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SPD-C82E9

Manufactured by ACROBiosystems
Sourced in United States, Germany

The SPD-C82E9 is a high-speed centrifuge designed for laboratory applications. It features a maximum speed of 82,000 rpm and a maximum relative centrifugal force (RCF) of 501,000 x g. The centrifuge is capable of processing a wide range of sample volumes and can accommodate various rotor types to meet the needs of diverse research and analysis requirements.

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8 protocols using SPD-C82E9

Bicycle bacteriophage (phage) libraries, consisting of linear peptides (10 to 20 amino acids in length) containing 3 cysteines which are cyclized in situ to form thioether-bonded bicyclic peptide libraries were used in selections against either S1 (ACROBiosystems S1N-C82E8), S1-RBD (ACROBiosystems SPD-C82E9), S2 (ACROBiosystems, S2N-C52E8) or Spike Trimer (pCOV55) recombinant proteins. Four rounds of selection were performed, using decreasing target concentrations of protein immobilized onto streptavidin magnetic beads. Binders were eluted at low pH. After round 4, phage clones were isolated, sanger sequenced, and tested for binding to proteins using AlphaScreen assay. Binders were chemically synthesized as peptides for further characterization. Certain lead clones subsequently returned to the phage platform for further affinity maturation. For these, custom Bicycle phage libraries were constructed based on the initial sequence, retaining residues thought to be important for binding and randomizing others. Phage selections using these custom libraries were carried out as before to identify binders with improved affinity over the parent sequences.
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The ability of cells to bind SARS-CoV-2 spike and spike 1 proteins was analyzed by confocal microscopy using biotinylated spike proteins. Cells were prepared as described earlier and labeled with 250 ng of either biotinylated spike protein (RBD) (SPD-C82E9; ACROBiosystems, Newark, DE, USA) or biotinylated spike 1 protein (SIN-C82E8; ACROBiosystems) for 30 min. Unbound spike proteins were removed by washing cells twice with 200 μL of PermaCyte medium. Bound spike proteins were visualized by staining with streptavidin-Alexa Fluor 594 (S11227; Life Technologies). Cells were counterstained with DAPI to visualize the nuclei. This procedure allowed visualization of binding to surface and cytoplasmic ACE-2 and is outlined in Figure 1B.
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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.
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PMMA (polymethyl methacrylate) 8.2 μm microbeads coated with streptavidin were purchased from PolyAn (Cat#10652009). Each type of microbead presented a different fluorescence intensity (Red4 dye, excitation: 590–680 nm/emission: 660–780 nm). First, microbeads were washed with cold phosphate-buffered saline (PBS) pH 7.2 (Gibco Cat#14190-094) by centrifugation at 2000 r.p.m. for 5 min and resuspended in PBS. Then, biotinylated recombinant RBD, S1, and N (Acrobiosystems Cat#SPD-C82E9, Cat#S1N-C82E8, and Cat#NUN-C81Q6, respectively) were added to the tubes (RBD at 11 μg/mL, S1 at 30 μg/mL, N at 19,5 μg/mL) and kept at 4 °C on a rotating head over tail for an hour, protected from light. Positive control beads were coated with biotinylated human IgG (Novus Biologicals Cat#NBP1-96855) and IgM (Novus Biologicals Cat#NBP1-96989) on the same microbead at 15 μg/mL each. Negative control beads were not coated with protein. After the coupling reaction, microbeads were washed three times with PBS. Then, d-biotin (2 µM) (Sigma-Aldrich Cat#8512090001) was added and incubated for 15 min at room temperature (RT) to inactivate residual streptavidin. After three additional washes, equal amounts of each microbead were combined in the same vial.
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Vaccine-induced mucosal IgA specific to SARS-CoV-2 variant spike and RBD were measured with the Mesoscale Discovery (MSD) 4-spot U-PLEX Development Pack (MSD cat#K15229N). Spots were linked with anti-Syrian hamster IgA antibody (Brookwood Biomedical cat#sab3001a) biotinylated with the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Fisher Scientific cat#A39257) and either biotinylated Wuhan, delta, or omicron variant SARS-CoV-2 spike trimer and RBD proteins (ACROBiosystems cat#SPD-C82E9, SPN-C82Ec, SPD-C82Ed, SPN-C82Ee, SPD-C82E4). Plates were coated, blocked, washed, and incubated with sample and detection antibody according to the manufacturer’s recommendations. Nasal samples were diluted at 1:15 and oral samples were diluted at 1:5 in Diluent-100 (MSD cat#R50AA). The anti-Syrian hamster IgA antibody was sulfo-tagged with the MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack (MSD cat#R31AA) and diluted with Diluent-100 to 2 µg/mL for the nasal samples and 1 µg/mL for the oral samples. Plates were read on a Meso QuickPlex SQ 120 instrument (MSD). Samples were reported as relative light units (RLUs). Due to the variability in mucosal sampling, samples were normalized by total IgA and expressed as fold change was reported as vaccinated over unvaccinated sample. Data analysis was performed in GraphPad Prism (Version 9.4.1).
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Bio-layer interferometry was used to measure the affinity binding constants of purified VHH clones. Assay buffer is defined as 0.1% BSA (w/v) in 1xPBS. All assay conditions are prepared in a Greiner 96-well plate (#655209) in a volume of 300 µL. Biotinylated SARS-CoV-2 S protein RBD (SPD-C82E9, ACRO Biosystems) with a C-terminal AviTag was used to ensure uniform directionality of the protein. Biotinylated-RBD was diluted into assay buffer at 1 µg/mL and immobilized onto streptavidin coated biosensors (#18-5019, BioForte) to a minimum response value of 1 nm on the Octet Red96 System (ForteBio). A baseline response was established in assay buffer prior to each association. The purified VHH clones were diluted into assay buffer at the specified concentrations (typically 1000–0 nM). The VHH clones were allowed to associate for 180–240 s followed by dissociation for 300–600 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 ForteBio Data Analysis suite, the data was normalized to the association curves following background normalization and Savitzky-Golay filtering. Curve fitting was applied using global fitting of the sensor data and a steady state analysis calculated to determine the association and dissociation constants.
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Custom-made Becton Dickinson (BD) functional cytometric bead array (CBA) beads were used in preparation of streptavidin beads as described in the Functional Bead Conjugation Buffer Set Instruction Manual (BD 558556). Briefly, 1 mg of SMCC-modified streptavidin per mL (in 50 mM sodium phosphate, 0.2 M sodium chloride, pH 7.4) was used for conjugation to CBA beads as described in the above protocol. The streptavidin beads were suspended in 1x PBS, 0.5% BSA, pH 7.2 before coating with biotinylated RBD proteins. Streptavidin microbeads were then coated with antigen as described before (18 (link)). Briefly, streptavidin coated microbeads were incubated with 11 μg/mL of biotinylated RBD proteins from the following SARS-CoV-2 variants: RBD Wuhan-1 (Acrobiosystems SPD-C82E9), RBD-B.1.351 (β, Acrobiosystems SPD-C82E5), RBD- B.1.617.1 (κ, Acrobiosystems SPD-C82Ec) and RBD-B.1.1.529 (ο, Acrobiosystems SPD-C82E4). Uncoated microbeads were used as negative controls. All microbeads were incubated with an excess of D-biotin to saturate the remaining free streptavidin sites.
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ELISA-positive clones were further assessed by bio-layer interferometry binding to RBD. Assay buffer was defined as 0.1% BSA (w/v) in 1xPBS. All assay conditions were prepared in a Greiner 96-well plate (#655209) in a volume of 300 µL. Biotinylated SARS-CoV-2 S protein RBD (SPD-C82E9, ACRO Biosystems) with a C-terminal AviTag was used to ensure uniform directionality of the protein. Biotinylated-RBD was diluted into assay buffer at 1 µg/mL and immobilized onto streptavidin coated biosensors (#18-5019, BioForte) to a minimum response value of 1 nm on the Octet Red96 System (ForteBio). A baseline response was established in assay buffer prior to each association. Candidate clone supernatant prepared previously was diluted 1:1 with assay buffer and allowed to associate for 60 s and dissociate for 60 s, which provided a sufficient interval to detect positive binding. Biosensors were regenerated in 250 mM imidazole in assay buffer for 45 s which removed residual bound candidate nanobody and returned to the baseline well. This method allowed for detection of clones that bind RBD in both ELISA and a qualitative estimate of binding based on response curve dynamics. Clones positive on both the RBD ELISA and bio-layer interferometry measurement were selected for sequencing and further characterization.
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