Advertisement

PCR of the V Region

  • Rakesh Verma
Part of the Methods in Molecular Medicine book series (MIMM, volume 40)

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).

Keywords

Guanidinium Thiocyanate Kappa Chain Reverse Transcriptase Buffer Constant Segment Versus Region Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    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.Google Scholar
  2. 2.
    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. Methods 165, 81–91.CrossRefPubMedGoogle Scholar
  3. 3.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  4. 4.
    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. USA 86, 5728–5732.CrossRefPubMedGoogle Scholar
  5. 5.
    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.PubMedGoogle Scholar
  6. 6.
    Carroll, W. L., Mendel, E., and Levy, S. (1988) Hybridoma fusion cell lines contain an aberrrant Kappa transcript. Mol. Immunol. 25, 991–995.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Rakesh Verma
    • 1
  1. 1.Department of ImmunologyImperial College School of MedicineLondonUK

Personalised recommendations