Preparation Monoclonal Antibody Of Anti-Shiga Toxin ⅡB And Using For Immunoaffinity Chromatography

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, an emerging pathogen, causes severe hemorrhagic colitis and the life-threatening extraintestinal complication of hemolytic uremic syndrome (HUS) in 5 to 10% of patients. The source of EHEC O157:H7 is mainly of contaminated food or drinking water. Treatment of infection with EHEC O157:H7 has been difficult because antibiotics could induce bacteriolysis and then Shiga toxins release in intracellular.The Stxs consist of an A-subunit monomer and a B-subunit pentamer. The A subunit's N-glycosidase activity is activated, resulting in the removal of the adenine group from position 4324 in the eukaryotic 28S rRNA of the 60S ribosomal subunit. The resulting A subunit-mediated inhibition of protein biosynthesisis cytotoxic to the target cell. The B subunit is involved in binding to target cells via its glycolipid receptor, globotriaosylceramide (Gb₃-Cer). The Stxs includes two groups, Stx1 and Stx2. The Stx2, but not Stx1, is thought to be a major cause of toxity that induces HUS in humans based on epidemiological analyses and studies on EHEC-infected animals. So the Stx2 is the better antigen for preparing antibody treating the infection of EHEC O157:H7.Meanwhile, obtaining the natural Stx2 is the base about studying its toxicity mechanisms, screening the better target for producing vaccine, preparing specific gene engineering antibody and analyzing its crystal structure.In view of these, we constructed anti-Stx2B monoclonal antibody and use the antibody to purify the natural Stx2.1. Preparation and identification biological activity of anti-Stx2B McAbMethods The hybridoma cell lines that can secrete anti-Stx2B McAb with high affinity and specificity were established by hybridoma technique. Single cell lines which stably produce antibodies against Stx2B were picked out and identified by enzyme linked immunosorbent assay(ELISA). Injected them to abdominal cavity of Balb/c mice for ascites, and obtained a mount of McAbs from ascites. The McAbs was purified with Protein A-Sepharose from ascites respectively following precipitating by saturated ammonium sulfate. The Ig hypotype of McAbs is identified by Mouse Monoclonal Antibody Isotyping Kit. The affinity constant of McAbs was examined by non-competitive enzyme immuno-assay. Based on above studies, the ability of each Stx2B specific McAb to neutralize the effects of Stx2 was examined both in vitro with a Vero cell cytotoxicity assay. There is very significant difference between three McAbs and 1D₁₀(independent antibody)( p<0.01) and not significant difference between three McAbs and 1H10 is the best. Results Obtained three hybridoma cell lines named 1H9,1H10 and 3E5 respectively specificity against Stx2B, each Ig hypotype is IgG₁κ, IgG_2aκ, IgG_2bK, the affinity constant is 7.36×10⁸,6.94×10⁸, 5.85×10⁸. Neutralization of 20pg(induce 50% mortality in Vero cells) of Stx2 in vitro in the presence of 0.2-0.3μg of McAb was determined by the Vero cell cytotoxicity assay. Conclusions The three McAb all can neutralize the cytotoxicity of Stx2 and 1H10 is the best.2. Initial identification the epitope of anti- Stx2B McAbMethods The epitope of Stx2B(1-72aa) is predicted by using bioinformatics. The Stx2B is cut off at 23aa and 54aa. The genes of Stx2B50(23-72aa) and Stx2B54(1-54aa) were amplied from EHEC O157:H7 EDL933 genome, cloned into expression vector pET-22b(+) and the recombinant plasmids were transformed into Escherichia coli BL21(DE3), the productions were induced by the introduction of IPTG. Tris-Tricine-PAGE was used for the expression of proteins. The epitope domains were identified by Western blot analysis. Results In Western blot analysis, the Stx2B50 is discriminated by McAb 1H9 and 1H10; the Stx2B54 is discriminated by McAb 3E5, the Stx2B is discriminated by McAb 1H9, 1H10 and3E5. Conclusions The epitopes recognized by McAb 1H9, 1H10 are located in 55-72aa and The epitopes recognized by McAb 3E5 is located in 23-54aa.3. Purification the natural Stx2 by Immunoaffinity ChromatographyMethods The CNBr-activated Sepharose 4B matrix is coupled with the purified McAb 1H10. The culture of EHEC O157:H7 99A021 is induced by mitomycin C. And the culture supernatant is used as sample to be purified. Then the purified production is identified by many methods. Results SDS-PAGE of the purified protein have two protein strap: 32KD and 8KD and is identified as natural Stx2 by Western blot analysis, Duopath Verotoxins Kit, Vero cell toxicity test and animal toxicity test. The CD₅₀ for Vero cell is 2pg and LD₁₀₀ is 5ng per mouse. Conclusions The natural Stx2 is purified by Immunoaffinity Chromatography . In a word, we obtain the McAb could neutralize the toxicity of Stx2 and Initial identify its epitope, then establish Immunoaffinity Chromatography and purify the natural Stx2. So all of these could make substantial foundation for the following study about the Stx2.