Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus

Abstract

High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice.

Figure 1. GMR biosensor autoantigen microarrays.

(a) Optical images of a GMR biosensor chip and a cartridge with a reaction well (left). The sensor chip measures 10 × 12 mm and consists of an array of 8 × 10 sensors (total 80 sensors). Each sensor size is 100 × 100 μm (right). (b) A schematic of assaying antibody reactivity to autoantigens (not to scale). (1) Autoantigens were printed on the surface of the chip’s sensors. (2) The sample was added to the reaction well, allowing antibodies to bind to their corresponding antigens. (3) After washing, species-specific, biotinylated anti-IgG antibodies were used as a secondary reagent. (4) Streptavidin-coated MNPs bind to the biotinylated detection antibodies, and the respective sensor detects stray field from the bound MNPs.

Figure 2. Validation of GMR biosensor autoantigen microarrays with commercial antibodies, including PTM-specific antibodies.

Serial dilutions of monoclonal antibodies to (a) FLAG, (b) Ro52, (e) H2B K5Ac, and (f) H2B K20Ac, as well as polyclonal antibodies to (c,d) U1-70K, were used to probe individual GMR biosensor autoantigen microarrays printed with the cognate antigens and controls. The target epitopes K5Ac and K20Ac, are indicated in red in (e,f) respectively. Each data point is the average of signals from four replicate sensors on the microarray, and error bars represent standard deviations. The concentration of anti-H2B K20Ac was not provided by the manufacturer, and is presented as a relative concentration to the stock solution.

Figure 3. GMR biosensor autoantigen microarrays identify autoantibodies associated with dysregulation of the IFN pathway in SLE.

Serum samples from SLE patients with high (n = 15) or low (n = 15) chemokine scores were evaluated using GMR biosensor autoantigen microarrays. A single microarray was used to measure each sample. Significance analysis of microarrays (SAM) was used to identify autoantigens with significantly different antibody reactivity between the groups (q < 0.001, Wilcoxon). An unsupervised hierarchically clustered heatmap (Euclidean distance, complete linkage) shows the candidate autoantigens identified by SAM.

Figure 4. Antibodies to U1-70K are associated with high chemokine scores in SLE.

Serum samples from SLE patients with high (n = 24; red) and low (n = 20; blue) chemokine scores were assessed for IgG reactivity to U1-70K by indirect ELISA. P-value was determined using a Mann-Whitney test. Bars represent medians.

Figure 5. Antibodies to post-translationally modified forms of the H2B N-terminal tail are associated with dysregulation of the IFN pathway.

Sera from SLE patients with high (n = 15) and low (n = 15) IFN signatures as well as healthy controls (n = 15) were analyzed for IgG reactivity to peptides from the N-terminal tail of histone H2B by indirect ELISA. The peptides corresponded to acetylated (blue), dimethylated (red) and unmodified (black) forms of H2B 1-7 (a) and H2B 8-14 (b). P-values were determined using Mann-Whitney tests. Bars represent medians.