Article Figures & Data
Figures
- Figure 1 Optimization of the α3-nAChR Cell-Based-Assay
(A) Treatment of α3-nAChR–transfected HEK293 cells, with RIC3, NACHO, and nicotine increases significantly the expression of these receptors. Cells expressing α3β4-, α3β2-nAChRs, or control molecules AQP4 and α4β2-nAChR were cotransfected with 1 (RIC3) or 2 (RIC3 and NACHO) chaperons and cultured in the presence or absence of 1 nM nicotine. Cells were then incubated with the serum of a patient with autoimmune autonomic ganglionopathy with positive RIPA titer for α3-nAChR. Scale bar: 20 μm. (B) Colocalization of patient 2 serum with the rat anti-β2 mAb. (C) Colocalization of α3-nAChR antibody binding and cell nucleus/DNA staining. The fluorescent signal of patient 13 antibodies was counterstained with Hoechst (for cell nucleus staining). α3-nAChR = α3-subunit–containing nicotinic acetylcholine receptor; RIPA = radioimmunoprecipitation assay.
- Figure 2 The Novel CBA With α3β4-nAChR–Transfected HEK293 Cells Detects Antibodies in Sera With High or Low RIPA Antibody Levels Specifically in Patients With AAG
HEK293 cells expressing α3β4, α3β2-nAChRs, or control AQP4 molecule were incubated with sera from patients with AAG (of high or low RIPA antibody levels) or from a patient with POTS and stained with anti-human IgG (red). It is shown that both patients with AAG showed positive staining, independently of the RIPA antibody level, whereas the patient with POTS gave no staining. The AAG RIPA-high, AAG RIPA-low, and POTS RIPA-low patients are patients no. 6 (RIPA 1.4 nM, CBA 1/1,000), 18 (RIPA 0.05 nM, CBA 1/10), and 16 (RIPA 0.13 nM, CBA negative) of Table 1, respectively. Scale bar: 20 μm. AAG = autoimmune autonomic ganglionopathy; CBA = cell-based-assay; POTS = postural orthostatic tachycardia syndrome; RIPA = radioimmunoprecipitation assay.
- Figure 3 Correlation of α3 nAChR Antibody Levels by RIPA vs CBA Titers for the 15 Patients With Autoimmune Autonomic Ganglionopathy
The normality Shapiro-Wilk test showed that the RIPA data follow normal distribution, whereas the CBA data do not (RIPA Shapiro-Wilk p = 0.109; CBA Shapiro-Wilk p = 0.002). Pearson correlation coefficient r = 0.71 and coefficient of determination R2 = 0.50. We also plotted the values using log-log transformation of the RIPA and CBA values, which resulted in improved r (=0.88) and R2 (=0.77). CBA = cell-based-assay; RIPA = radioimmunoprecipitation assay.
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