Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sjogren's syndrome

Abstract

Sjogren's syndrome (SjS) patients often have a variety of extraglandular manifestations including neurological and gastrointestinal involvement. In this study we evaluated the diagnostic performance of luciferase immunoprecipitation system (LIPS) that employs mammalian cell-produced recombinant antigens for analyzing SjS autoantibody responses. LIPS testing of mammalian cell-produced La, Ro60 and Ro52 recombinant antigens with defined commercial antibodies demonstrated highly specific immunoprecitation of each antigen without cross-reactivity. Next, sera from 57 SjS and 25 volunteers were evaluated by LIPS against a panel of human autoantigens. LIPS detected robust anti-La antibody levels in 43/57 SjS patients (75% sensitivity) and markedly outperformed an ELISA (46% sensitivity). Profiling of other SjS-associated autoantigens revealed the presence of anti-Ro60, anti-Ro52 in 63% and 61%, of SjS patients, respectively. Interestingly, a C-terminal fragment of Ro52 (Ro52-Delta2), a protein fragment not previously found to be antigenic by ELISA, also showed positive immunoreactivity in 42/57 SjS patients (65% sensitivity). Additional profiling of other autoantigens revealed that certain SjS patients also showed positive immunoreactivity with thyroid peroxidase (14%), AQP-4 (12%) and the H(+)/K(+) gastric ATPase (16%) suggesting potential autoantibody attack of thyroid, neuronal and gastric parietal cells, respectively. These heterogeneous autoantibody responses detected by LIPS in SjS will likely be useful for diagnosis and for evaluating extraglandular manifestations.

Figure 1

Validation of LIPS tests for detecting anti-La, anti-Ro52 and anti-Ro60 antibodies. As described in the material and methods, commercial antibodies to La, Ro52 and Ro60 were mixed with extract from lysates containing Ruc-La (A), Ruc-Ro52 (B) and Ruc-Ro60 (C) for 1 hour, incubated with protein A/G beads for an additional hour, processed and light units measured. The mean LU plus standard error is shown for each immunoprecipitation determination.

Figure 2

LIPS detection of autoantibodies against La in 25 normal volunteers and 57 primary SjS patients. (A). Each circle or square symbol represents the anti-La antibody titer of a normal volunteer (NV) or SjS patient sample, respectively. For determining sensitivity and specificity for this anti-La antibody test, the dashed line represents the cut-off level derived from mean plus 3 SD of the antibody titer of the 25 normal volunteers, while the solid line is the cut-off for the mean plus 5 SD. p-values were calculated using the Mann Whitney U-test. (B). Roc plot analysis of anti-La antibodies detected by the LIPS test. Area under the curve (AUC) = 0.88.

Figure 3

LIPS detection of autoantibodies against Ro60, Ro52 and a C-terminal fragment of Ro52 in 25 normal volunteers and 57 primary SjS patients. Each circle or square symbol represents an individual normal volunteer (NV) or SjS patient sample, respectively. For determining sensitivity and specificity, the dashed line represents the cut-off level derived from mean plus 3 SD of the antibody titer of the 25 normal volunteers, while the solid line is the cut-off for the mean plus 5 SD. (A) The anti-Ro60 antibody test. (B) The anti-Ro52 antibody test. (C) The anti-Ro52-Δ2 antibody test. p-values were calculated using the Mann Whitney U-test.

Figure 4

LIPS detection of anti-TPO, gastric H/K ATPase and anti-AQP4 antibodies. Each circle or square symbol represents an individual normal volunteer (NV) or SjS patient sample, respectively. For determining sensitivity and specificity, the dashed line represents the cut-off level derived from mean plus 3 SD of the antibody titer of the 25 normal volunteers, while the solid line is the cut-off for the mean plus 5 SD. (A) Anti-TPO autoantibodies, (B), Anti-H + /K + gastric ATPase autoantibodies (C). Anti-AQP-4 autoantibodies.

Figure 5

Heatmap representation of SjS patient autoantibody profiles. Autoantibody titers to 7 autoantigens are shown for each of the 25 NVand 57 SjS patients. The titer values greater than the mean of the 25 normal volunteers plus 3 standard deviations were color-coded from clear to dark blue to signify the relative number of standard deviations above these reference values.