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The development of high affinity VHH antibodies follows the well-established pathway shown above.
JIR Anti-Alpaca antibodies can be utilized at specific stages to expedite and enhance the development of high affinity VHH antibodies. The success and speed of discovering high quality VHH candidates is optimized by the fidelity and utility of the tools used at each step.
Use JIR Anti-Alpaca IgG, subclasses 2+3 to time PBMC harvest for VHH library construction. The VHH domain is located on these subclasses only. Subclass 2+3 response may mature differently from total IgG, and monitoring serum conversion of these antibodies can inform the decision to harvest PBMCs. Timing PBMC harvest based on total IgG titer might suggest a collection point at 150 days, but the IgG 2+3 response was not maximal until after 300 days post immunization. This ELISA data shows the antibody subclass repertoire in response to the immunogen over time. Antiserum was used to detect the coated immunogen, and peroxidase-conjugated secondary antibodies were used to compare total IgG to IgG subclasses 2+3.
Anti-Alpaca IgG, VHH domain antibodies show amplified signal compared to an anti-tag antibody. Polyclonal antibodies that broadly recognize many epitopes on VHH deliver additional signaling molecules (e.g. fluorophores or enzymes) vs. antibodies narrowly focused on small peptide sequences. In this figure, a His6 tagged recombinant VHH was detected by western blot using Goat Anti-VHH domain antibody or an anti-His6 antibody.
Screening VHH-expressing clones for solubility and binding activity is commonly done by ELISA. Significant signal can be obtained even at low VHH concentrations. In this panel, VHH was titrated across its coated immunogen and detected with Peroxidase conjugated Goat Anti-Alpaca VHH domain antibody.
JIR’s Anti-Alpaca antibodies recognize both alpaca and llama IgG (H+L), subclasses 2+3, or VHH domain.
**These antibodies react primarily with the Fc region, and are not recommended for detection of VHH antibodies.
AffiniPure™ Antibodies is trademark of Jackson ImmunoResearch Laboratories, Inc.
*Nanobody is a registered trademark of Ablynx N.V
DyLight™ fluorescent dyes is a trademark of Thermo Fisher Scientific.
Cy™ is a registered trademark of GE Healthcare.
Rhodamine Red™-X is a trademark of Invitrogen.
Alexa Fluor® is a trademark of Life Technologies Corp.