Immunization of alpacas (Lama pacos) with protein antigens and production of antigen-specific single domain antibodies

K. Martin Chow, Sidney W. Whiteheart, Jeffrey R. Smiley, Savita Sharma, Kathy Boaz, Meggie J. Coleman, Alvina Maynard, Louis B. Hersh, Craig W. Vander Kooi

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

Original languageEnglish
Article numbere58471
JournalJournal of Visualized Experiments
Volume2019
Issue number143
DOIs
StatePublished - Jan 2019

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health (P30GM103486).

Publisher Copyright:
© 2019 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

Keywords

  • Antigen
  • Biochemistry
  • Blood collection
  • Camelid
  • Immunization
  • Issue 143
  • Phage display
  • Purification
  • Recombinant expression
  • Screening

ASJC Scopus subject areas

  • Neuroscience (all)
  • Chemical Engineering (all)
  • Biochemistry, Genetics and Molecular Biology (all)
  • Immunology and Microbiology (all)

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