Preparation And Application Of Monoclonal Antibodies Against Staphylococcus Aureus Protein A

Staphylococcus aureus has emerged as one of the important contemporaryhuman food borne pathogens capable of inflicting various disease symptoms inhumanbeing. It is widely distributed in environment. At present, the frequent incidentsof food poisoning caused by Staphylococcus aureus. Therefore, accurate, rapid andsimple detecting assay is of great significance to prevent and control Staphylococcusaureus infection. In this study SPA and monoclonal antibodies against SPA wereprepared to establish a sandwich ELISA for the detection of the organism. Theprincipal researchs are as the following.Isolate and purify Staphylococcal protein A (SPA) and study it’s specificity. TheSPA was use heat PBS isolated and purified by SephadexS-200Gel Chromatography.and protein contents of SPA was0.506mg/mL. SDS-PAGE showed single proteinband with relative molecular mass of about12Kd. A high purity, activity natural SPAwas successfully acquired.Produce Monoclonal antibodies (McAbs)directed against the SPA. Hybridomacells were obtained by fusion of myeloma cells with lymphocytes from BALB/c miceimmunized with heat-killed Staphylococcus aureus cells. Postive clones were selectedusing indirect ELISA with Staphylococcus aureus and SPA as antigens. Finally, nineMcAbs specific for the SPA of Staphylococcus aureus were obtained. The six McAbsreacted with Staphylococcus aureus and SPA, no cross-reactivity was observed withother tested strains. Two of the McAbs3B11and4F7were proved to be specific forthe, and agglutinated Staphylococcus aureus wol-04cells well. They are of IgG1andIgG3subtype, and have high specificity stability and titer.3B11was conjugated with horseradish peroxidase (HRP) by using an improvedsodium periodate oxidation method. The sandwich ELISA format was developed todetect Staphylococcus aureus using4F7as the capture-antibody and3B11-HRP as thedetection antibody. This assay is specific for Staphylococcus aureus and showed nocross reaction with the tested non-Staphylococcus aureus strains. The detection limitis1×10~5CFU/mL in pure culture of Staphylococcus aureus. The sandwich ELISAprovides a reliable, sensitive and rapid assay for the detection of Staphylococcusaureus, and would be used to detect Staphylococcus aureus combined with other assays.