Single-cell antibody nanowells: a novel technology in detecting anti-SSA/Ro60- and anti-SSB/La autoantibody-producing cells in peripheral blood of rheumatic disease patients

Abstract

Background: Anti-SSA/Ro60 and anti-SSB/La are essential serological biomarkers for rheumatic diseases, specifically Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Currently, laboratory detection technology and platforms are designed with an emphasis on high-throughput methodology; therefore, the relationship of sensitivity with specificity remains a significant area for improvement. In this study, we used single-cell antibody nanowells (SCAN) technology to directly profile individual B cells producing antibodies against specific autoantigens such as SSA/Ro60 and SSB/La.

Methods: Peripheral blood mononuclear cells were isolated using Ficoll gradient. Fluorescently labeled cells were added to fabricated nanowells and imaged using a high-speed epifluorescence microscope. The microengraving process was conducted using printed slides coated with immunoglobulins. Printed slides were hybridized with fluorescence-conjugated immunoglobulin G (IgG), SSA/Ro60, and SSB/La antigens. Microarray spots were analyzed for nanowells with single live B cells that produced antigen-specific autoantibodies.

Results: Our results indicate that SCAN can simultaneously detect high frequencies of anti-SSA/Ro60 and anti-SSB/La with a specific IgG isotype in peripheral blood mononuclear cells of patients, as well as measure their individual secretion levels. The data showed that patients with SS and SLE exhibited higher frequency and greater concentration of anti-SSA/Ro60- and anti-SSB/La-producing B cells in the IgG isotype. Furthermore, individual B cells of patients produced higher levels of IgG-specific anti-SSA/Ro60 autoantibody, but not IgG-specific anti-SSB/La autoantibody, compared with healthy control subjects.

Conclusions: These results support the application of SCAN as a robust multiparametric analytical bioassay that can directly measure secretion of autoantibody and accurately report antigen-specific, autoantibody-producing cells.

Keywords: Anti-SSA/Ro60; Anti-SSB/La; Antinuclear autoantibody; Single-cell analysis; Sjögren’s syndrome; Systemic lupus erythematosus.

Fig. 1

Single-cell antibody nanowell process. Arrays of nanowells with dimensions of 50 μm × 50 μm × 50 μm were used for microengraving. Peripheral blood mononuclear cells were loaded into the nanowells. Cells in the nanowells were imaged using an automated epifluorescence microscope. Micrograving was performed by hybridizing nanowells with capture slides containing antihuman immunoglobulins for 1 h at 37 °C with 5 % CO₂. After incubation, nanowells containing intact live cells and capture slides were separated. A mixture of goat antihuman immunoglobulin G (IgG)-Alexa Fluor 647 (AF647) and fluorochrome-conjugated SSA/Ro60-AF488 and SSB/La-AF550 were added to the capture slides. Micrographs of microarrays were generating by scanning using a GenePix Autoloader 4200AL microarray scanner. The schematic has been modified from a previous study [17]

Fig. 2

Profiling anti-SSA/Ro60- and anti-SSB/La-producing B cells using single-cell antibody nanowell technology. a Representative micrographs of cells (bright field [BF]) in nanowells labeled with calcein (live cells) and CD19-Alexa Fluor 488 (AF488). Micrographs of matching microarray by microengraving showing detection signals for immunoglobulin G (IgG)-AF647, anti-SSA/Ro60-AF488, and anti-SSB/La-AF550. b Profiling of autoantibody-producing B cells in nanowells (n = 9 healthy control subjects, n = 13 pSS/SLE patients). Data extracted by image processing with GenePix software were used to identify the appropriate signals. The data were correlated with the nanowell image data in which nanowells contained a single cell positive for both calcein and CD19. The percentage of CD19⁺ B cells producing IgG, anti-SSA/Ro60, anti-SSB/La, anti-SSA/Ro60 in IgG isotype, and anti-SSB/La in IgG isotype was determined by using the count of positive signals from wells with single cells and the total number of wells with single cells. *p < 0.05, **p < 0.01 by unpaired t test

Fig. 3

Quantifying the concentration of anti-SSA/Ro60 and anti-SSB/La produced by individual B cells. A standard curve for the antibodies produced by each cell was constructed by applying a series of concentrations of corresponding fluorochrome-conjugated antihuman immunoglobulin G (IgG) (e.g., 1 nM to 10 μM) to the set of replicate microarrays and then measuring the mean fluorescence intensity of captured anti-SSA/Ro60 and anti-SSB/La as a function of concentration using data processing of micrographs of microarrays. a Concentration of each signal spot from nanowells with single live CD19⁺ B cell producing IgG-specific anti-SSA/Ro60. ^{^}Seronegative patients for anti-SSA/Ro60 autoantibody. b Combined concentrations of individual positive signal spots (live CD19⁺ B cells secreting IgG-specific anti-SSA/Ro60 autoantibody) from patients and healthy control subjects. c Concentration of each signal spot from nanowells with single live CD19⁺ B cell producing IgG-specific anti-SSB/La. ^{^}Seronegative patients for anti-SSB/La autoantibody. d Combined concentrations of individual positive signal spots (live CD19⁺ B cells secreting IgG-specific anti-SSB/La autoantibody) from patients and healthy control subjects. X-axis in (a) and (c) denotes healthy and patient coded personal identification. *p < 0.05. NS not significant by unpaired t test