Powerwave x microplate reader

The 6C407 and 3D8 Ab series (1 μg/ml) in phosphate-buffered saline (PBS) were incubated in a 96-well polystyrene microtiter plate (ThermoFisher Scientific; cat# 439454) coated with 10 μg/ml HSET-N peptide or 5 μg/ml ss-(dN)₄₀ antigen, respectively, for 1 h at room temperature. If necessary, Ab proteins were heated for 10 min to 4 h at 30–90 °C. Mouse IgG Abs bound to wells were detected using a rabbit anti-mouse IgG (Rockland; cat# 610-4503) followed by an alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (ThermoFisher Scientific; cat# 31341). Chimeric MH and MC Abs bound to wells were detected using rabbit anti-human IgG (ThermoFisher Scientific; cat# 31142) and rabbit anti-chicken IgY (Dianova; cat# 303-035-008) Abs, respectively, followed by an AP-conjugated goat anti-rabbit IgG. Color was developed by adding p-nitrophenyl phosphate substrate solution (1 mg/ml prepared in 0.1 M glycine, 1 mM ZnCl₂, and 1 mM MgCl₂, pH 10.3) to each well. The absorbance at 405 nm was read using a PowerWavex microplate reader (BioTek).

This assay was conducted in a 96-well UV microplate, as described previously [17 (link)]. A total of 0.24 mg MAP-rich tubulin was mixed with various concentrations of drugs and incubated at 37 °C in 120 μL reaction buffer (100 mM PIPES, pH 6.9, 1.5 mM MgCl₂, 1 mM GTP, and 1% (v/v) DMSO). A₃₅₀ was monitored every 30 s for 30 min, using the PowerWave X Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). The increase in A₃₅₀ indicated the increase in tubulin polymerization; 100% polymerization was defined as the AUC of the untreated control.

Commercial ELISA kits were used to quantify C1q (Hycult Biotech, Uden, The Netherlands, #HK356-02), MBL (Hycult Biotech, #HK323), C3 (Abcam, Cambridge, UK, #ab108823), and C1 inhibitor (R&D Systems, Minneapolis, MN, USA, #DY2488-05) in serum and milk samples, following the instructions provided by the manufacturer. The absorbance was read by a PowerWave X Microplate Reader (Bio-Tek Instruments) spectrophotometer.

The cell surface hydrophobicity of WT and chi mutants was determined using a Pasteur pipette with 1 ml of phenyl sepharose fast flow resin (GE Healthcare), washed with 50 mM phosphate buffer (pH 7.2) containing 150 mM NaCl. Bacteria (cultured overnight) were diluted 10-fold in TSBC, incubated for 4 h for mid-log phase culture. Bacteria were washed and resuspended with 50 mM phosphate buffer (pH 7.2), 150 mM NaCl. Bacteria (0.3 ml) were loaded onto column, washed with 0.9 ml of the same buffer. OD₆₀₀ was measured (PowerWave X microplate reader, BioTek Instruments), and the percentage of bacteria retained in the hydrophobic column was calculated from the absorbance of a ¼ dilution of the original bacterial suspension as follows. Equation 1: % adsorption  =  [(A₀-A₁)/A₀] x 100, where A₀  =  OD of ¼ diluted bacterial suspension, and A₁  =  OD of the eluted bacterial suspension.

Patient sera (1:50) and milk (1:2) were incubated in microplate wells coated with SARS-CoV-2 S recombinant protein (S1/S2), as described above. In order to assess the capability of anti-SARS-CoV-2 antibodies to activate the C system, wells were then incubated with AB Rh+ pooled sera (1:100 in PBS + 2% w/v BSA + 0.7mM Ca⁺⁺Mg⁺⁺) for 30 min at 37 °C. After washing with PBS-T, the binding of C1q was evaluated using rabbit anti-human C1q polyclonal antibody (1:2000, Dako, Santa Clara, CA, USA) for 1 h at 37 °C and anti-rabbit IgG AP-conjugated (1:10,000, Sigma-Aldrich, St. Louis, MO, USA) as a secondary antibody. Simultaneously, the deposition of C3 was detected using goat anti-human polyclonal antibodies (1:5000, Quidel, San Diego, CA, USA) for 1 h at 37 °C and anti-goat IgG AP-conjugated (1:30,000, Sigma-Aldrich). The formation of the terminal C complex C5b-9 was assessed using anti-human C5b-9 (1:50, clone: aE11, Dako) for 1 h at 37 °C and anti-mouse polyvalent Igs (G,A,M)-AP (1:30,000, Sigma Merck). The binding was revealed with pNPP and the absorbance was read at 405 nm using a PowerWave X Microplate Reader (Bio-Tek Instruments).

The recombinant globular head regions of the human C1q A, B, and C chains (ghA, ghB, and ghC, respectively) were produced as fusion proteins linked to maltose-binding protein (MBP) in Escherichia coli BL21 and purified, as described previously (22 (link)). The collagen like-regions (CLR) were obtained as described by Defendi et al. (23 (link)). Microtiter wells were coated with 4 μg/well of ghA, ghB or ghC in sodium carbonate/bicarbonate buffer (0.035 M NaHCO₃, 0.015 M Na₂CO₃, pH 9.6) or 1 μg/well of CLR in sodium borate buffer (0.2 M H₃BO₃, 75 mM NaCl, pH 8.2) overnight at 4°C. The wells were washed with PBS+0.1% Tween 20 and blocked with 2% (w/v) BSA for 1h at 37°C. Patient sera were incubated at a dilution of 1:25-1:50 in PBS/0.75 M NaCl overnight at 4°C. Anti-C1q were detected using an anti-human IgG-alkaline phosphatase (AP)-conjugate (1:50000, Sigma Merck). The binding of secondary antibodies was revealed using the substrate p-nitro phenyl phosphate (pNPP). The absorbance was read at 405 nm using PowerWave X Microplate Reader (Bio-Tek Instruments).

Human Complement FH DuoSet ELISA kit (#DY4779; R&D Systems, Inc., Minneapolis, Canada) was used to quantify FH in serum samples, following the manufacturer's instructions. The plate was read by the PowerWave X Microplate Reader (Bio-Tek Instruments) spectrophotometer at 450 nm.