All procedures with the alpacas were performed in accordance with protocols (2017-2627 and 2018-2925) approved by the University of Kentucky's Institutional Animal Care and Use Committee (IACUC).
1. Generation of Antigen-specific Alpaca Antibodies
2. Purifying Lymphocytes for Single Domain Antibody Library Construction
3. Single Domain Antibody Panning
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.
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. Please click here to view a larger version of this figure.
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 *). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
GERBU FAMA adjuvant | Biotechnik, Heidelberg, Germany | 3003,6001 | |
Serum collection tube | Becton Dickinson | 367983 | |
Blood collection tube | Becton Dickinson | 366643 | |
Vacutainer blood collection set | Becton Dickinson | 368652 | |
Maxisorp Immuno plates | Nunc | 439454 | |
BSA | Sigma-Aldrich | A7906 | |
HRP-conjugated goat anti-llama IgG antibody | Bethyl Labs | A160-100P | |
TMB reagent chromogenic peroxidase substrate | KPL | 50-76-03 | |
Plate Reader | Spectramax | M5 | Any UV/VIS capable reader is acceptable |
Uni-SepMAXI+ lyphocyte separation tube | Novamed | U-17 | |
RNeasy Mini Kit | Qiagen | 74104 | |
QIAshredder column | Qiagen | 79654 | |
Superscript IV reverse transcriptase | Invitrogen | 18064014 | |
AEBSF solution | Biosynth | A-5440 | |
TG1 phage display competent cells | Lucigen | 60502 |
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.
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.
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.