title: An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens.
abstract: Construction of a monoclonal antibody (mAb) bank containing a vast variety of antibodies against human tissue proteins is important for proteomic research. A novel strategy of subtractive immunization using fractionated native proteins was developed for high throughput generation of mAb against human plasma proteins. By this novel approach, the bottleneck of antigen preparation can be overcome by combining repeated immunization of animals with subtracted fractions of plasma or tissue proteins and identification of target antigen by immunoprecipitation/mass spectrum strategies. Plasma freshly collected from healthy adults was pooled and three fractions were prepared by size exclusion chromatography. Mice were immunized with the fractionated plasma proteins, and 205 strains of hybridomas secreting mAb were obtained after two-round subtractive immunizations and cell fusions. In the first round, 110 strains of hybridomas were established, in which 77 strains secreting mAb were identified against 10 human plasma high-abundant proteins. In the second round, plasma fraction I was absorbed with mAb against IgM, IgG, ceruloplasmin and haptoglobin. The absorbed fraction I was used as immunogen for the second round immunization and cell fusion. Ninety-five strains of hybridomas secreting mAb were obtained. Although the target antigens of mAb from 82 strains of hybridomas were identified as IgM, IgA, alpha2-macroglobulin and fibrinogen, about 85% antibodies obtained from this round were identified as new antibodies when compared with mAb obtained in the first round immunization with plasma fraction I. The results suggest that subtractive immunization with fractionated plasma proteins followed by identification of antigens with immunoprecipitation/mass spectrum may be an effective approach for rapid preparation of mAb against high-and medium-abundant plasma or tissue proteins.
authors.affiliation: School of Biotechnology, Southern Medical University, Guangzhou, PR China.
authors.forename: Yunshan
authors.initials: Y
authors.lastname: Ning
authors.forename: Yundan
authors.initials: Y
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Hong
authors.forename: Dandan
authors.initials: D
authors.lastname: Peng
authors.forename: Yichu
authors.initials: Y
authors.lastname: Liu
authors.forename: Junxiang
authors.initials: J
authors.lastname: Wang
authors.forename: Wenbo
authors.initials: W
authors.lastname: Hao
authors.forename: Xuemei
authors.initials: X
authors.lastname: Tian
authors.forename: Fenfang
authors.initials: F
authors.lastname: Wu
authors.forename: Wenqi
authors.initials: W
authors.lastname: Dong
authors.forename: Lihong
authors.initials: L
authors.lastname: Wang
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Liu
authors.forename: Jianen
authors.initials: J
authors.lastname: Gao
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.forename: Qi-Hong
authors.initials: QH
authors.lastname: Sun
authors.forename: Ming
authors.initials: M
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000936:Antigen-Antibody Complex; D001798:Blood Proteins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007114:Immunization; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001798:Blood Proteins; D010446:Peptide Fragments