Piccolo ELISA

Sera from GBS (N = 8; A3, AI-AVII) and SCI (N = 4; DI-DIV) unrelated patients with similar medications, and healthy control individuals (N = 4; B3, CI-CIII), including the GBS patient (A3) and her healthy identical twin (B3) were included in the analysis (Table ​(Table1).1). Sera were albumin depleted with Pierce albumin depletion Kit (Thermo Scientific, San Jose, CA, USA) following manufacturer's instructions. ELISA plates were coated with 100 μl/well of albumin-depleted sera at concentration of 2 ng/μl in carbonate/bicarbonate buffer and incubated overnight at 4°C. Then, 100 μl of blocking buffer (5% skim milk powder in phosphate buffered saline, PBS) were added to each well and incubated 1 hr at room temperature (RT) followed by five washes with PBS supplemented with 0.05% Tween 20 (PBST) (Sigma-Aldrich, Madrid, Spain). The mouse monoclonal anti-human Piccolo antibodies [6H9-B6] (ab101654; Abcam, Cambridge, UK), which are specific for human Piccolo and without cross-reactivity to Bassoon (http://www.abcam.com/piccolo-antibody-6h9-b6-ab101654-references.html) were added at 1:1000 dilution and incubated for 1 hr at 37°C followed by five washes with PBST. For the detection of Piccolo, 100 μl of anti-mouse IgG (μ-chain specific)-peroxidase antibodies produced in goat (Sigma-Aldrich) were added at a 1:1000 dilution in blocking buffer. Plates were incubated for 1h at RT, and subsequently washed with PBST five times. Color was developed by the addition of 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) (Promega Biotech, Madrid, Spain) and protected from the light for 20 min at RT. The optical densities (OD) were measured at 450 nm with an ELISA reader. The average value of the blanks (wells without serum coating; N = 4) was subtracted from all reads. For Piccolo protein quantitation, the human recombinant protein (Abnova, Taipei, Taiwan) was used as standard. Two technical replicates were included for each sample. The Piccolo concentration in serum samples was compared between GBS patients, SCI patients and healthy individuals by Student's t-test with unequal variance (P = 0.05).