<scp>MAXTIA</scp> : A high‐throughput platform for rapid functional epitope mapping by kinetic screening of mutant libraries

Abstract Accurate identification of epitope residues is essential for developing biopharmaceuticals and understanding the mechanisms of immune recognition. However, experimental approaches for residue‐level epitope mapping remain time‐consuming and labor‐intensive, while accurate computational prediction of protein–protein interfaces remains challenging. Here, we present MAXTIA, a high‐throughput kinetic screening platform that integrates cell‐free protein synthesis with high‐throughput surface plasmon resonance and demonstrate its application to alanine scanning‐based functional epitope mapping. This workflow enables the rapid preparation and kinetic characterization of up to 384 protein variants, allowing the identification of functional epitope residues within 3 days while simultaneously providing binding affinity and kinetic parameters ( K D , k on , and k off ). We applied MAXTIA to map the epitope of the single‐domain antibody (VHH) N1 against the pentraxin domain of neuronal pentraxin‐2 (NP2 PTX). Alanine substitutions that cause substantial affinity losses clustered within a localized region on the AlphaFold3‐predicted NP2 PTX structure, defining a functional epitope site. These residues closely matched the interface observed in the NP2 PTX–VHH N1 crystal structure, validating the accuracy of MAXTIA. Beyond epitope identification, MAXTIA provides a simple and versatile platform for the quantitative analysis of protein–protein interactions, including high‐throughput screening of antibody variants for affinity optimization. This approach should accelerate biopharmaceutical development and facilitate mechanistic studies of molecular recognition.