Label-free detection of immune complexes with myeloid cells

Abstract

SummaryThe aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins.

Detection and functional resolution of soluble multimeric immune complexes by a comprehensive FcγR reporter cell panel

“…Our methodology provides a comprehensive system, supporting the assessment of essentially all FcγRs, which presents an advantage over previously developed sIC detection and FcγR activation assays (Aoyama et al, 2019;Cheng et al, 2014;Hsieh et al, 2017;Stopforth et al, 2018;Szittner et al, 2016;Tada et al, 2014). In contrast to currently available commercial assays detecting sICs by C1q-CIC or C3d ELISA in the micromolar range, our assay measures overall sIC bioactivity in the nanomolar range and has a sole specificity for IgG sICs.…”

Detection of Experimental and Clinical Immune Complexes by Measuring SHIP-1 Recruitment to the Inhibitory FcγRIIB

“…Recently developed assays(14–17) designed to detect autoantigen or viral IC interactions with FcγRs, with the exception of the use of those employing tetrameric FcγRs(14), are plate-bound methodologies limited by the fact that antibodies require immobilization and are captured in all orientations (i.e. via both F(ab) and Fc regions).…”

“…These reporter-based assays do not require primary human immune cells as an effector cell source, are highly reproducible and so ideal for high-throughput screening purposes. There have also been recent advances in technologies for the detection of IC, focused on either the use of cell-free tetrameric(14) or dimeric(15) FcγRs to detect IgG-antigen-coated beads or surface immobilized IgG/IC, respectively, or cell-based methods to detect plate-bound IgG/IC(16, 17). Although potentially useful, these assays are designed to detect surface-immobilised IC binding to FcγRs, as opposed to IC in solution.…”

Why current quantitative serology is not quantitative and how systems immunology could provide solutions

“…By loading white blood cells onto protein microarray chips (Fig. 3 b), we were able to identify antigens against which antibodies were present and characterize the cell-activating property of those antibodies in antigen specific manner (Szittner et al 2013 , 2016 ; Kecse-Nagy et al 2016 ). This approach is particularly exciting when cells from the examined individual are used, since in this case in vivo conditions are simulated in vitro: interactions between own immunoglobulins and cells reveal such subtle differences that are completely concealed by traditional serological assays.…”