Immunization of Alpacas (<em>Lama pacos</em>) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

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. Video Link The video component of this article can be found at https://www.jove.com/video/58471/ 7. These unique camelid antibodies possess the same ability to specifically bind foreign substances with high affinity but are only made up of one protein chain 8. Furthermore, camelid antibodies can be experimentally truncated to an even smaller unit the V H H fragment, also called a single domain antibody 9. Due to their small size, single domain antibodies are useful for a wide range of biomedical research applications and are available from a variety of academic and commercial

Development, Screening, and Validation of Camelid‐Derived Nanobodies for Neuroscience Research

“…These may differ in the immunization vehicle (e.g., whether and which type of adjuvant is used) and the route and frequency of injections. Detailed example protocols have been published previously (e.g., Chow et al., 2019; Pardon et al., 2014).…”

Development and characterization of nanobodies specifically targeting the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3)

“…The anti-PRL-3 nanobodies were successful in both PRL-3 ELISA and immunoprecipitation of PRL-3, suggesting that they bind to the protein very well. The small size, high stability, and high specificity of nanobodies lends them well towards acting as chaperones in structural studies (31), and our data suggest that the anti-PRL-3 nanobodies may be useful to stabilize the PRLs in a single conformation to aid in crystallization studies.…”

Generation and characterization of a laforin nanobody inhibitor

“…Primary screening for nanobody binding was achieved by a small-scale pulldown method in which Affi-gel 10 affinity resin (Bio-Rad) was bound to laforin and subsequently to each purified VHH clone 34 . Expression and purification of laforin was performed as described above in 50 mM HEPES, 100 mM NaCl, 10% glycerol, 2 mM DTT, pH 7.5.…”