Abstract
Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We performed a proof-of-concept using mixed mouse hybridoma cells and we also showed that our method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19+ B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization. We anticipate broad applicability of our method for providing insights into adaptive immune responses associated with various diseases, vaccinations, and cancer immunotherapies.
Highlights
High-throughput sequencing of immunoglobulin repertoires from B cells has emerged as a powerful tool to investigate repertoire changes for antibody discovery, vaccine efficacy studies, and in other healthcare applications [1,2,3]
Microsome-associated messenger RNAs (mRNA) can be used to retain native antibody heavy chains (HC)-LC pairs with high pairing accuracy Our approach is based on the concept that each B cell contains rough endoplasmic reticulum with bound ribosomes for co-transcriptional translocation of secretory proteins
We reasoned that rough endoplasmic reticulum (rER) microsomes obtained after cell lysis should retain the correctly paired HC and LC mRNAs of each individual B cell and represent the smallest subcellular entity comprising both types of mRNAs
Summary
High-throughput sequencing of immunoglobulin repertoires from B cells has emerged as a powerful tool to investigate repertoire changes for antibody discovery, vaccine efficacy studies, and in other healthcare applications [1,2,3]. Retaining paired HC-LC data from bulk B cell populations at single-cell level remained a major obstacle for a long time. To this end, several single-cell paired sequencing technologies were reported more recently, which were initially limited by low cell numbers (< 400–105 cells) and sometimes required the use of complex microfluidic systems [8,9,10,11,12]; cellular throughput is improving through newer developments, such as droplet-based systems and the 10× Genomics platform [13, 14]. Two emulsion-based methods reported paired HC-LC repertoire sequencing from 2–3 × 106 B cells at single-cell
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