Identification and Characterization of Novel Autoantibody Biomarkers for Rheumatoid Factor-Negative and Accp-Negative Rheumatoid Arthritis
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease in which 30% of patients are seronegative for the two serological RA markers, rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (ACCP). However, both markers also have lower sensitivities in early stages of the disease. The lack of effective biomarkers for this subpopulation of RA patients causes a delayed diagnosis. Therefore, discovery of novel circulating autoantibody biomarkers for these early RF-negative and ACCP-negative RA patients are advance planning of these subsets. 136 enriched antigenic markers were identified by high-throughput screening an RA library with autoantibodies in the sera of RA patients (positive selection) and healthy control (negative selection). Phage-ELISA-based re-screening determined 13 out of 136 enriched antigenic targets in which increased antibody levels rised up. Two of the 13 identified targets, namely special A-T rich DNA binding protein (SATB1) and EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), which had highest increased antibody levels, were selected for further characterization. Reactivity of serum antibodies against these proteins was tested in a protein ELISA using the sera of 137 RA patients and 159 healthy controls. Autoantibodies against SATB1 were detected with 79% sensitivity and 82% specificity, while 87% sensitivity and 52% specificity were obatained for antibodies directed against EFEMP1. With regard to cytokine assay, interestingly, pro-inflammationary cytokines including TNF (tumour necrosis factor) increased over 1000 pg/ml or IL-4 (interleukin-4) or IL- 13 similarly increased up to 90 pg/ml and 102 pg/ml, respectively for co-culture with anti-SATB1 monoclonal antibody. Under treatment with EFEMP1 mAb resulting IL-1, TNF and IL-4 triggered up to 1300 pg/ml, 1250 pg/ml and 180 pg/ml, respectively. In T cell phenotype characterization, two subsets of CD4 + T cell and CD8 + cell were stimulated and differentiated from peripheral blood mononuclear cells (PBMCs) at 29.7% and 1.2% for anti-SATB mAb; 31.3% and 2.8% for anti-EFEMP1 mAb.
KeywordsSATB1 EFEMP1 biomarkers rheumatoid arthritis phage display
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