The secondary screening consisted of: (1) blocking the RBD:hACE-2 interaction in an Intellicyt® iQue3 system (Sartorius; Göttingen, Germany) as described by Mendoza-Salazar et al. [8 (link)] and (2) an alternative assay called competition assay, which was similar to the blocking assay except that the biotinylated RBD protein (SPD-C82E9, Acro Biosystems) was captured by the SAv (streptavidin) beads (iQue Qbeads® DevScreen, Sartorius) and 20 µL of each antibody dilution plus 20 µL of 50 ng/mL biotinylated hACE-2 (AC2-H82E6, Acro Biosystems) were transferred to a 96 V-wells plate. Next, 10 µL of RBD-Qbeads were added. The Qbeads-RBD-hACE-2-biotin were detected with 10 µL of 1:500 dilution of Streptavidin-PE.
Ac2 h82e6
Manufactured by ACROBiosystems
Sourced in Germany, United States
The AC2-H82E6 is a laboratory equipment product manufactured by ACROBiosystems. It is designed to perform a specific function, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Octet red96 system, by Molecular Devices (2 mentions)
Spd c82e9, by ACROBiosystems (1 mentions)
Tetramethylbenzidine, by Merck Group (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Data analysis software 10, by Molecular Devices (1 mentions)
Kinetics buffer, by Sartorius (1 mentions)
Data analysis suite, by Molecular Devices (1 mentions)
Streptavidin coated biosensors, by Molecular Devices (1 mentions)
6 protocols using ac2 h82e6
1
RBD-hACE2 Interaction Blocking Assay
2
SARS-CoV-2 RBD Nanobody Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SARS-CoV-2 RBD (#SPD-C52H3, ACRO Biosystems) was coated at 0.5 µg/mL in sodium carbonate buffer (pH 9.6), 100 µL per well, onto Nunc Maxisorp plates overnight at 4 °C. Coating solution was removed, and plate blocked with 300 µL 2% BSA (#5217, Tocris) in 1xPBS for 1 h at room temperature. Purified nanobodies were diluted at specified concentrations in 0.2% BSA in 1xPBS and transferred to the blocked plate in triplicate. The nanobodies were incubated for 45 min to allow for association with RBD. Biotinylated ACE2 (AC2-H82E6, ACRO Biosystems) was prepared at 0.2 µg/mL in 0.2% BSA solution. 10 µL of the ACE2-biotin solution was transferred into each well of the assay and allowed to incubate for 15 min with at 600 rpm. The assay plate was washed and 100 µL of poly-streptavidin (#85R-200, Fitzgerald Industries International) diluted 1:2000 in 2% BSA solution was transferred to each well and incubated with at 600 rpm for 30 min. After a final wash the plate was developed by addition of 100 µL tetramethylbenzidine (#T5569, Sigma-Aldrich). Assay development was stopped by addition of 50 µL 1 M sulfuric acid and the assay absorbance measured at 450 nm on a Biotek Synergy 2 plate reader. The assay plate was washed with 1xPBS 5 times between each step of the assay.
3
SARS-CoV-2 RBD-ACE2 Binding Kinetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RBD-ACE2 binding measurements were performed as described before73 (link) using an Octet® RED96 system (ForteBio) with integrated data acquisition software 10.0. Streptavidin (SA) biosensors (Sartorius #18-5019) were pre-hydrated in 1X Kinetics Buffer (Sartorius #18-1105) and were then loaded with biotinylated human ACE2/ACEH recombinant protein (Acrobiosystems #AC2-H82E6) for up to 0.5-1 nm thickness. The ACE2 loaded SA biosensors were then dipped in RBD or its mutants in the concentration of 0.07-50 μg/mL in 1X Kinetics Buffer for 600 s for association, followed by being immersed in 1X Kinetics Buffer alone for another 600 s for dissociation. All steps were conducted at 26°C with constant shaking at 1,000 RPM. Collected data were analyzed using ForteBio Data Analysis software 10.0. The Kon and Koff values were determined using a built-in 1:1 global curve-fitting model and the Kd values were calculated. In antibody blocking experiments, diluted human sera were pre-incubated with 10 μg/mL of RBD or its mutants at 37°C for 60 min before being applied to ACE2-loaded SA biosensors as described above.
4
Affinity Binding of RBD-ACE2 Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bio-layer interferometry was used to measure the affinity binding constants of the RBD spy tag. All assay conditions were prepared in a Greiner 96-well plate (#655209) in a volume of 250 µL using Kinetic assay buffer (1X PBS containing 0.05% Tween-20 including 0.5%BSA, pH 7.2). Biotinylated ACE-2 human protein (AC2-H82E6, ACRO Biosystems) with a C-terminal AviTag was diluted into assay buffer at 10 µg/mL and immobilized onto streptavidin-coated biosensors (#18-5019, Forte Bio) to a minimum response value of 1 nm on the Octet Red96 System (Forte Bio). A baseline response was established in the assay buffer before each association. The RBD spy tag was diluted into assay buffer at a 10–300 nM grade concentration. The RBD spy tag was allowed to associate for 200 s, followed by dissociation for 600 s in the same baseline wells. The assay included one biosensor with only assay buffer, which was used as the background normalization control. Using the Forte Bio Data Analysis suite, the data were normalized to the association curves following background normalization and Savitzky–Golay filtering. Curve fitting was applied using a 1:1 interaction model with the global fitting of the sensor data, and a steady state analysis was used to determine the association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD).
5
Isolation of SARS-CoV-2 spike-specific scFv
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of single-chain Fv fragments (scFv) clones specifically reacting with the human SARS-CoV-2 spike protein was performed according to our previous report with some modifications (23 (link)). Biotinylated human SARS-CoV-2 spike protein (#HAK-SPD _BIO-1, Hakarel Co., Ltd., Ibaraki, Japan) and biotinylated human ACE2 protein (#AC2-H82E6, AcroBiosystems, Inc., DE, U.S.A.) were used as antigens. First, the biotinylated ACE2 protein was mixed with the scFv phage display library constructed from naïve donors to remove scFv reacting with the ACE2 protein non-specifically (negative panning). Subsequently, the resultant library was mixed with SARS-CoV-2 protein to enrich for specific scFv (positive panning). After two-rounds of negative and positive panning, soluble scFv expression in Escherichia coli infected with the phage was induced. The resulting supernatant was immediately used for enzyme-linked immunosorbent assay (ELISA) screening. One scFv clone that reacted with SARS-CoV-2 but not with ACE2 was isolated. Sequences containing the IgH region, peptide linker, and IgK region are shown in Supplementary Data Sheet 2
6
Recombinant ACE2 Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant dimeric ACE2 proteins were purchased or produced from commercial sources. Recombinant human ACE2 (Uniprot: Q9BYF1-1) was purchased from ACROBiosystems (AC2-H82E6), consisting of residues 18-740 spanning an intrinsic dimerization domain, followed by a His tag and biotinylated Avitag used for downstream detection. Civet (Paguma larvata) ACE2 (Uniprot: Q56NL1-1) was purchased from ACROBiosystems (AC2-P5248), consisting of residues 18-740 spanning an intrinsic dimerization domain, with an N-terminal His tag used for downstream detection. Mouse (Mus musculus) ACE2 (Uniprot: Q8R0I0-1) was purchased from Sino Biological (50249-M03H), consisting of residues 18-740 spanning an intrinsic dimerization domain, followed by a His tag and human IgG1 Fc domain used for downstream detection.
The remaining ACE2s were produced by Genscript. Specifically, pangolin (Manis javanica, Genbank: XP_017505746.1), R. affinis 787 (Genbank: QMQ39222), R. affinis 9479 (Genbank: QMQ39227), R. sinicus 3364 (Genbank: QMQ39219), and R. sinicus 1434 (Genbank: QMQ39216) ACE2 residues 19-615 were cloned with a C-terminal human IgG1 Fc domain for dimerization and downstream detection. pcDNA3.4 expression plasmids were transfected into HD 293F cells for protein expression. ACE2-Fc fusions were purified from day six culture supernatants via Fc-tag affinity purification.
The remaining ACE2s were produced by Genscript. Specifically, pangolin (Manis javanica, Genbank: XP_017505746.1), R. affinis 787 (Genbank: QMQ39222), R. affinis 9479 (Genbank: QMQ39227), R. sinicus 3364 (Genbank: QMQ39219), and R. sinicus 1434 (Genbank: QMQ39216) ACE2 residues 19-615 were cloned with a C-terminal human IgG1 Fc domain for dimerization and downstream detection. pcDNA3.4 expression plasmids were transfected into HD 293F cells for protein expression. ACE2-Fc fusions were purified from day six culture supernatants via Fc-tag affinity purification.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!