Polyclonal antibody‐based farmer‐friendly flow‐through test for the detection of acute hepatopancreatic necrosis disease in shrimp

Abstract

Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND)is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method.In this study, a simple, sensitive, rapid and polyclonal antibody-based farmer-friendly flow-through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect-related (Pir) A toxin-like protein of AHPND pathogen was used to immunize rabbits at 21-day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross-reactivity with AHPND-free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer-friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirA VP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8-10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost-effective. Collectively, sensitive FTA test would help shrimp farmers to take real-time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.

Development of Monoclonal Antibody against PirB and Establishment of a Colloidal Gold Immunochromatographic Assay for the Rapid Detection of AHPND-Causing Vibrio

“…Through a series of immunizations, screening, and hybridoma cell fusion techniques, we successfully obtained hybridoma cell lines capable of secreting monoclonal antibodies against PirB. Compared to polyclonal antibodies [ 21 , 22 ], monoclonal antibodies have a better specificity and a more stable production capacity because of hybridoma cell lines. This achievement underscores the significance of monoclonal antibodies in the accurate detection of AHPND-causing Vibrio .…”

“…Therefore, there is an urgent need for a detection method targeting virulence proteins. A small number of studies have developed antibodies and immunological methods targeting PirA or PirB [ 15 , 18 , 19 , 20 , 21 , 22 ], primarily focusing on AHPND-causing V. parahaemolyticus strains without a comprehensive evaluation of other AHPND-causing Vibrio species.…”

Development of real‐time fluorescence saltatory rolling circle amplification for rapid detection of Vibrio parahaemolyticus in seafood

“…In recent years, various antibody‐based immunoassays and molecular biological methods have been developed as diagnostic tools (Lee et al ., 2020a). Although immunological methods have the inherent advantages of simplicity and rapidity, a pre‐culture process is still needed, and suitable sample morphology and detection environment are also highly required (Hanumanthappa et al ., 2020; Li et al ., 2020). As the earliest DNA‐based molecular method, polymerase chain reaction (PCR) has been widely applied to detect pathogens in safety‐ and health‐related areas, such as food safety, disease prevention, clinical diagnosis and environmental analysis (Zhang et al ., 2020).…”

“…Besides, more PCR‐based methods are being used in many studies, including quantitative real‐time PCR (qPCR) (Ling et al ., 2020), digital PCR (Lei et al ., 2020), and multiplex PCR (Kim & Lee, 2014). However, these methods require complicated thermal cycling instruments, competent personnel and expertise laboratory systems, which are not equipped in resource‐limited conditions (Hanumanthappa et al ., 2020). Recently, substantial research interest in isothermal amplification techniques has been inspired greatly for nucleic acid analysis, such as rolling circle amplification (RCA; Song et al ., 2019) and loop‐mediated isothermal amplification (LAMP; Shim et al ., 2020).…”