Liver powder removes anti-AQP1 antibodies.

<p>Two exclusively anti-AQP1-positive and two exclusively anti-AQP4-positive serum samples were pretreated with guinea pig liver powder, then the supernatants were tested by RIPA using indirectly radiolabeled AQP1 (left panel) or AQP4 (right panel). Key: +, pretreated serum; -, untreated serum.</p

Anti-AQP1 antibody binding to synthetic peptides corresponding to AQP1 extracellular and cytoplasmic domains.

<p><b>A.</b> Synthetic peptides corresponding to human AQP1 extracellular loops A (residues 36–49), C (116–137), and E (183–188, 199–210) or to the intracellular N-terminus (1–22), loop B (81–100), and the C-terminus (248–270) are shown (from Uniprot/Swissprot entry P29972, for the human AQP1). Extracellular residues are shown in bold and underlined. Loop-E is formed by two segments separated by a 14 residue gap; in “pept-Loop-E”, 10 of these 14 residues (between TG and SE) were omitted (marked by:..). In the first 4 peptides, an extra tyrosine residue was added to the N-terminus for future radioiodination studies. Below the AQP1 peptide sequences are shown the corresponding AQP4 sequences; (*), identical amino acid residues; (:) and (.), homologous residues. <b>B.</b> Peptide mapping with anti-AQP1 positive sera. 22 sera from patients with known clinical characteristics (17 NMOsd and 5 MS) were tested for binding to 6 synthetic peptides corresponding to the 3 extracellular loops (loops A, C and E) and to the 3 cytoplasmic segments (N-terminal, Loop-B and C-terminal) by an ELISA assay. Positive values are considered those above O.D.₄₅₀ 0.45 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#s2" target="_blank">Methods</a>). It is shown that most NMOsd sera bind to the Loop-A peptide.</p

Detection of binding of anti-AQP1 antibody to denatured AQP1 by Western blotting.

<p>Yeast-expressed AQP1 was electrophoresed and transferred onto nitrocellulose membranes, which were then incubated with the test sera (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#s2" target="_blank">Methods</a>). Lanes 1-3: Three sera from healthy controls at dilutions 1/250, 1/60, and 1/30, respectively; lane 4: commercial rabbit anti-AQP1 antibody; lanes 5-8: representative anti-AQP1-positive sera with titers of 132, 25, 10.5, and 5.8 nM, at dilutions of 1/500, 1/250, 1/30, and 1/30, respectively. None of the test anti-AQP1 sera bound to the control protein MuSK (not shown).</p

Laboratory and clinical data for anti-AQP1 seropositive patients.

<p>F, female; LETM, longitudinally extensive transverse myelitis; M, male; MS, multiple sclerosis; NMO, neuromyelitis optica; NT, not tested.</p>a<p>All 22 patients tested negative for serum anti-AQP-4 autoantibodies in the two-step RIPA and confirmed by both a commercial and an in-house CBA.</p>b<p>The anti-AQP1 positivity of the sera was also confirmed by the more sensitive two-step RIPA for anti-AQP1 antibodies (not shown).</p>c<p>According to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#s2" target="_blank">Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#pone-0074773-g008" target="_blank">Figure 8</a>.</p>d<p>Calculated by multiplying anti-AQP1 titers (in 6^th column) by the percentages of the extracellularly binding antibodies (in 7^th column).</p>e<p>Binding of sera to synthetic peptides corresponding to AQP1 segments (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#pone-0074773-g009" target="_blank">Figure 9B</a>). Underlined loops are extracellular.</p

Anti-AQP1 specificity of the identified autoantibodies.

<p>(A) Patient’s autoantibodies recognize the AQP1 moiety of AQP1-GST. Five sera that had tested positive for binding to the AQP1-GST fusion protein were preincubated with an excess of GST immobilized on Sepharose-glutathione beads, then were tested by RIPA using ¹²⁵I-streptavidin labeled AQP1-GST. (B) Binding of anti-AQP1 autoantibodies is specifically inhibited by an extract from AQP1-expressing HEK293 cells, but not control HEK293 cells. Four anti-AQP1-positive sera were preincubated with extracts prepared from either EGFP-transfected or AQP1-GFP-transfected HEK293 cells, then were tested by RIPA for binding to the commercial AQP1 preparation. (C) Binding of anti-AQP1 autoantibodies is specifically inhibited by yeast-expressed human AQP1. Four exclusively anti-AQP1-positive sera were preincubated with human AQP1 or AQP4 that had been expressed in yeast and purified or with BSA as control, then were tested by RIPA using ¹²⁵I-streptavidin-labeled commercial AQP1-GST fusion protein. (D) AQP1 autoantibody binding is independent of the source of AQP1. Both the commercial AQP1-GST fusion protein and the in house AQP1 purified from yeast were biotinylated, indirectly labeled by preincubation with ¹²⁵I-streptavidin, and used in the RIPA. Five anti-AQP1-positive sera and one serum sample from a healthy control (HC) were tested.</p

Detection of anti-AQP1 antibodies by an ELISA with immobilized purified human yeast-expressed AQP1.

<p>Sera previously tested by RIPA for AQP1 antibodies were tested for binding to immobilized AQP1 by ELISA. First column contains 31 sera found positive by the RIPA (including a double-positive anti-AQP1/AQP4, empty square, and 3 sera from anti-AQP1-positive MS patients, empty circles). The following 3 columns contain 5 anti-AQP4-positive/anti-AQP1-negative sera, 30 sera from anti-AQP1-negative MS patients and 44 sera from healthy controls). The dashed horizontal line denotes the cut-off (O.D.450: 0.36) for positive values.</p

Anti-AQP1 antibodies and anti-AQP4 antibodies do not bind to the other antigen.

<p>(A and B). Search for cross-reactivity of anti-AQP1 and anti-AQP4 antibodies with AQP4 and AQP1, respectively. Three (A) or five (B) double-positive serum samples were incubated with immobilized AQP1 (A) or AQP4 (B) to immunoadsorb the corresponding antibodies, then were tested by RIPA for binding to ¹²⁵I-labeled AQP1 or AQP4 (white bars) in parallel with the untreated sera (black bars). HC, healthy control. (C). Lack of correlation of the amount of radiolabeled AQP1 precipitated by anti-AQP1 antibodies (y axis) or AQP4 (x axis) precipitated anti-AQP4 antibodies by double-positive sera in identical regular RIPAs performed using the two labeled antigens.</p

Determination of the percentage of antibodies directed against the extracellular domain of membrane-embedded AQP1.

<p>Four anti-AQP1-positive sera were left untreated or were preincubated with increasing numbers of AQP1-GFP- (filled symbols) or EGFP-transfected (shown only for serum 3; empty triangle) HEK293 cells treated with secretin to increase surface expression of AQP1; the untreated and treated samples were tested in the usual RIPA for anti-AQP1 antibodies. The sera were also treated with AQP4-transfected HEK293 cells (shown only for serum 3; open circle). Serum 4 was also treated with an extract of AQP1-transfected HEK293 cells (open square).</p

Ig class and IgG subclass of the anti-AQP1 antibodies.

<p>The Ig class and IgG subclass of the anti-AQP1 antibodies from 7 positive human sera was tested by RIPA using anti-class/subclass second antibodies. Sera no. 1-4 are from patients 11-14 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074773#pone-0074773-t001" target="_blank">Table 1</a> and sera no. 5-7 are from the remaining anti-AQP1 antibody-positive sera.</p

Supp_1 – Supplemental material for Cytokeratin-18 fragments predict treatment response and overall survival in gastric cancer in a randomized controlled trial

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