An improved method for generating single-chain antibodies from hybridomas.
Cloning the correct VL kappa gene from hybridomas derived from MOPC-21 can be problematic because such cell lines variably express a transcript which is aberrantly rearranged at the VJ recombination site. Cellular levels of the aberrant transcript can exceed that of productive light chain RNA, so a large proportion of the VL gene-derived products obtained on PCR amplification of hybridoma cDNA may not encode a functional protein. We have developed a method in which antibody variable region genes are recovered from hybridoma cDNA using a unique set of V gene family-specific primers; the V region genes are then spliced by PCR, in the form 5'-VL-LINKER-VH-3' (where the linker encodes [GlyGlyGlyGlySer]3), and cloned into an expression vector under control of T7 RNA polymerase. Plasmid DNA is isolated from colonies, and the insert is expressed in an in vitro rabbit reticulocyte lysate-based coupled transcription/translation system, in a microtiter plate format. Since aberrantly rearranged VL kappa genes contain a translation termination codon at amino acid position 105, only constructs containing the correctly rearranged gene produce a protein of the predicted size. We demonstrate the method by producing the single-chain form of OKT9, a murine IgG1 which binds to the human transferrin receptor, and extend the results to show that the protein generated by the in vitro expression system retains the antigen binding properties of the parent antibody. Our method will be generally useful for screening single-chain antibodies for function prior to large scale production in vivo.
Nicholls
P J
PJ
Biochemistry Section, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892.
Johnson
V G
VG
Blanford
M D
MD
Andrew
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011990:Receptors, Transferrin / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured; D014709:Vero Cells
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011990:Receptors, Transferrin; D004247:DNA