Binding kinetics of mAb P4A2 to RBDs from various VOCs by ELISA and biolayer interferometry (BLI).

(A) P4A2 was immobilized on anti-mouse Fc biosensor and was tested using three-fold serial dilutions of RBD (starting with 300 nM and going down to 3.3 nM; the five concentrations tested are indicated). Data shown is after the reference was subtracted and aligned using Octet Data Analysis software v11.1 (Forte Bio). Curve fitting was done with a 1:1 binding model, and k_on, k_off and K_d values were calculated using a global fit. (B) Cross reactive binding potential (with half-maximal effective concentration, EC₅₀) of P4A2 mAb to RBD proteins of different VOCs was tested by indirect ELISA. (C) The epitope specificity of P4A2 was evaluated for epitope competition using BLI. RBD-Fc was captured using anti-human Fc biosensor and saturated with P4A2 and the indicated mAbs at a concentration of 40 μg/ml and unbound P4A2 was washed, followed by incubation with 20 μg/ml of ACE2, P4A2 and P5B3 (binds to a topologically distinct, non-competing epitope and served as a control). No binding signal was observed for P4A2 and ACE2.

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