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

Pathogens ◽  
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.

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

2021 ◽  
Vol 8 ◽  
Author(s):  
Xuexiang Yu ◽  
Xianjing Zhu ◽  
Xiaoyu Chen ◽  
Dongfan Li ◽  
Qian Xu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Confidence Interval ◽  
Roc Analysis ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Diagnostic Sensitivity ◽  
Cutoff Value ◽  
Blocking Elisa

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.

A solid-phase enzyme linked immunosorbent assay using monoclonal antibodies, for the detection of african swine fever virus antigens and antibodies

1997 ◽  
Vol 66 (2) ◽  
pp. 211-218 ◽  
Author(s):  
Monica I. Vidal ◽  
Matthias Stiene ◽  
Jörg Henkel ◽  
Ursula Bilitewski ◽  
João V. Costa ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Solid Phase ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Virus Antigens

scholarly journals A solid-phase enzyme linked immunosorbent assay for the detection of African swine fever virus antigen and antibody

1979 ◽  
Vol 83 (2) ◽  
pp. 363-370 ◽  
Author(s):  
R. C. Wardley ◽  
E. M. E. Abu Elzein ◽  
J. R. Crowther ◽  
P. J. Wilkinson
Keyword(s):  
Immunosorbent Assay ◽  
Solid Phase ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Virus Antigen ◽  
Fever Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Use

summaryA solid-phase enzyme-linked immunosorbent assay was developed to measure both African swine fever virus (ASFV) antigen and antibody. Experiments showed it to be reproducible and able to detect limiting antigen concentrations of 50–500 HAD50/ml. The assay was more sensitive than those used at present to detect ASFV antibody and it is suggested that it might be of great diagnostic use in countries where African swine fever has recently appeared.

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

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.

scholarly journals Techniques of blood sampling for detection of African swine fever virus in wild boar and domestic pigs in the field conditions

2021 ◽  
pp. 285-294
Author(s):  
A. S. Pershin ◽  
A. R. Shotin ◽  
E. O. Morozova ◽  
A. S. Igolkin ◽  
O. A. Manuylova ◽  
...  
Keyword(s):  
Wild Boar ◽  
Immunosorbent Assay ◽  
Filter Paper ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Blood Sampling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoperoxidase Technique ◽  
Domestic Pigs

It is thought that due to the high virulence of the African swine fever virus its circulation in the Russian Federation is accompanied by a low seroprevalence. However taking into account a long-term ASF unfavourable situation, the introduction of the virus into the wild boar population, and the occurrence of attenuated viral variants, the significance of serological testing aimed at the detection of viral antibodies is increasing. To collect field samples of biological material from animals for molecular genetic, virological, and serological tests, filter paper, as well as swabs, can be used. The specificity and sensitivity of enzyme-linked immunosorbent assay when testing blood absorbed by filter paper are worse than those shown when testing sera, but they allow effective detection of African swine fever virus antibodies. It was demonstrated that blood absorbed on filter paper can be used for the immunoblot analysis, but the optimum performance could be achieved when the immunoperoxidase technique in combination with samples, taken by swabs was used. When comparing results of enzyme-linked immunosorbent assay performed on sera collected from domestic pigs (infected with ASFV isolates Antonovo 07/14 and Sobinka 07/15), and blood from ear veins absorbed on filter paper the sensitivity was 88.9%, specificity – 90.6%. However, the use of the immunoperoxidase technique for testing blood from swabs showed 100% coincidence with ELISA, while testing of sera with immunoperoxidase technique was superior to ELISA in sensitivity. This means blood sampling using swabs may be recommended for tests after proper validation. This technique can be especially useful for collecting data about infected wild boars because effective eradication strategies are impossible without such data.

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.

scholarly journals Unreliable Measurement of Basophil Maximum Leukotriene Release with the Bühlmann CAST 2000 Enzyme-Linked Immunosorbent Assay Kit

2006 ◽  
Vol 13 (3) ◽  
pp. 420-422 ◽  
Author(s):  
S. E. Burastero ◽  
C. Paolucci ◽  
D. Breda ◽  
G. Monasterolo ◽  
R. E. Rossi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
False Positive ◽  
Positive Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Positive Results

ABSTRACT The Bühlmann CAST 2000 enzyme-linked immunosorbent assay is a potentially useful assay for measuring sulfidoleukotrienes released in vitro by allergen-challenged basophils. However, we observed that the positive-control reagent yielded positive signals in cell-free systems. These false-positive results depended on using a mouse anti-FcεRI monoclonal antibody and were prevented by degranulation-inducing reagents other than mouse monoclonal antibodies.

Detection of antibodies against African swine fever virus using infected monolayers and monoclonal antibodies

1988 ◽  
Vol 122 (22) ◽  
pp. 536-539 ◽  
Author(s):  
G. Wensvoort ◽  
C. Terpstra ◽  
M. Bloemraad
Keyword(s):  
Monoclonal Antibodies ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus
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