scholarly journals Development of a Dual ELISA for the Detection of CD2v-Unexpressed Lower-Virulence Mutational ASFV

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1214
Author(s):  
Changjie Lv ◽  
Ya Zhao ◽  
Lili Jiang ◽  
Li Zhao ◽  
Chao Wu ◽  
...  
Keyword(s):  
Differential Diagnosis ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epidemiology Study ◽  
Wild Type ◽  
Serum Samples ◽  
Viral Pathogen ◽  
Positive Serum

African swine fever virus (ASFV) is an important viral pathogen infecting pigs worldwide throughout the pig industry. CD2v (an outer-membrane glycosylated protein of ASFV)-unexpressed lower-virulence mutants have appeared in China and other countries in recent years. Using OIE-recommended quantitative PCR and ELISA methods, people can accurately judge whether pigs are infected with wild-type ASFV. However, the strategy has failed to distinguish ΔCD2v lower-virulence mutants and wild-type ASFV infection. Here, we expressed and purified the CD2v and p30 proteins via CHO cells and successfully established a dual enzyme-linked immunosorbent assay (ELISA), which can be used to differentiate pigs infected with wild-type ASFV or with CD2v-unexpressed lower-virulence mutants. The dual ELISA showed excellent specificity without cross-reactions with antibodies of PRRSV, CSFV, JEV, PRV, or PPV. The dual ELISA could detect ASFV-infected positive serum samples up to dilutions of 5120 times, possessing high sensitivity. Therefore, the application of this dual ELISA approach can play an important role in ASFV epidemiology study and fill the gaps in differential diagnosis.

scholarly journals Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies

2022 ◽  
Vol 8 ◽  
Author(s):  
Kexin Zhong ◽  
Mengmeng Zhu ◽  
Qichao Yuan ◽  
Zhibang Deng ◽  
Simeng Feng ◽  
...  
Keyword(s):  
Indirect Elisa ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Viral Disease ◽  
Fever Virus ◽  
Serum Samples ◽  
Swine Industry ◽  
Detection Techniques ◽  
Positive Serum ◽  
Great Reactivity

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

scholarly journals A Sensitive Dot Immunobinding Assay for Serodiagnosis of African Swine Fever Virus with Application in Field Conditions

1992 ◽  
Vol 4 (3) ◽  
pp. 254-257 ◽  
Author(s):  
Maria J. Pastor ◽  
Marisa Arias ◽  
Carlos Alcaraz ◽  
Maribel De Diego ◽  
Jose M. Escribano
Keyword(s):  
African Swine Fever Virus ◽  
Protein A ◽  
African Swine Fever ◽  
Fever Virus ◽  
Field Conditions ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Soluble Antigen ◽  
Serum Samples ◽  
Epitope Binding

The present work describes a simple dot immunobinding assay (DIA) for African swine fever virus (ASFV) antibody detection that can be used under field conditions. The assay uses nitrocellulose strips dotted with a cytoplasmic soluble antigen (CS-P) of ASFV. The nitrocellulose strips are adhered to a plastic handle. The test serum samples react with the CS-P, and antibodies are detected using a protein A-peroxidase conjugate. Both incubations are carried out at 20 C. The efficacy of the DIA as a screening test for ASFV was compared to an enzyme-linked immunosorbent assay (ELISA) and an immunoblotting (IB) test using 343 sera collected from natural African swine fever epizootics and from inapparent ASFV carriers. The DIA had comparable sensitivity to both reference techniques, and all samples positive in the ELISA and IB test were also positive in the DIA. False-positive reactions were not detected when whole blood or poorly preserved serum samples were tested by DIA. Some poorly preserved sera that were positive initially by the ELISA were no longer ELISA positive in a later run, although they were positive in IB and DIA. These positive DIA and IB test results could be caused by the differences in antibody epitope binding.

A Semi-automated System of Luciferase Immunoprecipitation Assay for Rapid and Easy Detection of African Swine Fever Virus Antibody

2021 ◽  
Author(s):  
Huan Liu ◽  
Ping He ◽  
Fei Meng ◽  
Mengwei Jiang ◽  
Jin Xiong ◽  
...  
Keyword(s):  
Disease Surveillance ◽  
Magnetic Beads ◽  
African Swine Fever Virus ◽  
Protein A ◽  
African Swine Fever ◽  
High Sensitivity ◽  
Automated System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoprecipitation Assay ◽  
Elisa Kit

African swine fever (ASF) is a highly contagious viral disease of domestic pigs and wild boars. For the disease surveillance and control, we developed a rapid and easy luciferase immunoprecipitation assay (MB-LIPS) to detect ASF virus (ASFV) antibody. The MB-LIPS is based on magnetic beads modified with protein A/G and the recombinant fusion protein of ASFV p30 and luciferase, where p30 functioned as the recognition element and the luciferase as the signal component. Incubation and washing could be finished automatically on a machine with magnetic rods. Under the optimal conditions, the MB-LIPS showed 96.3% agreement to a commercial enzyme linked immunosorbent assay (ELISA) kit for detecting ASFV antibody in swine sera. Analyzing serial dilutions of a swine serum sample showed that the MP-LIPS assay was 4 times more sensitive than the ELISA kit. The whole run of the MB-LIPS could be completed within 30 min. With its high sensitivity and simple operation, the MB-LIPS platform has great potentials to be used for the detection of ASFV antibody and ASF control in small labs and farms.

scholarly journals A novel multi-epitope recombinant protein for diagnosis of African swine fever

2020 ◽  
Author(s):  
ZHAN GAO ◽  
JUNJUN SHAO ◽  
GUANGLEI ZHANG ◽  
SUDAN GE ◽  
YANYAN CHANG ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
African Swine Fever Virus ◽  
Expression System ◽  
African Swine Fever ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Swine Diseases

Abstract Background: African swine fever(ASF) is an acute, severe and highly fatal infectious disease of pigs. The disease spreads rapidly, causing huge economic losses to the pig industry in infected areas. The structural proteins p30 and p54 in African swine fever virus(ASFV) have been verified as diagnostic antigens.Methods: In this study, we constructed a novel multi-epitope fusion antigen gene based on P30 and P54 proteins, induced expression in a prokaryotic expression system and analyzed the reactivity of the recombinant fusion protein. The purified recombinant protein m35 was used as the coating antigen to establish an indirect enzyme-linked immunosorbent assay (ELISA) detection method for ASFV. 116 serum samples and positive sera of other swine diseases were detected by indirect ELISA.Results: Our results indicate that the m35 gene fragment with a length of 558bp was successfully constructed. SDS-PAGE and Western Blotting analysis showed that the protein had a band at 22kDa, proving its good reactogenicity. ROC analysis was performed to validate the assay, the area under the ROC curve is 0.9738 (95% confidence interval, 0.9336 to 1.014), and does not cross-react with other swine diseases.Conclusion: Our results show that its sensitivity and specificity were highly accurate. It is feasible to use this recombinant protein as a diagnostic antigen to distinguish ASFV infection.

scholarly journals Tetanus Immunity among Women Aged 15 to 39 Years in Cambodia: a National Population-Based Serosurvey, 2012

2016 ◽  
Vol 23 (7) ◽  
pp. 546-554 ◽  
Author(s):  
Heather M. Scobie ◽  
Bunsoth Mao ◽  
Sokhal Buth ◽  
Kathleen A. Wannemuehler ◽  
Charlotte Sørensen ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Population Based ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cluster Sampling ◽  
Serum Samples ◽  
Nulliparous Women ◽  
Vaccination Campaigns ◽  
Antibody Levels ◽  
Multiple Pathogens ◽  
Tetanus Immunity

To monitor progress toward maternal and neonatal tetanus elimination (MNTE) in Cambodia, we conducted a nationwide serosurvey of tetanus immunity in 2012. Multistage cluster sampling was used to select 2,154 women aged 15 to 39 years. Tetanus toxoid antibodies in serum samples were measured by gold-standard double-antigen enzyme-linked immunosorbent assay (DAE) and a novel multiplex bead assay (MBA). Antibody concentrations of ≥0.01 IU/ml by DAE or the equivalent for MBA were considered seroprotective. Estimated tetanus seroprotection was 88% (95% confidence interval [CI], 86 to 89%); 64% (95% CI, 61 to 67%) of women had antibody levels of ≥1.0 IU/ml. Seroprotection was significantly lower (P< 0.001) among women aged 15 to 19 years (63%) and 20 to 24 years (87%) than among those aged ≥25 years (96%), among nulliparous women than among parous women (71 versus 97%), and among those living in the western region than among those living in other regions (82 versus 89%). The MBA showed high sensitivity (99% [95% CI, 98 to 99%]) and specificity (92% [95% CI, 88 to 95%]) compared with DAE. Findings were compatible with MNTE in Cambodia (≥80% protection). Tetanus immunity gaps should be addressed through strengthened routine immunization and targeted vaccination campaigns. Incorporating tetanus testing in national serosurveys using MBAs, which can measure immunity to multiple pathogens simultaneously, may be beneficial for monitoring MNTE.

scholarly journals Comparison of Recombinant Trypanosoma cruzi Peptide Mixtures versus Multiepitope Chimeric Proteins as Sensitizing Antigens for Immunodiagnosis

2009 ◽  
Vol 16 (6) ◽  
pp. 899-905 ◽  
Author(s):  
Cecilia Camussone ◽  
Verónica Gonzalez ◽  
María S. Belluzo ◽  
Nazarena Pujato ◽  
María E. Ribone ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Serum Sample ◽  
Chagas Disease ◽  
Single Molecule ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chimeric Proteins ◽  
Serum Samples ◽  
Peptide Mixtures ◽  
Positive Serum ◽  
Recombinant Peptides

ABSTRACT The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

scholarly journals Rapid and sensitive RPA-Cas12a-fluorescence assay for point-of-care detection of African swine fever virus

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254815
Author(s):  
Jinyu Fu ◽  
Yueping Zhang ◽  
Guang Cai ◽  
Geng Meng ◽  
Shuobo Shi
Keyword(s):  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
High Sensitivity ◽  
Fluorescence Assay ◽  
Fever Virus ◽  
Diagnostic Methods ◽  
High Morbidity ◽  
Swine Industry ◽  
Control And Prevention

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.

scholarly journals Horseradish Peroxidase-Conjugated-Nanobody-Based cELISA For The Rapid and Sensitive Clinical Detection of African Swine Fever Virus Antibodies

2021 ◽  
Author(s):  
Huijun Zhao ◽  
Jiahui Ren ◽  
Shuya Wu ◽  
Yongkun Du ◽  
Bo Wan ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Expression System ◽  
African Swine Fever ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hemorrhagic Disease ◽  
Serum Samples ◽  
Promising Alternative ◽  
Technical Requirements ◽  
Pig Serum

Abstract Background: African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that affects pigs and has the potential to cause mortality in almost 100% of domestic pigs and wild boars. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, cost-effective detection and strict control and elimination strategies. Traditional molecular and serological testing methods are generally associated with high testing costs, complex operations and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. The application of Nbs in the detection of ASFV antibodies in the serum has not yet been reported, to the best of our knowledge. Results: Using a phage display technology, one specific Nb against the ASFV p54 protein that exhibited high specificity and affinity to the protein, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. The cut-off value for the cELISA was 15.78%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The specificity of the cELISA was 98.97%, and the limit of detection was 1:320 in inactivated ASFV antibody-positive reference serum samples, with the coincidence rate between the two methods being 98.56%. Conclusions: A specific, sensitive and repeatable cELISA was successfully developed based on the unique Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.

scholarly journals Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts

2009 ◽  
Vol 16 (12) ◽  
pp. 1774-1780 ◽  
Author(s):  
Eduardo A. F. Coelho ◽  
Laura Ramírez ◽  
Mariana A. F. Costa ◽  
Vinicio T. S. Coelho ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Ribosomal Proteins ◽  
High Sensitivity ◽  
Leishmania Species ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Leishmania Chagasi ◽  
Serum Samples ◽  
Potential Tool

ABSTRACT In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

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