Journal: Npj viruses

Article Title: Automated and virus variant-programmable surrogate test qualitatively compares to the gold standard SARS-CoV-2 neutralization assay.

doi: 10.1038/s44298-024-00083-9

Figure Lengend Snippet: Fig. 1 | Schematic diagram of the sVNT for SARS- CoV-2. The capture reagent is a DIG-labeled RBD (red) that binds to the anti-DIG antibody attached to the GNR wall (blue). Capture-protein-specific antibodies (gold) in the serum compete with His- tagged ACE-2 (brown and orange) to decrease the signal. The ACE-2-His bound to the DIG-labeled RBD is measured using a biotinylated anti-His antibody (pink) that produces a fluorescent signal when the biotin binds to a fluorescent streptavidin conjugate (green).

Article Snippet: We determined RBD concentrations using a bicinchoninic acid (BCA) protein assay (Kit-23227, PierceTM, Thermo Fisher Scientific Rockford, IL) andprepared theDIG-labeled stocks at 10 μg/mL in sample diluent 13 (SD13; part # 896069, ProteinSimple).We determined the half maximal effective concentration at 50% (EC50) of ACE2-His (R&D Systems 933-ZN) for the WT and Delta DIG-S1-RBDs using a Simple PlexTM customizable 48-DIG open cartridge with a dilution series of ACE2-His and 10 μg/mL of each RBD, measuring the relative fluorescence unit (RFU) response with a biotinylated mouse anti-His-tag IgG antibody (R&D Systems BAM050) that was prepared at 3.33 μg/mL in SD13.

Techniques: Labeling

Journal: Gels

Article Title: Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

doi: 10.3390/gels9030243

Figure Lengend Snippet: The production yield, activity, and purification of recombinant Cripto produced in the 3D microcarriers as compared to the 2D method. ( A ) SDS-PAGE analysis of the purification steps of recombinant Cripto: lane M is the protein molecular weight marker; lane 1 is the protein solution from ultrafiltration; lane 2 is the flowthrough after the first passage through the His-tag affinity Ni-NTA resin; lane 3 is the flowthrough after the second passage through the same resin; lane 4 is the first wash step; lane 5 is the second wash step; lane 6 is the first elution from the His-tag affinity Ni-NTA resin; and lane 7 is the second elution from the same resin. The band at approximately 27 kDa is the Cripto protein (indicated by the red arrow). ( B ) Quantitative amounts of Cripto protein produced in the 3D batch (Cripto (3D) ) with HEK293 cells encapsulated in PF microcarriers and incubated in bioreactors after three rounds of harvesting are compared to the maximum amount of Cripto produced in the 2D batch (Cripto (2D) ) with HEK293 cells adherent to cell culture plates and cultured to their density threshold limits. An initial cell seeding of 3.2 × 10 6 cells was used for both techniques. ( C ) The biological activity of recombinant Cripto was compared for Cripto produced in PF microcarriers versus the 2D method by measuring binding affinity to the AlK4 receptor. Four independent experiments were carried out for the 3D system and three independent experiments were performed for the 2D cultivation method. Results are shown as mean ± S.D. *** indicates p < 0.001.

Article Snippet: The plates were then washed three times and Cripto samples were added (300 μg) and incubated for 2 h. The plates were incubated with 0.5 μg/mL His-tag biotinylated antibodies (R&D systems BAM 050) in PBS-Tween for 1 h at 37 °C and then for 1 h at RT.

Techniques: Activity Assay, Purification, Recombinant, Produced, SDS Page, Molecular Weight, Marker, Incubation, Cell Culture, Binding Assay