Following stimulation with LPS and/or adiponectin for 24 h and 48 h, cells were incubated with XTT reaction solution for 4 h

June 21, 2017

ups were determined using Receiver Operator Characteristics and the sensitivity and specificity was calculated. The dynamic range and the statistical difference was determined for each antigen between the SLE and control samples. From LIPS testing, antibodies against the RNP-70k antigen demonstrated the greatest sensitivity in the antigen panel with 71% sensitivity and 94% specificity. The related RNP-A protein was only 59% MedChemExpress AZ-505 sensitive, and detected SLE patients who were all also seropositive for RNP-70k autoantibodies by LIPS. Detecting antibodies against the Sm antigen in SLE also showed a sensitivity of 59% and a specificity of 94%. Analysis of autoantibodies against Ro52 and Ro60, comprising the SSA antigen, using immunodominant protein fragments from each protein demonstrated 50% and 57% sensitivity, respectively and autoantibodies against SSB/La were Statistical analysis GraphPad Prism software was used for statistical analysis. Mann-Whitney U-tests were used to compare antibody titers among the different clusters. Cutoffs for sensitivity and specificity were determined using optimal separation based on receiver operator characteristics. Fisher’s exact test was employed to evaluate differences in autoantibody frequency between clusters. Heatmap assembly and determination of autoantibody enriched clusters To compare autoantibody titers between different antigens, a colored heatmap based on the Z score of each titer was employed. First, a cutoff was calculated based on the mean plus three standard deviations of the seronegative healthy controls for each antigen. This cutoff was subtracted from the autoantibody titer measured for each patient, and the resulting value was divided by the standard deviation of the control cluster to yield the Z score. Patients were then color coded by the number of standard deviations above the calculated cutoff. Sensitivity Core SLE antigens Ro52 Ro60 La Sm RNP-A RNP-70k Histone 2B Overall sensitivity of core antigens Interferons IFN-a IFN-v IFN-l IFN-c Overall prevalence of anti-IFN antibodies Neuronal proteins GAD65 AQP-4 TH GFAP Overall prevalence of anti-neuronal antibodies 30 12 30 16 47 13 29 17 10 42 50 57 49 59 59 71 42 88 Specificity Mean titer HC Mean titer SLE P 89 83 83 94 94 94 89 72 90,000 178,000 126,000 3,000 16,000 7,900 22,000 408,000 898,000 265,000 26,000 121,000 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189475 103,000 50,000 6 ,0.001{ 0.0019{ 0.0193{,0.0001{,0.0001{,0.0001{ 0.0047{ 94 100 100 94 94 1,600 2,600 19,000 7,200 2,900 7,400 66,000 7,500 0.1118 0.0030{ 0.0345{ 0.5708 89 94 89 100 77 2,500 5,000 13,000 5,000 9,400 10,000 22,000 0.0739 0.1441 0.1739 0.0763 Excluding Histone 2B. { Statistically significant, P,0.05. doi:10.1371/journal.pone.0032001.t001 3 Autoantibody Clusters in SLE detected in 49% of the SLE patients. Finally, in the LIPS assay, anti-histone 2B autoantibodies were the least sensitive, detecting only 42% of the SLE patients, but did not add to the overall diagnostic performance because seropositive patients were already positive for at least one of the other nuclear antigens by LIPS. Overall, a six antigen panel consisting of Sm-D3, RNP-A, RNP-70k, La, Ro52 and Ro60 detected at least one statistically significant SLE antibody in 88% of this SLE pilot cohort. As shown in Anti-interferon and anti-neuronal autoantibodies in Lupus patients Based on our previous ability to detect anti-cytokine and neuronal autoantibodies in other diseases by LIPS, a select panel of Ruc-antigen fusion proteins was evaluated in the