By Dr. Kushal Suryamohan, Director of Bioinformatics Services & Aditya Pai, Vice President, Corporate and Business Development, MedGenome Inc, USA
Antibody discovery has gained immense prominence with the COVID-19 pandemic. Numerous technologies are available to produce antibodies. For several decades now, hybridoma technology has been the mainstay to generate monoclonal antibodies by immortalization of antigen-specific B-lymphocytes.
Hybridomas are cells formed by a fusion between an antibody-producing B-cell which is short-lived and an immortal myeloma cell. Each hybridoma expresses a large amount of one specific antibody (monoclonal antibody). If a hybridoma is stable, it can be used and cryopreserved as a long-term source of monoclonal antibody production. The workflow for creating hybridomas involves several technical procedures, including antigen preparation, animal immunization, cell fusion, hybridoma screening and sub-cloning as well as characterization and production of specific antibodies. Despite the fact that hybridomas have fueled the discovery and production of antibodies for a multitude of applications, the technology has some disadvantages. These include contamination of hybridoma cultures, the range of useful antibodies generated given the losses that occur during the fusion process, genetic drift over time leading to batch effects, time, cost to maintain and the limitations in use in rat and mice. Furthermore, generation and identification of high-quality hybridoma clones is a fairly labor-intensive, low-throughput process, and requires months of work during the time frame from immunization to specific hybridoma identification.
Newer antibody discovery methods such as phage display technologies overcome some of the limitations of hybridoma technology. However, with evolving sequencing technology, MedGenome has developed and standardized, HitMab (High-throughput antibody discovery using single cell sequencing), a streamlined workflow for antibody discovery using high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired full-length variable regions in a massively parallel fashion. This method allows for the rapid discovery of thousands of antibodies leveraging single cell BCR sequencing and bypasses hybridoma-based antibody discovery. scBCR-seq can be used for rapid discovery of large, diverse panels of high-affinity antigen-specific antibodies with natively paired heavy- and light-chains when combined with high-quality antigen-specific B-cell sorting. By interrogating each individual B-cell, HitMab maximizes the antibody repertoires that we can access making it inherently superior to existing antibody discovery methods. HitMab provides users with a prioritized list of candidate antibody sequence panels with a fast turnaround time of 12 weeks that includes immunization of mice or rats with antigen of interest, isolation of individual B cells, scBCR-seq, antibody cloning, recombinant IgG expression and purification, and initial testing of binding properties (Figure 1). Briefly, mice or rats are immunized with a specific antigen of interest. From the antibody forming cells, B-cells are then isolated and each B-cell is processed through the 10X Chromium platform for single cell library preparation and sequencing for identification of paired heavy (Vh) and light (Vl)chain antibody sequences.
HitMab has some key advantages over traditional hybridoma-based antibody discovery:
- Does not rely on immortalizing B-cells and instead relies on isolating B-cells from spleen or lymph nodes of immunized rats or mice
- Cost effective and time efficient – obtain a panel of prioritized paired antibody sequences for testing/screening in 12 weeks compared to 6-8 months for hybridoma-based antibody discovery.
- Maximal antibody diversity compared to hybridoma where there is a lower B-cell repertoire due to fusion loss.
- scBCR-seq not restricted to mice and humans. MedGenome offers custom species including horse and rat.
For more information to customize a project to your needs, please contact email@example.com