title: [Preparation of monoclonal antibodies against flagellin core protein of Vibrio cholerae and its application in establishing double-antibody sandwich ELISA for testing Vibrio cholerae from food products].
abstract: OBJECTIVE
  To prepare the monoclonal antibodies (mAbs) against flagellin core protein of Vibrio cholerae and establish the double-antibody sandwich ELISA method for testing Vibrio cholerae from food products.
  
  
  METHODS
  BALB/c mice were immunized with flagellin extracted from Vibrio cholerae Vc75 by differential centrifugation. The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells when the antibody titer in serum reached 1:32 000. The hybridoma cell lines were obtained by regular subcloning and used to generate ascites. And mAbs reacting to Vibrio cholerae flagellin were achieved by purified from the ascites.
  
  
  RESULTS
  Six hybridoma cell lines stably secreting mAbs against Vibrio cholerae flagellin were taken and named VcNo.1-VcNo.6. The mAb titer in serum by indirect ELISA was 1:2 × 10(6). SDS-PAGE showed that the flagellin protein molecular weight (Mr) was 44 000 and the purity was high. Double-antibody sandwich ELISA method was set up using VcNo.6 antibody for detecting Vibrio cholerae. The sensitivity reached 10(3) CFU/mL. The ELISA method showed high specificity to Vibrio cholerae through testing 100 Vibrio cholerae (100% positive) and 101 non-Vibrio cholerae strains (100% negative). The detection limit was 1 CFU/g sample in artificial contaminated samples.
  
  
  CONCLUSIONS
  The mAbs against flagellin core protein of Vibrio cholerae was successfully prepared and used to set up the double-antibody sandwich ELISA. The mAb of VcNo.6 was highly specific to Vibrio cholerae. The sensitivity of the established ELISA was as high as 10(3) CFU/mL. Moreover, it did not react to non-Vibrio cholerae strains. Therefore, the mAbs of VcNo.6 could be widely used in Vibrio cholerae detection from food samples as well as clinical samples.
authors.affiliation: Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing 100026, China.
authors.forename: Jinxia
authors.initials: J
authors.lastname: Cheng
authors.forename: Jing
authors.initials: J
authors.lastname: Zeng
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.forename: Haiyu
authors.initials: H
authors.lastname: Zhang
authors.forename: Xuesong
authors.initials: X
authors.lastname: Liu
authors.forename: Dong
authors.initials: D
authors.lastname: Cao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005408:Flagellin / Q000276:immunology*; D005516:Food Microbiology / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D014734:Vibrio cholerae / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D005408:Flagellin