ABSTRACT
Objective To develop a PCR-ELISA assay for the rapid, specific and sensitive detec-tion of human seasonal influenza virus ( H1, H3 and B) by using molecular biological and immunological methods in combination.Methods The primers were designed according to the genes encoding the matrix protein ( M) , the H1 and H3 hemagglutinin ( HA) of influenza A virus and the nonstructural proteins ( B-NS) of influenza B virus and then were labeled with biotin.The PCR products were detected by ELISA by use of an internal catching probe labeled with DIG.Results The minimum copy numbers of genes encoding the M, H1, H3 and B-NS proteins detected by the established assay were 1.43?103 , 8.67?102 , 3.86?103 and 5.45?103 copies/μl, respectively, which indicated that the PCR-ELISA assay was about 10 times more sensitive than agarose gel electrophoresis in the detection of PCR products.No cross-reactions between the different subtypes of influenza virus or different species of virus were observed.Moreover, a total of 104 clin-ical specimens of influenza virus were examined by the PCR-ELISA assay, the results of which were consist-ent with those of the virus isolation method.Conclusion The newly developed PCR-ELISA assay was a highly sensitive and specific method for the rapid detection and subtyping of influenza virus, suggesting the possibility of using it in laboratory for the surveillance and detection of influenza virus.
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Objective To establish a double-antibody sandwich ELISA for the rapid detection of shiga toxin typeⅡ ( StxⅡ) in shiga toxin-producing Escherichia coli ( STEC) infection. Methods A pool of murine hybridomas was used to screen out the optimal antibody pair for the establishment of double-anti-body sandwich ELISA. The established ELISA system was used to detect StxⅡin the culture supernatants of 16 clinical strains of STEC. Specificity and sensitivity of the established ELISA system were also evaluated. Results Two antibodies, S2D8 and S2C6, were successfully screened out, based on which the double-anti-body sandwich ELISA was set up. StxⅡand its variants rather than StxⅠwas detected in the culture super-natants of STEC with a lowest detection limit of 4 ng/ml. Its performance was consistent with that of commer-cial colloidal gold test kit, indicating the characteristics of good specificity and sensitivity. Conclusion The S2D8/S2C6-based ELISA laid a foundation for researches which designates the shiga toxin as a potential can-didate on the diagnosis and therapy of STEC infection.
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One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like HIN1, NS from classical swine H1NI, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.
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Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production.The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay.The isotype and specificity of resultant clone was determined,and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model.Lastly,its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model.It was demonstrated that one of the 12 positive MAb clones against Stx2,designating $2C4 had neutralizing activity.S2C4 belongs to the immunoglobulin G1 subclass and has a K light chain,and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1.S2CA could efficiently neutralize the cytotoxicity of Stx2 to Veto cells and mice.It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha.S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx-producing Escherichia coli(STEC)infection.
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Objective To purify Shiga toxin Ⅱ (STX Ⅱ) of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX Ⅱ A subunit-specific antibody S1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157:H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose (CD50) for Vero cell line and 100% lethal dose (LD100) for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157:H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32 000 and 7 500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively.The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5 ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.
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Objective To prepare and study the immunogenicity of hepatitis B virus surface anti-gen (HBsAg)-tetanus toxoid (TT) conjugate vaccine. Methods Tr was activated by cyangen bromide and reacted with adipic acid dihydrazide, then HBsAg-TT conjugate was prepared by carbediimide. Conjugate, HBsAg or hepatitis B vaccine was injected subcutaneously into mice. Anti-HBsAg and HBsAg-specific T cell response elicited by these immunogens were assayed. Results New HBsAg-TT conjugate elicited higher levels of anti-HBsAg and HBsAg positive conversion rates after the immunization than did HBsAg alone or hepatitis B vaccine. Conjugate induced mesdy antibodies of the IgG2a subclass, while HBsAg alone or hepa-titis B vaccine mainly elicited anti-HBsAg in the IgG1 subclass. The number of IFN-γand IL-2 secreting T cells induced by conjugate was also significantly higher than that did by HBsAg or hepatitis B vaccine. Con-clusion This study indicated new HBsAg-TT conjugate can induce both stronger humoral and TH1 type of cellular immune response.