M1000 pro

A FRET-based fluorescence quenching assay was developed to monitor hydrolysis activity of nsp5. Upon cleavage of the substrate 2-Aminobenzoyl is no longer quenched by N-Tyrosine and able to fluorescence. Any changes in fluorescence were monitored in the excitation spectra of 330 nm and emission spectra of 420 nm. Unless otherwise stated, the reaction master mix used in all FRET experiments contributed to a final concentration of; 10 nM nsp5, 50 mM HEPES-KOH pH 7.6, 1 mM EDTA, 2 mM DTT, 10% glycerol, and 0.02% Tween. Reactions were initiated by the addition of FRET substrate to a final concentration of 20 µM (unless otherwise stated) to reaction master mix. A Tecan M1000 pro or Tecan Spark were used to take fluorescence readings for all FRET experiments with the following settings: Kinetic cycle: 10, Interval time: 2 min, Excitation bandwidth: 5 nm, Emission bandwidth: 15 nm, Gain: 140, Number of flashes: 50, Flash frequency: 400 Hz, Integration time: 20 µs and Z-position: 22 470 µm. All nsp5 protease reactions were done at room temperature (19°C–23°C) however, ambient temperature inside the Tecan M1000 pro or Tecan Spark plate readers fluctuated between 25°C and 27.5°C.

1 µM of Xyn-Doc fusion proteins (wild-type Q1, Q3, QQ Doc fusions) bearing either wild-type or mutant Doc domains were adsorbed onto surfaces of the wells of a 96-well nunc maxi sorp plate (Thermo Scientific, Pittsburgh, PA). After blocking (2% (w/v) BSA, 0.05% Tween 20 in TBS buffer) and several rinsing steps, a red fluorescent protein-cohesin (StrepII-TagRFP-Coh2 (C.t.), Addgene ID 58,710 (Otten et al., 2014 (link))) fusion construct was incubated to the unspecifically immobilized Doc fusion proteins over a range of concentrations. After further rinsing, the fluorescence of the TagRFP domain was measured with a multi-well fluorescence plate reader ( M1000 PRO, Tecan Group Ltd., Männedorf, Switzerland). Fluorescence values were plotted against their corresponding concentration values for each protein variant, and 4 parameter logistic nonlinear regression model functions were fitted to the data to determine the transition point of the curve.

CAC measurements were performed on samples, which were not thawed before.
The measurement was performed on a 96-well Nunc Maxisorp (ThermoFisher, USA) plate, coated by rabbit anti-human C1-inhibitor IgG antibody purified by affinity chromatography. Coating was performed on 37 °C for 2 h or on 4 °C overnight. After coating and threefold washing, the plate was blocked by 2% BSA-PBS (bovine serum albumin—phosphate buffered saline) for 2 h.
After a threefold washing step, the blank, the controls and the samples were placed on the plate for 1 h. 1% BSA-PBS-Tween was used as blank and for diluting the samples and controls. Samples and the normal serum control were measured in 1000-fold dilution. After a threefold washing step, the conjugates were horseradish peroxidase labeled rabbit anti-human antibodies against IgG, IgM and IgA antibodies, which were incubated on the plate for 1 h on room temperature. Threefold washing step was followed by TMB (3,3′5,5′-Tetramethylbenzidine) detection, and after a few minutes of incubation on room temperature the stop solution (0,4 M sulfuric acid) was added. Absorbance was measured on 450 nm, reference measurement was performed on 620 nm by Tecan device (Tecan M1000pro, Group Ltd).
The method is summarized on Fig. 2.Fig. 2

Measuring method for the C1-INH/C1-INH antibody complexes

Aliquots of ricin (10 μg/mL in 0.1 M NaHCO₃, pH 8.6) and RCA120 (10 μg/mL in 0.1 M NaHCO₃, pH 8.6) were coated on a 96-well plate and incubated at 4 °C overnight [36 (link),37 (link)]. The excess target molecules were removed and a blocking solution was filled, which was blocked at 4 °C for 2 h. After removing the blocking solution, the plate was washed six times with a TBST-5 (50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween-20, pH 7.5) buffer. Phage clones were incubated in ricin or RCA120-coated ELISA plates at room temperature for 2 h with shaking. Then, the HRP-labeled M13 antibody was added to each well and incubated at room temperature for 1 h. After washing the plate six times with TBST-5, the TMB solution (100 μL/cell) was reacted for 30 min, and the absorbance at 450 nm was measured by a microplate reader (Tecan M1000 PRO, Tecan, Männedorf, Switzerland).