title: [Preparation and characterization of the monoclonal antibodies against human CA15-3 and establishment of a sandwich CLIA system].
abstract: OBJECTIVE
  To prepare the monoclonal antibodies (mAbs) against human carbohydrate antigen 15-3 (CA15-3) and establish a double-antibody sandwich chemiluminescent immunoassay (CLIA) system for detecting CA15-3 in the human serum.
  
  
  METHODS
  BALB/c mice were immunized with human CA15-3 antigen. Spleen cells of the immunized mice were fused with Sp2/0 cells and the positive hybridoma cells were selected and subcloned. The supernatant was taken for purify mAbs using protein A chromatography. The purity, titer, epitope and subtype of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity, repetitiveness and specificity.
  
  
  RESULTS
  Five hybridoma cell lines named #3-1-3, #5-2-2, #11-2-2, #12-1-3 and #16-1-3 were obtained respectively, which are of strong positive signal and high secretion. The titers of the mAbs secreted by these hydridomas were above 10(-8); g/mL. All of the mAbs expressed κ light chains, and their heavy chains were as follows: #3-1-3 mAb had IgG2a, #5-2-2 mAb and #12-1-3 mAb had IgG2b, #11-2-2 mAb and #16-1-3 mAb had IgG3. The double-antibody sandwich CLIA system had a good linear relationship between 0.59 U/mL and 300 U/mL. The recovery rate for accuracy was 97.45% and the limit of detection was 0.59 U/mL. The assay was highly linear (correlation coefficient 0.9978) and highly repeatable [coefficient of variation (CV) <10%]. This CLIA system was also highly specific without cross-reactivity to the tumor marker AFP, CEA, CA50, CA19-9 and CA72-4.
  
  
  CONCLUSIONS
  The mAbs against human CA15-3 have been prepared and a sandwich CLIA system for detecting CA15-3 in the human serum has been established successfully, which provides a basis for CA15-3 quantification and clinical application.
authors.affiliation: College of Life Science, Northeast Forestry University, Harbin 150040; Daqing MabCom, Inc., Daqing 163316, China.
authors.forename: Linggai
authors.initials: L
authors.lastname: Cao
authors.affiliation: College of Life Science, Northeast Forestry University, Harbin 150040, China.
authors.forename: Xingguo
authors.initials: X
authors.lastname: Lan
authors.affiliation: College of Life Science, Northeast Forestry University, Harbin 150040; 2Daqing MabCom, Inc., Daqing 163316, China.
authors.forename: Deqiang
authors.initials: D
authors.lastname: Wei
authors.affiliation: College of Life Science, Northeast Forestry University, Harbin 150040; Daqing Biotechnology Research Institute, Northeast Forestry University, Daqing 163316, China.
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000097:blood* / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D016014:Linear Models; D008163:Luminescent Measurements; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes