Expression and Purification of Monospecific and Bispecific Recombinant Antibody Fragments Derived from Antibodies That Block the CD80/CD86-CD28 Costimulatory Pathway
| Autor: | Fons Bosman, Linda Celis, Veronique Van Doorsselaere, Marie-Ange Buyse, Marc de Boer, Rosina Degrieck, Stéphanie Dincq, Erwin Sablon |
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| Rok vydání: | 2001 |
| Předmět: |
Models
Molecular Blotting Western Protein domain Immunoglobulin Variable Region Heterologous Enzyme-Linked Immunosorbent Assay Protein Engineering medicine.disease_cause Cell Line law.invention Mice CD28 Antigens Antigen Antibody Specificity Antigens CD law Antibodies Bispecific Immune Tolerance medicine Animals Immunoglobulin Fragments Escherichia coli CD86 B-Lymphocytes Membrane Glycoproteins biology Antibodies Monoclonal Flow Cytometry Molecular biology Recombinant Proteins B7-1 Antigen biology.protein Recombinant DNA B7-2 Antigen Binding Sites Antibody Antibody CD80 Signal Transduction Biotechnology |
| Zdroj: | Protein Expression and Purification. 22:11-24 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.2001.1417 |
| Popis: | The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb. |
| Databáze: | OpenAIRE |
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