2017;169:891C904.e15. two\fold serially diluted antibodies ranging from (50?g?mL?1C1.5?ng?mL?1) against 10?g antigen per well through ELISA. BSA was used as a control. EC50 values for scFvs D4, D8, D20, and D31 are 0.21?M, 0.74?M, 0.30?M, and 3.35?nM, respectively. All four antibodies except D31 have EC50 in the micromolar range. D31 has an EC50 in the nanomolar range (Physique?3a). Bio\Layer Interferometry (BLI) was used to quantitate the binding affinities of antibodies to 12 mer epitope. The affinity constants were calculated as equilibrium dissociation constant, for protein expression. Transformed cells were inoculated in fantastic broth media (Hi Media) and induced at OD600 of 1 1.5 with 1?mM IPTG at 18C overnight. Folded protein was extracted from periplasm of bacteria. For periplasmic extraction, 1?L of harvested cells were dissolved in 50?mL of Extraction buffer 1 (100?mM tris pH?8.0, 20% glucose, 1?mM EDTA) and incubated on ice for 1?h. Cells were pellet down and dissolved in extraction buffer 2 (MgCl2) and incubated on ice for 30?min. Supernatant of both the extraction were mixed and loaded on Ni\NTA column (Cytiva) overnight at 4C. Column was washed with Buffer A (50?mM tris pH?8.0, 150?mM NaCl, 20?mM imidazole). Protein was eluted using gradient of imidazole with Buffer A using Akta FPLC (GE Healthcare). Eluted fractions were analyzed on 12% SDS\PAGE gel. Proteins were concentrated and purified further using gel filtration chromatography with Sephacryl S\75 column (GE Healthcare). 4.6. Titration ELISA with soluble scFvs BSA\S2 peptide and BSA in 1 PBS was coated with 10? g/mL concentration in 96\well ELISA plate overnight at 4C. BSA was coated as control. Plates were washed Rabbit Polyclonal to ZNF682 three times with 1 PBS and blocked using 2% skim milk in 1 PBS for 2?h at RT. Purified antibodies were titrated from 50?g/mL up to 16 dilutions and incubated for 1.5?h at 37C. Plates were washed three times with PBST answer and secondary antibody, anti\his HRP conjugated antibody (Santa Cruz Biotechnology, Cat# sc\8036) in 1:5000 dilution was incubated for 1?h at 37C. Plates were washed three times with PBST answer. Color was developed using OPD (o\phenylenediamine) (HiMedia) and H2O2 (SigmaCAldrich). The color intensity was measured by OD at 490?nm using spectramax (Molecular Devices). EC50 were calculated with GraphPad Prism version 6.01. 4.7. Binding kinetics using biolayer interferometry All affinity measurements were carried out using BLI. BSA\S2 epitope and SARS\CoV\2 spike protein was biotinylated using 0.2?L of biotin 10?mg/mL stock solution (Thermo scientific) and immobilized on streptavidin biosensor (SA). BSA was immobilized as control. Buffer utilized for immobilization was 1 PBS with 0.05% Tween 20. Antibodies as analytes were diluted in 1 PBS buffer with two\fold serial dilution starting with 10?M. For regeneration, 10?mM glycineCHCl\pH?2.5 was used. Associations and dissociations were recorded for 120?s and 200?s, respectively. The data was analyzed using Forte Bio Data analysis software 10.0.0.1. Global fit 1:1 model was utilized for the analysis. 4.8. Circulation cytometry Human Embryonic Kidney 293?T (HEK293T; procured from ATCC) cells were seeded in 60?mm Dish and transfected with 2?g of plasmid DNA using PEI 25?K reagent (Polysciences). Cells harvested after 48?h were stained with scFvs for 1?h on ice. His\Tag (D3I1O) XP? Rabbit mAb (Alexa Fluor? 647 Conjugate, CST Cat# 14931) was used as secondary antibody to detect his\tag K252a scFvs. Stained cells were analyzed on a BD LSRFortessa using Diva software K252a (BD Bioscience). 4.9. Data collection and crystallography of scfv and S2 peptide complex For co\crystallization, S2 peptide was dissolved in 100% DMSO, and scFv proteins?were K252a mixed in 1:5 (protein: peptide) molar K252a ratio and incubated for 1?h at 20C prior to setting up the crystallization plates using hanging drop vapor diffusion method using Mosquito LCP nano\dispenser (TTP Labtech). The final concentration of scFv protein was 8?mg/mL in 30?mM Tris pH?8.0, 50?mM NaCl. S2 peptide\scFv complex crystals were obtained in 0.2?M sodium thiocyanate at pH?5.9, 20% PEG 1000 at 20C. The crystals were subjected to an X\ray beam on a home source with Rigaku FR\E+ SuperBright rotating\anode X\ray generator equipped with an R\AXIS IV++ detector at heat in 100?K. Collected data set was processed with the HKL 3000 (Minor et al.,?2006). Phases were decided through molecular replacement using PDB: 6DSI as the.
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