Plates were washed four times with 1 PBST and then incubated with TMB substrate (BD Biosciences) for ten minutes. a SARS-CoV-2 neutralizing cocktail. This work may offer a strategy for using plants to quickly develop mAb cocktail-based therapeutics against emerging viral diseases with high efficacy and low costs. Keywords: SARS-CoV-2, COVID-19, monoclonal antibody (mAb), plant-made antibody, antibody cocktail, neutralization synergy, plant-made pharmaceutical 1. Introduction The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an unprecedented public health crisis. SARS-CoV-2 is a betacoronavirus of the family for agroinfiltration, as described previously [41]. Six-week-old plants were agroinfiltrated and leaves were harvested at peak recombinant protein expression (7 days post infiltration). 2.2. mAb Extraction and Purification MAbs were extracted from leaves and subjected to Protein A affinity chromatography, as previously described [41,42,43]. Briefly, plant leaves expressing a mAb were blended in a 1:1.5 ratio of fresh leaf mass: extraction buffer. Extraction buffer consisted of 1 phosphate-buffered saline (PBS), pH 5.2, 10 mg/mL of sodium L-ascorbate, 1 mM ethylenediaminetetraacetic acid Fedovapagon (EDTA), and 2 mM phenylmethylsulfonyl fluoride (PMSF). The plant extract was clarified by centrifugation and host protein precipitation overnight at pH 5.2. The clarified extract was filtered through a 0.22 m filter prior to enrichment and purification using MabSelect (GE Healthcare, now Cytiva, Chicago, IL, USA) resin for affinity chromatography. 2.3. SDS-PAGE and Western Blotting Purified, plant-made CA1 and CB6 were assessed by SDS-PAGE and Western blot analysis, as described [41,44]. Briefly, purified CA1 and CB6 were subjected to SDS-PAGE under reducing and non-reducing conditions and separated on 4C20% gradient polyacrylamide gels (Bio-Rad). The gels were then stained with Coomassie Brilliant Blue R-250 or proteins were transferred to PVDF membranes. The membranes were then probed with either goat anti-human kappa chain (Southern Biotech, Birmingham, AL, USA, Cat. No. 2060-05) or goat anti-human IgG (Southern Biotech, Cat. No. 2040-05) conjugated to horseradish peroxidase (HRP), prior to development with the Pierce ECL Western blotting substrate (Thermo Scientific, Waltham, MA, USA) and image capture with an ImageQuant instrument. 2.4. Temporal Expression Rabbit polyclonal to ADAMTSL3 ELISA Temporal analysis of mAb expression in plant leaves was performed, as described previously [30]. Briefly, goat anti-human IgG (Southern Biotech, Cat. No. 2040-01) was coated in a carbonate-bicarbonate buffer on high-binding, 96-well plates at a concentration of 2 g/mL at 4 C overnight. Wash steps were performed with phosphate-buffered saline with 0.05% Tween?20, pH 7.4 (PBST), four times between each step. Leaves harvested at one-day intervals after agroinfiltration were blended as described above and a two-fold dilution series was added to the plate, alongside a human IgG standard curve dilution series. Goat anti-human kappa chain conjugated to HRP (Southern Biotech, Cat. No. 2060-05) was used to detect the captured plant-made mAbs. A KPL 3,3,5,5-tetramethylbenzidine (TMB) substrate (SeraCare Life Sciences Inc., Milford, MA, USA) was used in conjunction with a 1M H2SO4 stop solution. Absorbance data were analyzed with GraphPad Prism to calculate the microgram of recombinant antibody per gram of fresh leaf weight. 2.5. RBD-Binding ELISA Recombinant RBD from the WA1/2020 strain was coated in a carbonate-bicarbonate buffer on high-binding 96-well plates at a concentration of 2 g/mL Fedovapagon at 4 C overnight. Wash steps were performed with 1 PBST, four times between each step. Purified, Fedovapagon plant-made CA1 or CB6 dilutions were added to the plate, and antibodies bound to RBD were detected with goat anti-human IgG conjugated to HRP (Southern Biotech, Cat. No. 2040-05). A KPL TMB substrate was used in conjunction with a 1M H2SO4 stop solution to develop the plates. Absorbance data were analyzed with GraphPad Prism software with a value calculated by nonlinear regression analysis using a one-site binding model. 2.6. Competitive Sandwich ELISAs Serial dilutions of each antibody (plant-made CA1 and CB6, and hybridoma-made 3C4 and 11D7) were coated overnight at 4 C in Fedovapagon 96-well plates. The plates were then incubated with recombinant WA1/2020 RBD at 2 g/mL for 1 h at 37 C. After washing with 1 PBST, the plates were further incubated with either CR3022 or.