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 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 1

Validation of serum factor array specificity and dynamic range.

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Validation of serum factor array specificity and dynamic range. (A) Qual...
(A) Qualitative visualization of array results showing the specificity of serum factor–targeted probes for cognate antigen printed in triplicate. 5 separate arrays were probed with Abs against IL-2, TNF, GMCSF, bFGF, and BAFF R/Fc (see Methods and Supplemental Table 1). Reactivity was detected using Cy5-conjugated secondary Abs. Fluorescent images of indicated array features are shown for each probing condition, with each antigen shown in triplicate. (B) Heatmap showing proportion of array-wide maximum MFI at the indicated antigens for arrays probed separately with 16 different Abs. (C–E) Range of detection for reagents (C) TNF mAb, (D) IL-2 pAb, and (E) BAFF R/Fc binding to cognate targets on serum factor microarrays. Data are mean ± SEM. (F) Array reactivity of a mixture of Abs targeting bFGF and BAFF, either uncleared (No clear) or subjected to 3 rounds of preclearing (1×, 2×, 3×) with BAFF-conjugated beads. Data (mean ± SEM) are shown as percent maximum MFI signal (relative to uncleared). (G) Array reactivity of a mixture of Abs targeting IL-2 and BAFF, either without competition (No comp) or incubated with 0.5:1, 1:1, or 5:1 molar ratios of unconjugated BAFF cytokine/Ab (0.5×, 1.0×, 5.0×, respectively). Data (mean ± SEM) are shown as percent maximum MFI signal (relative to no competition).