Development and Preliminary Field Evaluation of an Indirect ELISA for Detecting Tomato Yellow Leaf Curl Virus

Tomato yellow leaf curl virus (TYLCV) is a major threat to tomato production, creating a need for sensitive, low-cost detection methods that can be applied to early symptomatic or low-viral-load samples. Recombinant antigen configuration may influence serological assay development, although the specific contribution of multiple-cloning-site (MCS)-derived intermediate sequences remains uncertain. In this study, a recombinant Trx-His-coat protein (CP) fusion antigen was produced using an MCS-free direct-fusion construct that retained the Trx-His tag while removing the MCS-derived intermediate sequence, followed by gradient refolding. No direct comparison with linker-containing, tag-cleaved, or tag-free antigen constructs was performed. The purified antigen was used to immunize rabbits and generate a high-titre polyclonal antibody (pAb). The resulting indirect enzyme-linked immunosorbent assay (ELISA) achieved a theoretical limit of detection of 1.8 ng/mL and an estimated pre-dilution equivalent the limit of detection (LOD) of 72 ng/mL after sample dilution. The assay showed favourable tolerance to crude tomato leaf matrices, with spike-recovery rates of 95.45-100.40%. In a preliminary evaluation using a balanced panel of 32 field-collected samples, ELISA absorbance correlated with droplet digital PCR quantification (R² = 0.9819) and plant disease index values (R² = 0.9774). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping and AlphaFold2-based modelling were used only to provide preliminary computational context for antigen interpretation. The assay showed cross-recognition toward Tobacco curly shoot virus (TbCSV), indicating that it should not be considered strictly TYLCV species-specific. Therefore, this assay may support preliminary serological screening under the tested conditions, whereas molecular confirmation remains necessary when species-level identification is required.