title: Isolation, identification and expression of specific human CD133 antibodies.
abstract: CD133, a 120 KDa glycoprotein is a transmembrane glycoprotein which has been recently used as a cancer stem cell (CSCs) marker in a variety of carcinomas. CD133(+) cells possess strong tumorigenicity, responsible for tumor initiation and maintenance. Therefore, the goal of our study was to develop a novel CD133 humanized antibody as a promising target for cancer therapy. CD133 purified proteins were used for panning the naive human-semi-synthetic Tomlinson I + J phagemid library. The second extracellular domain (loop1) and the third extracellular domain (loop2) of CD133 were expressed in E. coli. In this study, we adopted a novel five-round selection strategy based on moderate stringent selection during the first rounds. This unique strategy was aimed at avoiding the loss of rare phages with high affinity to target proteins. After the five rounds of specific panning, six phage-antibody clones which specifically recognized recombinant human CD133 protein were obtained. The desirable phage clone named CD133-scFv-1 was cloned into the expression vector, then induced and purified. We show that CD133-scFv-1 and commercial murine antibody 293C3 could compete with each other in the indirect competitive immunoassay. Our work may lay the groundwork for future studies involving biological functions and applications of the CD133 humanized antibody.
authors.affiliation: Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Department of Microbiology and Immunology, Medical School, Southeast University, 210009, Nanjing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xia
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jun
authors.initials: J
authors.lastname: Qian
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhu
authors.forename: Yafen
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jianqiong
authors.initials: J
authors.lastname: Zhang
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
mesh_terms: D000071916:AC133 Antigen; D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics / Q000276:immunology; D006528:Carcinoma, Hepatocellular / Q000276:immunology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D006639:Histidine / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D014411:Neoplastic Stem Cells / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA
chemical_list: D000071916:AC133 Antigen; D000906:Antibodies; D015703:Antigens, CD; D014408:Biomarkers, Tumor; D006023:Glycoproteins; C471213:His-His-His-His-His-His; D008565:Membrane Proteins; D009842:Oligopeptides; C000606023:PROM1 protein, human; D010455:Peptides; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D006639:Histidine