Preparation antibodies against recombinant bovine IFN-gamma and development of sandwich ELISA for bovine IFN-gamma detection / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 40-45, 2007.
Artigo
em Chinês
| WPRIM
| ID: wpr-325421
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Plasmídeos
/
Proteínas Recombinantes
/
Ensaio de Imunoadsorção Enzimática
/
Células Cultivadas
/
Western Blotting
/
Interferon gama
/
DNA Complementar
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Alergia e Imunologia
/
Genética
Tipo de estudo:
Estudo diagnóstico
Limite:
Animais
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2007
Tipo de documento:
Artigo
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