Supersignal elisa pico

Expression of CCKAR protein was determined quantitatively using CCKAR antibody (Santa Cruz, USA). Initially, standard curve was plotted with known concentrations of antigen (0.312 ng/mg–60 ng/mg). In brief, 96-well immunoassay plates were coated with 100 μL/well of diluted antigen for 2 hours followed by blocking step with blocking solution (1% BSA). Plates were incubated overnight with 100 μL/well of diluted (1 : 1000) anti-human CCKAR antibody at 4°C. After washing away any unbound antibody, 100 μL/well of secondary antibody (1 : 2000) was added, and incubation was done for 3 hours. This was followed by dispensation of 100 μL/well of substrate solution (Super Signal ELISA Pico, Thermo Scientific). The enzyme-substrate reaction was stopped after sufficient colour development by adding 50 μL/well of 0.5 M H₂SO₄. Colour change was measured spectrophotometrically at a wavelength of 450 nm.

Three µg/mL of α-synuclein and TDP-43 diluted in PBS were coated on separate polystyrene plates (ThermoFisher, Waltham, MA, USA), and incubated overnight at 4 °C. The plates were then washed three times with PBST and blocked with 200 µL of 3% bovine serum albumin (BSA; Millipore, Burlington, MA, USA) diluted in PBS for 1 h at room temperature. After washing, 100 µL of serially diluted (50, 10, and 2 ng/mL) of recombinant TDP-43 and α-synuclein in PBST were applied on to α-synuclein and TDP-43 coated wells, respectively. The plates were then left to incubate for 1 h at 37 °C. After washing, 100 µL of 0.1 µg/mL of biotinylated TDP-43 antibodies (10782-2-AP and 12892-1-AP; Abcam, Cambridge, UK) diluted in PBST with 10% BlockAce, and anti-α-synuclein antibodies (FL-140; Santa Cruz Biotechnology, Dallas, TX, USA) were applied on wells treated with TDP-43 and α-synuclein, respectively, and left to incubate for 1 h at 37 °C. After washing, the wells were treated with 100 µL of HRP-conjugated streptavidin diluted 10,000 times in PBST with 10% BlockAce and left to incubate for 30 min at room temperature. After washing, chemiluminescent substrate (SuperSignal ELISA Pico, Thermo Scientific) was added to achieve greater sensitivity. The resulting luminescence was then measured using a Victor 3 multilabel reader.

PBS was used as the microplate coating buffer. Two percent w/v skim milk in PBS (PBS-M) and PBS containing 0.05 % v/v Tween 20 (PBS-T) were used as blocking solution and washing buffer, respectively. Sample or reagent dilutions were prepared in 2 % w/v skim milk, 0.05 % v/v Tween 20 in PBS (PBS-MT). Immunopure Avidin was purchased from Pierce, and Avidin–Horseradish Peroxidase (HRP) and rabbit anti-human IgG-HRP were purchased from Jackson ImmunoResearch Laboratories, Inc. The 3,3’,5,5’-tetramethyl-benzidine/H₂O₂ (Single Component TMB Peroxidase EIA Substrate Kit, BioRad, Hercules, CA, USA) was employed as the chromogenic substrate and the SuperSignal ELISA Pico (ThermoScientific, Rockford, IL, USA) as the chemiluminescent substrate. Except when otherwise indicated, incubations were performed at RT, washing steps were performed with PBS-T and 50 μL per well were added in each incubation step.