Abstract
To engineer or manipulate antibody or Fv-based molecules, isolation of the V region of the antibodies is necessary. A number of strategies can be adopted for amplification; one such approach is to use subgroup-specific oligonucleotides for the amplification of the V-region genes. Small differences in the conserved regions of variable genes have allowed the division of murine VH and VL genes into subgroups and families, with the members of one family having more homology with each other than members of another family. Therefore, it is possible to categorize the variable genes of particular hybridoma, and there are fewer chances of amplifying VL genes of myeloma partners of the hybridoma with the family-specific primers as compared to a set of highly degenerate primers. Therefore, many (up to six) different 5’ primers can be designed from the FR1 of the kappa chain using the Kabat database (1). Similarly, four different 5’ primers are usually designed for the heavy chain. The 3’ primers are designed from constant segments of the chains (2).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest. US Department of Health and Human Services, Bethesda, MD.
Nicholls, P. J., Johnson, V. G., Blanford, M. D., and Andrew, S. M. (1993) An improved method for generating single-chain antibodies from hybridomas. J. Immunol. Methods165, 81–91.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sastry, L., Alting-Mees, M., Huse, W. D., Short, J. M., Sorge, J. A., Hay, B. N., Janda, K. D., Benkovic, S. J., and Lerner, R. A. (1989) Cloning of the immuno-globulin repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library. Proc. Natl. Acad. Sci. USA86, 5728–5732.
George, A. J. T., Titus, J. A., Jost, C. R., Kurucz, I., Andrew, S. M., Nicholls, P. J., Huston, J. S., and Segal, D. M. (1994) Redirection of T cell mediated cyto-toxicity by a recombinant single-chain Fv molecule. J. Immunol.152, 1802–1811.
Carroll, W. L., Mendel, E., and Levy, S. (1988) Hybridoma fusion cell lines contain an aberrrant Kappa transcript. Mol. Immunol.25, 991–995.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc.
About this protocol
Cite this protocol
Verma, R. (2000). PCR of the V Region. In: George, A.J.T., Urch, C.E. (eds) Diagnostic and Therapeutic Antibodies. Methods in Molecular Medicine, vol 40. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-076-4:453
Download citation
DOI: https://doi.org/10.1385/1-59259-076-4:453
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-0-89603-798-4
Online ISBN: 978-1-59259-076-6
eBook Packages: Springer Protocols