Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). A sensitive and reliable serological diagnostic assay is required, so laboratories can effectively and quickly detect ASFV infection. The p30 protein is abundantly expressed early in cells and has excellent antigenicity. Therefore, this study aimed to produce and characterize p30 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Three monoclonal antibodies against p30 protein that were expressed in E. coli were generated, and their characterizations were investigated. Furthermore, a blocking ELISA based on a monoclonal antibody was developed. To evaluate the performance of the assay, 186 sera samples (88 negative and 98 positive samples) were analyzed and a receiver-operating characteristic (ROC) analysis was applied to determine the cutoff value. Based on the ROC analysis, the area under the curve (AUC) was 0.997 (95% confidence interval: 99.2 to 100%). Besides, a diagnostic sensitivity of 97.96% (95% confidence interval: 92.82 to 99.75%) and a specificity of 98.96% (95% confidence interval: 93.83 to 99.97%) were achieved when the cutoff value was set to 38.38%. Moreover, the coefficients of inter- and intra-batches were <10%, indicating the good repeatability of the method. The maximum dilution of positive standard serum detected by this ELISA method was 1:512. The blocking ELISA was able to detect seroconversion in two out of five pigs at 10 Dpi and the p30 response increasing trend through the time course of the study (0–20 Dpi). In conclusion, the p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity. The assay could be a useful tool for field surveillance and epidemiological studies in swine herd.

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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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A comparison of two indirect immune electron microscopy procedures using monoclonal antibody to localize specific African swine fever virus (ASFV) proteins in infected cells and virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077578 ◽

1983 ◽

Vol 41 ◽

pp. 800-801

Author(s):

G. J. Letchworth III ◽

T. C. Whyard ◽

S. H. Wool

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Grid Method ◽

Viral Proteins ◽

Fever Virus ◽

Immune Electron Microscopy ◽

Infected Cells

The primary purpose of this study was to devise a procedure in which a large number of monoclonal antibodies could be tested against several African swine fever virus (ASFV) preparations (virus, infected cells). The procedure had to be rapid, use a small sample and provide for some TEM.Monoclonal antibodies were produced by standard methods using myeloma cells and lymphocytes from mice immunized against ASF viral proteins. Antibodies were characterized by immunoprecipitation of radio-labelled viral proteins (Fig 1).The viral preparations used in this study were either infected macrophages, culture fluid supernatant from ASFV-infected macrophages or red blood cells from infected pigs. Each of the three viral preparations was tested by two indirect immune electron microscopy (IEM) methods; a grid method and a test tube method. In the grid method, formvar coated grids were floated on a viral preparation then for one hour each on the mouse monoclonal antibody and finally on ferritin tagged sheep anti-mouse IgG with washes between each step.

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