Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection ofAphanomyces invadansin the field

Abstract

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 µg/mL for A. invadans compared to 56 µg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10-11 dilution compared to a 10-8 dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8℃. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish.

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

“…Furthermore, the FTA test reagents were found stable till the end of the experiment (one month). Similar results were reported in studies where EUS and WSV kits were developed (Adil et al, 2013; Patil et al, 2013). Overall, the FTA test developed in the present study was more sensitive, specific and time saving.…”

“…Many research groups have developed sensitive, monoclonal antibody‐based famer‐friendly immunoassay test kits, such as to detect EUS in finfish and WSV in shrimp (Adil et al, 2013; Patil et al, 2013). In similar manner, this study used PirA VP ‐specific polyclonal antibodies to develop FTA test (Figure 5a) and standardized according to Patil et al, (2013) with slight modifications.…”

Design standards for experimental and field studies to evaluate diagnostic accuracy of tests for infectious diseases in aquatic animals

“…For presumptive and confirmatory purposes: use clinically affected cases from infected source population or apparently healthy animals from pathogen‐free source population (see Adil et al. () and McClure, Hammell, Stryhn, Dohoo, and Hawkins (), McClure et al. ())…”