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 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 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.

Evaluation of Coagulase Activity and Protein A Production for the Identification of Staphylococcus aureus

1995 ◽  
Vol 58 (8) ◽  
pp. 858-862 ◽  
Author(s):  
TSUNG C. CHANG ◽  
SU H. HUANG
Keyword(s):  
Public Health ◽  
Staphylococcus Aureus ◽  
Protein A ◽  
High Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
American Public Health Association ◽  
Bacterial Strains ◽  
Test Sensitivity ◽  
Microtiter Plates ◽  
Health Association

The activities of coagulase and thermostable nuclease (TNase) and the production of protein A were studied in 338 bacterial strains. These included 213 isolates of Staphylococcus aureus to determine which characteristic was most specific for the identification of S. aureus. The evaluation of different protocols for interpretation of coagulase results was also undertaken. Protein A was analyzed by a sandwich enzyme-linked immunosorbent assay using microtiter plates coated with antiprotein A antibodies. Coagulase activities were determined according to the criteria recommended by Association of Official Analytical Chemists (AOAC; any degree of clot formation is a positive reaction), American Public Health Association (APHA; coagulase activities ≥ 3+ are positive reactions), and the Bacteriological Analytical Manual (BAM; only 4+ reaction is positive). It was found that the AOAC protocol, which had a test sensitivity of 97.7% and a specificity of 95.1% and could be completed within six hours, was more practical than the methods used by APHA and BAM. Compared with coagu1ase and TNase, protein A was a better marker of S. aureus; a high sensitivity (100%) and specificity (96.8%) were obtained by using protein A for the identification of S. aureus.

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.

An electronic enzyme-linked immunosorbent assay platform for protein analysis based on magnetic beads and AlGaN/GaN high electron mobility transistors

The Analyst ◽  
2020 ◽  
Vol 145 (7) ◽  
pp. 2725-2730 ◽  
Author(s):  
Jin Wang ◽  
Zhiqi Gu ◽  
Xinsheng Liu ◽  
Lei Zhao ◽  
Huoxiang Peng ◽  
...  
Keyword(s):  
Electron Mobility ◽  
Magnetic Beads ◽  
High Sensitivity ◽  
High Electron Mobility Transistors ◽  
High Electron Mobility Transistor ◽  
Fast Response ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Electron ◽  
High Electron Mobility ◽  
Detection Of Biomolecules

The AlGaN/GaN high electron mobility transistor (HEMT) biosensors have the characteristics of high sensitivity, stability and fast response in the detection of biomolecules.

scholarly journals Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against African Swine Fever Virus

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 760
Author(s):  
Fangfeng Yuan ◽  
Vlad Petrovan ◽  
Luis Gabriel Gimenez-Lirola ◽  
Jeffrey J. Zimmerman ◽  
Raymond R. R. Rowland ◽  
...  
Keyword(s):  
Time Course ◽  
Serological Test ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Internal Quality ◽  
Enzyme Linked Immunosorbent Assay ◽  
Post Inoculation ◽  
Swine Industry ◽  
Control Serum

The incursion of African swine fever virus (ASFV) into Eurasia presents a threat to the world’s swine industry. Highly sensitive and specific diagnostic assays are urgently needed for rapid detection during an outbreak, post-outbreak investigation, and disease surveillance. In this study, a highly specific and repeatable blocking ELISA (bELISA) was developed using a recombinant p30 protein as the antigen combined with biotinylated mAb against p30 as the detection antibody. Initial test validation included sera from 810 uninfected animals and 106 animals experimentally inoculated with ASFV or recombinant alphavirus/adenovirus expressing p30. Receiver operating characteristic (ROC) analysis of the data calculated an optimal percentage of inhibition (PI) cutoff value of 45.92%, giving a diagnostic sensitivity of 98.11% and diagnostic specificity of 99.42%. The coefficient of variation of an internal quality control serum was 6.81% for between runs, 6.71% for within run, and 6.14% for within plate. A time course study of infected pigs showed that bELISA was able to detect seroconversion as early as 7 days post-inoculation. Taken together, these results demonstrate that bELISA can be used as an alternative serological test for detecting ASFV infection.

scholarly journals Detection of E. coli O157:H7 in Food Using Automated Immunomagnetic Separation Combined with Real-Time PCR

Processes ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 908
Author(s):  
Ji Young Park ◽  
Min-Cheol Lim ◽  
Kisang Park ◽  
Gyeongsik Ok ◽  
Hyun-Joo Chang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Magnetic Beads ◽  
High Sensitivity ◽  
Immunomagnetic Separation ◽  
Sample Volume ◽  
Food Samples ◽  
Automated System ◽  
Bacterial Strains ◽  
Foodborne Bacteria

In this study, we describe the development of an automated immunomagnetic separation device combined with real-time polymerase chain reaction (PCR) for detecting foodborne bacteria. Immunomagnetic separation (IMS) is a well-known method for the separation and concentration of target bacteria from a large volume of food samples. Magnetic beads functionalized with an antibody provide selectivity for target bacteria such as Escherichia coli O157:H7. Moreover, compared to conventional methods, real-time PCR enables high-sensitivity detection of target bacteria. The method proposed in this study involves three steps: (1) pre-enrichment, (2) automated IMS and concentration of target bacteria, and (3) detection of target bacteria by real-time PCR. Using food samples with a working sample volume as large as 250 mL, the whole process only requires 3 h. As a result, target bacteria in the range of 101–102 colony-forming units per mg or g of sample can be detected in food samples, such as milk, ground beef, and cabbage, by using the proposed approach. We anticipate that the automated IMS system combined with real-time PCR will contribute to the development of a fully automated system for detecting foodborne bacteria and serve as a multi-tester for a variety of bacterial strains in the capacity of a sample-to-answer device in the near future.

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.

scholarly journals The C962R ORF of African Swine Fever Strain Georgia Is Non-Essential and Not Required for Virulence in Swine

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 676 ◽  
Author(s):  
Elizabeth. Ramirez-Medina ◽  
Elizabeth. A. Vuono ◽  
Ayushi. Rai ◽  
Sarah. Pruitt ◽  
Ediane. Silva ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Field Isolate ◽  
Protein A ◽  
African Swine Fever ◽  
Virus Genome ◽  
Economic Losses ◽  
Virus Protein ◽  
Swine Industry ◽  
Reading Frame

African swine fever virus (ASFV) is the causative agent of the African swine fever (ASF) epizootic currently affecting pigs throughout Eurasia, causing significant economic losses in the swine industry. The virus genome encodes for more than 160 genes, of which only a few have been studied in detail. Here we describe the previously uncharacterized ASFV open reading frame (ORF) C962R, a gene encoding for a putative NTPase. RNA transcription studies using infected swine macrophages demonstrate that the C962R gene is translated as a late virus protein. A recombinant ASFV lacking the C962R gene (ASFV-G-ΔC962R) demonstrates in vivo that the C962R gene is non-essential, since ASFV-G-ΔC962R has similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔC962R produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the C962R gene from the ASFV genome does not impact virulence.

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