A synthetic nanobody phage display library with high diversity was prepared as previously described.30 (link) Screening for nanobodies was performed by panning in both immunotubes and with magnetic bead-conjugated antigen, using SARS-CoV-2 variants P.1 and B.1.617 derived recombinant RBD proteins. Briefly, for the 2nd and 4th panning rounds, the purified SARS-CoV-2 RBD proteins were coated on Nunc MaxiSorp immunotubes (ThermoFisher) at 5 µg/mL in PBS overnight. For the 1st and 3rd panning rounds, RBD protein was first biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin (ThermoFisher) and then selected with streptavidin-coated magnetic Dynabeads™ M-280 (ThermoFisher). The panning was performed according to a standard protocol.30 (link) After four rounds of panning, phage ELISA identification was performed with 960 individual colonies using Anti-CM13 antibody in the plates coated with recombinant RBDs. The absorbance was measured using a SpectraMax M5 plate reader from Molecular Devices (San Jose, CA, USA). The positive clones were sent for sequencing. After sequence alignments, the distinct sequences were chosen for protein expression.
Nunc maxisorp immunotubes
Manufactured by Thermo Fisher Scientific
Nunc MaxiSorp immunotubes are a type of laboratory equipment used for enzyme-linked immunosorbent assay (ELISA) procedures. They feature a MaxiSorp surface, which provides high protein-binding capacity. The tubes are made of polystyrene material.
Automatically generated - may contain errors
2 protocols using nunc maxisorp immunotubes
1
Synthetic Nanobody Screening for SARS-CoV-2
2
Screening for Broadly Neutralizing Nanobodies
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A synthetic nanobody phage display with high diversity was prepared by our laboratory as previously described (32 (link), 35 (link)). To screen for broadly neutralizing nanobodies, three recombinant RBD proteins were used, and four rounds of panning were performed in both immunotubes and with magnetic bead-conjugated antigens. Briefly, for the second and fourth panning rounds, SARS-CoV RBD proteins were coated on Nunc MaxiSorp immunotubes (Thermo Fisher) at 5 μg/mL in PBS overnight. For the first and third panning rounds, SARS-CoV-2 variants Alpha or Omicron BA.5 RBD was first biotinylated with EZ-Link Sulfo-NHS-LCBiotin (Thermo Fisher) and then selected with streptavidin-coated magnetic Dynabeads M-280 (Thermo Fisher). Panning was performed following the standard procedure as previously described (32 (link), 35 (link)). After biopanning, phage ELISA was performed with 480 individual colonies using anti-CM13 antibody (HRP) in plates coated with recombinant RBDs. The absorbance was measured using a SpectraMax M5 plate reader from Molecular Devices (San Jose, CA, USA). Positive clones were sent for sequencing, and after sequence alignments, distinct sequences were chosen for protein expression.
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