Using rabbit as a model, we profiled changes in expression levels of IgH and Igk sequences before and after immunization with a human recombinant protein. We generated over 100 million sequences of the full variable region for IgG heavy and light chain mRNA transcripts. From the raw sequence data we measured changes in expression frequency and somatic hypermutation of variable region sequences.
Our immune repertoire analysis identifed the top 96 IgH and Igk sequences likely to be involved in the immune response and also provided a statistical likelihood of the pairing of IgH and Igk sequences. We synthesized and cloned these variable region sequences into our custom IgH and Igk expression vectors. Based on our pairing predictions for heavy and light chains, we expressed full IgG antibody proteins in a high-throughput plate-based format and screened for antigen binding with a custom bead-based ELISA assay. For the top binding antibodies identified, we validated binding specificity by western blot and successfully created humanized versions of the antibodies.
The antibody repertoires shown here are from a rabbit before and after injection with a human protein. We used a force-directed layout to display the CDRH3 sequences as nodes for which the most similar sequences are located next to one other. The frequency of each sequence is calculated before and after immunization, and the area of the node is proportional to the frequency of that sequence at that time point. As seen in these network maps, some sequences expand significantly after immunization.
Antibody repertoire sequence analysis identified the top 96 heavy:light sequence pairs to express and test for binding. Using our high-throughput gene synthesis and assembly pipeline, we cloned each variable region sequence into corresponding custom pAB_IgH and pAB_Igk expression vectors and expressed the full IgG antibodies in plate-based format. We then harvested the supernatants containing the secreted antibody protein and measured antigen binding using a custom bead-based ELISA assay. Figure 3 shows that 5 of the 96 antibodies show significant binding ranging from 10 to 80-fold over background.
For the top hits identified in our primary screen, we selected additional similar sequences from the sequence dataset to express and test for antigen binding. We paired 3 heavy chain sequences with 18 light chain sequences (CDRs shown). The binding results shown here highlight interesting sequence-activity relationships. Using this combination of approaches we rapidly identified a large set of rabbit monoclonal antibodies that strongly bind our antigen of interest.