ArticleApplied microbiology and biotechnology2026
Development of a double-monoclonal antibody sandwich ELISA for the detection of Citrus tristeza virus.
Article in Applied microbiology and biotechnology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Citrus tristeza virus (CTV) poses a severe and economically significant threat to global citrus production. While serological detection offers a valuable tool, existing immunoassays often lack sufficient sensitivity and specificity, and the precise epitope information recognized by antibodies remains inadequately characterized. In this study, we developed an efficient double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for CTV detection. Purified CTV particles were used to generate monoclonal antibodies (mAbs) in mice. Among six obtained hybridoma lines, mAbs 10A2 and 10D9 exhibited high titers (up to 3.28 × 10⁷ by indirect ELISA; HRP-conjugated 10D9 reached 1.64 × 10⁷ by direct ELISA). Epitope mapping revealed that both mAbs specifically target linear epitopes (aa 1-15 and aa 166-180) within the major coat protein (CP), corresponding to two surface-exposed loop regions, with high conservation confirmed by phylogenetic analysis. For the DAS-ELISA, mAb 10A2 was selected as the capture antibody, and HRP-10D9 was used as the detection antibody. This antibody combination demonstrated high efficiency and specificity for CP, achieving a detection limit of 15.63 ng/mL for recombinant CP. Field surveys in major citrus-growing regions of China (Guangxi, Jiangxi, Zhejiang) using this DAS-ELISA showed positive detection rates of 40.95%, 52.71%, and 27.5%, respectively, exhibiting strong correlativity with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) (Kappa = 0.938). The developed mAbs and the sensitive, specific DAS-ELISA provide a powerful tool for CTV surveillance and hold significant promise for both agricultural production and research. KEY POINTS: Two monoclonal antibodies were generated using high-quality CTV particles, which target complementary epitopes on the major coat protein. A double-monoclonal antibody sandwich ELISA was developed based on two antibodies, which target complementary epitopes in the outer loop regions of the viral particle. A large-scale epidemiological survey conducted in citrus groves confirmed the method's effectiveness in identifying CTV-infected citrus.
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