Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system
| Autor: | Anke Krebber, Andreas Plückthun, Susanne Bornhauser, Jörg Willuda, Hans Rudolf Bosshard, Annemarie Honegger, Jörg Burmester |
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| Rok vydání: | 1997 |
| Předmět: |
Phage display
medicine.drug_class Recombinant Fusion Proteins Immunology Genetic Vectors Molecular Sequence Data Immunoglobulin Variable Region Biology Immunoglobulin light chain Monoclonal antibody Polymerase Chain Reaction law.invention Mice Structure-Activity Relationship law Peptide Library medicine Immunology and Allergy Animals Amino Acid Sequence Cloning Molecular Peptide library DNA Primers Cloning Hybridomas Base Sequence Genes Immunoglobulin Antibodies Monoclonal Restriction enzyme Biochemistry Recombinant DNA biology.protein Binding Sites Antibody Antibody Spleen |
| Zdroj: | Journal of immunological methods. 201(1) |
| ISSN: | 0022-1759 |
| Popis: | A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed. |
| Databáze: | OpenAIRE |
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