Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus
Jordan V. Price, … , Emily C. Baechler, Paul J. Utz
Jordan V. Price, … , Emily C. Baechler, Paul J. Utz
Published December 2, 2013; First published November 25, 2013
Citation Information: J Clin Invest. 2013;123(12):5135-5145. https://doi.org/10.1172/JCI70231.
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Research Article

Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus

Abstract

Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor–binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor–binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell–activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α–driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE.

Authors

Jordan V. Price, David J. Haddon, Dodge Kemmer, Guillaume Delepine, Gil Mandelbaum, Justin A. Jarrell, Rohit Gupta, Imelda Balboni, Eliza F. Chakravarty, Jeremy Sokolove, Anthony K. Shum, Mark S. Anderson, Mickie H. Cheng, William H. Robinson, Sarah K. Browne, Steven M. Holland, Emily C. Baechler, Paul J. Utz

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Figure 5

Detection of BAFF blocking activity in SLE and HC samples using FL-17 cells.

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Detection of BAFF blocking activity in SLE and HC samples using FL-17 ce...
(A) Caspase luminescence signal in wells containing FL-17 cells incubated with a serial dilution of soluble recombinant human and mouse BAFF (hBAFF and mBAFF, respectively), showing a sensitivity range of 1.0–250.0 ng/ml. CPS, counts per second. (B) Luminescent signal of FL-17 cells incubated with recombinant human BAFF in the presence of no inhibitor, BAFF R/Fc, or TNF R/Fc. (C) Inhibition of luminescence signal of FL-17 cells incubated with 100 ng/ml recombinant human BAFF in the presence of a serial dilution of IgG purified from individual anti-BAFF–high (n = 15), anti-BAFF–low (n = 15), and HC (n = 10) serum samples. P < 0.001 for all between-group comparisons, 2-way ANOVA and Tukey correction for multiple comparisons. (A–C) Data represent mean ± SEM of ≥4 replicate wells. (D) Relationship between BAFF binding and BAFF blocking level in each of 40 SCIDRR cohort samples in the FL-17 assay. Blocking percentage was calculated as average CPS per sample normalized to maximum signal (no sample condition). (E–H) Caspase luminescence signal in FL-17 cells incubated with 100 ng/ml BAFF in the presence of (E) 10 μg/ml BAFF R/Fc, or serum IgG derived from (F) 6 anti-BAFF–high, (G) 4 anti-BAFF–low, and (H) 4 HC samples from the SCIDRR cohort, with or without preclearing with sepharose beads conjugated to BAFF or IFN-γ/IL-2. Data are mean ± SEM. P values (2-tailed) were calculated by Mann-Whitney U test.