Preparation Of Monoclonal Antibodies Against Enterobactor Sakazakii LPS And Development Of A Sandwich ELISA

F.sakazakii(ES) is an emerging opportunistic pathogen associated with life-threatening illnesses in infants. It is widely existed in environment. ES can cause sepsis,meningitis and nerotizing enterocolitis in neontates. Therefore, rapid, accurate and simple detection assay is very important to prevent and control ES infection. ES lipopolysaccharide (LPS) and monoclonal antibodies against ES-LPS were prepared in this study.Meanwhile, a double-antibody sandwich-ELISA was developed to detect ES.LPS of ES was extracted by the hot phenol water and cold phenol water methods, the aqueous and phenol phases were collected, respectively. The purity, yield and immunoreactivity were investigated. All samples have high immunogenicity and specificity. Meanwhile, which molecular weigh is similar between14-45KD. However,compared with cold phenol water method,the hot phenol water way with higher yield and protein content.Spleen cells of BALB/C mice immunized with the antigen of ES ATCC51329and LPS were fused with murine SP2/0myeloma cells. The indirect ELISA with ES ATCC51329and LPS as coating antigen was used to screen specificity hybridoma cell. Nine hybridoma cell lines which stably secreted monoclonal antibodies against ES ATCC51329LPS were obtained by three times fusions. Three of the McAbs1A11,2A7and4B10were proved to be specific to the core polysaccharide and agglutinated ES ATCC51329cells well. The results of the ELISA showed subtypes of the three McAbs belong to class IgGl,IgG3and IgG3.The ELISA titers of the ascites were104to105while the ELISA titers of the1A11supernatants is l:2560,2A7and4b10are1:1280. Antigen dots and specific tests of the three McAbs were analysed.The three McAbs are against different epitopes of ES.The results of the SDS-PAGE showed molecular weight of heavy chains of the three McAbs is50KD roughly and the light chain is25KD approximately. The OD450values of the three McAbs in time of cloning culture were ascending and the ELISA titers of the ascites were104to105,which showed the three hybridoma cell lines are stable.Interspecies and exterspecies reactivity of the three McAbs were analysed.Cross reactivity of the three McAbs with other antigens were not observed,except with Escherichia coli.1A11was conjugated with horseradish peroxidase(HRP)by using an improved sodium periodate oxidation method.A double-antibody sandwich-ELISA was developed to detect ES,using2A7as the capture-antibody and1A11-HRP as the detection antibody.The assay established in the present study was specific for ES ATCC51329and showed high specificity and sensitivity.The detection limit of this method was103CFU/ml in pure culture of ES ATCC51329, and has lower cross reactions with other bacteria.The sandwich ELISA provides a reliable, sensitive and rapid assay for the detection of ES C. Muytjensh, and would be used to detect ES combined with other assays.