The cloning and expression of an anti-peptide antibody: a system for rapid analysis of the binding properties of engineered antibodies

The cloning of the light and heavy chain genes of an antilysozyme monoclonal antibody is described and the application of site-directed mutagenesis for reconstruction of complete cDNAs from incomplete cDNA clones is discussed. The behaviour of the RNA transcripts produced from these cDNAs after thei...

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Bibliographic Details
Published in:Protein engineering 1986-10, Vol.1 (1), p.59-65
Main Authors: Roberts, S, Rees, A R
Format: Article
Language:English
Subjects:
Animals
Antibodies - genetics
Base Sequence
cDNA
cloning
Cloning, Molecular
DNA - genetics
Female
gene expression
gene products
genes
Genes, Immunoglobulin
lysozyme
microinjection
monoclonal antibodies
Oocytes
Peptides - immunology
Protein Biosynthesis
Protein Engineering
RNA
site-directed mutagenesis
Transcription, Genetic
Xenopus
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Summary:The cloning of the light and heavy chain genes of an antilysozyme monoclonal antibody is described and the application of site-directed mutagenesis for reconstruction of complete cDNAs from incomplete cDNA clones is discussed. The behaviour of the RNA transcripts produced from these cDNAs after their subcloning into an appropriate Salmonella typhimurium, SP6, vector and subsequent injection into Xenopus oocytes has been analysed. The antibody secreted by oocytes after RNA injection has been quantitated and its antigen-binding properties compared with wild-type hybridoma antibody. The results show that this route for expression of antibodies, though limited in its capacity to produce more than a few micrograms of protein, is potentially a means for rapid evaluation of the antigen-binding properties of engineered antibodies.
ISSN:0269-2139