Generation and evaluation of nanobodies specific for pirA and pirB from Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease

Abstract

Acute hepatopancreatic necrosis disease (AHPND) is a bacterial disease of farmed shrimp caused by Vibrio parahaemolyticus. In recent years, AHPND has caused significant damage to the shrimp aquaculture industry, resulting in a notable decline in shrimp production. Given the lack of effective treatment options, it is crucial to develop an early, quick and accurate method of detecting the AHPND pathogen to help prevent disease in shrimp aquaculture. The present study was aimed to generate and evaluate specific nanobodies against the pathogenic factors of V. parahaemolyticus causing AHPND. Anti-pirA and anti-pirB nanobodies were obtained using phage display technology from a natural phage library derived from alpacas, and subsequently produced via the prokaryotic expression system. The specificity and affinity of the nanobodies were validated through western blot and ELISA. The nanobody colloidal gold test strips for the detection of AHPND have been preliminarily developed using colloidal gold immunochromatography technology. The application of colloidal gold test strips enables the identification of AHPND to be reduced to a few minutes, with satisfactory sensitivity and specificity. The detection limits were 125 μg/mL for pirA and 62.5 μg/mL for pirB. These works provided a rapid, inexpensive, and convenient option for the detection of diseases in shrimp aquaculture production.