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Journal of clinical microbiology2000; 38(5); 1854-1859; doi: 10.1128/JCM.38.5.1854-1859.2000

Development of a fluorescence polarization-based diagnostic assay for equine infectious anemia virus.

Abstract: The control of equine infectious anemia virus (EIAV) infections of horses has been over the past 20 years based primarily on the identification and elimination of seropositive horses, predominantly by a standardized agar gel immunodiffusion (AGID) assay in centralized reference laboratories. This screening for EIAV-seropositive horses has been to date hindered by the lack of a rapid diagnostic format that can be easily employed in the field. We describe here the development of a rapid solution-phase assay for the presence of serum antibodies to EIAV based on fluorescence polarization (FP) (patent pending). Peptides derived from antigenic regions of EIAV core and envelope proteins were initially screened for their utility as probes in an FP assay to select the best peptide antigen candidates. The FP assay was optimized to detect the presence of EIAV-specific antibodies by a change in the FP of a fluorescein-labeled immunoreactive peptide diagnostic antigen. The most sensitive and specific peptide probe was a peptide corresponding to the immunodominant region of the EIAV transmembrane protein, gp45. This probe was tested for its reactivity in the optimized FP assay with 151 AGID-positive horse sera and 106 AGID-negative serum samples. The results of these studies demonstrated that the FP assay reactivity correlated with reported AGID results in 106 of 106 negative serum samples (100% specificity) and in 135 of 151 positive serum samples (89.4% sensitivity). The FP assay was also found to have a very low background reactivity and to readily detect antibodies produced early in infection (</=3 weeks postinfection). The developed EIAV FP assay is rapid (5 to 20 min) and simple to perform and is equally suitable for use both in the field and in the diagnostic laboratory, thus providing the basis of an improved commercial diagnostic assay for EIAV infection of horses.
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Publication Date: 2000-05-02 PubMed ID: 10790112PubMed Central: PMC86607DOI: 10.1128/JCM.38.5.1854-1859.2000Google Scholar: Lookup
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  • Journal Article

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 research focused on developing a fast and efficient method, called the Fluorescence Polarization (FP) assay, to detect equine infectious anemia virus (EIAV) in horses. The assay uses fluorescence to identify the presence of EIAV-specific antibodies in the horse’s serum.

Assay Development

  • The existing method of controlling EIAV involved identifying and eliminating horses that tested positive for EIAV using an agar gel immunodiffusion (AGID) assay.
  • The AGID assay, however, was not suitable for quick field testing and the study aimed to address this problem by developing a rapid solution-phase assay that used fluorescence polarization (FP) to detect EIAV.
  • The FP assay worked by monitoring the change in the FP of a labeled immunoreactive peptide diagnostic antigen. This change was used to ascertain the presence of EIAV-specific antibodies in the serum of horses.

Selection of Peptide Antigen

  • To select the best peptide antigen for the FP assay, peptides derived from antigenic regions of EIAV core and envelope proteins were screened.
  • The peptide representing the immunodominant region of the EIAV transmembrane protein, gp45, was found to be the most sensitive and specific peptide probe.

Testing and Results of the FP Assay

  • The effectiveness of this test was evaluated using 151 AGID-positive horse sera and 106 AGID-negative serum samples.
  • The results of these studies showed a 100% correlation in specificity when tested on AGID-negative serum- there were no false positives.
  • The sensitivity of the test, when tested on AGID-positive serum samples, was found to be 89.4%, indicating a high true positive rate.
  • The FP test was also capable of early detection of EIAV, as it could identify antibodies produced in less than or equal to 3 weeks post-infection.

Added Benefits of the FP Assay

  • The FP assay is a quick test which can provide results in as little as 5 to 20 minutes.
  • The simplicity of the test also makes it suitable for both laboratory and field use.
  • Given these factors, the FP assay shows promise as a robust commercial diagnostic tool to detect EIAV infection in horses.

Cite This Article

APA
Tencza SB, Islam KR, Kalia V, Nasir MS, Jolley ME, Montelaro RC. (2000). Development of a fluorescence polarization-based diagnostic assay for equine infectious anemia virus. J Clin Microbiol, 38(5), 1854-1859. https://doi.org/10.1128/JCM.38.5.1854-1859.2000

Publication

Journal of clinical microbiology
ISSN: 0095-1137
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 38
Issue: 5
Pages: 1854-1859

Researcher Affiliations

Tencza, S B
  • Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Islam, K R
    Kalia, V
      Nasir, M S
        Jolley, M E
          Montelaro, R C

            MeSH Terms

            • Amino Acid Sequence
            • Animals
            • Antigens, Viral / chemistry
            • Antigens, Viral / immunology
            • Enzyme-Linked Immunosorbent Assay / methods
            • Equine Infectious Anemia / diagnosis
            • Equine Infectious Anemia / prevention & control
            • Fluorescence Polarization Immunoassay / methods
            • Fluorescence Polarization Immunoassay / veterinary
            • Horses
            • Immunoglobulin G / blood
            • Infectious Anemia Virus, Equine / isolation & purification
            • Mass Screening / veterinary
            • Molecular Sequence Data

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            Citations

            This article has been cited 5 times.
            1. Eremin SA, Mukhametova LI, Krylov VB, Nifantiev NE. Fluorescence Polarization Assay for Infection Diagnostics: A Review. Molecules 2024 Oct 5;29(19).
              doi: 10.3390/molecules29194712pubmed: 39407640google scholar: lookup
            2. Zhang Z, Guo K, Chu X, Liu M, Du C, Hu Z, Wang X. Development and evaluation of a test strip for the rapid detection of antibody against equine infectious anemia virus. Appl Microbiol Biotechnol 2024 Dec;108(1):85.
              doi: 10.1007/s00253-023-12980-9pubmed: 38189948google scholar: lookup
            3. Hu Z, Guo K, Du C, Sun J, Naletoski I, Chu X, Lin Y, Wang X, Barrandeguy M, Samuel M, Wang W, Lau PI, Wernery U, Raghavan R, Wang X. Development and evaluation of a blocking ELISA for serological diagnosis of equine infectious anemia. Appl Microbiol Biotechnol 2023 May;107(10):3305-3317.
              doi: 10.1007/s00253-023-12504-5pubmed: 37039847google scholar: lookup
            4. Takeda Y, Yonezawa Y, Asake S, Ogawa H, Imai K. A fluorescence polarization immunoassay for the rapid detection of antibody against influenza A virus in chicken and goat sera. J Vet Diagn Invest 2020 Nov;32(6):887-891.
              doi: 10.1177/1040638720960046pubmed: 33025860google scholar: lookup
            5. Bonham AJ, Paden NG, Ricci F, Plaxco KW. Detection of IP-10 protein marker in undiluted blood serum via an electrochemical E-DNA scaffold sensor. Analyst 2013 Oct 7;138(19):5580-3.
              doi: 10.1039/c3an01079apubmed: 23905162google scholar: lookup
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