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

Abstract

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.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

“…However, upon their adjustment to prolonged mammalian assays, they require multilayer fabrication, either involving high-pressure cell loading or not allowing for different substrates to be used (26,27). Several single-cell platforms that encapsulate cells in stationary droplet arrays are available (5,(28)(29)(30)(31)(32)(33)(34)(35); however, the ability to culture adherent cells for long periods remains difficult (36).…”

Single-cell analysis tools for drug discovery and development

“…For both SCBCs and microengraving, cells that exhibit unique or desirable protein signatures may be further analyzed. 34 CyTOF can capture large panels of ‘secrete-able’ proteins, but protein secretion must be blocked and the cells fixed prior to analysis, and so the detected proteins are not actually secreted, and the cells cannot be further analyzed 13 . For the microfluidics tools, the cells can be imaged in situ , so that factors such as cell motility or morphology can be correlated with the secretion of specific proteins 94 .…”

“…Microfluidic designs can also permit cell analysis within highly controlled, custom environments, 31-33 or can allow for non-destructive cell analysis, so that cells identified as interesting, such as B cells producing specific antibodies, can be harvested for further use. 34, 35 Two recent tissue staining methods, in situ RNA profiling via sequential hybridization 36-38 , and proteomic analysis via ion beam profiling 39 can enable the analysis of single cells within fixed, intact tissues, with a level of multiplexing that significantly exceeds traditional immunohistochemical staining methods. The level of analyte quantitation varies from measurements that yield copy numbers per cell 22, 36, 40 , to relative quantitation between cells.…”

Microscale Technologies for High-Throughput Analysis of Immune Cells

“…The methodology has proven useful for the detection of anti-insulin secreting B cells isolated from the blood of an adult recent-onset type-I diabetic patient who had not yet received insulin therapy [ 58 ]. This nanowell microengraving approach was further advanced to incorporate antibody V H and V L pairing by scRT-PCR following micromanipulation extraction of cells exhibiting the desired phenotype [ 51 ]. Using this technique Sendra et al were able to identify and isolate antibodies specifi c for anti-citrullinated protein, a diagnostic and disease marker for rheumatoid arthritis [ 51 ].…”

“…This nanowell microengraving approach was further advanced to incorporate antibody V H and V L pairing by scRT-PCR following micromanipulation extraction of cells exhibiting the desired phenotype [ 51 ]. Using this technique Sendra et al were able to identify and isolate antibodies specifi c for anti-citrullinated protein, a diagnostic and disease marker for rheumatoid arthritis [ 51 ]. Additionally, the microengraving technique is easily multiplexed for probing specifi city to multiple antigens and classifi cation of antibody isotype [ 52 ].…”