title: [Preparation and characterization of monoclonal antibodies against cytolethal distending toxin protein of Campylobacter jejuni].
abstract: OBJECTIVE
  To express Campylobacter jejuni cytolethal distending toxin B protein (CdtB) in a prokaryote to prepare monoclonal antibodies (mAbs) against the protein, and to study their antitoxic effects.
  
  
  METHODS
  The C. jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a( + ) and pGEX-6p-1. The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein. The mAb titers were determined with an indirect enzyme-linked immunosorbent assay (ELISA), and their specificity with a Dot-ELISA and western blotting analysis. We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells.
  
  
  RESULTS
  Recombinant expression plasmids pET-30a (+)-cdtB and pGEX-6p-l-cdtB were successfully constructed, and fusion proteins rHis-CdtB and rGST-CdtB expressed, respectively. Five hybridoma cell lines, designated 1F3, IF5, 2E4, 2E11, and 2F2, were screened for the stable secretion of mAbs against CdtB. The immunoglobulin subclass of 2E11 was IgG2b and that of the other mAbs was IgG1. The mAb titers in the ascites fluids were 1:1 x 10(8) on indirect ELISA. Dot-ELISA demonstrated that the five mAbs reacted specifically with C. jejuni. Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein. The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells (P < 0.01).
  
  
  CONCLUSIONS
  The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C. jejuni.
authors.forename: Lei
authors.initials: L
authors.lastname: Lu
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Shang
authors.forename: Fangzhe
authors.initials: F
authors.lastname: Ren
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Xin'an
authors.initials: X
authors.lastname: Jiao
authors.forename: Jinlin
authors.initials: J
authors.lastname: Huang
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002169:Campylobacter Infections / Q000382:microbiology*; D016123:Campylobacter jejuni / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; C067044:cytolethal distending toxin