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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]

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 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]

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]

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]