LIPS for gAChR in the sera from patients with autoimmune autonomic ganglionopathy (AAG) and controls.

<p>We tested the sera from patients with AAG, disease controls (DC), and healthy controls (HC). a) Anti-gAChRα3 antibodies were detected in 23 samples. The mean anti-gAChRα3 antibody level in the HC was 0.305 antibody index (A.I.), which was significantly lower than in the AAG samples with a mean level of 1.210 A.I. (<i>p</i> < 0.001). b) Anti-gAChRβ4 antibodies were also detected in seven samples, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118312#pone.0118312.g004" target="_blank">Fig. 4B</a> (<i>p</i> = 0.005). The mean anti-gAChRβ4 antibody level in the HC was 0.367 A.I., which was significantly lower than the measn level of 0.618 A.I. in the AAG samples (<i>p</i> < 0.001).</p

Schematic representation of the acetylcholine receptor (AChR) α3-<i>Gaussia</i> luciferase (GL) ⁸⁹⁹⁰.

<p>For the ganglionic AChR (gAChR)-LIPS assay, human embryonic kidney (HEK) 293 cells were transfected with an expression plasmid for the gAChRα3 or β4-GL reporter.</p

Autonomic function tests at baseline of anti-gAChR Ab positive AAG patients.

<p>Anti-gAChRα3 Ab = ganglionic acetylcholine receptor α3 antibody; Anti-gAChRβ4 Ab = ganglionic acetylcholine receptor β4 antibody; A.I. = Antibody Index; OH = orthostatic hypotension; HUTT = head-up tilt test; CV R-R = CV = coefficient of variation R-R interval; H/M ratio = heart-to-mediastinum ratio in ¹²³I-MIBG myocardial scintigraphy; TST = thermoregulatory sweat test; NE = norepinephrine; SSR = sympathetic skin response; QSART = quantitative sudomotor axon reflex test; CSF = cerebrospinal fluid</p><p>Autonomic function tests at baseline of anti-gAChR Ab positive AAG patients.</p

Confirmation of the LIPS assay system for the gAChRα3 or β4 with ready-made antibodies.

<p>The anti-gAChRα3 antibody (H-100) and the anti gAChRβ4 antibody (S-15) bound the gAChRα3-GL and the gAChRβ4 reporters, respectively in a dose-dependent manner (a and b). The X-axis indicates the amount of ready-made gAChRα3 or β4 antibody used. The Y-axis indicates the gAChRα3 or β4-GL activity. The line with closed diamonds shows the results that were obtained in this experiment.</p

Clinical and autonomic characteristics at baseline of anti-gAChR Ab positive AAG patients.

<p>Initial symptoms were expressed in bold.</p><p>AE = antecedent event; OH = orthostatic hypotension; OI = orthostatic intolerance; HI = heat intolerance; AH = anhidrosis; GI = gastrointestinal tract symptoms; α3 Ab = ganglionic acetylcholine receptor α3 antibody; β4 Ab = ganglionic acetylcholine receptor β4 antibody; A.I. = Antibody Index; AE = antecedent event; OI = orthostatic intolerance; OH = orthostatic hypotension; HI = heat intolerance; AH = anhidrosis; GI = gastrointestinal tract symptoms; Dept = department</p><p>a. Subacute = peak of autonomic failure within 3 months; gradual = gradual onset of chronic autoimmune autonomic ganglionopathy with the peak of autonomic failure after 3 months.</p><p>b. Patient 2 = fever up; 7 = epididymitis; 21 = influenza virus type A infection; 23 = fever up and cough.</p><p>c. Patient 4 and 7 = Adie’s tonic pupil. The other cases had the abnormality of papillary reflex to light bilaterally or unilaterally.</p><p>d. Patient 1 = constipation; 2 = constipation; 3 = early satiety and vomiting; 4 = constipation; 5 = constipation; 6 = constipation, ileus, and sigmoid volvulus suspected; 7 = early satiety, vomiting, alternate stool abnormality, abdominal pain, and taste impairment; 8 = constipation; 9 = constipation; 10 = constipation; 11 = anorexia and diarrhea; 12 = diarrhea and achalasia; 13 = constipation, early satiety, vomiting, and ileus; 14 = abdominal pain, anorexia, and diarrhea; 15 = diarrhea; 17 = constipation, anorexia, and achalasia; 18 = alternate stool abnormality, abdominal pain, and anorexia; 20 = constipation; 21 = constipation; 22 = early satiety, ileus, alternate stool abnormality, and abdominal pain; 23 = constipation, vomiting, and achalasia; 24 = constipation.</p><p>Clinical and autonomic characteristics at baseline of anti-gAChR Ab positive AAG patients.</p

Clinical features of patients with AAG/APD.

<p>a. We reviewed the 26 male patients only.</p><p>b. Numbness, mental symptom, dementia, character change, and back pain</p><p>c. Amenorrhea, eating disorder, SIADH (Syndrome of inappropriate secretion of antidiuretic hormone), and panhypopituitarism</p><p>d. Still disease, PBC (primary biliary cirrhosis), Hashimoto disease, PMR (polymyalgia rheumatica), SLE (systemic lupus erythematosus), SS (Sjögren's syndrome), Graves’ disease, RA (rheumatoid arthritis), fibromyalgia, and other autoantibodies positive</p><p>e. Ovarian tumor, pancreas cancer, mediastinal tumor, and paranasal cancer</p><p>Clinical features of patients with AAG/APD.</p

The Luciferase Immunoprecipitation Systems (LIPS).

<p>The soluble fractionated component from the solubilized HEK 293F cells, including the gAChRα3 or β4-GL, reacted with human serum, and the specific luciferase activities of the gAChRα3 or β4-GL were found with the luminometer. The <i>in vitro</i> LIPS assay can quantitatively evaluate an interaction between an antigen and an antibody with high sensitivity and without a radioisotope.</p