Detection and isolation of auto-reactive human antibodies from primary B cells


The isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (?105), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases.



Single-cell, antibodies, autoimmunity, nanowell array, ACPA


Copyright 2013 Methods. This is a post-print version of a published paper that is available at: Recommended citation: Sendra, Victor G., Anthony Lie, Gabrielle Romain, Sandeep K. Agarwal, and Navin Varadarajan. "Detection and isolation of auto-reactive human antibodies from primary B cells." Methods 64, no. 2 (2013): 153-159. doi: 10.1016/j.ymeth.2013.06.018. This item has been deposited in accordance with the publisher copyright and licensing terms and with the author's permission.