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An abrupt decrease in egg production and quality in laying hens can be brought on by the avian adenovirus known as the egg drop syndrome virus (EDSV). So far, there are no efficient commercial diagnostic approaches for EDSV. In the present study, we first purified two monoclonal antibodies (mAbs 5G4 and 6G6) specifically against the EDSV fibre protein. The mAb 5G4 was then employed as capture antibody. Additionally, the detection antibody 6G6 (HRP-6G6) was conjugated with horseradish peroxidase. Consequently, based on these two mAbs, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for EDSV detection. Specificity evaluation demonstrated that only EDSV gave a positive result while other avian viruses showed negative results. Sensitivity investigation showed that the limit of detection of EDSV was 102.9 TCID50/ml and the limit of detection of the purified His-Fiber was 5 ng/ml. Furthermore, the screening of samples from infected chickens showed that this method was very effective and could be applied to the detection of EDSV in infected samples. In summary, the sandwich enzyme-linked immunosorbent assay developed here provides an efficient, sensitive, and large-scale method for the specific diagnosis of EDSV. A sandwich ELISA was developed to detect EDSV using the mAbs 5G4 and HRP-6G6. The sandwich ELISA maintained high specificity and sensitivity. The sandwich ELISA had equivalent consistency with real-time PCR assay. [ABSTRACT FROM AUTHOR]

Simple Summary: Newcastle disease (ND) is an Office International Des Epizooties (OIE) notifiable terrestrial and aquatic animal disease that can infect chickens of all ages with a high mortality rate, causing ongoing damage to the poultry industry in China currently. In this study, we have developed a blocking lateral flow assay (bLFA) strip for the rapid detection of Newcastle disease virus (NDV) antibodies, using neutralizing monoclonal antibodies against hemagglutinin-neuraminidase. The results showed that the chicken NDV hyperimmunized serum had a complete blocking titer of 11 log 2, and half-blocking titer of 13 log 2, which are 4 times less than and the same as that of the Hemagglutination inhibition (HI) test (13 log 2), and 8 and 2 times less than that of the virus neutralization test (14 log 2), respectively. Additionally, the bLFA strip has no cross-reactivity with positive serum of other avian pathogens. A total of 510 clinical samples were tested for NDV antibodies. The coincidence rate between the results of the bLFA strip and HI test was 97.65%. Therefore, it is an ideal alternative method for assessing the clinical immunity of ND vaccines in the field in real-time. Newcastle disease (ND) is an acute septicemic infectious disease caused by Newcastle disease virus (NDV). Considering that vaccination is currently the main modality for the prevention of ND, it is essential to assess the effectiveness of clinical immunization. In this study, we have developed a blocking lateral flow assay (bLFA) strip for the rapid detection of NDV antibodies using the monoclonal antibody 9C1 against haemagglutinin-neuraminidase (HN), which allows for the determination of an NDV-specific antibody titer within 10 min at room temperature. In addition, the bLFA strip has no cross-reactivity with the positive serum of other avian pathogens including avian influenza subtypes H5, H7, and H9, MD, IBD, IB, EDS, and avian adenovirus. The ability of the bLFA strip for detecting a neutralizing antibody was also estimated. The results showed that the chicken NDV hyperimmunized serum had a complete blocking (100%) titer of 11 log 2, and half-blocking titer of 13 log 2, which are 4 times less than and the same as that of the HI test (13 log 2), and 8 and 2 times less than that of the VN test (14 log 2), respectively. A total of 510 clinical samples were tested for NDV antibodies. The coincidence rate between the results of the bLFA strip and HI test was 97.65%. Therefore, it is an ideal alternative method for assessing the clinical immunity of ND vaccines in the field in real-time. [ABSTRACT FROM AUTHOR]

A strip test is described for the optical determination of influenza virus H3 subtype. It utilizes gold nanoparticle (AuNP) coated polystyrene latex microspheres (PS) as the label and a sandwich format. The AuNP and PS particles were linked using monoclonal antibodies against influenza virus as the bridge. Under the optimal conditions, the visual detection limit of the AuNP-PS-based strip test was as low as 1/16 hemagglutination unit (HAU). It was 64 times higher than that of 10 nm (4 HAU) AuNP-based strip tests. Quantitative analysis showed that the detection limit of the AuNP-PS-based strip is 0.016 HAU. The AuNP-PS-based strip test showed no cross-reactivity to the other subtypes (H1, H5, H7, or H9) of influenza viruses. [ABSTRACT FROM AUTHOR]

We evaluated an immunochromatographic strip for the detection of avian avulavirus 1 (Newcastle disease virus, NDV) based on a high-affinity monoclonal antibody (mAb) that specifically recognizes the hemagglutinin-neuraminidase (HN) protein. The anti-HN mAb was labeled with colloidal gold as the detector. A chicken anti-NDV polyclonal antibody and staphylococcal protein A (SPA) were blotted on the nitrocellulose membrane for the test and control lines, respectively. The strip specifically recognized the NDV antigen with no cross-reactivity to other viruses that were examined. Furthermore, it specifically recognized a variety of NDV isolates, including virulent and attenuated strains. These results were confirmed using hemagglutination (HA) and RT-PCR assays. The NDV detection strip detected 104.9 EID50 viruses/0.1 mL in the NDV-infected sample, which is comparable to the classical HA test (105.2 EID50/0.1 mL). Following experimental infection, NDV was detected using the detection strip in infected tissues as early as 36 h after experimental infection and prior to development of clinical signs and appearance of gross anatomic lesions. The diagnostic sensitivity and specificity of the NDV detection strip for NDV infection were 83.3% and 100%, respectively, as confirmed by RT-PCR. [ABSTRACT FROM AUTHOR]

The authors describe an ultrasensitive method for simultaneous detection of neomycin (NEO) and quinolones antibiotics (QNS). It is based on the use of (a) two immuno-nanoprobes (a probe for NEO and a probe for QNS), (b) surface-enhanced Raman scattering (SERS) detection, and (c), a portable lateral flow assay (LFA). The two probes consist of gold nanoparticles (AuNPs) conjugated to the Raman active molecule 4-aminothiophenol (PATP), and to monoclonal antibody against NEO (NEO mAb) or against NOR (NOR mAb). Quantitative detection of NEO and QNS was realized via SERS of the PATP-coated AuNPs captured in the test line of a LFA. Under optimized condition, the visual limits of LFA are 10 ng·mL for NEO and 200 ng·mL for NOR, and with LODs down to 0.37 pg·mL and 0.55 pg·mL by using SERS. The NEO test line is not interfered by the NEO analogues gentamycin, streptomycin and tobramycin, but the NOR test line suffers from different degrees of cross-reactivity (CR) to 12 common other QNS, the CRs ranging from 1.5% to 136%. The recoveries of NEO and NOR from spiked milk samples ranged between 86% and 121%, with relative standard deviations (RSD) from 3% to 6%. The method is highly sensitive, accurate and effective. It may be applied to simultaneous detection of NEO and 8 QNS, including NOR, enoxacin, ciprofloxacin, ofloxacin, fleroxacin, marbofloxacin, enrofloxacin, and pefloxacin. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) caused by African swine fever virus (ASFV) is becoming a serious threat to the swine industry worldwide. CD2v is a key pathogenic factor of ASFV and the protective antigen with low immunogenicity, whereas viral protein-based nanoparticles have advantages of precise assembly and high immunogenicity. In this study, the CD2v protein fused with Norovirus (NoV) P particle assembled into nanoparticle for improved immunogenicity. Then, CD2v protein nanoparticle and monomer CD2v protein were expressed in HEK293F cells. The former induced higher levels of antibodies, and thus highly potent monoclonal antibodies (mAbs) were generated and characterized. The highest antibody titration of mAb 10A3 reached 1:2048000, and mAb 2E9 had the highest inhibition percent of 84% when competed with ASFV positive serum. Meanwhile, all mAbs reacted specifically with the denatured CD2v protein, and the linear epitope with the location of amino acids 28th to 51st of CD2v extracellular domain sequence was identified. In summary, this study produced a highly immunogenic CD2v protein and generated high-titer mAbs, the precise location of linear epitope on the CD2v was further determined. These findings may provide a powerful help for etiology and serological detection of ASFV. • Highly immunogenic CD2v protein nanoparticle was obtained. • High-titer mAbs induced by CD2v protein nanoparticle were generated, and mAb 10A3 had the highest antibody titration of 1:2048000. • The linear epitope from the 28th to the 51st position of amino acids locating in the CD2v extracellular domain sequence was first reported. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome coronavirus (MERS-CoV) is a coronavirus which can cause severe human respiratory diseases with a fatality rate of almost 36%. In this study, we report the generation, characterization and epitope mapping of several monoclonal antibodies against the spike receptor-binding domain (RBD) of MERS-CoV. Two monoclonal antibodies (4C7 and 6E8) that can react with linearized RBD have been selected for subsequent identification of RBD mAb-binding epitopes. Two distinct novel linear epitopes, 423FTCSQIS429 and 546SPLEGGGWL554,were precisely located at the outermost surface of RBD by dot-blot hybridization and ELISAs. Multiple sequence alignment analysis showed that these two peptides were highly conserved. Alanine (A)-scanning mutagenesis demonstrated that residues 423F, 428I, and 429S are the crucial residues for the linear epitope 423FTCSQIS429 while residues 548L, 550G, 553W, 554L for epitope 546SPLEGGGWL554. These findings may be helpful for further understanding of the function of RBD protein and the development of subsequent diagnosis and detection methods. [ABSTRACT FROM AUTHOR]

Classical swine fever virus (CSFV) is a member of the genus Pestivirus, which causes serious economic losses. The re-emergence of the disease in Japan in 2018 has increased awareness of CSFV. In this study, Balb/c mice were immunized with plant-derived E2 protein, and four monoclonal antibodies (mAbs) 4B11, 7B3, 11A5 and 6F3 were generated. Two of these mAbs, 4B11 and 7B3, effectively blocked CSFV infection of PK-15 cells. Both mAbs recognized a novel linear epitope, 256CLIGNTTVKVHASDER271. The neutralizing ability of anti-CSFV serum decreased 63%, when pre-incubated with the linear peptide at 200 μg/mL. Structural analysis showed that this linear epitope is present at the border of Domain C and Domain D on the surface of the E2 protein. Alignment of amino acid sequences showed that the epitope was conserved in different subgroups of CSFV but not in other members of the Pestivirus genus. Consistently with the analysis above, this epitope distinguished antibodies against CSFV from those against bovine viral diarrhea virus (BVDV). Our study provides an ideal candidate peptide for new vaccine design and differential diagnosis of CSFV. These findings will contribute to the control and eradication of classical swine fever. • Four mAbs against CSFV E2 were generated. Two of them presented neutralizing reactivity against CSFV. • A novel linear neutralizing epitope P33 located at the border of Domain C and Domain D of the E2 protein was identified. • Epitope P33 distinguished antibodies to CSFV from those to BVDV. [ABSTRACT FROM AUTHOR]

• 14 PEDV COE-specific mAbs were prepared. • A novel linear B-cell epitope 592TSLLASACTIDLFGYP607 was identified. • The 606Y, 605G and 604F were core residues for recognition. • The epitope and core residues are conserved among PEDV strains. • The difference of mAb reactivity suggested antigenic changes in the S protein. The porcine epidemic diarrhea virus (PEDV) collagenase equivalent domain (COE, residues 499–638), a crucial antigenic region within the viral spike (S) glycoprotein, has been widely utilized for the development of subunit vaccines to prevent viral infection. In the current study, we immunized BALB/c mice with recombinant truncated PEDV COE protein and obtained 14 COE-specific monoclonal antibodies (mAbs). Based on the reactivity analysis of the mAbs with two prevalent PEDV strains in G2 type and the attenuated CV777 strain in G1 type, 6 mAbs were selected for subsequent identification of COE mAb-binding epitopes. Dot-blot hybridization and enzyme-linked immunosorbent assays (ELISAs) identified the peptide 592TSLLASACTIDLFGYP607 as a novel linear B-cell epitope involved in binding of mAbs 4D8F10 and 6F3E3. Subsequently, alanine (A)-scanning mutagenesis demonstrated that residues 606Y, 605G and 604F were core residues involved in recognition. Importantly, this novel COE epitope, including core residues, is conserved among G1 and G2 type PEDV strains. Further experiment indicates that the mAbs 4D8F10 and 6F3E3 were suitable for PEDV detection via mAb binding to the conserved epitope. The current work actually provides potential uses for the development of diagnostic methods to detect PEDV. [ABSTRACT FROM AUTHOR]

• This assay can determine zeranol and its five family analogues in milk. • The cutoff values for zeranol and its analogues were all 10 ng/mL coincidentally. • The lower detection limits for zeranol and its analogues were all below 0.3 ng/mL. • The detection time of this assay is less than 15 min. Based on colloidal gold and broad-spectrum monoclonal antibody that binds to zeranol and its five analogues with high sensitivity, a lateral flow immunochromatographic assay (LFIA) in a competitive format was developed to specifically determine residues of zeranol, an illegal growth promoter in livestock. In this study, the assay had high sensitivity and was broad-spectrum only for zeranol and its five analogues, and the results were obtained within 10 min without needing sophisticated procedures. The cutoff values for zeranol and its five analogues were 10 ng/mL, and the IC 50 values for zeranol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol and zearalenone were 1.250, 1.800, 1.775, 1.225, 1.709 and 1.319 ng/mL, respectively. The recovery rates were ranged from 85.6 to 93.9%, with the coefficient of variations less than 12.4%. The results demonstrated that the LFIA could be used for rapid, simultaneous, semi-quantitative and quantitative detection of residues of zeranol and its five analogous in milk. [ABSTRACT FROM AUTHOR]