Horseradish peroxidase conjugated anti mouse igg antibody

Ribose (30 mM) was mixed with 2 mg/mL gelatin in 200 mM NaPB in the presence of AMG (0.1, 1, 10 and 100 µM) or TBE (0.01, 0.1, 1, 10, 100 µg/mL), and incubated at 37 °C for 7 days. MG-gelatin was prepared by incubating 2 mg/mL gelatin with 100 µM MG in PBS at 37 °C for 3 days in the presence of AMG or TBE. MG-H1 formation was measured by ELISA as previously described [37 (link)]. In brief, for noncompetitive ELISA, each well of a 96-well immune plate (Thermo Fisher Scientific, Waltham, MA, USA) was coated with 0.1 mL of the 1 µg/mL sample in PBS and blocked with 0.5% gelatin hydrolysate in PBS. The wells were incubated for 1 h with 0.1 mL of 1 µg/mL MG-H1 antibody [19 (link)]. Antibodies bound to the wells were detected using horseradish peroxidase-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA). Then, stained with 100 μL of 500 µg/mL O-phenylenediamine dihydrochloride (Fuji Film Wako Pure Chemical, Japan) in citrate-phosphate buffer (pH 5.0) containing 5.9 mM hydrogen peroxide for 3 min. The reaction was terminated with 100 μL of 1.0 M sulfuric acid, and the absorbance was measured at 492 nm using a Sunrise RAINBOW THERMO RC system (TECAN, Männedorf, Switzerland).

After cell transfection, the supernatant was discarded, and the cells were washed twice with PBS before lysing with lysis buffer (20 mM Tris HCl, 150 mM NaCl, 1% NP-40). After centrifugation, the protein concentration in the supernatant was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). An aliquot of protein was denatured by boiling in a 100°C water bath for 5 min. SDS-polyacrylamide gel electrophoresis of the denatured proteins (20 μg protein per well) was performed using an 8% separating gel and 5% stacking gel. The separated proteins were transferred onto a polyvinylidene fluoride membrane, which was blocked before incubating with mouse anti-CaSR ab19347 antibody (Abcam) followed by horseradish peroxidase-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific; Product # 31430). Enhanced chemiluminescent reagent (Thermo Fisher Scientific) was added, and the fluorescent signals were imaged using a Chemiluminescence Fluorescence Image Analyzer (FUJIFILM, Osaka, Japan).

SARS-CoV-2 RBD-binding serum IgGs were measured by enzyme-linked immunosorbent assay (ELISA). 96-well high binding plates (Thermo Scientific) were coated with 100 μL of 0.1 μg/mL prototype RBD solution per well for 12 h at 4 °C. Plates were washed and blocked with 1% bovine serum albumin (BSA) for 1 h at 37 °C. Then, 100 μL of serially diluted sera were added to the plates and incubated for 1 h at 37 °C. Following washing steps, the plates were incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody ((Thermo Fisher Scientific) for 1 h at 37 °C. After washing, color development was initiated with 3,3’,5,5’-tetramethylbiphenyldiamine and stopped with 1 M H₂SO₄. The absorbance was measured at 450 nm with Spectramax ABS (Molecular Devices, San Jose, CA, USA).