Preparation Of Monoclonal Antibody And Establishment Of Block ELISA Against Pasteurella Multocida OmpH

Duck pasteurellois is caused an acute septic infectious disease by Pasteurella multocida, asduck hemorrhagic septicemia or cholera. Pasteurella multocida is a Gram-negative bacteria thatinfect ducks of all ages, almost all of the poultry and wild birds are susceptible, especiallychickens, ducks and geese. The outer membrane protein H is the major outer membrane proteinsof Pasteurella multocida, high homology among the serotypes. OmpH is a pore-forming proteinrelated to the superfamily of the nonspecific bacterial porins. Purified natural OmpH induced goodprotection rate as the protection rate of whole cell lysate induced; OmpH and whole cell lysatecould induce high levels of ELISA antibody. So Pasteurella multocida OmpH recombinantprotein as immunogen, preparation of OmpH monoclonal antibodies, Using this antibody, a blockELISA method for the detection of Pasteurella multocida was developed, that is of greatsignificance for diagnosis and detection of duck pasteurellois.6-week-old female BALB/c mice were immunized by Pasteurella multocida OmpHrecombinant protein. spleen cells were fused with myeloma cell Sp2/0in50%polyethylene glycol3350after four immunizations, established hybridoma anti-Pasteurella multocida OmpH antibody.The test of indirect ELISA is used to screen hybridoma cells and limited dilution method wasperformed to subclone the positive clones. Three strains of hybridoma secreted MAb againstPasteurella multocida OmpH are obtained after three subcloning, and are designated as1A9,1E9and3G7. The titers of obtained MAbs from the mouse ascitic fluid are1.6×10~4～3.2×10~4inindirect ELISA test. By using the immunoglobulin subtypes kit, the antibodies are all IgG1with κlight chain. Western blot and IFA Confirme three Mabs are specific to Pasteurella multocidaOmpH.1A9,1E9and3G7in vitro continuous30generations, every5detection efficiency showthat they could secrete stably antibodies.A blocking ELISA based on the3G7monoclonal antibody was developed to detectantibodies to Pasteurella multocida. The best concentration of antigen that was selected byphalanx experiment was1.0μg/mL; serum dilution and monoclonal antibody dilution were1:5and1:500; the best concentration dilution of second antibody marked with HRP was1:30,000. Thethreshold value of the blocking ELISA was conformed according to the detection result of60negative sera. It was positive when PI was more than37.6%, it was negative when PI was lessthan29.13%. Positive serums to6usual pathogens and of duck were tested by blocking ELISA,the results showed that the blocking ELISA with good specificity. Intro-batch test and Inter-batchtest were less than10%, with good reproducibility.10positive serums and10negative serumswere tested by blocking ELISA, the results showed that the blocking ELISA with good sensitivity.60sera samples collected in helongjiang province were tested by blocking ELISA, the coincidencerate between of ELISA and Western blot was91.3%.