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Discovery of new autoantibodies in a serum as a biomarker for the diagnosis of Sjogren’s syndrome

  • 김승주 (SEUNG-JU KIM, 아주대학교 일반대학원)

초록/요약

Sjogren's syndrome (SS) is one of the autoimmune diseases characterized by sicca symptoms caused by the damage or destruction of exocrine glands by lymphocytic infiltration. SS is distinguished into primary SS (pSS) and secondary SS (sSS). According to the 2016 ACR/EULAR classification criteria, a total score ≥ 4 is classified as pSS with 5 weighted items in patients with at least 1 symptom of ocular or oral dryness. Considering the known positive rate of 50-70% for the anti-Ro antibody in serological tests, this study was performed to identify additional diagnostic biomarkers characterized by high sensitivity and specificity for SS. Based on the protein microarray results from patients with pSS and normal controls (NCs), we have identified 22 autoantibodies associated with pSS and have selected two protein candidates as potential biomarkers. Through protein microarray, anti-transmembrane 185B (TMEM185B) showed a positive rate of 78% in patients with pSS and anti-lysosomal protein transmembrane 4 alpha (LAPTM4A) showed a positive rate of 67%. Glutathione-S-Transferase (GST)-LAPTM4A and GST-TMEM15B were prepared as autoantigens, and the autoantibody was evaluated through enzyme-linked immunosorbent assay (ELISA) using sera from patients with pSS, sSS, and total SS (tSS) and the NCs. The titer was compared using the t-test. The anti-LAPTM4A antibody showed higher titer in patients with pSS, sSS, and tSS than the NCs. Also, the anti-TMEM185B antibody showed higher titer in patients with sSS and tSS than the NCs. The combined anti-LAPTM4A and anti-TMEM185B antibody showed higher titer in patients with pSS, sSS, and tSS than the NCs. The positive rate was 52% for the anti-LAPTM4A antibody and 45% for the anti-TMEM185B antibody. The positive rate of the combined anti-LAPTM4A and anti- TMEM185B antibody was 50%, and lower positive rate compared to the anti-LAPTM4A antibody alone. The positive rates for the anti-LAPTM4A antibody, the anti-TMEM185B antibody, and the combined anti-LAPTM4A and anti-TMEM185B antibody was 43% in tSS, 30% in pSS, and 48% in sSS in patients negative for the anti-Ro antibody. Therefore, the anti- LAPTM4A antibody is expected to play a role as a useful biomarker for SS. Keywords: Sjogren’s syndrome, Autoimmune disease, Biomarker, Autoantibody, Protein

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목차

I. INTRODUCTION 1
II. MATERIALS AND METHODS 4
A. Sampling of patients with Sjogren's syndrome 4
B. Detection of autoantibodies for Sjogren's syndrome 7
1. Protein microarray 7
C. Production of autoantigens for Sjogren's syndrome 9
1. Gene amplification by polymerase chain reaction (PCR) and purification 9
2. TA cloning 9
3. Production of Glutathione-S-Transferase (GST) tagged recombinant plasmids through LR reaction 12
4. Baculovirus expression system 13
5. GST-fusion protein production 14
6. Enzyme-linked immunosorbent assay (ELISA) 16
7. Statistical analysis 17
III. RESULTS 19
A. Screening of autoantibody candidates for Sjogren's syndrome using protein microarray 19
B. GST-LAPTM4A protein and GST-TMEM185B protein production 19
C. Analysis of anti-LAPTM4A and anti-TMEM185B antibodies for Sjogren's syndrome 26
D. Analysis of the positive rate of anti-LAPTM4A and anti-TMEM185B antibodies in patients with Sjogren's syndrome who have negative anti-Ro antibody 28
IV. DISCUSSION 30
V. CONCLUSION 34
REFERENCE 36

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