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White spot syndrome virus (WSSV) is extremely pathogenic and causes huge economic losses in the shrimp farming industry. Neutralizing antibodies against WSSV is expected to be an effective means of preventing infection with the virus. In the present study, eight monoclonal antibodies (mAbs) against VP28 were developed by immunizing BALB/c mice with WSSV-VP28 recombinant protein. Among them, three mAbs named 3B7, 2G3 and 5D2 were determined to be able to delay the mortality of WSSV-infected shrimp in vivo neutralization assay, suggesting their neutralizing ability against WSSV infection. Immunoblotting results showed that the three mAbs reacted specifically with native VP28 of WSSV, and could also recognize the virions in the gills of WSSV-infected shrimp by IFA. Furthermore, the single chain variable fragment (scFv) genes specific for WSSV-VP28 were cloned from the three hybridoma cells and expressed in Escherichia coli. After purification and refolding, three biologically active scFv recombinant proteins were all capable of recognizing the native VP28 of WSSV and delayed the mortality of WSSV-infected shrimp, indicating their neutralizing capacity against WSSV. Subsequently, the eukaryotic expression plasmids of three scFv genes were constructed and the transcriptional properties of expression vectors in shrimp were analyzed. Animal experiments also proved that the scFv eukaryotic expression plasmids were able to partially neutralize WSSV infection. Thus, the production of neutralizing mAb and recombinant scFv antibodies against WSSV has a promising therapeutic potential in prevention and treatment of white spot disease of shrimp. • Three specific mAbs against WSSV-VP28 were developed. • Three scFv antibody genes against WSSV-VP28 were cloned from hybridoma cells producing the mAbs. • The three mAbs and scFvs all had a partial neutralizing ability against WSSV infection. • Eukaryotic expression plasmids of the scFv genes could partially neutralize WSSV infection. [ABSTRACT FROM AUTHOR]

The pre-aflatoxin protein biomarker PAB-01 is a key target for early predicting of aflatoxin occurrence risk. Improving its detection sensitivity is significant for the early evaluation of potential risks. In this study, we obtained the monoclonal antibody 6A6 against PAB-01 through hybridoma technology. A sandwich ELISA was developed using pairwise combinations of PAB-01 nanobody, monoclonal antibody, and polyclonal antibody. It was found that the nanobody as the capture antibody and monoclonal antibody as the detection antibody exhibited the highest sensitivity, with a detection limit of 0.92 ng/mL. Based on this, a rolling circle amplification-based immunoassay (RCA-EIA) was established, with a linear range of 0.056–41.152 ng/mL and a detection limit of 0.033 ng/mL. Recovery rates in spiked peanut and corn samples ranged from 94.49% to 127.50%. This method successfully identified the presence of the PAB-01 protein in maize samples 10 days prior to the aflatoxin production. Based on PAB-01 concentration classifications, the maize samples were categorized into different risk levels. This development is significant for early warning and risk stratification management of aflatoxin occurrence, providing a highly effective tool in ensuring food safety. • A new monoclonal antibody against PAB-01 protein was obtained. • VHH capture and mAb detection yielded the highest sensitivity in sandwich ELISA. • A rolling circle amplification (RCA)-based sandwich ELISA method was established. • RCA-EIA detected PAB-01 giving a 10-day early aflatoxin warning in corn. • Potential risk of aflatoxin occurrence was classified based on PAB-01 concentration. [ABSTRACT FROM AUTHOR]

Nervous necrosis virus (NNV) has caused severe diseases in global aquaculture industry with tremendous economic and environmental damages. While red-spotted grouper NNV (RGNNV) is the most catastrophic genotype because of plentiful susceptible hosts and expansive distribution ranges. As the only structural protein, capsid protein enables automatic assembly into virus-like particles (VLPs) remaining native infectivity and immunogenicity. In this study, abundant RGNNV VLPs were artificially manufactured and immunized in BALB/c mice, followed by development and characterization of three monoclonal antibodies (mAbs, 2B7, 3A6 and 6E11) subtyped as IgG with a titer of 1:32,000. These mAbs precisely examined two bands (approximately 40 kDa and 120 kDa) of capsid protein by western blot, demonstrating the recognition targets as linear epitopes instead of conformational epitopes. The results of immunofluorescence, immunohistochemistry, immunoprecipitation and immunoelectron microscopy exhibited that all mAbs not only reacted specifically with intracellular viruses, but also exactly labeled free virions, suggesting promising prospects in the detection, localization and tracking of RGNNV. In addition, mAbs 2B7 and 3A6 possessed modest capacity to neutralize RGNNV infection. The pre-incubation of mAbs 2B7 and 3A6 with RGNNV served to suppress the multiplication of virus with 27.99% and 34.37% inhibition ratio, delay the development of cytopathic effect and enhance cell viability by 28.60% and 31.35%, respectively. Taken together, this article proposes several mAbs, which aims at sharing powerful tools for the establishment of potent diagnostics for RGNNV and further investigation into potential pathogenic mechanism. While mAbs 2B7 and 3A6 may advance the intensive development of antibody-based therapeutic countermeasures for RGNNV infection in fish. • VLPs of CNPgg2018 were artificially expressed, assembled and produced in E. coli. • Three mAbs specific for RGNNV were developed successfully. • All mAbs precisely recognized linear epitopes rather than conformational epitopes. • All mAbs specifically reacted with capsid protein and virion of RGNNV. • MAbs 2B7 and 3A6 served to neutralize RGNNV infection and enhance cell viability by 28.60% and 31.35%. [ABSTRACT FROM AUTHOR]