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

Abstract

AbstractBACKGROUNDDevelopment 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.ResultsWe 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.ConclusionThe 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).