Objective To prepare hightiter monoclonal antibodies against STX2A1 subunit of enterohemorrhagic E.coli  (EHEC) O157∶H7.  Methods BALB/c mice were immunized with GST-STX2A1 fusion protein and the spleen cells of BALB/c mice which were not immunized were used as feeder cells. Hybridoma technique

natural STX2A protein and ELISA test were used to prepare and screen the hybridoma cell lines of monoclonal antibodies against STX2A1. The ascites developed by injecting the hybridoma cells into abdominal cavity of the BALB/c mice and was purified with Protein A-Sepharose.

The subclasses and isotypes were identified by mouse monoclonal antibody isotyping kit. The antigenic epitopes that can be recognized by STX2-1A3

STX2-1E10 and STX2-3A7 were analyzed by the ELISA additivity test.  Results Three hybridoma cell strains were obtained and named as STX2-1A3

STX2-1E10 and STX2-3A7

respectively

all of which produced monoclonal antibodies specifically against STX2A1. The isotypes of the monoclonal antibodies were IgG1λ

IgG1κ

and IgG3κ and the affinity constant was 5.76×10

9

1.21×10

9

and 3.97×10

8

respectively.  Conclusion We have successfully prepared three hybridoma cell strains which secrete high-titer and highly specific monoclonal antibodies against STX2A1. Our study provides a basis for researching the early diagnosis

prevention and cure of the disease induced by EHEC O157:H7.