| Interferon-γ(IFN-γ) is a pleiotropic cytokine, especially in cell-mediated immunity. The level of endogenous IFN-γis an important index to evaluate the cellular immune status. So, measurement of the production of this cytokine would be a good indicator of immunity. Also, the quantitative determination of IFN-γconcentrations is highly valued both in theory and practice, which can facilitate the studies on immune mechanism, immune function, vaccine evaluation, transplant operation,anaphylactoid reaction and intracellular pathogen diagnosis. My study focuses on the establishment of some detection methods to equine interferon-γ(EIFN-γ), such as sandwich ELISA, intracellular cytokine staining (ICS) assay and ELISPOT assay.Total RNA was extracted from the equine peripheral blood mononuclear cells (PBMCs) stimulated with ConA and used as the template. The full-length ofΕΙFN-γencoding gene was amplified by RT-PCR and cloned into the vector PCR2.1-TOPO. Then the gene encoding mature EIFN-γ(mEIFN-γ) was amplified from the recombinant plasmid named pCR-EIFN-γand sub-cloned into pET-28a (+). After sequencing conformation, the recombinant plasmid pET-mEIFN-γwas transformed into E. coli BL21 (DE3). Subsequently, the fusion protein was expressed with the induction of IPTG and purified with Ni2+NTA column. The antiviral activity of the recombinant mEIFN-γwas evaluated by using a Vesicular Stomatits Virus expressing GFP (rVSV-GFP) system in EFK-78 cells. The GFP expression level dramatically decreased in the EIFN-γtreated EFK-78 cells in a dose-dependent manner, comparing with the mock-treated cells. The titer of antiviral activity was 1×103 AU/mL.BALB/c mice (Mus musculus) were immunized intraperitoneally with purified recombinant mEIFN-γprotein. Murine myeloma cells were fused with the splenocytes originated from the immunized mice. An indirect ELISA with recombinant mEIFN-γderived from the baculovirus expression system as antigen was employed to screen antibody-producing hybridomas. After 3 cycles of subcloning, twelve hybridomas cells were obtained, which could produce McAbs steadily. Also, all twelve McAbs showed positive reaction to mEIFN-γin indirect fluorescence assay (IFA) tests. Among these twelve McAbs, four were IgG1, two were IgG2a, three were IgG2b and the rest three were IgM isotype respectively. Additionally, the light chains of all twelve McAbs wereκchains. Furthermore, seven McAbs showed positive reaction to GST-mEIFN-γ(1~84) protein, and the others showed positive reaction to GST-mEIFN-γ(80~146) protein in the specific ELISA tests. The results indicated these 12 McAbs could recognize two or more mEIFN-γepitopes.To measure the EIFN-γproduct from equine lymphocytes, a quantitative ELISA was developed in this study. The mAb A5 and biotinylated mAb SB10 were used as the capture and detection antibody respectively to establish the Sandwich ELISA for equine IFN-γ. The limit of detection for the sandwich ELISA of equine IFN-γwas 1 ng/mL and no cross-reactivity was observed with recombinant equine IL-18. Using the ELISA established in this study, equine IFN-γcan be detected and quantified easily from culture medium of the PBMCs after being stimulated with ConA or PMA/Ionomycin.In order to develop the ICS assay to detect the IFN-γin equine lymphocytes, purified McAb (SB10) was labeled with FITC. During the development of ICS, comparative trail with anti-bovine IFN-γMcAb (cc302) was carried side by side. All the results indicated that the FITC-conjugated monoclonal antibody SB10 could be used to detect equine IFN-γeffectively for ICS assay.The ELISPOT assay for equine IFN-γwas developed using the mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. After that, this ELISPOT assay was employed to detect the EIFN-γin PBMC stimulated with PMA/Ionomycin. The result showed the ELISPOT assay established in this study can be used to measure the EIFN-γsuccessfully.All these methods developed in this study, Sandwich ELISA, ICS, ELISPOT, can not only be used as the effective tools to monitor immune status of equines, but also facilitate the understanding of the immune responses to infection versus vaccination on equines and some related immune mechanisms.
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