Abstract
von Willebrand factor-cleaving protease/ADAMTS13 is a plasma metalloproteinase that degrades unusually large von Willebrand factor multimers (UL-vWF) derived from endothelial cells (ECs) and megakaryocytes (MCs) into small peptides circulating in blood. It is documented that transcript mRNAs of the protease are present in many human tissues; however, the protein expression of ADAMTS13 remains to be elucidated. In the present work, the gene of metalloproteinase domain of human ADAMTS13 was cloned into the multiclone site of pET28a(+). After induced by IPTG, the recombinant protein was purified using a Ni-NTA column and the Bal b/c mice were immunized with the protein. Screened with ELISA, three monoclonal antibodies against the metalloproteinase domain of ADAMTS13 were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belonged to IgG1 subclass. The quantity of them in ascites were 4 mg/ml, and their titers were as high as 1×10−5. The data of competitive ELISA showed that SZ-112 and SZ-113 recognized different epitopes of the recombinant protein. Western blot results demonstrated that SZ-112 not only reacted against the recombinant protein, but also recognized the full-length recombinant ADAMTS13 protein that expressed in CHO cell line (the vectors containing the ADAMTS13 cDNA sequences were provided by Prof. Sadler JE). The immunoprecipitation results showed that the two antibodies could react to an approximately 200 KDa protein in platelet lysate. Then, the expression panels of ADAMTS13 in human normal tissues were investigated using immunohistochemistry with the monoclonal antibodies. And the protease was found to be present in many kinds of tissues such as liver, spleen, ovary, prostate, bladder, small intestine, thyroid and thymus with significantly positive staining. The protease was also present in lung, uterus, large intestine and heart but stained weakly. We did not found the protease in brain. In most of these organs, the protease was expressed in epithelium of the tissues. While in liver, spleen and thymus, it was mainly presented in a subgroup of the solid tissue cells. Moreover, the preliminary results showed that the expression of ADAMTS13 slightly decreased in liver tissues of patients suffering form hepatitis type B and cirrhosis. In conclusion, our data indicated that two novel monoclonal antibodies against the metalloproteinase domain of human ADAMTS13 were successfully prepared, and the expression of ADAMTS13 in different tissue and specific locality might be associated with the regulation and function of the protease, which would contribute to the further research of the deficiency mechanism of the proteases in some disorders.
Production and characterization of osteoclast-selective monoclonal antibodies that distinguish between multinucleated cells derived from different human tissues.