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Plant defensins (PDFs) are cysteine‐rich antimicrobial peptides (AMPs) that are important components of plant immunity. They occur constitutively in various plant tissues but are also upregulated upon stress. Therefore, these molecules are of great interest as markers for the diagnosis of early forest stress response in plants at the molecular level. PDFs are small peptides (~5 kDa) with a compact tertiary structure, requiring specific protocols and dedicated antibodies for detection by quantitative ELISA. We developed monoclonal recombinant antibodies using phage display in solution against the correctly folded antigen defensin Fs PDF2 from beech ( Fagus sylvatica ) and analysed the antibody–antigen interaction in silico with AlphaFold 3. In a proof‐of‐principle study, we investigated the Fs PDF2 stress response to abiotic (drought) and biotic (gall midge) stresses. Notably, we established an assay for defensin quantification in crude plant extract, detecting for the first time natively folded proteins in a specific sandwich ELISA. Our antibody generation strategy can be transferred by practitioners to other small antimicrobial peptides (AMP), paving the way to study this group of proteins and their corresponding stress response comprehensively.

Plant defensins (PDFs) are cysteine‐rich antimicrobial peptides (AMPs) that are important components of plant immunity. They occur constitutively in various plant tissues but are also upregulated upon stress. Therefore, these molecules are of great interest as markers for the diagnosis of early forest stress response in plants at the molecular level. PDFs are small peptides ( 5 kDa) with a compact tertiary structure, requiring specific protocols and dedicated antibodies for detection by quantitative ELISA. We developed monoclonal recombinant antibodies using phage display in solution against the correctly folded antigen defensin FsPDF2 from beech (Fagus sylvatica) and analysed the antibody–antigen interaction in silico with AlphaFold 3. In a proof‐of‐principle study, we investigated the FsPDF2 stress response to abiotic (drought) and biotic (gall midge) stresses. Notably, we established an assay for defensin quantification in crude plant extract, detecting for the first time natively folded proteins in a specific sandwich ELISA. Our antibody generation strategy can be transferred by practitioners to other small antimicrobial peptides (AMP), paving the way to study this group of proteins and their corresponding stress response comprehensively.