Shark-derived single-domain antibodies for the detection of decapod iridescent virus

Decapod iridescent virus 1 (DIV1) has emerged as a novel threat to shrimp farming. The development of a rapid, convenient, and accurate method for diagnosing DIV1 is urgently needed, serving as a key factor in the reduction of the risk of viral outbreaks. This study aimed to develop a new variable antigen receptor (vNAR), derived from shark heavy-chain antibodies, directed against DIV1 for use in detection assays. White-spotted bamboo sharks were immunized with DIV1 virions, and an immune phage-display library was constructed. After three rounds of panning, the lead D02 vNAR was isolated from the library. D02 vNAR was identified to target the viral antigen ORF064L through western blotting, LM-SC/SC, and pull-down analyses. Furthermore, indirect enzyme linked immunosorbent assay (indirect ELISA) and dot-blot assays were developed based on D02 vNAR. The detection limits of the indirect ELISA and dot blot methods were 5 ng/mL and 6.25 ng/spot, respectively. Furthermore, in the indirect ELISA method, a standard curve describing the log concentration of DIV1 versus OD value was established, which was linear in the concentration range of 80-10,240 ng/mL, and the linear regression equation was derived as y = 0.3565x -0.51601. Furthermore, 300 clinical samples were analyzed by the two methods using quantitative real-time polymerase chain reaction (qRT-PCR) as a reference. Compared to qRT-PCR, the diagnostic sensitivity and specificity of the indirect ELISA and dot blot methods were 96.7 % and 100 % and 93.5 % and 100 %, respectively, demonstrating the practicability and reliability of the vNAR-based indirect ELISA and dot blot methods for the robust detection of DIV1. Overall, this is the first study in which a vNAR has been developed against DIV1. The developed vNAR offers a robust tool with great potential for the diagnosis of DIV1 during shrimp cultivation.

Automated summary

In this study, DIV1 detection methods were developed using a variable antigen receptor (vNAR) derived from shark heavy-chain antibodies. The lead vNAR, D02, was identified to target the viral antigen ORF064L. Indirect ELISA and dot-blot assays were developed based on D02 vNAR, demonstrating high sensitivity and specificity compared to qRT-PCR. This is the first study to develop a vNAR against DIV1, providing a robust tool for its diagnosis.