Tmb substrate

ELISA to examine the binding of mAb B38 and H4 to SARS-CoV-2 RBD protein was performed. Three biological replicates were performed, and each sample was subjected to three technical replicates to assess the mAb binding efficiency. Briefly, RBD of SARS-CoV-2 protein (His tagged RBD produced in Sf9 insect cells, Z03479; Genscript Biotech, United States) was bound to 96-well microplates (Greiner Bio-One GmbH, Frickenhausen, Germany). After overnight incubation, the plates were blocked with 5% skim milk (BD, Franklin Lakes, NJ, United States) in 1 × PBS for 2 h, washed three times with PBST, and incubated with plant-produced mAbs B38 and H4. After 2-h incubation at 37°C, sheep anti-human kappa LC conjugated with HRP (Southern Biotech, United States) at a dilution of 1:1,000 in 1 × PBS was added, and samples were incubated for 1 h at 37°C. The plate was then washed three times with PBST, developed using TMB substrate (Promega, United States), and the absorbance was read at 450 nm. The commercially available human IgG1 (Abcam, United Kingdom) and plant-produced human anti-PD1 antibody (Rattanapisit et al., 2019b (link)) was used as negative controls.

Serum IgM and IgG antibodies against ZIKV were detected by ELISA using formaldehyde-inactivated ZIKV. Briefly, 96-well microtiter plates were coated overnight with 10³ PFU of inactivated ZIKV. Diluted plasma samples (1:50) were incubated with the coated antigen for 2 h, followed by 1 h incubation of either HRP-conjugated goat-anti-monkey IgM or IgG (Abcam, UK). After washing, TMB-substrate (Promega, USA) was added to the wells and the plate was incubated for 10 min at room temperature in darkness. Then, 2.0 M H₂SO₄ was added to stop the reaction, and plates were measured at 450 nm using a microplate reader (Beckman, USA). Endpoint titers were considered the highest dilution that resulted in a value two-fold greater than the absorption of the control serum, with a cut-off value of 0.05.
Neutralizing antibody titers were determined by a constant virus-serum dilution 50% plaque reduction neutralization test (PRNT₅₀) as previously described. Briefly, serial 2-fold dilutions of inactivated serum were mixed with equal volumes of ZIKV in DMEM supplemented with 2% FBS. After incubation at 37 °C for 1 h, virus-antibody mixtures were added to plates containing BHK-21 cells. The concentration of infectious virus was determined using the plaque assay described above. The endpoint neutralization titer was calculated according to the method of Reed and Muench.

The binding affinity of the plant-produced RBD to ACE2 was analyzed by ELISA. Briefly a 96-well plate (Greiner Bio-One GmbH, Germany) was coated with 2 μg/ml of commercially available ACE2 (ab273687, Abcam, UK) and incubated overnight at 4ºC. After incubation, the coating buffer was discarded and the plates were blocked with 5% skim milk in 1XPBS for 2 h at 37 °C. The plate was then washed three times with 1X PBST and incubated with the plant-produced RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD protein (R & D Systems, USA) for 2 h at 37 °C and non-infiltrated (WT) plant protein was used as a control. Then the plates were washed three times with 1X PBST followed after which an anti 6xHis antibody (ab1187, Abcam, UK) diluted (1:1000) in 1X PBS was added to the plate which was then incubated for 1 h at 37 °C. Finally the plate was washed with 1X PBST, and the signal developed with TMB substrate (Promega, USA) and the absorbance read at 450 nm.

Western blotting was carried out on whole cell lysates extracted using RIPA buffer (Thermo, USA) on ice. Whole protein was extracted from 4 patients’ and 4 healthy controls’ fibroblasts and quantified using BCA quantification kit (Abcam, UK). The details were as follows: 20 μg of each sample were denatured in Laemmli buffer for 5 minutes at 95°C. Protein separation was achieved using SDS polyacrylamide gel electrophoresis and transferred onto PVDF membrane using Trans-Blot^® SD semi dry transfer system (BioRad, USA). Membranes were blocked with 5% skimmed milk in TBST buffer for 1 hour and then incubated with primary antibodies, diluted in 1% blocking buffer at 4°C overnight. After that, they were washed and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Washed immunoblots were then incubated with TMB substrate (Promega, USA) for 2 minutes. Image acquisition was performed using ChemiDoc imaging system (BioRad, USA). Antibodies used: rabbit anti-aprataxin (1:500; Abcam, ab192598) and mouse anti-β-actin (1:500; Abcam, ab8227), anti-rabbit IgG H&L (1:1000, Abcam, ab6721), and anti-mouse IgG H&L (1:1000, Abcam, 6728).

A 96-well ELISA plate (Corning, New York, USA) was coated with 25 μl of 2 μg/ml human ACE2 protein (Sino Biological, Beijing, China) and incubated overnight at 4°C. The coated plate was washed three times with 1xPBST (1xPBS with 0.05% Tween20) and blocked with 5% skim milk for 1 h at 37°C. Then, the plate was washed three times with 1xPBST and incubated with 0.02–5.00 μg/ml of purified plant-produced SARS-CoV-2 RBD-Fc or Alpha RBD-Fc or Beta RBD-Fc or Fc protein for 2 h at 37°C. The plate was washed three times with 1xPBST and incubated with HRP-conjugated goat anti-human IgG diluted 1:2,000 in 1xPBS for 1 h at 37°C. The plate was washed and the signal was detected with 25 μl of TMB substrate (Promega, Wisconsin, USA) and stopped with 25 μl of 1M H₂SO₄. The absorbance was measured at 450 nm using SpectraMax M5 (Molecular devices, California, USA). The experiment was performed in three replicates and the data are presented as mean±SD.

The microplate (Corning, Corning, NY) wells were coated with 100 μl of PA of anthrax (BEI resources, Manassas, VA) at concentration of 2.5 μg/ml for 1 h in 1X PBS buffer, and then the plate was blocked with 5% nonfat milk for 30 min. The rCMG2-Fc samples in 1X PBS with 1 mM MgCl₂ (controls and 37°C incubated samples) were loaded to the plate, 100 μl per well (starting from 2.5 μg/ml, 2.5X serial dilutions). The functional rCMG2-Fc bound to the PA was detected with an HRP-conjugated goat anti-human IgG (Southern Biotech, Birmingham, AL) at concentration of 0.5 μg/ml for 1 h. Plates were washed for three times with 1X PBST between steps above, and all the steps were done at room temperature. The 37°C incubation was eliminated to avoid potential effects on protein activity. The plate was developed with 100 μl of TMB substrate (Promega, Fitchburg, WI) and stopped by the 100 μl of 1 N HCl. The absorbance at 450 nm was read with a microplate reader (Molecular Devices, San Jose, CA).