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. 2016 Jul;185(1):72-80.
doi: 10.1111/cei.12788. Epub 2016 May 12.

Label-free Detection of Immune Complexes With Myeloid Cells

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Free PMC article

Label-free Detection of Immune Complexes With Myeloid Cells

Z Szittner et al. Clin Exp Immunol. .
Free PMC article

Abstract

The 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.

Keywords: ACPA; Fc receptor; IgG; imaging SPR; monocyte.

Figures

Figure 1
Figure 1
U937 cells bind efficiently to human immunoglobulin (Ig) G1, IgG3 and IgG4, but not IgG2. (a) IgG subclasses, coupled equally, detected with kappa chain‐specific antibody. (b) Representative sensogram of IgG subclasses detected with U937 cells. (c) Cell binding expressed as percentage of the highest signal in each cycle. Data in (a,c) shows the mean of three replicate runs with three spots per run. Resonance unit values shown were recorded in the last 10 s of the 15 min incubation, marked with the dotted frame in (b). Bars represent means with error bars showing standard deviation.
Figure 2
Figure 2
Comparison of healthy serum donors (NHS) and rheumatoid arthritis (RA) sera reactivity towards immunoglobulin (Ig) G multiple antigenic peptides (MAPs) and subsequent recognition by U937 cells. (a) Representative sensograms recorded for NHS and RA samples, on various ligands, showing phases of the measurement (depicted in (b)], with dashed frames indicating the response values evaluated. (c) Results of serum association measured on IgG1 and IgG3, and (d) MAPs: histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2), and (e–f) the following cell binding response on these ligands are shown. Symbols in (c–f) mark the mean response unit obtained on the ligand subtracted by phosphate‐buffered saline (PBS) background. Statistical significance (c–f) was calculated by Mann–Whitney U‐test. (g) Comparison of serum and cell binding response on MAPs; here Spearman's rank correlation coefficients (r) were calculated.
Figure 3
Figure 3
Immunoglobulin levels against citrullinated peptides correlate with U937 responses in RA patients. (a) Histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2)‐specific immunoglobulin (Ig) A, IgG and IgM measured by microarrays and (b) IgG subclasses as determined by enzyme‐linked immunosorbent assay (ELISA) from rheumatoid arthritis (RA) samples were compared to cell response values obtained from the biosensor experiments measured on the same antigens. Symbols represent the mean of replicate measurements after subtraction of background values. Cell response on MAPs was expressed in ratio of cell response on hIgG1 10mM spots in the same cycle. Spearman's rank correlation coefficients (r) were calculated to evaluate the correlations.

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