Preparation Of Monoclonal Antibodies Against B Subunit Of Shiga-like Toxin Ⅱ Variant

Edema disease(ED) of weaning piglets is mainly caused by verotoxigenic Escherichia coli (VTEC) which lead to tetraplegia,titubation,spasm and coma.It's named "Edema disease" because edema of the stomach submucosa and the mesentery is often a prominent feature of the disease.Following the reseachs of these years,people become grasp the pathogenic mechanism of edema disease.F18ab fimbria and shiga-like toxinâ…¡variant(SLT-â…¡e) of VTEC were two pathogenic factors to weaning pigs.SLT-â…¡e is the most important virulent factor.In this study,the SLT-â…¡e B gene was amplified by PCR and cloned into pET-30a vector after sequencing and comparing with that of reference strains.The recombinant plasmid was transformed into BL₂₁(DE₃) and induced to express by IPTG.The purified protein was used to immunize the BALB/c mice.SP2/0 cells were fused with spleen cells from BALB/c mice immunized.The supernatants of hybridoma cell were selected by indirect ELISA and the selected cells were subcloned four times by limiting dilution.Six hybridoma series secreting monoclonal antibodies(McAbs) against SLT-â…¡e B were obtained and named 2B₁₀,3A₈,3C₁₁,3D₇,3G₇ and 4F₃,respectively.The subtypes of the McAbs were IgG2a,IgG2b,IgA and IgM,and all McAbs belonged toκchain.The results of analyzing chromosome number displayed that the chromosome numbers of six hybridoma cell were from 90 to 115.The results of ELISA indicated that the titers of six hybridoma cell's supematants were from 1:128 to 1:512.The titers of hydroperitoneum prepared by hybridoma cells 3G₇ and 4F₃ were 1:10⁵.Blocking ELISA confirmed that all McAbs were able to reacted with native SLT-â…¡e and not able to reacted with native LT and ST.The results of addition ELISA displayed that McAbs 3G₇ and 4F₃ was not to the same epitope.