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Home / Equine Research Bank / Clin Vaccine Immunol / Development of a fluorescent-microsphere immunoassay for det...
Clinical and vaccine immunology : CVI2007; 15(1); 76-87; doi: 10.1128/CVI.00388-07

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

Abstract: The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.
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Publication Date: 2007-11-21 PubMed ID: 18032597PubMed Central: PMC2223870DOI: 10.1128/CVI.00388-07Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study discusses the creation and verification of a microsphere immunoassay (MIA), a test created to detect certain horse antibodies associated with the equine arteritis virus (EAV). It was found that the MIA correlates strongly with traditional virus neutralization tests (VNT), but it is quicker, more convenient, and more cost-effective, though slightly less sensitive.

Creating and Testing the Microsphere Immunoassay (MIA)

  • The researchers created the MIA based on the cloning and expression of genes linked to the main structural proteins of the EAV. Some of these proteins tested include full-length proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), and some are partial sequences of those structural proteins, which were presumed to hold antigenic regions.
  • After cloning and expressing these proteins, they were then purified and attached to fluorescent polystyrene microspheres. These microspheres were then tested and analyzed using a specialized device called the Luminex xMap 100 instrument.
  • Out of the eight proteins explored, the best alignment with the virus-neutralizing test (VNT) results was obtained with the partial GP5(55-98) protein. This protein was then selected for the remaining validation process.

Validation of the Microsphere Immunoassay (MIA)

  • The MIA was validated by testing a total of 2,500 equine serum samples previously analyzed using the VNT. These prior results were then compared with those achieved using the new MIA.
  • By using an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were recorded as 92.6% and 92.9%, respectively. This suggests a significant correlation between the results of the traditional VNT and the newly developed MIA.
  • However, it was observed that while the MIA yields somewhat comparably accurate results, it was slightly less sensitive than the usual VNT.

Potential Benefits and Applications of the Microsphere Immunoassay (MIA)

  • The newly developed MIA provides a quicker, more convenient, and economically viable alternative for initial screening of equine sera for antibodies associated with EAV.
  • Despite being slightly less sensitive than the standard VNT, the high level of correlation between the two methods suggests that the MIA could be used to speed up initial testing processes, with the VNT then being used as a confirmatory test when the MIA returns a positive result.
  • Overall, the development of this new MIA could streamline the testing process, making it more efficient and cost-effective, especially in situations where large quantities of samples need to be tested rapidly.

Cite This Article

APA
Go YY, Wong SJ, Branscum AJ, Demarest VL, Shuck KM, Vickers ML, Zhang J, McCollum WH, Timoney PJ, Balasuriya UB. (2007). Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test. Clin Vaccine Immunol, 15(1), 76-87. https://doi.org/10.1128/CVI.00388-07

Publication

Clinical and vaccine immunology : CVI
ISSN: 1556-679X
NlmUniqueID: 101252125
Country: United States
Language: English
Volume: 15
Issue: 1
Pages: 76-87

Researcher Affiliations

Go, Yun Young
  • Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky 40546, USA.
Wong, Susan J
    Branscum, Adam J
      Demarest, Valerie L
        Shuck, Kathleen M
          Vickers, Mary L
            Zhang, Jianqiang
              McCollum, William H
                Timoney, Peter J
                  Balasuriya, Udeni B R

                    MeSH Terms

                    • Amino Acid Sequence
                    • Animals
                    • Antibodies, Viral / analysis
                    • Antibodies, Viral / immunology
                    • Arterivirus Infections / immunology
                    • Arterivirus Infections / veterinary
                    • Arterivirus Infections / virology
                    • Equartevirus / immunology
                    • Equartevirus / isolation & purification
                    • Fluorescent Antibody Technique / economics
                    • Fluorescent Antibody Technique / methods
                    • Horse Diseases / immunology
                    • Horse Diseases / virology
                    • Horses
                    • Immunoassay / economics
                    • Immunoassay / methods
                    • Microspheres
                    • Molecular Sequence Data
                    • Neutralization Tests / methods
                    • Polymerase Chain Reaction / methods
                    • Recombinant Proteins / genetics
                    • Sensitivity and Specificity
                    • Viral Structural Proteins / genetics
                    • Viral Structural Proteins / immunology

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