Secondary Antibodies For VHH Discovery – Jackson Immunoresearch

The development of high affinity VHH antibodies follows the well-established pathway shown. 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.

Test for VHH expression and binding activity with greater confidence

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.

AffiniPure™ Secondary Antibodies

Product Code

Goat Anti-Alpaca IgG (H+L)

128-005-003

Goat Anti-Alpaca IgG (H+L) (min X Bov, Hu, Ms, Rb, Rat Sr Prot)

128-005-160

Goat Anti-Alpaca IgG, subclasses 2+3 specific (min X Bov, Hu, Ms, Rb, Rat Sr Prot)

128-005-229

Goat Anti-Alpaca IgG, VHH domain (min X Bov Sr Prot)

128-005-232

Goat Anti-Alpaca IgG, VHH domain (min X Bov, Hu, Ms, Rb, Rat Sr Prot)

128-005-230

Horseradish Peroxidase

Alkaline Phosphatase

Biotin-SP

DyLight™ 405

Alexa Fluor® 488

Fluorescein (FITC)

Cy™ 3

R-Phycoerythrin (R-PE)

Rhodamine Red™-X

Alexa Fluor® 594

Alexa Fluor® 647

Cy™ 5