Comparative evaluation of novel African swine fever virus (ASF) antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests

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.

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Alternative sampling strategies for passive classical and African swine fever surveillance in wild boar – Extension towards African swine fever virus antibody detection

Veterinary Microbiology ◽

10.1016/j.vetmic.2014.09.018 ◽

2014 ◽

Vol 174 (3-4) ◽

pp. 607-608 ◽

Cited By ~ 4

Author(s):

Sandra Blome ◽

Katja V. Goller ◽

Anja Petrov ◽

Carolin Dräger ◽

Jana Pietschmann ◽

...

Keyword(s):

Wild Boar ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Antibody Detection ◽

Sampling Strategies ◽

Virus Antibody

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Highly specific confirmatory Western blot test for African swine fever virus antibody detection using the recombinant virus protein p54

Journal of Virological Methods ◽

10.1016/0166-0934(94)00150-f ◽

1995 ◽

Vol 52 (1-2) ◽

pp. 111-119 ◽

Cited By ~ 14

Author(s):

C. Alcaraz ◽

F. Rodriguez ◽

J.M. Oviedo ◽

A. Eiras ◽

M. De Diego ◽

...

Keyword(s):

Western Blot ◽

Recombinant Virus ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Antibody Detection ◽

Virus Protein ◽

Virus Antibody ◽

Western Blot Test ◽

Confirmatory Western Blot

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Characterization of Anti-p54 Monoclonal Antibodies and Their Potential Use for African Swine Fever Virus Diagnosis

Pathogens ◽

10.3390/pathogens10020178 ◽

2021 ◽

Vol 10 (2) ◽

pp. 178

Author(s):

Weldu Tesfagaber ◽

Lulu Wang ◽

Ghebremedhin Tsegay ◽

Yibrah Tekle Hagoss ◽

Zhenjiang Zhang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Immunosorbent Assay ◽

Roc Analysis ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Antibody Detection ◽

Effective Vaccine ◽

Enzyme Linked Immunosorbent Assay

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). Although a good advance has been made to understand the virus, a safe and effective vaccine against ASFV is still lacking and its eradication solely depends on its early and accurate diagnosis. Thus, improving the available diagnostic assays and adding some validated techniques are useful for a range of serological investigations. The aim of this study was to produce and characterize p54 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Five monoclonal antibodies against p54 protein expressed in Escherichia coli was generated and their characterizations were investigated. Furthermore, a competitive enzyme-linked immunosorbent assay (cELISA) based on a monoclonal antibody designated as 2A7 was developed. To evaluate the performance of the assay, a total of 365 pig serum samples (178 negative and 187 positive samples) were tested and a receiver-operating characteristic (ROC) analysis was applied to determine the cut-off value. Based on the ROC analysis, the area under the curve (AUC) was 0.982 (95% confidence interval: 96.9% to 99.4%), besides a sensitivity of 92.5% and a specificity of 98.9% was achieved when the percent inhibition of 20% was selected as a threshold. Moreover, the result showed an excellent agreement when compared to other commercially available blocking ELISA (kappa value = 0.912) and showed no reaction to other swine pathogens. Overall, the newly developed cELISA method offers a promising approach for a rapid and convenient ASFV serodiagnosis, which could be used as alternative to other serological assays for screening possible ASFV infection.

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Lateral Flow Assay for the Detection of African Swine Fever Virus Antibodies Using Gold Nanoparticle-Labeled Acid-Treated p72

Frontiers in Chemistry ◽

10.3389/fchem.2021.804981 ◽

2022 ◽

Vol 9 ◽

Author(s):

Wenzhuang Zhu ◽

Kaiwen Meng ◽

Yueping Zhang ◽

Zhigao Bu ◽

Dongming Zhao ◽

...

Keyword(s):

Gold Nanoparticle ◽

African Swine Fever Virus ◽

African Swine Fever ◽

High Sensitivity ◽

Lateral Flow ◽

Fever Virus ◽

Lateral Flow Assay ◽

Diagnostic Methods ◽

Detection Methods ◽

Antibody Detection

African swine fever is a widespread and highly contagious disease in the porcine population, which is caused by African swine fever virus (ASFV). The PCR and ELISA detection methods are the main conventional diagnostic methods for ASFV antigen/antibody detection in the field. However, these methods have limitations of expensive equipment, trained technicians, and time-consuming results. Thus, a rapid, inexpensive, accurate and on-site detection method is urgently needed. Here we describe a double-antigen-sandwich lateral-flow assay based on gold nanoparticle-conjugated ASFV major capsid protein p72, which can detect ASFV antibody in serum samples with high sensitivity and specificity in 10 min and the results can be determined by naked eyes. A lateral flow assay was established by using yeast-expressed and acid-treated ASFV p72 conjugated with gold nanoparticles, which are synthesized by seeding method. A high coincidence (97.8%) of the assay was determined using clinical serum compared to a commercial ELISA kit. In addition, our lateral flow strip can detect as far as 1:10,000 diluted clinically positive serum for demonstration of high sensitivity. In summary, the assay developed here was shown to be rapid, inexpensive, accurate and highly selective. It represents a reliable method for on-site ASFV antibody detection and may help to control the ASFV pandemic.

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

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063879200400305 ◽

1992 ◽

Vol 4 (3) ◽

pp. 254-257 ◽

Cited By ~ 8

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.

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