Production and characterization of monoclonal antibodies against recombinant extracellular domain of CD99
| Autor: | Thangarajan Rajkumar, Krishna Priya Thangaretnam, Gopal Gopisetty, Oviya Revathi Paramasivam, Priya Ramanathan |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
medicine.drug_class Immunology Enzyme-Linked Immunosorbent Assay 12E7 Antigen Cross Reactions Monoclonal antibody law.invention Gene product 03 medical and health sciences Mice 0302 clinical medicine law Antibody Specificity medicine Immunology and Allergy Animals Humans Molecular Targeted Therapy Cloning Molecular RNA Small Interfering Mice Inbred BALB C Vaccines Synthetic Hybridomas biology Chemistry Antibodies Monoclonal Fast protein liquid chromatography General Medicine Isotype Molecular biology Transmembrane protein Recombinant Proteins Extracellular Matrix 030104 developmental biology 030220 oncology & carcinogenesis Gene Knockdown Techniques Recombinant DNA biology.protein Hybridoma technology Antibody |
| Zdroj: | Human antibodies. 27(1) |
| ISSN: | 1875-869X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|