Abstract: Objective  To prepare the FITC labeled Escherichia coli　(E.coli) O157∶H7 antibody with  high titer,high purity,high specificity,high fluorescence intensity
 and stability,and to approach its application in immunofluorescence detection.Methods Standard E.coli O157∶H7 strain was cultured and the bacteria was inactivated by adding formaldehyde solution to prepare vaccine.After four immunizations,blood samples were collected from the marginal ear vein.After purified by the method of caprylic acid-sulfide saturated solution precipitation and HiTrap Protein G chromatography column on AKTA Protein purification device,the purity and content of protein and titer of antibody were detected using the methods of SDS-PAGE,BCA Fit,indirect ELISA.Different ratios of FITC to protein and different reaction conditions were selected to label E.coli O157∶H7 polyclonal antibody,the F485/535 was measured and t-test was used to identify the best ratio of FITC to  protein and the best reaction condition.The reaction of polyclonal antibody with the mixed liquid of E.coli O157∶H7 and Staphylococcus aureus (S.aureus) was observed.Results The best E.coli O157∶H7 polyclonal antibody purity method of HiTrap Protein
 G was 35% and 100% gradient elution;the titer of antibody was 1∶25 600.It showed no cross-reactivities with 8 kinds of enterobacters such as Salmonella,Enterobacterc loacae and so on.The best ratio of FITC to protein was 1∶10,and the best reac
tion condition was 20℃and 2 h.When this fluorescence antibody was applied to react with E.coli O157∶H7 and S.aureus,it could react obviously and stron
gly.It had stability with E.coli O157∶H7 after irradiated with strong fluorescence  for 15 min,without across-reaction with S.aureus.
Conclusion FITC labeled E.coli O157∶H7 polyclonal antibody which high  purity,fluorescence intensity and stability is prepared  at t
he first time.The study  completes the major step of establishing of immunofluorescence detection method of E.coli O157∶H7.

Key words: Escherichia coli O157∶H7, polyclonal antibody, immunofluorescence, fluorecein isothiocyante