Preparation Of Monoclonal Antibodies Against Recombinant Water Buffalo IFN-γ And Development Of DAS-ELISA For Bovine Tuberculosis Diagnosis

Bovine tuberculosis(TB) is a chronic bacterial disease of animals and humans caused by Mycobacterium bovis.The disease has a worldwide distribution.In many countries bovine TB is still a major infectious disease among domestic animals and the wildlife population,and transmission to humans constitutes a public health problem.Microbiological diagnosis of M.bovis is an extremely slow procedure which may take as long as 5 to 8 weeks,the sensitivity of culture is not 100%,and false-negative culture results may occur.The tuberculin skin test(TST) is the official test used in the eradication campaign for the detection of infected animals in the field,but the PPD can't distinguish between M.bovis infection and BCG vaccination.,it isn't a good BTB diagnosis method for water buffalo,goat and other wildlife.,The interferon gamma(IFN-g) assay is a recently developed whole-blood in vitro assay.Although early versions of IFN-γassays used purified protein derivative(PPD) as the stimulating antigen,PPD contains many mycobacterial antigens,some of which are shared among pathogenic mycobacteria belonging to the M.tuberculosis complex,environmental nontuberculous mycobacteria (NTM),and the vaccine substrain M.bovis BCG.Therefore,specific antigens expressed by M.bovis but absent from BCG constitute prime candidates for differential diagnostic reagents.Recently,two such antigens,ESAT-6 and CFP-10,have been reported to be promising candidates as diagnostic reagents for the detection of M.bovis infection in cattle.Using this peptide cocktail in T-cell assays,M.bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond.This study was aimed to develop McAbs against recombinant Water Buffalo IFN-γand established a sandwich indirect ELISA to detect water buffalo IFN-γ.The results of research are summarized as following:1.Total RNA was isolated from water buffalo peripheral blood lymphocytes stimulated with ConA.The water buffalo interferon-γ(WBIFN-γ) cDNA was amplified by RT-PCR,and then cloned and sequenced.The ORF of the cloned water buffalo IFN-γgene was 501bp in length,encoding a polypeptide with 166 amino acid.Compared with other IFN-γ,the nucleotide homologies between the water buffalo IFN-γgene and the cattle IFN-γgenes were higher than 97%,the amino acid homologies were higher than 96%.The Water Buffalo mature peptide IFN-γwas subcloned and constructed into the expression plasmid of pGEX-WBIFN-γand PET-WBIFN-γ,and then transformed into E.coli BL21(DE3),the rGST-WBIFN-γprotein and rHIS-WBIFN-γprotein was highly expressed under IPTG.The SDS-PAGE results showed that the predicted fusion protein rGST-WBIFN-γwas 43Ku in molecular weight with 30%of bacterial whole proteins,the fusion protein rHIS-WBIFN-γwas 37.4 Ku in molecular weight with 45%of bacterial whole proteins,the two existed in soluble portion and inclusion bodies.The soluble fusion protein was purified using GST and HIS affinity columns respectively.Western-blotting and ELISA analysis were employed to determine the expression,purification and activity identification of the expected protein and indicated that the two recombinant protein possess good immunological activity.2.Balb/c mice(Mus musculus) were immunized four times byrecombinant buffalo interferon-gamma(rGST-WBIFN-γ) expressed in E.coli BL21(DE3) at a dose of 100μg per mice.Murine myeloma sp2/0 cells were fused with splenocytes from the immunized mice. An indirect ELISA using recombinant protein rHIS-WBIFN-γand rGST-WBIFN-γas coating antigens were used to screen antibody-producing hybridomas.Two hybridomas cell stains could produce monoclonal antibody(McAbs) steadily after 3 cycles of subcloning.The titer of the two rWBIFN-γMcAbs in the culture Supernatant are 1:3200, the titer of the two rWBIFN-γMcAbs in the ascite are 1:819200.Both McAbs blonging to IgG1 isotype andκchain,showed positive reaction to rHIS-WBIFN-γin western blotting.3.The gene encoding protein CFP10-ESAT-6 was amplified from mycobacterium tuberculosis AN5 chromosomal DNA by PCR.The PCR product was cloned into the expression plasmid PET-32a to generate the recombinant plasmid PET-CFP10/ESAT-6.The recombinant expression plasmid was transformed into E.coli BL21(DE3).The fused protein CFP10/ESAT-6 with His-tag was expressed after inducing with IPTG.The SDS-PAGE results showed that the predicted fusion protein rHIS-CFP10/ESAT-6 was 42Ku in molecular weight with 40%of bacterial whole proteins.The soluble protein was purified with HIS affinity chromatography.Western-blotting analysis was employed to determine the activity identification of the expected protein and indicated that the recombinant protein possess good immunological activity.The protein was used to stimulate the whole blood to generate high level IFN-γ,the result showed that the IFN-γresponses to rHIS-CFP10/ESAT-6 is better than PPDa and PPDb.4.Double antibody Sandwich ELISA for IFN-γin water buffalo was developed.Using McAbs 1E4 as a capture antibody and HRP-labeled McAbs 4A11 as a detection antibody. The optimal reaction conditions for the assay were determined.The result indicated that the optimal concentration of capture antibody is 625ng/mL,the working concentration of HRP-labeled 4A11 was 1:100,the threshold for serum sample was 0.3,the detection limit of the sandwich ELISA for IFN-γwas 25ng/mL.The diagnosis specificity,sensitivity and coincidence test of DAS-ELISA compared with TST is 81.81%,71.05%,75%,respectivity. Therefore,we propose this DAS-ELISA assay will be a useful tool in the bovine tuberculosis diagnosis in water buffalo.