Elisa starter accessory kit

Blood was collected from each mouse at the end of the experiment. Total serum IgE levels were measured by enzyme-linked immunosorbent assay (ELISA) using the ELISA starter accessory kit (Bethyl Laboratories, Montgomery, TX, USA) and Mouse IgE antibody (Bethyl Laboratories) according to the manufacturer’s instructions. The absorbance was determined using a microplate reader.

Albumin in culture supernatants and blood of grafted mice were measured using the Human Albumin ELISA Quantitation Set (Cat No.E80-129, Bethyl Laboratories, TX, USA) and the ELISA Starter Accessory Kit (Cat No. E101, Bethyl Laboratories, TX, USA) as per manufacturer’s recommendations. Absorbance was measured at 450nm using Victor 3 plate reader (Model No. 1420–041, PerkinElmer, MA, USA). The results obtained were normalized with cell number.

Human Kappa ELISA Quantitation Set (Bethyl Laboratories, AL, USA), Mouse IgG2b ELISA Quantitation Set (Bethyl Laboratories, AL, USA), and ELISA Starter Accessory Kit (Bethyl Laboratories, AL, USA) were used to determine Ig secretion according to the manufacturer’s instructions. The results were measured based on the absorbance (450 nm) with a microtiter plate reader, and the content was determined using CurveExpert 1.4 software.

EV-packaged WNT was detected using ELISA starter accessory Kit (E101, Bethyl Laboratories, Montgomery, Texas) according to manufacturer's protocol. Biotin-conjugated antibodies against WNT5A, WNT9A (Bioss antibodies, Woburn, MA) and WNT6 (Novus Biologicals, Littleton, CO) was used for the ELISA. Purified EV with the concentration of 100 μg ml⁻¹ was used for each assay.

The cells were cultured for 72 h with medium exchange every 24 h. Albumin in the culture supernatant was measured using an enzyme-linked immunosorbent assay (ELISA) starter accessory kit and anti-albumin antibody (Bethyl Laboratories, Inc.) according to the manufacturer’s protocol. Albumin production was calculated per 24 h and corrected for cell number.

To citrullinate the recombinant proteins, proteins at a final concentration of 100 mg/ml were incubated with 5 units/ml of rabbit PAD (P1584, Sigma-Aldrich, St. Louis, MO) in working buffer (100 mM Tris–HCl, 5 mM CaCl2, pH 7.4) for 18 h at 37 °C [18 (link)]. Rabbit peptidyl arginine deiminase (PAD) is a counterpart of human PADI2 and is highly homologous to human PADI4. For the detection of serum IgG antibodies, the ELISA Starter Accessory kit (E101; Bethyl Laboratories, Montgomery, TX, USA) was used according to the manufacturer’s instructions. Briefly, microtiter plates were coated with 100 mL of 2 mg/mL recombinant protein, incubated at room temperature for 1 h, blocked, and incubated with 100 mL of diluted patient serum (1:100) for 60 min. After washing, the plate was incubated with 100 mL of horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody (1:50,000; ab6759; Abcam, Cambridge, UK) at room temperature for 60 min. After washing, bound reactants were detected by incubation with 3,3′3Cambrtetramethylbenzidine for 25 min. The absorbance was measured at 450 nm using a microplate reader (iMark™ Microplate Absorbance Reader, Bio-Rad Laboratories, Hercules, California, USA) [19 (link)].