For competitive ELISA used in epitope mapping of mAbs, 2 μg/mL recombinant RBD-his (Sino Biological, Beijing, China) was added in 384-well plates and incubated at 4 °C overnight. 50 μg/mL mAbs per well were added. The plates were incubated at 37 °C for 1 h and then washed. Biotinylation of mAbs (the top 20 neutralizing Abs and 81A11, previously reported SARS-CoV CR302224 (link)) were performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) according to the manufacturer’s protocol and purified using MINI Dialysis Unit (ThermoFisher, 69576). 500 ng/mL biotinylated mAbs were added to each well, and the plates were incubated at 37 °C for 1 h. ALP-conjugated streptavidin (Mabtech, Sweden, 3310-10) was added at 1:1000, followed by an incubation of 30 mins at 37 °C. For the quantification of bound IgG, PNPP (Thermo Fisher) was added at 1 mg/mL and the absorbance at 405 nm was measured by the MultiSkan GO fluoro-microplate reader (Thermo Fisher).
Mini dialysis unit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MINI Dialysis Unit is a compact device designed for sample preparation and buffer exchange. It utilizes a semi-permeable membrane to facilitate the removal or addition of small molecules, ions, or other solutes while retaining the larger target molecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
Octet r2 protein analysis system, by Molecular Devices (2 mentions)
Streptavidin sa biosensor, by Molecular Devices (2 mentions)
Rbd his, by Sino Biological (1 mentions)
Multiskan go fluoro microplate reader, by Thermo Fisher Scientific (1 mentions)
Alp conjugated streptavidin, by Mabtech (1 mentions)
Sa biosensor, by Molecular Devices (1 mentions)
H 600, by Hitachi (1 mentions)
L 2000, by Hitachi (1 mentions)
Ar2g biosensor, by Molecular Devices (1 mentions)
Hislink protein purification system, by Promega (1 mentions)
L arabinose, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
6 protocols using mini dialysis unit
1
Competitive ELISA for Epitope Mapping
2
Binding Kinetics of Anti-SARS-CoV-2 Antibody
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Bio-layer interferometry assays were conducted on Octet® K2 Protein Analysis System (Fortebio). Protein biotinylation was performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) and purified using MINI Dialysis Unit (ThermoFisher). After baseline adsorption of nonspecific binding, SA biosensors (Fortebio) were immersed with biotinylated WT and Omicron RBD to capture RBD to 0.3 nm and 1 nm, then sensors were immersed in kinetics buffer (0.02% Tween-20, 1 mg/mL BSA in PBS) to the baseline. After association with different concentrations of 510A5 for 180 s, disassociation was conducted for 300 s. For the detection of the affinity of 510A5 with Spike protein, biotinylated 510A5 Fab was captured to 0.3 nm. Different concentrations of WT and Omicron Spike were conducted for 180 s, and disassociation was conducted for 300 s. For ACE2 binding competition experiments, biotinylated WT or Omicron Spike protein was loaded at 1 μg/mL to 3 nm in kinetics buffer onto SA biosensors. Association of mAbs was performed in kinetics buffer at 20 μg/mL for 300 s, and then ACE2-Flag-His was loaded for 300 s at 40 μg/mL in kinetics buffer. Data were recorded and analyzed using Octet BLI Discovery (12.0).
3
Characterizing Liraglutide-Loaded Nanoparticles
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The particle size, size distribution and zeta potential of the NPs were determined by dynamic light scattering (DLS) analysis using a Zetasizer Nano ZS instrument (Malvern, Worcestershire, UK). The morphology of the NPs was determined using transmission electron microscopy (TEM; H-600; Hitachi, Tokyo, Japan) immediately after the samples were negatively stained with sodium phosphotungstate. To determine the drug encapsulation efficiency (EE) of the liraglutide-loaded NPs, a predetermined amount of NPs was dissolved in acetonitrile to release the liraglutide. The resulting mixture was agitated to ensure complete dissolution, then filtered and analyzed by reverse-phase HPLC (L-2000; Hitachi) with a C18 column (dimensions of 250×4.6 mm) to determine the liraglutide concentrations. The mobile phase consisted of acetonitrile (solvent A) and a 0.05-M aqueous KH2PO4 solution (pH 4.0, solvent B). The flow rate was set to 1.0 mL/min. NP samples (100 µL) at 0.02 mg/mL, enclosed in a mini dialysis unit (Thermo Fisher Scientific, Waltham, MA, USA), were incubated in 1 mL PBS (pH 7.4) at 37°C under 100-rpm orbital shaking. Thereafter, 100 µL samples were taken from the incubation medium and analyzed for liraglutide by HPLC as described earlier. After sampling, the incubation medium was replenished with blank PBS. The controlled release of liraglutide from the NPs was measured for 4 weeks.
4
Biolayer Interferometry Analysis of SARS-CoV-2 S Protein Variants
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Biolayer interferometry assays were carried out using the Octet R2 Protein Analysis System (Fortebio). Biotinylation and purification of 58G6 were performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) biotinylated and MINI Dialysis Unit (Thermo Fisher), respectively. The S proteins of SARS-CoV-2 and the Delta, Omicron BA.1 and Omicron BA.2 variants were immobilized onto AR2G biosensors (Fortebio) separately, with 58G6 used as an analyte to measure the affinities of 58G6 with four different sourced SARS-CoV-2 S proteins. After baseline adsorption of nonspecific binding, streptavidin (SA) biosensors (ForteBio) were immersed with biotinylated 58G6 to capture mAbs, and then the sensors were immersed in kinetics buffer (0.02% Tween-20, 1 mg/mL BSA in PBS) to the baseline. The disassociation was conducted after association with twofold diluted S proteins (diluted from 50 nM to 3.125 nM). Data were recorded using Octet BLI Discovery (12.0) and analyzed using Octet BLI Analysis (12.0).
5
Bacterial Expression and Purification of Recombinant RT Proteins
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The plasmids containing wild type or mutant RT p66 in pDEST 40 vector (Invitrogen, USA) were transformed into BL21 AI bacterial cells (Invitrogen, USA). The cells were cultured to the density of OD600 ~0.4 and the protein expression was induced by the final concentration of 1mM IPTG and 0.2% L-arabinose (Sigma, USA). The protein purification was carried out using the HisLink protein purification system according the product protocol (Promega, USA). The purified proteins were dialyzed using MINI dialysis unit (Thermo Scientific, USA) in PBS and examined by SDS PAGE and Coomassie blue staining.
6
Binding Kinetics of SARS-CoV-2 Antibody
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BLI assays were conducted on Octet R2 Protein Analysis System (Fortebio) as previously described (51 (link)). Protein biotinylation was performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce, A35358) and purified using MINI Dialysis Unit (Thermo Fisher Scientific). For measurement of the affinities of 55A8 for SARS-CoV-2 and Delta, Omicron BA.1, Omicron BA.2, and Omicron BA.4/5 variants, streptavidin (SA) biosensors (ForteBio) were immersed with biotinylated mAbs to generate capture mAbs. Then, the sensors were immersed in buffer (0.02% Tween-20, 1 mg/mL BSA in PBS) to the baseline. After association with 2-gold diluted proteins (diluted from 50 nM to 3.125 nM), disassociation was conducted.
For the mAb competition experiments, biotinylated S proteins were loaded at 1 nm onto SA biosensors, and mAb association was performed at 20 μg/mL for 300 seconds. For ACE2 competition experiments, the biotinylated RBD and S were loaded at 1 nm and 3 nm, respectively, at 1 μg/mL onto SA biosensors. The first antibody was allowed to associate for 600 seconds at 20 μg/mL, and the second protein (ACE2 [50 μg/mL] or a mixture of equal amounts of antibodies and ACE2) was allowed to associate for 300 seconds.
For the mAb competition experiments, biotinylated S proteins were loaded at 1 nm onto SA biosensors, and mAb association was performed at 20 μg/mL for 300 seconds. For ACE2 competition experiments, the biotinylated RBD and S were loaded at 1 nm and 3 nm, respectively, at 1 μg/mL onto SA biosensors. The first antibody was allowed to associate for 600 seconds at 20 μg/mL, and the second protein (ACE2 [50 μg/mL] or a mixture of equal amounts of antibodies and ACE2) was allowed to associate for 300 seconds.
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