[Preparation antibodies against recombinant bovine IFN-gamma and development of sandwich ELISA for bovine IFN-gamma detection].
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.
Li
Chuan
C
The State Key Laboratory of Agricultural Microbiology, Wuhan 430070, China.
Tan
Ya-Di
YD
Chen
Ying-Yut
YY
Hu
Qiao-Yun
QY
Yan
Zhang Gui-Rong
ZG
Qin
Bo
B
Chao
Yan-Jie
YJ
Chen
Huan-Chun
HC
Guo
Ai-Zhen
AZ
D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006825:Hybridomas; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011994:Recombinant Proteins; D007371:Interferon-gamma