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Abstract B cells in some fish were recently found to have potent phagocytic activities. Sea bass ( Lateolabrax japonicus ) as an important economical marine fish species, it could be used as an appropriate model to study the functions of B cells in phagocytosis. In the paper, three positive hybridomas designated as 1E11, 2H4 and 3F3 secreting monoclonal antibodies (MAbs) against sea bass immunoglobulin M (IgM) were produced and used as research tools. Indirect enzyme-linked immunosorbent assay showed that all the three MAbs had a high binding capacity with sea bass serum IgM. Western blotting analysis showed that all the three MAbs were specific for the heavy chain of sea bass IgM. Indirect immunofluorescence assay (IFA) analysis suggested that both MAbs 1E11 and 2H4 could recognize membrane-bound IgM (mIgM) molecule of sea bass. Specificity analysis showed that three MAbs had no cross-reactions with other six teleosts IgMs. Flow cytometric analysis exhibited that the percentages of sea bass mIgM + lymphocytes in peripheral blood, spleen and pronephros were 25.6%, 21.1%, and 17.5%, respectively. Moreover, we found that the mIgM + lymphocytes of sea bass could phagocytose fluorescence microspheres and Lactococcus lactis , but lower phagocytosis rates of L. lactis was observed. These results demonstrated that the MAbs produced in this paper could be used as tools to study secretory IgM and mIgM + lymphocytes of sea bass, and mIgM + lymphocytes might also play an important role in innate immunity of sea bass. Highlights • MAbs against IgM of sea bass are produced and characterized. • The produced MAbs are special for the sIgM and mIgM of sea bass. • The produced MAbs don't cross-react with other six teleosts' IgMs. • The mIgM + lymphocytes of sea bass possess phagocytic capacity. • The phagocytosis rates of L. lactis are lower than those of fluorescence microsphere. [ABSTRACT FROM AUTHOR]

Developing a rapid, accurate and quantitative method for detecting white spot syndrome virus (WSSV) is extremely urgent and critical for reducing the risk of white spot disease outbreaks. In the present work, an optimized double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for quantitative detection of WSSV. The method employed rabbit polyclonal antibodies against WSSV as the capture antibody and previously produced anti-WSSV monoclonal antibodies as the detector antibody. A standard curve of the log concentration of WSSV versus OD value was established, which was linear in the concentration range of 120–7680 ng/mL, and the linear regression equation was y = 0.166x − 0.151. Viral proteins in different tissues of crayfish ( Procambarus clarkia ) post artificial infection with WSSV were quantitatively measured using the DAS-ELISA. WSSV proliferated quickly within 60 h post infection and gradually slowed down afterwards. According to the linear regression relationship, the viral proteins in hemolymph, gut and gonad were firstly able to be quantified at 24 h post infection with the concentrations of 186, 158 and 128 ng/mL, respectively. These three tissues also contained higher viral proteins than the gill, heart, hepatopancreas and muscle during the entire infection period. The viral protein concentration in gut reached the highest level of 6220 ng/mL at 72 h post infection. Real time quantitative PCR was also used to detect the dynamic change of viral copies in crayfish hemolymph post WSSV infection, with similar results for both assays. The developed DAS-ELISA could detect WSSV propagation from initial to moribund stage in infected crayfish and demonstrated potential application for diagnosis of WSSV. [ABSTRACT FROM AUTHOR]

In this work, the two positive hybridomas designated as 1B2 and 2G9 secreting monoclonal antibodies (MAbs) against half-smooth tongue sole IgM were produced and then cloned by limited dilution. Western blotting showed that MAbs 1B2 and 2G9 could specifically react with the heavy and light chains of IgM, respectively. The indirect immunofluorescence assay (IFA) showed that green fluorescent signals were detected on the membrane of lymphocytes, which indicated that membrane-bound IgM (mIgM) could be recognized by both MAbs 1B2 and 2G9. Specificity analysis showed that no cross-reactions were detected between IgM of half-smooth tongue sole and other five teleosts IgMs using MAbs 1B2 and 2G9. Flow cytometric analysis (FCA) demonstrated that the percentages of mIgM+ lymphocytes in peripheral blood, spleen and pronephros of health half-smooth tongue sole were 21.1%, 17.6% and 16.1%, respectively. Moreover, the phagocytosis rates of fluorescence microspheres by mIgM+ lymphocytes in isolated leukocytes from peripheral blood, spleen and pronephros of health half-smooth tongue sole were determined to be 4.6 ± 0.5%, 3.6 ± 0.4% and 4.2 ± 0.3% by FCA, respectively, and the proportions of mIgM+ phagocytic lymphocytes in total mIgM+ lymphocytes in the peripheral blood, spleen and pronephros were calculated to be 21.6 ± 1.1%, 20.6 ± 1.8% and 26.0 ± 0.8%, respectively. These results demonstrated that the MAbs produced in this study could specifically recognize the serum IgM and mIgM on B lymphocytes of half-smooth tongue sole. Meanwhile, partial mIgM+ lymphocytes have the phagocytic capacity, which indicates mIgM+ lymphocyte play important role in innate immune response. [ABSTRACT FROM AUTHOR]

Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD. [ABSTRACT FROM AUTHOR]

The 27.8kDa membrane protein from flounder (Paralichthys olivaceus) gill (FG) cells was previously identified as a putative cellular receptor involved in lymphocystis disease virus (LCDV) infection. In this paper, the expression of receptor-27.8kDa (27.8R) and LCDV loads in FG cells and hirame natural embryo (HINAE) cells were investigated upon LCDV infection and anti-27.8R monoclonal antibody (MAb) treatment. The results showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cell lines was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry, providing important new insights into the LCDV replication cycle and the interaction between this virus and the host cells. [ABSTRACT FROM AUTHOR]

A monoclonal antibody (MAb) 6H7 raised specifically against granulocytes of scallop (Chlamys farreri) was employed to observe granulocyte occurrence successively in blastulae, gastrulae, trochophore larvae, D-shape larvae, umbo-veliger larvae and creeping larvae of C. farreri by immunohistochemistry assay contrasted with H&E stain using semi-thin sections. Moreover, the reactivity of the MAb with granulocytes of C. farreri, Bay scallop Argopecten irradians, Japanese scallop Patinopecten yessoensis, Blue mussel Mytilus edulis, Pacific oyster Crassostrea gigas and Manila clam Ruditapes philippinarum, was detected by immunofluorescence assay (IFA) with differential interference contrast and fluorescent microscopy and flow cytometric immunofluorescence assay (FCIFA). The results showed that positive signals were first observed at D-shape larval stage, about 28 h post fertilization, after that, umbo-veliger larvae exhibited the positive cells with a diameter of 3–5 μm distributed in velum, digestive gland and esophagus. Then in creeping larvae, the number of positive cells increased with average diameter of 5–7 μm, and widely distributed in foot, digestive gland, gills and adductor muscles. No positive signal was found in blastulae, gastrulae and trochophore larvae. The results of IFA and FCIFA showed MAb 6H7 reacted to granulocytes of C. farreri, A. irradians, P. yessoensis and C. gigas, and the positive percentage reactivity were 53 ± 2.5%, 15 ± 2.5%, 12 ± 2.1% and 19 ± 2.1%, respectively, however, no cross-reaction was detected in hemocytes of R. philippinarum and M. edulis. [ABSTRACT FROM AUTHOR]

The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC50 against ZEA of 0.02 µg L−1. The mAb 2D3 exhibited a high recognition of ZEA (100%) and β-zearalenol (β-ZOL, 88.2%). Its cross-reactivity with α-zearalenol (α-ZOL) and β-zearalanol (β-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83–93%) and HPLC (94–108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize. [ABSTRACT FROM AUTHOR]

Hirame rhabdovirus (HIRRV) is one of the most important viruses of fish, posing a great threat to the fish industry in Asia and Europe. The glycoprotein (G) of HIRRV is known to play important roles in virus attachment and entry, making it an ideal target for both diagnosis and therapy. In this study, a truncated G of HIRRV was expressed as a fusion protein in Escherichia coli. Using the recombinant G protein (rG), monoclonal antibodies (mAbs) were prepared by the hybridoma technology. Subsequently, positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA) and further characterized by Western blot and immunofluorescence assay (IFA). ELISA results showed that two mAbs (3E5 and 4D10) could react with the rG, as well as the purified HIRRV. Western blot analysis showed that the mAbs belong to the IgG isotype and could recognize a 60 kDa viral protein, which is consistent with the molecular weight of G protein and determined to be the G protein of HIRRV by mass spectrometry. The virions in HIRRV-infected EPC could also be recognized by two mAbs in IFA. Moreover, neutralization assay showed that mAb 4D10 could significantly inhibit the proliferation of HIRRV and delay the development of cytopathic effect in viral-infected EPC cells, and in vivo neutralization assay also showed that mAb 4D10 could significantly reduce the mortality of HIRRV-infected flounder, indicating that mAb 4D10 can partially neutralize the HIRRV infection. Western blot analysis showed that mAb 4D10 could specifically bind the C-terminal domain of HIRRV-G protein. These results demonstrated that the produced mAbs could specifically recognize the G protein of HIRRV and displayed virus-neutralizing activity in vitro and in vivo , which could serve as effective detection probes and potential neutralizing antibodies for HIRRV. • Two mAbs were produced with the specificity to the G protein of HIRRV. • The mAb 4D10 could partly neutralize the HIRRV infection in vivo and in vitro. • MAb 4D10 could specifically bind the C-terminal domain (CTD) of HIRRV-G protein. [ABSTRACT FROM AUTHOR]

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]

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV–host interaction and might be promising inhibitors of LCDV infection in fish. [ABSTRACT FROM AUTHOR]

In our previous work, two monoclonal antibodies (Mabs) against granulocytes of shrimp ( Fenneropenaeus chinensis ) had been produced, in this paper, haemocyte subpopulations were analyzed by flow cytometry (FCM) using the Mabs. Then immunomagnetic bead (IMB) method was applied for separation hyalinocytes and granulocytes using the Mabs. The separated hyalinocytes and granulocytes were analyzed by FCM, indirect immunofluorescence assay, Giemsa staining and transmission electron microscopy, respectively. The results showed the proportion of hyalinocytes in haemolymph of F . chinensis was 15.14 ± 1.22%, and that of granulocytes was 75.43 ± 2.31%. After two times separation by IMB, the purity rate of hyalinocytes and granulocytes was 96.27 ± 1.06% and 98.13 ± 0.86%, respectively. The hyalinocytes possessed 0.60–0.85 in nucleus/cytoplasm (N/C) ratio and had few granule in cytoplasm, whereas the separated granulocytes with N/C ratio of 0.12–0.36 and high electronic density of double membrane granules. The results reported the separation of haemocyte subpopulations using Mabs in shrimp for the first time, and the hyalinocytes and granulocytes isolated by IMB could be used for their differential protein analysis. [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]

To simultaneously detect two metabolites of Aspergillus flavus, namely, cyclopiazonic acid (CPA) and aflatoxin (AFT), an ultrasensitive monoclonal antibody (mAb) YTT-2 against CPA was developed and characterized, with sensitivity to CPA of 1.32 ng mL−1. Along with the previously homemade mAb 1C11 against AFT, two mAbs were used to develop time-resolved fluorescence immunoprobes or gold immunoprobes. We developed two multiple-analyte paper immunosensors including time-resolved fluorescent immunochromatographic assay (TRFICA) and gold immunochromatographic assay (GICA) for the simultaneous determination of CPA and AFT. The TRFICA showed limits of determination (LODs) of 0.21 and 0.004 ng mL−1, while the GICA showed LODs of 0.33 and 0.01 ng mL−1 for CPA and AFT, respectively. To validate the specificity of the two rapid immunoassays, rice, corn and peanut samples were spiked with different concentrations of CPA and AFT. The two methods showed satisfactory recoveries (76.39~90.82% for CPA and 84.60~94.45% for AFT) and coefficients of variation of 3.50~7.80% for CPA and 4.12~13.90% for AFT. The results indicated that the TRFICA could complete the test within 5 min and had lower LODs and linear ranges, compared with that of GICA. The method developed in this work can be widely applied to the rapid and quantitative simultaneous determination of multiple harmful metabolites in fungi for food safety and health care. [ABSTRACT FROM AUTHOR]

Anti-idiotypic nanobodies, usually expressed by gene engineering protocol, has been shown as a nontoxic coating antigen for toxic compound immunoassays. We here focused on how to increase immunoassay sensitivity by changing the nanobody's primary sequence. In the experiments, two anti-idiotype nanobodies against monoclonal antibody 1H2, which is specific to ochratoxin A, were obtained and named as nontoxic coating antigen 1 (NCA1) and nontoxic coating antigen 2 (NCA2). Three differences between the nanobodies were discovered. First, there are six amino acid residues (AAR) of changes in the complementarity determining region (CDR), which compose the antigen-binding site. One of them locates in CDR1 (I–L), two of them in CDR2 (G–D, E–K), and three of them in CDR3 (Y–H, Y–W). Second, the affinity constant of NCA1 was tested as 1.20 × 108 L mol−1, which is about 4 times lower than that of NCA2 (5.36 × 108 L mol−1). Third, the sensitivity (50% inhibition concentration) of NCA1 for OTA was shown as 0.052 ng mL−1, which was 3.5 times lower than that of nontoxic coating antigen 2 (0.015 ng mL−1). The results indicate that the AAR changes in CDR of the anti-idiotypic nanobodies, from nonpolar to polar, increasing the affinity constant may enhance the immunoassay sensitivity. In addition, by using the nontoxic coating antigen 2 to substitute the routine synthetic toxic antigen, we established an eco-friendly and green enzyme-linked immunosorbent assay (ELISA) method for rapid detection of ochratoxin A in cereals. The half-maximal inhibitory concentration (IC50) of optimized ELISA was 0.017 ng mL−1 with a limit of detection (LOD) of 0.003 ng mL−1. The optimized immunoassay showed that the average recoveries of spiked corn, rice, and wheat were between 80% and 114.8%, with the relative standard deviation (RSD) ranging from 3.1–12.3%. Therefore, we provided not only basic knowledge on how to improve the structure of anti-idiotype nanobody for increasing assay sensitivity, but also an available eco-friendly ELISA for ochratoxin A in cereals. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is one of the most serious viral pathogens causing major economic losses to the shrimp farming industry worldwide. Rapid diagnostic test is critical for disease prevention and control. Previously we have developed a rapid immunochromatographic test strip for WSSV detection, but it is limited to the qualitative testing. In this study, a semiquantitative immunochromatographic test strip (SIT-strip) was developed for WSSV detection by observing the number of positive test lines to determine the viral load levels. To achieve semiquantitative analytical ability, three test lines were designed on NC membrane to form the test zone, on which different concentration of anti-WSSV monoclonal antibody (MAb) 4G9 in a decreasing sequence from test line (TL) I to TL-III was dispensed, instead of the traditional application of a single test line. The anti-WSSV MAb 2E6 served as colloidal gold conjugate and goat anti-mouse IgG antibody on control line was used to validate the test performance. The threshold concentrations for TL-I, -II and -III were determined to be 783 ng/ml, 3.13 μg/ml and 6.26 μg/ml of WSSV, respectively, and the visual detection limit of the SIT-strip was 783 ng/ml. The developed SIT-strip was used to detect WSSV at different time points post injection of Procambarus clarkia , the results indicated that one red band was present on TL-I at 12 h post infection (hpi) in the gills, two bands in muscle and three bands in gills and hemolymph were produced at 24 hpi although there was no death at that time, and thereafter three bands were visible in all tested tissues and dead individuals. The mortality was low from 36 to 48 hpi and increased rapidly during 48–72 hpi. Meanwhile, WSSV copy numbers were detected by real time qPCR for severity grading of infection. These results revealed that this SIT-strip could provide confirmation of viral presence and concentration levels prior to mortality, which was early enough to achieve the goal of monitoring for WSSV infection loads predictive of impending disease. [ABSTRACT FROM AUTHOR]

In our previous work, granulocytes and hyalinocytes were successfully separated by immunomagnetic bead (IMB) method using monoclonal antibodies (mAbs) against granulocytes of shrimp ( Fenneropenaeus chinensis ). In order to elucidate the proteomic differentiation between granulocytes and hyalinocytes, in this paper, the differentially expressed proteins were analyzed between non-fixed/un-permeabilized (NFP) haemocytes and fixed/permeabilized (FP) haemocytes using two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS). Then the FP haemocytes were separated into two haemocyte subpopulations using IMB method, and the comparative proteome between granulocytes and hyalinocytes was investigated. The results showed that 10 differentially expressed protein spots were detected and identified as 4 proteins in the NFP haemocytes. Twenty one differentially expressed proteins were successfully identified between granulocytes and hyalinocytes, which include 4 unique expressed proteins in granulocytes, 4 significantly highly expressed proteins in granulocytes, and 13 significantly high expressed proteins in hyalinocytes. According to Gene Ontology annotation, the identified proteins between granulocytes and hyalinocytes were classified into six categories, including binding proteins, proteins involved in catalytic activity, enzyme regulator activity, structural molecule activity, translation regulator activity, and ungrouped proteins. Furthermore, quantitative PCR confirmed that the trend of transcription levels of three selected genes were consistent with the proteomic data from 2-DE. The results may lead to better understanding of the functions of haemocyte subpopulations. [ABSTRACT FROM AUTHOR]

Abstract: Mycotoxins are highly toxic contaminants and have induced health threat to human and animals. Aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEA) commonly occur in food and feed. A multi-component immunochromatographic assay (ICA) was developed for rapid and simultaneous determination of these three mycotoxins in agro-food. The strategy was performed based on the competitive immunoreactions between antibody-colloidal gold nanoparticle conjugate probes and mycotoxins or mycotixin antigens. Each monoclonal antibody specially recognize its corresponding mycotoxin and antigen, and there was no cross reactivity in the assay. Three mycotixin antigens were immobilized as three test lines in the nitrocellulose membrane reaction zone, which enable the simultaneous detection in one single test. The visible ICA results were obtained in 20min. The visual detection limits of this strip test for the AFB1, OTA and ZEA were 0.25ng/mL, 0.5ng/mL and 1ng/mL, respectively. The assay was evaluated using spiked and naturally contaminated peanuts, maize and rice samples. The results were in accordance with those obtained using enzyme-linked immunosorbent assay. In summary, this developed ICA could provide an effective and rapid approach for onsite detection of multi-mycotoxin in agro-food samples without any expensive instrument. [Copyright &y& Elsevier]

Abstract: To investigate the interaction between white spot syndrome virus (WSSV)-VP37 and gill membrane proteins (GMPs) of Pacific white shrimp (Litopenaeus vannamei), the VP37 protein was expressed and purified, and a distinct 53 kDa VP37-binding protein band was identified in GMPs by virus overlay protein binding assay and GST pull-down assay. By electroelution, the VP37 binding protein was purified and identified as F1ATP synthase β (F1ATPase β) subunit by Mass Spectrometry. The purified F1ATPase β subunit was used to immunize BALB/C mice to produce monoclonal antibodies (Mabs). After cell fusion, sixteen hybridomas secreting Mabs against F1ATPase β subunit of L. vannamei were screened by enzyme-linked immunosorbant assay (ELISA), three of which designated as 1D5, 1E8 and 2H4 were cloned by limiting dilution and further characterized by indirect immunofluorescence assay (IIFA) and western blotting. The results of IIFA showed that specific fluorescence signals located at the peripheral zone of the gills of L. vannamei. Western blotting demonstrated that three Mabs reacted specifically with the 53 kDa protein band in GMPs of L. vannamei. By IIFA, the Mabs could also cross-react with the gill cells of three other WSSV-susceptible shrimps Fenneropenaeus chinensis, Penaeus monodon and Marsupenaeus japonicus. Furthermore, the three anti-F1ATPase β subunit Mabs could partially block the binding of WSSV to GMPs by ELISA in vitro, and also exhibited direct anti-WSSV activity in shrimp by neutralization assay in vivo. These findings suggested that F1ATPase β subunit involved in WSSV infection in L. vannamei. [Copyright &y& Elsevier]

Abstract: A monoclonal antibody (MAb 6H7) specific to granulocytes of scallop Chlamys farreri was produced by immunising mice with separated granulocytes as an antigen. Characterised using a flow cytometric immunofluorescence assay, MAb 6H7 reacted to granulocytes by 87.1% of total positive haemocytes. At the ultrastructural level, MAb 6H7 demonstrated epitope in cytoplasmic granules of granulocytes. Western blotting analysis indicated that a peptide of 155 kDa was recognised by MAb 6H7. It was therefore used to investigate granulocyte variation in C. farreri after acute viral necrobiotic virus (AVNV) infection using an enzyme-linked immunosorbent assay. The result illustrated that granulocytes varied greatly by AVNV infection, and their amount significantly increased on day 1 post-injection, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8. [Copyright &y& Elsevier]

• A duplex phage-display immuno-polymerase chain reaction (PD-IPCR) was developed. • PD-IPCR was shown to be a sensitive method for mycotoxins detection. • The PD-IPCR was used successfully to evaluate aflatoxins and zearalenone in grains. An anti-idiotypic nanobody-phage display-mediated immuno-polymerase chain reaction (PD-IPCR) method was developed for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals. Two phages, displaying the variable domain of the heavy chain anti-idiotypic nanobody that binds aflatoxin- or zearalenone-specific monoclonal antibody (1C11 or 2D3), were used as competitors for corresponding analytes. Specific DNA sequences encoding anti-idiotypic nanobodies were used to design the primers for PCR amplification. The results indicated that detection limits for total aflatoxins and zearalenone in a sample were 0.03 and 0.09 ng mL−1, respectively. Recoveries of spiked aflatoxins and zearalenone were 80–118% and 76.7–111%, respectively. Validation results were in good agreement with the gold-standard high-performance liquid chromatography method. This report is the first to describe PD-IPCR for simultaneous quantitative detection of total aflatoxins and zearalenone in cereals. [ABSTRACT FROM AUTHOR]

C-type lectins, as types of pattern recognition receptors (PRRs), have crucial roles in innate immunity, and their conserved carbohydrate recognition domain (CRD) is recognized as the binding dominant to pathogen-associated molecular patterns (PAMPs). In this study, a C-type lectin in the scallop Chlamys farreri (lectin B , GenBank No. DQ209290) was heterologously expressed and the recombinant protein (rlectin B) with a molecular mass of approximately 42 kDa was purified. The rlectin B protein could induce the aggregation of Escherichia coli , Vibrio anguillarum and Staphylococcus aureus. Six monoclonal antibodies (mAbs) against rlectin B was developed, among which two mAbs (1A7 and 2B6) could react with the gills, hepatopancreas, adductor muscle, and haemocytes of C. farreri by indirect immunofluorescence assay and bound to proteins with molecular masses of 21, 28, 37, 45, 58, and 70 kDa in western blot assays. Using the two mAbs, C-type lectins protein levels were detected in C. farreri haemocytes after V. anguillarum infection, showing that that lectin levels rapidly increased, peaking at 12 h before gradually decreasing to the control group levels. Furthermore, the FACS results also confirmed that V. anguillarum infection significantly increasing C-type lectins expression in haemocytes. These data suggest that the mAbs can recognize various C-type lectins in C. farreri and are a useful immunological tool for the detection C-type lectins in scallops. • Recombinant C-type lectin (rlectin B) can induce the aggregation of E. coli, V. anguillarum and S. aureus. • Six monoclonal antibodies (mAbs) against rlectin B were developed. • Two specific mAbs reacted with all tissues, especially the haemocytes, gills, hepatopancreas and adductor muscle. • C-type lectin proteins significantly increased in Chlamys farreri haemocytes after V. anguillarum infection. [ABSTRACT FROM AUTHOR]