Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli

Abstract

A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

Development of a lateral flow assay for rapid and accurate detection of chicken anemia virus

“…In our study, the sensitivity threshold of the lateral flow assay was determined to be 0.625 µg/mL for the Δ60VP1 antigen. This sensitivity is lower than the immunochromatography strip developed by Lee et al (2021) . They utilized paired polyclonal antibodies and MAB specific to VP1 and VP2 of the Sacbrood virus, an insect virus.…”

Development of a colloidal gold immunochromatographic strip for rapid detection of avian coronavirus infectious bronchitis virus

“…The colloidal gold-labeled antibody-based immunochromatographic strip is rapid, easy to operate, low cost, is not dependent on professional equipment, and is widely used in poultry disease detection ( Li et al, 2013 ; Qian et al, 2015 ; Yu et al, 2018 ; Wang et al, 2019 ; Yu et al, 2020 ), as well as in certain other fields ( Zhao et al, 2008 ; Li et al, 2012 ; Meng et al, 2014 ; Li et al, 2020 ; Lee et al, 2021 ; Li et al, 2021 ; Guo et al, 2023 ). Zhang et al, (2010) reported the use of IBV colloidal gold immunochromatography for the first time, but their method was only used to detect the antibodies against IBV.…”