Design and characterization of a plasmid vector system capable of rapid generation of antibodies of multiple isotypes and specificities |
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Authors: | Matthew P Morrow Panyupa Pankhong David B Weiner |
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Institution: | (1) Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 422 Curie Blvd, 505 Stellar Chance Labs, Philadelphia, PA 19104, USA |
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Abstract: | We present a detailed method for constructing a vector system composed of plasmids encoding immunoglobulin genes that have
been constructed in such a fashion so as to allow for the generation and secretion of antibodies of multiple specificities
and isotypes via a rapid and easy cloning-and-ligation scheme. Restriction sites within each plasmid allow for the removal
of variable domains, constant domains, leader sequences, or the entire immunoglobulin gene. Degenerate primers are used to
clone variable regions from hybridoma cDNAs, allowing for the creation of antibodies with varying binding specificities. Sequence-specific
primers are used to clone antibody constant domains, allowing for the creation of antibodies of multiple isotypes from a variety
of lineages. A high-efficiency leader sequence has been inserted into the start of each gene to improve secretion. Antibodies
constructed through this system are efficiently secreted by transfected cells, and are fully functional in antigen-specific
binding assays. |
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Keywords: | Antibody Expression vectors Hybridoma cDNA Immunoglobin Plasmid Vector |
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