VHH Antibody Discovery: Mixed Immunization

Biointron has developed a successful method for discovering alpaca VHH for over 200 projects, using flexible immunization strategies and a robust phage display screening platform. The alpacas used in the immunization process are all healthy and naïve and reside in an owned breeding base. Depending on the target, Biointron can use purified protein, DNA, over-expressing target cell lines, or a hybrid of these methods to immunize and achieve optimal serum antibody titer levels. Biointron creates an immunized phage library using PBMC samples for phage panning. After two to three rounds of solid and liquid-based panning in parallel, Biointron selects antigen-specific binders to obtain VHH sequence. The candidates are then used to express and purify on Biointron's high-throughput mammalian cell expression platform for further characterization.

This case study demonstrates a key feature of our VHH antibody discovery service: the successful discovery of VHH sequences for individual targets through co-immunization of up to four target proteins in a single alpaca. This involved injecting an alpaca with four different target proteins A, B, C, and D. In order to increase antibody diversity for all targets, the injection of target C was stopped during the fourth immunization, and PBMC from the third and fourth immunizations were used for library construction. As a result, a large number of unique VHH binder sequences with good affinity diversity were obtained for all four targets.