ABSTRACT
OBJECTIVE: To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2. METHODS: The coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2. RESULTS: PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP). CONCLUSION: The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.
Subject(s)
Breast Neoplasms/genetics , Inhibitor of Differentiation Protein 2/biosynthesis , Recombinant Proteins/biosynthesis , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Humans , Immune Sera/biosynthesis , Inhibitor of Differentiation Protein 2/genetics , Inhibitor of Differentiation Protein 2/immunology , Rabbits , Recombinant Proteins/geneticsABSTRACT
AIM: To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. METHODS: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. RESULTS: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. CONCLUSION: This method can be used to help understand the role of this cytokine in various equine diseases and develop specific cell-mediated immunity assay.
