Alexa fluor nhs ester kit

A competitive flow cytometry-based assay was performed to characterize nAbs binding profiles to SARS-CoV-2 S-protein as previously described^{22 (link)}. Briefly, magnetic beads (Dynabeads His-Tag, Invitrogen) were covered with His-tagged S-proteins, following manufacturers’ instructions. Then, 40 mg/mL of beads-bound-S-protein were incubated with unlabeled nAbs for 40 min at RT. Following incubation, samples were washed with PBS and incubated with fluorescently labeled Class 1/2 (J08-A647), Class 3 (S309-A488), or Class 4 (CR3022-A647) S-protein nAbs binders. Antibodies labeling was performed using Alexa Fluor NHS Ester kit (Thermo Scientific). Following 40 min of incubation at RT, the beads-antibodies mix was washed with PBS, resuspended in 150 μl of PBS-BSA 1%, and acquired using BD LSR II flow cytometer (Becton Dickinson). Results were analyzed using FlowJo™ Software (version 10). Beads with or without S-protein incubated with labeled antibodies were used as positive and negative controls respectively.

To classify mAbs candidates on the basis of their interaction with Spike epitopes, we performed a flow cytometry-based competition assay as previously described^{3 (link),9} . Briefly, magnetic beads (Dynabeads His-Tag, Invitrogen) were coated with histidine-tagged S protein according to the manufacturer’s instructions. Then, 20 µg/mL of coated S protein beads were pre-incubated with unlabeled nAbs diluted 1:2 in PBS for 40 min at RT. After incubation, the mix of Beads-antibody was washed with 100 µL of PBS-BSA 1%. Then, to analyze epitope competition, mAbs able to bind RBD Class 1/2, (J08), Class 3 (S309), and Class 4 (CR3022) were labeled with three different fluorophores (Alexa Fluor 647, 488, and 594) using Alexa Fluor NHS Ester kit (Thermo Scientific), were mixed and incubated with S protein-beads. Following 40 min of incubation at RT, the mix of Beads-antibodies was washed with PBS, resuspended in 150 µL of PBS-BSA 1%, and analyzed using BD LSR II flow cytometer (Becton Dickinson). Beads with or without S protein incubated with labeled antibodies mix were used as a positive and negative control, respectively. FACSDiva Software (version 9) was used for data acquisition and analysis was performed using FlowJo (version 10).

To classify mAb candidates on the basis of their interaction with S epitopes, we performed a flow cytometry-based competition assay.In detail, magnetic beads (Dynabeads His-Tag, Invitrogen) were coated with histidine-tagged S protein according to the manufacturer’s instructions. Then, 20 µg/ml of coated S protein beads were pre-incubated with unlabelled nAb candidates diluted 1:2 in PBS for 40 min at room temperature. After incubation, the mix beads–antibodies was washed with 100 µl of 1% PBS-BSA. Then, to analyse epitope competition, mAbs that are able to bind to the RBD (J08 and S309), NTD (4A8) or S2 domain (L19) of the S protein were labelled with four different fluorophores (Alexa Fluor 647, 488, 594 and 405) using the Alexa Fluor NHS Ester kit (Thermo Scientific), were mixed and incubated with S protein beads. Following 40 min of incubation at room temperature, the mix beads–antibodies was washed with PBS, resuspended in 150 µl of 1% PBS-BSA and analysed using the BD LSR II flow cytometer (Becton Dickinson). Beads with or without S protein incubated with labelled antibodies mix were used as positive and negative controls, respectively. FACSDiva Software (version 9) was used for data acquisition and analysis was performed using FlowJo (version 10).