Hi! This is my first post on Blopig as I joined OPIG in July 2017 for my second rotation project and DPhil.
During immune reactions to foreign molecules known as antigens, surface receptors of activated B-cells undergo somatic hypermutation to attain its high binding affinity and specificity to the target antigen. To discover how somatic hypermutation occurs to adapt the antibody from its germline conformation, we can compare the naive and antigen-experienced antibody repertoires. In this paper, the authors developed a protocol to carry out such comparison, detected, synthesised, expressed and validated the observed antibody genes against their target antigen.
What they have done:
- Mice immunisation: Naive (no antigens), CGG (a large protein), NP-CGG (hapten attached to a large protein).
- Sequencing: Total RNA was extracted from each spleen, cDNA was synthesised according to standard procedures, and amplified with the universal 5’-RACE primer (as oppose to the degenerate 5’-Vh primers) and the 3’-CH1 primer to distinguish between immunoglobulin-classes (IgG1, IgG2c and IgM). High throughput pyrosequencing was then used to recover the heavy chain sequences only.
- VDJ recombination analysis: V, D and J segments were assigned and the frequency of the VDJ combinations were plotted in a 3D graph.
- Commonality of the VDJ combination: For each VDJ combination, the “commonality” was counted from the average occurrence if n mice have the combination: if n=1, it’s the average occurrence if any 1 mouse has the combination; if n=5, the combination must be observed in all mice to generate a degree of commonality – otherwise it’s 0.
- The effect of increasing n on commonality scores in IgG1 class: As we tighten the requirement for the commonality calculation, it becomes clear that IGHV9-3 is likely to target the CGG carrier, while IGHV1-72 is against the NP hapten.
- IGHV9-3 can accommodate a wider range of D gene when targeting CGG alone. IGHV1-72 only uses IGHD1-1.
- Clustering V gene usage: Sequences were aligned to the longest sequence in the set (of VDJ combination), and the pairwise distance between sequences in the set were used to cluster the sequences using the UPGMA method.
- A number of sequences were commonly found in different individuals. Among these sequences, one was randomly selected to proceed to the next step.
- Synthesis and validation of the detected antibody against the NP hapten: by comparing the antibody repertoires against the CGG and NP-CGG, the gene of the antibody against NP can be recovered. The authors in this paper chose to pair 3 different light chains to the chosen heavy chain, and assess the binding of the 3 antibodies.
- NP-CGG bind well to both IGHV1-72 and IGHV9-3 antibodies; NP-BSA to IGHV1-72 only; and CGG to IGHV9-3 only.
- The binding capabilities are affected by the light chain in the pair.
This work presented a metric of defining the “commonality” between individuals’ antibody repertoire and validated the identified antibody against its target antigen by combining with different light chains.