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Published on: March 8, 2012
[Preparation and characterization of monoclonal antibodies against chicken interferon-gamma]
Hua Dai1, Xin-an Jiao, Zhi-ming Pan
1Key Laboratory for Animal Infectious Disease, Ministry of Agriculture, Yangzhou University, Yangzhou 225009, China. daihua@yzu.edu.cn
Aim:
To prepare monoclonal antibodies (mAb) against chicken interferon-gamma (ChIFN-gamma).
Methods:
By using lymphocyte hybridoma technique, the inclusion body of the recombined bacteria, BL21(DE3) (pET-ChIFN-gamma), was harvested and used to immunize BALB/c mice. With the purified GST-ChIFN-gamma as detecting antigen, mAbs against ChIFN-gamma were prepared, and positive hybridoma clones were screened by indirect ELISA. The specificity of the mAb was characterized by indirect ELISA, Dot-ELISA and Western blot.
Results:
Two hybridoma cell lines secreting mAbs against ChIFN-gamma named 1G5, 5E3 were obtained. The immunoglobulin subclasses of both 2 mAbs were IgG2a, and the ELISA titers of 2 mAbs ascitic fluids were 1:160,000, 1:12,000 respectively. In Dot-ELISA test, the 2 mAbs could only react with BL21 (DE3) (pET-ChIFN-gamma), BL21 (pGEX-6P-1-ChIFN-gamma), which expressed His-ChIFN-gamma, GST-ChIFN-gamma, respectively. Western blot analysis confirmed that the 2 mAbs could only react with GST-ChIFN-gamma and His-ChIFN-gamma proteins.
Conclusion:
Two mAbs specific to the protein of chicken interferon gamma are obtained, which may have important application value in further studies on immune detection, the functions of immune cells and immune regulation.
