The technology enables rapid and accurate sequencing of paired VH and VL antibody transcripts from single antigen-specific B cells. It involves isolating B cells, capturing their mRNA and antibodies, forming complexes with affinity agents, purifying these complexes, and performing RT-PCR to generate and sequence cDNA, facilitating various immunological and biotechnological applications.
The ability to monitor the antigen-specific immune response, particularly the depth and breadth of B cell repertoires, has been a long-standing challenge in immunology. Traditional methods for isolating and characterizing antigen-specific B cells, such as flow cytometry, are hampered by issues like false positives due to nonspecific binding and the need for expensive, complex equipment. Additionally, these methods often fail to adequately sort rare cells or those with low membrane-bound immunoglobulins, such as plasmablasts and plasma cells.
Current techniques, including microengraving and microfluidic platforms, also face limitations such as high costs, technical complexity, and difficulties in handling and processing cells. These challenges are particularly pronounced in vaccine research and the study of immune responses to pathogens, where understanding the diversity and specificity of antibody responses is crucial. A more efficient, high-throughput method for isolating and sequencing antigen-specific antibody transcripts from individual B cells could significantly advance our ability to study and manipulate immune responses.
The technology involves a method for high-throughput sequencing of paired VH (variable heavy) and VL (variable light) antibody transcripts from single antigen-specific B cells. The process starts by isolating individual B cells in compartments containing capture agents that bind both mRNA and antibodies. After the cells secrete antibodies and are lysed, the capture agents, now bound with mRNA and antibodies, are incubated with affinity agents that bind to the antibodies to form complexes. These complexes are then purified using techniques such as magnetic separation or density-based centrifugation.
The purified complexes are emulsified, and reverse transcription polymerase chain reaction (RT-PCR) is performed to generate linked amplification products containing the VH and VL cDNAs. The final step involves sequencing these products to identify the antibody sequences from each single cell. This method allows for the rapid and accurate determination of paired antibody repertoires from many individual B cells, which is beneficial for various applications in immunology and biotechnology.
This technology is differentiated by its ability to capture and sequence paired VH and VL transcripts from single B cells in a high-throughput manner, overcoming significant limitations of previous methods. Traditional techniques often involve bulk mRNA isolation and separate sequencing of heavy and light chains, which do not provide information on the natural pairing of VH and VL chains within individual cells. The described method uses a combination of capture agents, affinity agents, and advanced purification techniques to ensure that both VH and VL transcripts from the same cell are linked and sequenced together. This approach minimizes false positives and negatives, reduces the need for complex and expensive equipment like flow cytometry, and allows for the analysis of cells with little to no surface immunoglobulin.
Moreover, the technology can be adapted for various species and antibody isotypes, making it highly versatile and valuable for in-depth studies of immune responses and antibody discovery.
https://patents.google.com/patent/US10513733B2/en?oq=+15%2f078%2c507