Affiliations: Department of Molecular Oncology, Cancer Institute (WIA), Chennai, Tamil Nadu, India
Correspondence:
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Corresponding authors: Gopal Gopisetty and Thangarajan Rajkumar, Department of Molecular Oncology, Cancer Institute (WIA), 38, Sardar Patel Road, Chennai 600 036, Tamil Nadu, India. Tel.: +91 4422350340; Fax: +91 4424912085; E-mail: [email protected]@gmail.com.
Abstract: BACKGROUND AND OBJECTIVE: CD99/MIC2 gene product is a heavily glycosylated transmembrane protein which plays a major role in homotypic cell adhesion, apoptosis of double positive T cells and vesicular protein trafficking. It is over expressed in various cancers and has been considered as an ideal therapeutic target. The present study focused at developing monoclonal antibodies against the extracellular domain (ECD) of CD99 using hybridoma technology. MATERIALS AND METHODS: In order to generate monoclonal antibodies, the recombinant ECD of CD99 was used for immunizing the mice. Resulting hybridomas were screened through indirect ELISA. Clones which gave high absorbance values were sub cloned by limiting dilution followed by isotype determination, IP, WB and FACS. The monoclonal antibody 547F2 4F12 was purified from culture supernatant using FPLC and further screened using IF. Finally, the antibodies were validated for specificity using siRNA knock-down. RESULTS: We were able to establish stable hybridoma clones secreting CD99 antibodies. The antibodies reacted with both the recombinant ECD as well as the wild type CD99 and their isotype’s were determined as IgM. CONCLUSION: Based on these results, we propose that the purified monoclonal antibody 547F2 4F12 could be possibly used for targeting tumors which over express CD99.
Keywords: CD99, recombinant protein, antibody purification and validation
