Phage display selections, direct phage ELISAs and competitive phage ELISAs were performed as described [50 ] with minor modifications. Briefly, phage particles from the libraries were cycled through rounds of binding selections with SEB coated directly on 96-well Maxisorp immunoplates (Fisher Scientific, Nepean, ON, Canada) or biotinylated SEB captured by immobilized neutravidin (Thermo Fisher Scientific, Rockford, IL, USA) as the capture target. After four or five rounds of selections, phage particles were produced from individual clones grown in a 96-well format, and the culture supernatants were used in phage ELISAs to detect specific binding clones. Clones that bound specifically to SEB were subjected to DNA sequencing to decode the sequences of the displayed Fabs. To assess competition between Abs for binding to SEB antigen, a competitive phage ELISA was used, whereby binding of Fab-phage to immobilized SEB was assessed in the presence of solution-phase Fab protein, as described [50 ].
Immobilized neutravidin
Manufactured by Thermo Fisher Scientific
Sourced in Canada, United States
Immobilized neutravidin is a protein-based reagent that can be used for the selective capture and purification of biotinylated molecules. It provides a stable and reversible interaction between the immobilized neutravidin and the biotin-labeled target, enabling efficient separation and recovery of the desired analyte.
Automatically generated - may contain errors
2 protocols using immobilized neutravidin
1
Phage Display for Anti-SEB Antibodies
2
Immobilized NeutrAvidin Protein Assay
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Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). VEGF was purchased from R&D Systems (Minneapolis, MN, USA). Immobilized NeutrAvidin was purchased from Thermo Scientific (Waltham, MA, USA). Biotin was purchased from Sigma-Aldrich. All oligonucleotides (Table 1 ) were obtained from Life Technologies (Carlsbad, CA, USA). PNAs were purchased from Panagene (Daejeon, Korea).
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