Preparation antibodies against recombinant bovine IFN-gamma and development of sandwich ELISA for bovine IFN-gamma detection.
- Author:
Chuan LI
1
;
Ya-Di TAN
;
Ying-Yut CHEN
;
Qiao-Yun HU
;
Zhang Gui-Rong YAN
;
Bo QIN
;
Yan-Jie CHAO
;
Huan-Chun CHEN
;
Ai-Zhen GUO
Author Information
1. The State Key Laboratory of Agricultural Microbiology, Wuhan 430070, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Animals, Newborn;
Antibodies, Monoclonal;
biosynthesis;
immunology;
Antibody Specificity;
immunology;
Blotting, Western;
Cattle;
Cells, Cultured;
DNA, Complementary;
genetics;
Enzyme-Linked Immunosorbent Assay;
methods;
Female;
Hybridomas;
Interferon-gamma;
genetics;
immunology;
metabolism;
Mice;
Mice, Inbred BALB C;
Plasmids;
genetics;
Rabbits;
Recombinant Proteins;
immunology;
metabolism;
Reverse Transcriptase Polymerase Chain Reaction
- From:
Chinese Journal of Biotechnology
2007;23(1):40-45
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to establish ELISA for recombinant bovine IFN-gamma (BovIFN-gamma) detection and provide a new method for diagnosis of pathogenic infection. The total RNA was isolated from peripheral blood leucocytes cultured with PHA mitogen stimulation. Then bovine IFN-gamma (BovIFN-gamma) gene cDNA was amplified by RT-PCR and cloned into pET28a to obtain the expression plasmid designated as pETBovIFN-gamma. The pETBovlFN-gamma was further transformed into competent E. coli BL21 cells and a 18kD His-tagged protein as expected was expressed after IPTG induction. By using purified recombinant BovIFN-gamma as antigen and lymphocyte-hybridoma technique, four hybridoma cell lines which stably secreted monoclonal antibodies against rBovIFN-gamma were generated, designated as A7, A10, G6, and G10. The immunoglobin subset was identified as IgG1 . Western-blotting analysis and ELISA demonstrated that the monoclonal antibodies secreted by all the four hybridoma cell lines could react specifically to the recombinant BovIFN-gamma, but not irrelative proteins such as Ag85B, ESAT-6-CFP-10 and GM-CSF, suggesting that the four hybridoma cell lines were rBovIFN-gamma specific monoclonal antibodies. A sandwich ELISA was established by using A10 secreted monoclonal antibody and rabbit polyclonal antibodies against BovIFN-gamma, HRP labeled goat anti-rabbit IgG. The results indicated that the sensitivity was 2ng/mL. This sandwich ELISA to detect BovIFN-gamma paved the way to develop a sensitive method for specific infection detection such as bovine tuberculosis diagnosis.
