Development of a lateral flow immunochromatographic assay (LFIA) for the detection of hirame novirhabdovirus (HIRRV) in olive flounder (Paralichthys olivaceus)

Abstract

This study developed and standardized a lateral flow immunochromatographic assay (LFIA) for the detection of hirame novirhabdovirus (HIRRV) in olive flounder (Paralichthys olivaceus). Monoclonal antibodies (mAbs) (11-2D9) recognizing the matrix protein of HIRRV were bio-conjugated with gold nanoparticles and printed on the test line of a paper-based LFIA strip. The test results could be viewed within 10 min after the application of virus culture supernatants and spleen tissue lysates diluted in sample buffer in separate tests. The limit of detection of the LFIA was found to be 105.08 TCID50/100 μL with three different HIRRV isolates. In terms of specificity, the strip exhibited no cross-reactivity with five other fish viruses including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Interestingly, in vivo testing using spleen tissue decreased the sensitivity of LFIA. However, this problem could be solved by increasing the sample buffer dilution in ratio from 1:10 to 1:30. Field-level validation of the assay using spleen tissues from HIRRV-infected and non-infected olive flounder showed a specificity of 100% and sensitivity of 96%, with a diagnostic effectiveness of 98%. Therefore, the developed LFIA strip test facilitates the rapid detection (within 10 min) of HIRRV in olive flounders in farming facilities.