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However, experimental approaches for residue\u2010level epitope mapping remain time\u2010consuming and labor\u2010intensive, while accurate computational prediction of protein\u2013protein interfaces remains challenging. Here, we present MAXTIA, a high\u2010throughput kinetic screening platform that integrates cell\u2010free protein synthesis with high\u2010throughput surface plasmon resonance and demonstrate its application to alanine scanning\u2010based 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\u2009days while simultaneously providing binding affinity and kinetic parameters (\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>D<\/jats:sub>\n                    ,\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>on<\/jats:sub>\n                    , and\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>off<\/jats:sub>\n                    ). We applied MAXTIA to map the epitope of the single\u2010domain antibody (VHH) N1 against the pentraxin domain of neuronal pentraxin\u20102 (NP2 PTX). Alanine substitutions that cause substantial affinity losses clustered within a localized region on the AlphaFold3\u2010predicted NP2 PTX structure, defining a functional epitope site. These residues closely matched the interface observed in the NP2 PTX\u2013VHH N1 crystal structure, validating the accuracy of MAXTIA. Beyond epitope identification, MAXTIA provides a simple and versatile platform for the quantitative analysis of protein\u2013protein interactions, including high\u2010throughput screening of antibody variants for affinity optimization. This approach should accelerate biopharmaceutical development and facilitate mechanistic studies of molecular recognition.\n                  <\/jats:p>","DOI":"10.1002\/pro.70769","type":"journal-article","created":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T13:08:01Z","timestamp":1786972081000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["<scp>MAXTIA<\/scp>\n                    : A high\u2010throughput platform for rapid functional epitope mapping by kinetic screening of mutant libraries"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-6208-9142","authenticated-orcid":false,"given":"Kihoon","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biotechnology School of Engineering, The University of Tokyo  Tokyo 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