Novel sandwich immunoassay detects a shrimp AHPND-causing binary PirABVp toxin produced by Vibrio parahaemolyticus.

Introduction: The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp. Methods: We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp. Results: Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp. Discussion: These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

Physicochemical and sensory characteristics of commercial, frozen, dry, and wet-aged Hanwoo sirloins.

Objective: The objective of this study was to evaluate the physicochemical, sensory and taste characteristics of commercial, frozen, dry, and wet aged Hanwoo sirloin. Methods: Grade 2 sirloin from 6 Hanwoo steers (about 30 months old) were obtained after 5 days postmortem. Samples assigned into the following four groups commercial beef (CON), frozen beef (HF/40 days in -18°C freezer), wet-aged beef (HW/21 days), and dry-aged beef (HD/40 days) were stored in a 80±5% relative humidity cooler at 1 °C. Results: The HF group showed a significantly higher cooking loss and expressible drip with significantly higher pH compared to other groups. In addition, protein and fat contents in the HD group were higher than those in other groups (p < 0.05). The shear forces in the HW and HD groups were significantly lower than those in the CON group. The HD group had significantly higher omega-3 and polyunsaturated fatty acids compared with other groups. Glutamic acid levels in the HD group were significantly higher compared with those in other groups. Electronic tongue analysis revealed that sourness of the HD group was lower than that of other groups, whereas the HD group showed significantly higher umami, richness, and saltiness compared to other groups (p < 0.05). Sensory test results revealed that the HW group had significantly higher tenderness, while the HD group had significantly higher chewiness, juiciness, and overall acceptability scores. Conclusion: These results suggest that both wet- and dry-aging treatments can effectively improve sensory characteristics, and dry-aging was much more useful to enhance umami tastes and meat quality of 2 grade Hanwoo sirloins.