Generation of fluorescent minibodies for rapid FLISA-based detection of pine wilt disease marker

Abstract

BACKGROUND: Development of a rapid and accurate detection method for a pine wilt disease (PWD) biomarker is crucial to prevent the spread of this disease in forests. In this study, we produced a recombinant minibody-type antibody against Bursaphelenchus xylophilus peroxiredoxin (BxPrx), which is a protein secreted by the PWD-causing B. xylophilus pathogen. Results: We labeled the anti-BxPrx minibody using two different fluorescent dyes, Alexa Fluor (AF) 488 and AF647, and established a fluorescence-linked immunosorbent assay (FLISA) system using these probes. Both fluorescent minibodies showed limit of detection values of 10.2 and 14.6 ng for the AF488- and AF647-labeled antibodies, respectively, suggesting the usefulness of these probes for FLISA-based sensitive detection of BxPrx. Conclusion: The entire duration for performing FLISA was 3 h, and enzyme-conjugated secondary antibody incubation, washing and enzymatic reaction steps were not required, which was comparable to traditional ELISA. Both AF488- and AF647-labeled minibodies showed high sensitivity, suggesting that these probes are useful for various fluorescence measurement techniques, which typically use green or red filters. © 2023 Society of Chemical Industry (SCI). © 2023 Society of Chemical Industry (SCI).