3 3 5 5 tetramethylbenzidine substrate

Recombinant moesin (MSN) was expressed and purified using a His-tagged bacterial expression vector obtained from Arizona State University’s plasmid repository (ASU Biodesign Institute, Tempe, AZ, USA) [20 (link)]. A sandwich ELISA technique was utilized using a previously described technique [13 (link)]. Briefly, polyclonal moesin antibody (sc-6410, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was incubated at 4 °C overnight on a standard 96-well plate to serve as a capture antibody. Following routine washing and blocking, recombinant moesin (0.2 µg per well) was added and incubated for 2 h. Diluted sera (1:200) and sera-free controls were added in duplicate and incubated for 1 h, followed by washing. HRP-conjugated anti-human IgG (sc-2769, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was added to each well at a concentration of 1:1000 for 30 min, followed by washing. Finally, 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Scientific, Waltham, MA, USA) was added for 15 min and allowed to develop while protected from light. Sulfuric acid (Thermo Scientific, Waltham, MA, USA) was used to stop the reaction, and the optical density (OD) was immediately measured at 450 nM and normalized at 570 nM. The OD of the sera-free control was subtracted from the measured OD of all samples to reduce non-specific reactivity.

CHO-K1 cells overexpressing the human GCGR (with or without RAMP2) were seeded overnight in 96-well plates (30,000/well). Following fixation (2% paraformaldehyde), an in-cell enzyme-linked immunosorbent assay (ELISA) was performed in nonpermeabilized cells to detect surface GCGR expression. Antibodies used were rabbit primary vs GCGR N-terminus (1:200, ab137649; Abcam, United Kingdom) and anti-rabbit IgG horseradish peroxidase–conjugated secondary (1:2000, #15015; Active Motif, United Kingdom), with 2% bovine serum albumin block used during in all incubations. 3,3′,5,5′-Tetramethylbenzidine substrate (Thermo Fisher Scientific, United Kingdom) was added and absorbance read at 450 nm after addition of 1 M HCl. Surface GCGR expression was calculated as absorbance after subtraction of nonspecific binding (determined in the absence of primary antibody) and normalization to protein content (bicinchoninic acid assay).

Plasma anti-HSP70, ENO1 and RNH1 IgG autoantibodies were measured by indirect ELISA in 66 plasma samples from healthy individuals (n = 23), patients with cholangitis (n  = 12) and CCA (n = 31). The 96-well MaxiSorp immunoplates (Nunc, Roskilde, Denmark) were coated with 100 µl of recombinant human full-length HSP70, ENO1 or RNH1 (each, 1 µg/ml in 1× PBS) at 4°C overnight and washed once with 1× PBST. The plates were blocked with 100 µl of 5% skim milk for 2 h at room temperature and washed two times with 1× PBST. Then, the reactions were added with 100 µl of each 250-fold diluted plasma samples in dilution buffer (1× PBS pH 7.4, 5% skim milk, 0.05% Tween 20) and incubated for 1 h at 37°C. After washing three times with 1× PBST, 100 µl of 1∶4,000 goat anti-human IgG-HRP conjugated antibody was added and incubated for 1 h at room temperature. The plates were then washed five times with 1× PBST. Finally, 100 µl of 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Scientific, West Palm Beach, FL, USA) was added and the reaction was stopped with 3M H₂SO₄. The absorbance was read at 450 nm using ELISA reader (Tecan AG, Switzerland) and PBS was used as blank control.

The 96-well ELISA plates (Thermo Fisher) were coated with 100 ng recombinant αS proteins in 100 μl PBS per well. Wells were washed with PBS and blocked with TBS/5% skim milk powder. Primary antibodies were added to blocking solution and incubated at room temperature. After TBS washes, plates were incubated with horseradish peroxidase-conjugated anti-mouse antibody (Jackson Immuno Research Labs) in TBS/5% skim milk powder for 30 min. Plates were washed with TBS and 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Fisher Scientific) was added to each well. The reactions were stopped by adding 0.5 M HCl and the optical density was measured at 450 nm with a plate reader.

In our study, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated with purified recombinant protein at 4 °C overnight. The final concentrations were 0.1 μM for NcMIC4 and NcSAG1, and 0.5 μM for NcPrx2, in a 50 mM carbonate-bicarbonate buffer (pH 9.6). The plates were washed once with PBS containing 0.05% Tween 20 (PBS-T) and then blocked with 3% skimmed milk in PBS (PBS-SM) for 1 h at 37 °C. Following the blocking step, the plates were washed again with PBS-T, and 50 μL of the test sera (each sample in duplicate) diluted at 1:200 was added to the wells. The plates were incubated at 37 °C for 1 h. After washing, a secondary antibody, peroxidase-conjugated anti-mouse or bovine IgG (Invitrogen, Rockford, IL, USA), diluted at 1:4000 in PBS-SM, was added to the wells and incubated at 37 °C for 1 h. The plates were washed six times, and then 100 μL of 3,3′, 5,5′-tetramethylbenzidine substrate (Thermo Fisher Scientific, Frederick, MD, USA) was added to each well. The reaction was stopped by adding 100 μL of 1-M HCl. The absorbance at 450 nm was measured using the SUNRISE microplate reader (Tecan Austria GmbH, Grödig, Austria). The cut-off value was determined as the mean value of the standard N. caninum-negative control sera plus five times the standard deviation.