Tetramethylbenzidine

In a 96-well plate (nerbeplus, Winsen, Germany), 0.5 µg of rshCR-1 in PBS was adsorbed per well by incubation at 4 °C overnight. After washing three times with PBS, wells were blocked with 5% skim milk in PBS containing 1 mM EDTA by the incubation at 25 °C for 1 h. After three subsequent washes with PBS, sequentially diluted recombinant antibodies were added and incubated for 2 h at 25 °C. After treatment with the primary antibody, the plates were washed five times with PBS containing 0.1% Tween 20 (PBST). Then, anti-human IgG goat/rabbit polyclonal antibody conjugated with horse radish peroxidase (HRP) (abcam, Cambridge, UK) was incubated for 2 h at 25 °C. After washing five times with PBST, tetramethylbenzidine (Thermo Fisher Scientific, Waltham, MA, USA) was added at 100 µL/well as the substrate of HRP and incubate for 30 min and the reaction was stopped with the equal volume of 2 N sulfuric acid. The developed color at 450 nm was measured by microplate reader SH-9000 (Hitachi, Tokyo, Japan).

ELISA plates were coated with each antibody at 10, 8, 6, 4, 3, 2, 1 and 0 μg/ml in triplicate and incubated at 4 °C over-night. The plates were washed three times with PBS and then blocked with 1% BSA in PBS at 50 μl/well. C1q (Sigma-Aldrich, Taufkirchen, Germany) was added to each well at 2 μg/ml in blocking buffer and incubated for one hour at RT. The plates were then washed three times with 200 μl of PBS. Anti-C1q-HRP polyclonal antibody conjugate (Thermo Fisher Scientific) was added at a 1:250 dilution in blocking buffer (50 μl/well) for one hour. The plates were washed three times with 200 μl of PBS. 50 μl of Tetramethyl Benzidine (Thermo Fisher Scientific) was added to each well for 2 minutes. 50 μl of stop solution (1 M Sulfuric Acid) was added to each well before reading the absorbance at 450 nm.
A FortéBio BLItz system (Pall ForteBio LLC, Fremont, USA) was used to measure the antibody Fcγ-receptor binding affinities. 100 μg/ml of biotinylated CD16a, CD32a and CD64a proteins (ACROBiosystems, Cambridge, UK) were loaded onto the streptavidin sensors. Antibody association and disassociation to the receptors were measured at five different antibody concentrations (400 μg/ml, 200 μg/ml, 100 μg/ml, 50 μg/ml and 25 μg/ml). K_D values (and k_a, k_d rates) were calculated by means of the Blitz program, provided by the manufacturer.

Serum levels of circulating mouse anti-rabbit IgG were determined by ELISA using mouse IgG quantification sets (Bethyl, Montgomery, Texas, USA). In detail, each well was coated with 250 ng normal rabbit IgG. After blocking, a series of diluted samples was added and incubated for 60 min. Bound Abs were detected by HRP-conjugated goat anti-mouse IgG (Bethyl) and tetramethylbenzidine (Thermo Fisher Scientific). The enzymatic color reaction was stopped by 2 M sulfuric acid (Carl Roth, Karlsruhe, Germany), and the change in OD was measured with a plate reader (PerkinElmer, Waltham, Mass., USA) at 450 nm. Standard reference curves were established using the provided mouse reference sera (Bethyl).

The GloSensor cAMP Assay (E1290) and Dual-Luciferase Reporter Assay System (E1960) were purchased from Promega. Tetramethylbenzidine was purchased from Thermo Fisher. Coelenterazine 400a (FP-BB839B) was purchased from Interchim. Gq/11 selective inhibitor YM-254890 (AG-CN2-0509-M001) was purchased from Adipogen. All other reagents or chemicals were purchased from Sigma-Aldrich unless otherwise specified.

Serum levels of circulating total mouse IgG were determined by ELISA using mouse IgG quantification sets (Bethyl, Montgomery, Texas, USA) as described by the manufacturer's protocol using 1:50,000 diluted samples. For detection of circulating anti‐mCOL7^vWFA2‐IgG by ELISA, each well was coated with 250‐ng recombinant mCOL7^vWFA in coating buffer (0.05‐M carbonate–bicarbonate buffer, pH 9.6). After blocking, diluted samples (1:20,000) were added and incubated for 60 min. Standard reference curves were established by using the mouse reference sera (pooled serum of mice 12 weeks after immunization with mCOL7C^vWFA2; Hammers et al., 2011). For both ELISA protocols, bound antibodies were detected by HRP‐conjugated goat anti‐mouse IgG (Bethyl) and tetramethylbenzidine (Thermo Fisher Scientific). The enzymic colour reaction was stopped by 2‐M sulfuric acid (Carl Roth, Karlsruhe, Germany), and the change in OD was measured with a plate reader (GloMax™ Discover microplate reader, Promega, Mannheim, Germany) at 450 nm.