Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.
Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks.
Morphotek Inc., 210 Welsh Pool Road, Exton, PA, USA. Electronic address: jspidel@morphotek.com.
Jared L
JL
Spidel
Morphotek Inc., 210 Welsh Pool Road, Exton, PA, USA.
Benjamin
B
Vaessen
Morphotek Inc., 210 Welsh Pool Road, Exton, PA, USA.
Yin Yin
YY
Chan
Morphotek Inc., 210 Welsh Pool Road, Exton, PA, USA.
Luigi
L
Grasso
Morphotek Inc., 210 Welsh Pool Road, Exton, PA, USA.
J Bradford
JB
Kline
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D003587:Cytomegalovirus / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D005822:Genetic Vectors; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D057188:Workflow
D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D011061:Poly A
Bicistronic; High-throughput cloning; Monoclonal antibody; Stable cell line; Transfection