title: [Preparation of anti-B7-H4 monoclonal antibody to investigate B7-H4 expression in pancreatic cancer].
abstract: OBJECTIVE
  To prepare a monoclonal antibody (mAb) against extracellular domain of B7-H4 and to investigate the expression of B7-H4 in pancreatic cancer tissue with the prepared mAb.
  
  
  METHODS
  Balb/c mice were immunized with 3T3-B7-H4 cells and the splenic cells of the immunized mice were fused with Sp2/0 myeloma cells by conventional hybridoma techniques. An indirect ELISA method using 3T3-B7-H4 lysate as antigen was established to screen antibody-producing hybridoma cell lines. Western blott, immunoprecipitation (IP), and immunohistochemistry (IHC) were applied to characterize the mAb. Immunohistochemical staining was used to detect the expression of B7-H4 in human pancreatic cancer tissue. The correlation of B3-H4 expressions and pathological features of pancreatic cancer was analyzed.
  
  
  RESULTS
  A hybridoma cell line secreting mAb against B7-H4 was obtained. The subclass of this mAb was IgM, and the light chain was Kappa. Western blot and IP showed that the mAb specifically recognized B7-H4. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The B7-H4 was diffusely expressed in the cytoplasma and/or membrane of pancreatic cancer tissue, which was much higher than that expressed in normal pancreatic tissue (4.00 ± 1.44 compared with 1.12 ± 0.78, P ± 0.01). The expression of B7-H4 was higher in pancreatic cancer tissues with higher pathological grade or with lymph node metastasis as compared with that in pancreatic cancer tissues with lower grade or with no lymph mode metastasis (6.10 ± 0.72 compared with 3.55 ± 1.12,P<0.01: 6.14 ± 0.66 compared with 3.70 ± 1.25,P<0.01). The expression level of B7-H4 was not related to patients'age and gender.
  
  
  CONCLUSIONS
  Monoclonal antibody against B7-H4 with high activity and specificity has been prepared successfully. The expression of B7-H4 in pancreatic cancer tissue is up-regulated,which is closely related to the tumor grade and lymph node metastasis in pancreatic cancer.
authors.affiliation: Department of Pathology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Hong
authors.forename: Yun
authors.initials: Y
authors.lastname: Qian
authors.forename: Hang-Ping
authors.initials: HP
authors.lastname: Yao
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D006801:Humans; D007150:Immunohistochemistry; D008207:Lymphatic Metastasis; D051379:Mice; D008807:Mice, Inbred BALB C; D060787:Neoplasm Grading; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D060907:V-Set Domain-Containing T-Cell Activation Inhibitor 1 / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D060907:V-Set Domain-Containing T-Cell Activation Inhibitor 1