiedrecombinant protein. High titers of specific antibodies were detected in serum of rabbitsafter the forth booster injection. And the titer of the antiserum reached1:106. We have alsogenerated monoclonal antibodies using hybridoma technology and the titre reached1:105.In western blot analysis, the band of the antibodies to the hK5protein appeared at28kD.By immunohistochemistry, the results showed that hK5protein expression is almost absentin the normal tissue but present in several tumor cytoplasm within the ovarian cancer andgastric cancer tissue.3．The detection limit of the hK5ELISA method，defined as the concentration of hK5can be distinguished, was from0.45ng/mL to125ng/mL. hK5was determined by ELISAin many kinds of tumor patients’ serum. The results showed that hK5is significantlyelevated in serum of ovarian cancer patients (n=40) compared with health female (n=40).The hK5serum concentration in ovarian cancer patients (3.77±1.47)ng/mL weresignificantly higher than those in healthy female individuals (0.86±0.45)ng/mL (P<0.01).In27of40ovarian cancer serum samples were positive. However, hK5concentration inserum of patients with colorectal cancer had depressed hK5levels (0.50±0.18)ng/mLcompared with nornormal adult (0.72±0.41)ng/mL.ConclusionsWe have successfully generated anti-hK5monoclonal and polyclonal antibodies. Wehave also developed a double antibody sandwich ELISA method by using mAb as captureand pAb as tracer antibody. Our data support the finding that hK5is elevated in mostserum of ovarian cancer patients and decreased in serum of patients with colorectal cancer.