Development and evaluation of an ELISA method for the measurement of kallikrein-related peptidase 5 (KLK5) in human serum

Abstract

Kallikrein-related peptidases (KLKs) have been proposed as potential cancer biomarkers. Kallikreinrelated peptidase 5 (KLK5) is a secreted trypsin-like protease of the KLKs. Until now, detection of KLK5 in both biological fluids and tissues has been described frequently due to the potential of being a new cancer biomarker. Our objective was to prepare KLK5 antibodies and establish an ELISA method for KLK5 to study the possible clinical application of KLK5 as a biomarker for malignancies. In this study, recombinant KLK5 protein was produced and purified using a prokaryotic expression system, and then used as immunogen to generate antibodies. High titers of specific antibodies were measured in serum of rabbits after the forth booster injection. And the titer of the antiserum reached 1:10 6 . We have also generated monoclonal antibodies using hybridoma technology and the titer reached 1:10 5 . The activity of KLK5 antibodies was characterized by Western blot and immunohistochemistry. To quantitatively examine KLK5 in serum samples, we established double antibody sandwich ELISA method using mouse mAb as capture and rabbit pAb as tracer antibody. We have detected KLK5 levels in ovarian cancer serum to ensure that our sandwich ELISA measurement to have high sensitivity and specificity. The ranges of linearity reached by the standard curves of the newly developed ELISA were 0.45 ng/mL to 125 ng/mL. The detection limit of the method, defined as the concentration of KLK5 can be distinguished, was 0.20 ng/mL. Median serum KLK5 levels were 3.77 ng/mL and 0.86 ng/mL in ovarian cancer patients and normal female, respectively (P < 0.001). In conclusion, we prepare anti-KLK5 antibodies and develop the sandwich-type ELISA assay for KLK5. Our preliminary findings prompt that KLK5 may be a new potential biomarker for the diagnosis and prognosis in patients with ovarian.

Identification and quantification of serum KIN17 protein based on ELISA assay and exploring its clinical diagnostic value in liver cancer

“…Protein expression was detected by treatment with an FDbio-Dura ECL kit (Hangzhou Fude Biological Technology Co., Ltd) with a Syngene Protein Luminescence Imaging System (GeneGnome5-75000, SYNGENE). 17–31…”

“…Protein expression was detected by treatment with an FDbio-Dura ECL kit (Hangzhou Fude Biological Technology Co., Ltd) with a Syngene Protein Luminescence Imaging System (GeneGnome5-75000, SYNGENE). [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] Enzyme-linked immunosorbent assay (ELISA) for KIN17 detection…”

Upregulation of kallikrein-related peptidase 5 is associated with the malignant behavior of colorectal cancer

“…Subsequently, tissue sections were incubated with 200 µl KLK5 primary antibody at a dilution of 1:400 in phosphate-buffered saline (PBS) at 4˚C overnight. This KLK5 rabbit polyclonal antibody was generated in our laboratory using recombinant KLK5 protein (22). Immunohistochemical staining of antibodies was performed using the Dako K5007 Envision Plus System (Dako, Glostrup, Denmark).…”

“…A monoclonal anti-KLK5 antibody generated in our laboratory (22) was used to coat 96-well white polystyrene plates in PBS (750 ng/well) overnight at 4˚C. Subsequently, the plates were washed three times with PBS plus 0.5% Tween 20 (PBS-T), and diluted serum samples or calibrators (100 µl/well; Sino Biological, Inc., Beijing, China) were added to each well and incubated at 37˚C for 1 h. The plate was then washed a further six times with PBS-T.…”