Abcore generates nanobodies most frequently using immunized llamas. This approach to custom llama nanobody discovery results in nanobodies which have undergone natural selection of various nanobodies during clonal expansion of B-cells in the lymphoid organs of the immunized llamas.
Abcore has developed proprietary procedures for nanobody discovery. Our optimized library construction and screening processes with phage display technology guarantee successful isolation of high affinity llama nanobodies in a short time. We have successfully produced multiple positive clones for a variety of antigens, ranging from large protein molecules (>400kD) to small haptens (small chemical molecules of ~200 dalton), many of the binders have sub-nanomolar affinities measured by ELISA.
Details of VHH single domain antibody production:
- Llama immunization: Animal immunization is handled by Abcore exclusively. Abcore is well established for llama immunization. Scientists at Abcore are very knowledgeable on polyclonal antibody production in a variety of animals. Their experiences are indispensable on generating good VHH antibodies since high antibody titer in the animal blood is the first step to guarantee the success of VHH antibody isolation.
- Phage display library construction: We monitor the anti-serum titer by ELISA and collect multiple production bleeds and PBMC cells throughout the immunization. The PBMC cells are preserved in RNA lysis buffer at different time point, so that the antibody repertoires are preserved through the immunization process. At the end of the immunization protocol, the client could choose the PBMC at any collection time point to construct the library.
- VHH library panning and screening: The constructed library will be screened through multiple rounds of affinity binding/selection with antigen. Positive clones will be identified through ELISA. Genes of these positive clones will be sequence analyzed. Affinity of each binder will be determined through ELISA. Depending on the complexity of the antigen, one to three clones with the best affinity will be delivered.
- We have one of the largest llama immunization facilities in the nation. Scientists and staff at Abcore are very knowledgeable on immunization of a variety of animals. Their experiences are indispensable on generating good VHH antibodies since high antibody titer in the animal blood is the first step to guarantee the success of VHH antibody isolation.
- We use the freshest cells and best quality RNA to make the library. Since VHH single domain library is constructed with mRNA that encodes the antibody genes, it is critical to capture the expression profile of the antibody producing B cells. It is also well known that expression profile and mRNA composition change rapidly once the cells are taken out of their native environment. Therefore, we make every effort to preserve the health of cells before lysing them for RNA isolation. All animal bleed are processed in the same day (usually within 2-4 hours) to purify the peripheral mononuclear cells (PBMC).
- We use the best quality RNA to make the cDNA. All RNAs are double-purified with phenol-chloroform extraction and spin columns. RNA qualities are examined by electrophoresis before being used to make cDNA.
- Our proprietary PCR primers ensure the maximum coverage of VHH repertoires. We have performed extensive bioinformatics research on VHH genes and designed novel primers for RT-PCR to amplify VHH cDNA. VHH libraries constructed with these primers have much higher diversity and larger coverage of the VHH repertoires. In fact, we have identified several pico-molar affinity VHH binders that have unique sequences and would have been missed if published primers were used.
- We deliver libraries with large numbers of independent clones. Our proprietary PBMC isolation and library construction protocol routinely yields >1×108 PBMC cells from each production bleed.
- We understand client’s need. Our principal scientist will discuss the project directly with clients to custom design each project. We offer flexible service schedules to fit client’s lab setup and budget (see additional services).