Selection Of Human Antibodies Targeting The Epithelial Cell Adhesion Molecule And Their Protein Expression

Objective:Screening human single domain antibodies against epithelial cell adhesion molecule by phage display technology, and the identified antibodies are expressed in Ecoli and purified.These antibodies may be tested in the treatment of cancers in the future.Methods:1. Prepare phage displayed human single domain antibody library;2. Analysis and selection of polypeptides that can be used in subsequent panning experiments,and synthesis of the selected peptide;3. Screen the antibody library with the epithelial cell adhesion molecule fragment;4. Randomly pick monoclonal phage antibodies from the screened library, and examine their antigen binding activity;5. Examine the binding specific of the candidate antibodies to the epithelial cell adhesion molecule fragment and determine their DNA sequences;6. Determine the binding of the selected phage antibodies to the full-length epithelial cell adhesion molecule;7. Express, purify and analyze the selected single domain antibodies.Results:1. Successfully prepared the phage displayed human single domain antibody library with a titer of 4.4 X 1013 pfu/ml;2. Selected the polypeptide encoded by the second exon of the gene of epithelial cell adhesion molecule as the antigen used in subsequent panning experiments, and artificially synthesize this polypeptide with a purity of more than 95%;3. Human single domain antibody library was screened for 5 rounds;4. A total of 478 phage antibodies were randomly picked and checked, and 30 antibodies were selected;5. Further analysis identified 17 antibodies. Their DNA sequences were determined and analyzed,and 5 different antibodies were obtained.6. Among 5 different antibodies, 3 phage displayed antibodies were confrim that could bind to the full-length epithelial cell adhesion molecule;7. These 5 single domain antibodies were successfully expressed in E-coli, and they could bind to the epithelial cell adhesion molecule fragment and the full-length protein.Conclusions:This study finally obtained 5 monoclonal single domain antibodies, which could specifically bind to the full-length epithelial cell adhesion molecule, and successfully expressed and purified these single domian antibodies.