A chemiluminescent magnetic enzyme-linked immunoassay method has been used to detect pathogens, but it is time-consuming and hard sledding in preparation of antibody and enzyme conjugated with antibody, especially in detecting multi-antigens simultaneously. In this paper, a system of chemiluminescent magnetic enzyme-linked immunoassay was developed. E. coli O157:H7 was sandwiched between rabbit anti-E. coli O157:H7 polyclonal antibody-coated magnetite nanoparticles (immunomagnetic nanoparticles or IMNP) and mouse anti-E. coli O157:H7 monoclonal antibody. Alkaline phosphatase conjugated horse anti-mouse immunoglobulin (ALP-Ab) was used to bond with the monoclonal antibody, finally the chemiluminescent signals were detected by adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD), which was the substrate reagent of ALP. Different solvents of AMPPD were compared to get an optimal chemiluminescent signal. The effect of sodium borohydride and glycine on blocking the aldehyde groups of IMNP was compared either, and the specificity and sensitivity of this system for detecting E. coli O157:H7 were researched. The results indicated that c buffer was the best solvent of AMPPD, sodium borohydride was better than glycine in blocking immunomagnetic nanoparticles, and this method was of good specificity when using E. coli Top 10f', D-group shigella sonnei, B-group shigella flexneri, Salmonella typhimurium, Staphylococcus aureus and Vibrio cholera as negative controls. The detection limit was 10³ cells/mL when the antigen solution was 1 mL, and the procedure duration was about 3 h.

Key words: Escherichia coli O157:H7, enzymoimmunoassay, chemiluminescence, magnetic nanoparticle