Journal
ANALYTICAL BIOCHEMISTRY
Volume 652, Issue -, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2022.114721
Keywords
Live cell -based flow cytometry assay; Simultaneous autoantibody detection; Anti-aquaporin-4 autoantibody; Anti-myelin oligodendrocyte glycoprotein; autoantibody; Autoimmune inflammatory disorders
Funding
- Dr. Toshiyuki Takahashi (Tohoku University)
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In this study, a live cell-based method for simultaneous detection of multiple autoantibodies was established. This method improves the efficiency of evaluating serum samples by using fluorescent proteins and fluorochromes.
Accurate serologic evaluation of autoantibodies in patients with autoimmune diseases is critical. In the present study, we established a live cell-based assay for simultaneous detection of multiple autoantibodies in a single serum sample. Autoantibody seropositivity was determined by 3-color flow cytometry using live Chinese hamster ovary cells transiently expressing a target protein of interest fused to enhanced green fluorescent protein and labeled with Alexa Fluor 647 and Hoechst 33342. As a representative example, we applied the strategy for simultaneous detection of 2 recently established biomarkers for central nervous system autoimmune inflammatory demyelinating disorders, anti-aquaporin-4 autoantibody and anti-myelin oligodendrocyte glycoprotein autoantibody, in a single serum sample. This analysis revealed the coexistence of these 2 autoantibodies. We demonstrated that this assay can simultaneously detect 3 different autoantibodies. We propose a quadrant gating strategy of flow cytometry contour plots to clearly distinguish seropositive sera from seronegative sera regardless of the extent of the background signal level or the autoantibody titer. This novel and practical method using a combination of fluorescent proteins and fluorochromes to simultaneously detect multiple autoantibodies improves the efficiency of evaluating serum samples, and therefore provides significant benefits to both the patient and the healthcare professionals performing autoantibody testing.
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