Name: Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies (Video) | JoVE Journal
Uploaded: 2019-01-26T12:30:00.000+00:00
Duration: 5 min 27 s
Description: Learn to produce single-domain antibodies from alpacas using immunization, blood collection, and phage display selection. This method generates diverse antibodies for therapeutic and diagnostic applications.

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

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Drs. Whiteheart and Hersh are at the University of Kentucky and operate a core in the Center for Molecular Medicine that produces single domain antibodies in alpaca as a fee-for-service.

As noted, the high affinity and specificity of single domain antibodies, combined with their facile expression and stability, make them ideal reagents for the applications in biomedical research, as critical biochemical reagents, diagnostic tools, or therapeutic agents18. For commercial applications, there are some issues related to intellectual property that must be considered. Additionally, single domain antibodies can be engineered to contain specific markers or tags that can be used as reporters in cellular assays or in diagnostics. The key to these uses is the ability to produce specific single domain antibodies against the antigens of interest. A method is described here to produce single domain antibodies using alpacas, which produces a robust immune response against a wide variety of protein antigens. Considerations for animal handling and regulatory compliance are described. These are key for the success in this part of the process.
There are other strategies for producing single domain antibodies that do not require the immunization, but instead use semisynthetic libraries with different systems for the selection and iterative optimization of affinities19,20. However, the advantage of using libraries derived from immunized animals is the ability to reliably isolate highly enriched, diverse, and high-affinity single domain antibodies. Additionally, not only are significant sequence variations observed, but a significant majority of specific single domain antibodies isolated had unique insertions and deletions, particularly in CDR3. 
Further, single domain antibodies can be produced via a straightforward process that only requires small injections of the antigen, and after this process, the animals can be returned to the herd. Of note, the animal can be freely used for fiber production, but must be excluded from use as meat (human food chain) due to the use of the test antigens and non-FDA approved adjuvant for single domain antibody production. When the procedure is completed, the animals should be monitored for one week, given a final veterinary exam, and then can be either returned to their home farm or rested for six months prior to another round of single domain antibody production.

Alpacas, and other members of the camelid family, have become a popular source for generating antibodies for biomedical research1,2,3. Camelids have a unique immune system in that they produce both normal antibodies as well as heavy-chain only antibodies which allow the production of much smaller single domain antibodies, while retaining high specificity and high affinity. Thus, camelid antibodies provide a versatile reagent useful for a variety of purposes. A method to immunize alpaca and produce single domain antibodies to a variety of protein antigens is here described. These antibodies can be produced in camelids via a relatively straightforward process that only requires the immunization via small injections of the target protein (antigen) and a blood draw4. Subsequently, library construction, M13 phage display5 and panning versus the recombinant antigen6 is used to isolate and produce single domain antibodies with the desired characteristics. Because the process takes advantage of the power of phage display technology, five or more antigens can be simultaneously utilized per animal, thereby reducing the number of animals used and associated costs.
Typical mammalian antibodies or immunoglobulins are large molecules consisting of two types of chains (2 heavy chains and 2 light chains), which are linked together through disulfide bonds. These antibodies are relatively large, exhibiting molecular weights of ~140-190 kDa for the more abundant IgG species from humans, mice, goats, and rabbits. Due to their multi-subunit structure, high molecular weight, and disulfide bonds, immunoglobulins can be rather challenging to produce in large quantities, making their cost high. In the 1990&#39;s, it was discovered that camelids, which includes camels, llamas and alpacas make, in addition to the typical mammalian type immunoglobulin, a novel form of immunoglobulin that is simpler in structure, possessing only heavy chains7. These unique camelid antibodies possess the same ability to specifically bind foreign substances with high affinity but are only made up of one protein chain8. Furthermore, camelid antibodies can be experimentally truncated to an even smaller unit the VHH fragment, also called a single domain antibody9. 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 sources1,10. An exception appears to be Western blotting since single domain antibodies are more often directed against conformation dependent epitopes, which are usually lost under the denaturing conditions used in Western blots.
Since they are made up of a single polypeptide chain, specific single domain antibodies can be selected and easily produced in large quantities as recombinant proteins in bacteria. Single domain antibodies can also be genetically engineered to contain specific markers or tags that can be used as &#34;reporter groups&#34; for diagnosing diseases11. This ease of manipulation coupled with their flexibility of use makes single domain antibodies an important resource for researchers at universities and elsewhere.
As part of a National Institutes of Health supported core, existing methods have been adapted to produce single domain antibodies from alpacas3,4. Alpacas have significant advantages in both ease of handling relative to larger camelid family members as well as accessibility. Alpacas are widely raised for fiber and meat and thus can be obtained regionally from local alpaca farmers, who can be identified through their websites or through state breeder associations. Two breeds of alpacas are available, Suri and Huacaya. Huacaya are more common and were used for this protocol, but the protocol is generally applicable to either breeds and also more widely applicable to other camelids.

<table><tbody><tr><td>GERBU FAMA adjuvant</td><td>Biotechnik, Heidelberg, Germany&amp;nbsp;</td><td>3003,6001</td><td></td></tr><tr><td>Serum collection tube</td><td>Becton Dickinson</td><td>367983</td><td></td></tr><tr><td>Blood collection tube</td><td>Becton Dickinson</td><td>366643</td><td></td></tr><tr><td>Vacutainer blood collection set</td><td>Becton Dickinson</td><td>368652</td><td></td></tr><tr><td>Maxisorp Immuno plates</td><td>Nunc</td><td>439454</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>HRP-conjugated goat anti-llama IgG antibody&amp;nbsp;</td><td>Bethyl Labs</td><td>A160-100P</td><td></td></tr><tr><td>TMB reagent chromogenic peroxidase substrate&amp;nbsp;</td><td>KPL</td><td>50-76-03</td><td></td></tr><tr><td>Plate Reader</td><td>Spectramax</td><td>M5</td><td>Any UV/VIS capable reader is acceptable</td></tr><tr><td>Uni-SepMAXI+ lyphocyte separation tube</td><td>Novamed</td><td>U-17</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>QIAshredder column</td><td>Qiagen</td><td>79654</td><td></td></tr><tr><td>Superscript IV reverse transcriptase&amp;nbsp;&amp;nbsp;</td><td>Invitrogen</td><td>18064014</td><td></td></tr><tr><td>AEBSF solution</td><td>Biosynth</td><td>A-5440</td><td></td></tr><tr><td>TG1 phage display competent cells</td><td>Lucigen</td><td>60502</td><td></td></tr></tbody></table>

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

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

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.

The protocol presented here was utilized to generate single domain antibodies against a range of protein antigens. Five antigens were utilized per alpaca. Immune monitoring indicated that the majority of antigens showed robust response beginning at three weeks (Figure 1). Production bleeds and library construction after approximately six weeks gives the best balance for the animals that have multiple antigens injected. Two rounds of panning were performed for each antigen, and isolated colonies screened (Figure 2). Sequencing of positive colonies identified diverse single domain antibody sequences for the different antigens. For examples, three unique clones were isolated to the reference antigen Maltose Binding Protein (MBP) (Figure 3A). As is frequently observed, the single domain antibodies contain significant sequence diversity and highly variable CDR3 length (Figure 3). These features are particularly important in single domain antibody/antigen interactions as seen in the reference single domain antibody/CX3CL1 complex17 (Figure 3B).
The majority of full-length single domain antibody sequences can be expressed and purified at 1-10 mg/L of culture (Figure 4A). Because of the diversity in CDR3 length, different single domain antibodies show slight variation in molecular weight. Confirmation of direct binding and application specific performance is critical. For example, after two rounds of selection against Antigen B, a panel of single domain antibodies were identified (Figure 2). Multiple identical sequences were identified, and the single domain antibody was produced and purified. It was then tested via direct pull down with antigen affinity resin and showed robust dose-dependent binding (Figure 4B). Importantly, the single domain antibody showed no binding to control resin. These data demonstrate a direct binding interaction, and one that is well suited for affinity capture in both pull-down and ELISA-based formats.
<img alt="ELISA result chart, Absorbance vs. Serum dilution, Antigen A/B, Preimmune/Immunized, data analysis." class="xfigimg" src="/files/ftp_upload/58471/58471fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Representative immune monitoring of two distinct antigens showing the significant specific immune response to distinct antigens in the same animal. Many antigens show robust response as early as three weeks after commencing immunization, with the majority showing maximal response after six weeks. Six weeks also allows for affinity maturation and is the recommended time for the production bleed and B-cell isolation. <a href="https://www.jove.com/files/ftp_upload/58471/58471fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="DNA electrophoresis results, PCR gel showing clone analysis, 100-500bp bands for genetic validation." class="xfigimg" src="/files/ftp_upload/58471/58471fig2.jpg" data-altupdatedat="1788250015000" data-alt="Figure 2"> 
Figure 2: PCR-based confirmation of positive clones. Primers span the MCS of the pMES4 vector, and a positive nanobody clone produces an amplicon of ~500 bp (marked with *). <a href="https://www.jove.com/files/ftp_upload/58471/58471fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Protein structure analysis; sequence alignment, 3D protein diagram, CDR1-3 regions, bioinformatics." class="xfigimg" src="/files/ftp_upload/58471/58471fig3.jpg" data-altupdatedat="1788250015000" data-alt="Figure 3"> 
Figure 3: Antigen-specific sequences highlight alpaca single domain antibody diversity. (A) Alignment of select single domain antibody sequences isolated to MBP with a reference single domain antibody 4XT1, with framework and complementarity-determining regions (Kabat) highlighted below. (B) Structure of alpaca single domain antibody 4XT1 bound to CX3CL1 (PDB 4XT1), emphasizing the critical role of variable CDR loops in specific antigen binding. <a href="https://www.jove.com/files/ftp_upload/58471/58471fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="SDS-PAGE electrophoresis results showing protein bands; analysis with antigen concentration gradient." class="xfigimg" src="/files/ftp_upload/58471/58471fig4.jpg" data-altupdatedat="1788250015000" data-alt="Figure 4"> 
Figure 4: Purification and validation of single domain antibodies. (A) SDS-PAGE of IMAC purified single domain antibodies, comparing diverse single domain antibodies. Different molecular weights are primarily due to varying length of the CDR3 loop. Differing levels of expression are also observed, with a minority of single domain antibodies showing poor yields of purified protein. (B) Verification of direct binding using an antigen affinity pull-down. Robust dose-dependent single domain antibody binding is observed with antigen-coupled resin but not with control resin. <a href="https://www.jove.com/files/ftp_upload/58471/58471fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies ...

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Learn to produce single-domain antibodies from alpacas using immunization, blood collection, and phage display selection. This method generates diverse antibodies for therapeutic and diagnostic applications.

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

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