Development Of Double Antidody Sandwich ELISA For Shiga Toxin Type ? And ? In Shiga Toxin-producing E.Coli

Shiga toxin-producing E.coli(STEC)infection in humans can lead to hemorrhagic colitis(HC),diarrheaand other gastrointestinal diseases.These diseases are often complex and produce potentially fatal systemic sequelae,such as nervous system damage andhemolytic uremic syndrome(HUS).STEC can secrete two kinds of shiga toxin(Stx),Stx? and Stx?.The production of Stx in the intestinal lumen is closely related to the gastrointestinal diseases.A small portion of toxinenters the blood circulation,and causes vascular tissue damage in brain and kidney target organs.Once the STEC infection outbreaks,it is difficult to contain,since the epidemics can cause a high morbility and lethality in humans.So,STEC infection has became a worldwide public health issue.Therefore,it is urgent to develop a rapid and accurate method to detect toxins from the blood of patients,to facilitate clinical diagnosis and treatment.In this study,a kind of unique double-antibody sandwich enzyme-linked immunosorbent assay(ELISA)has been set up to detect Shiga toxin type ?(Stx?)and shiga toxin type ?(Stx?),respectively.For Stx?,recombinant Shiga toxin type ? A subunit(Stx?A)was used to immunize mice to develop monoclonal antibodies(MAbs),and two un-competitive MAbs were selected out to set up double-antibody sandwich ELISA.This system was further evaluated by detecting Stx? from supernatant of STEC culture.Four strains of hybridomas which can secrete MAbs against StxIA,namely,1H2-G7,2H1-C12,8E7-E6 and 2F6-F8,respectively,were obtained.Of them,only the combination including 8E7-E6 as coating antibody,and 2F6-F8 as detection antibody gives the highest OD value in double-antibody sandwich ELISA system.In its application,this system can detect Stx?in all Stx?-positive samples,rather than Stx?.For Stx?,A pool of murine hybridomas was used to select the antibody combination to compose double-antibody sandwich ELISA.Its specificity and sensitivity were also evaluated.Two antibodies,called S2D8 and S2C6,respectively,were successfully screened out,and double-antibody sandwich ELISA was successfully set up.Stx? and its variants rather than Stx? have been detected from STEC culture supernatant,and its lowest detection limit is 4ng toxin per ml,indicating this system's good specificity and sensitivity.In summary,the development8E7-E6/2F6-F8 and S2D8/S2C6-based ELISA laid a solid foundation for the research which designates the shiga toxin as a potential candidate in diagnosis and therapy of STEC infection.