FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / J Clin Microbiol / Development of a fluorescence polarization-based diagnostic ...
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.
Get Full Text
Publication Date: 2000-05-02 PubMed ID: 10790112PubMed Central: PMC86607DOI: 10.1128/JCM.38.5.1854-1859.2000Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • 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

            References

            This article includes 18 references
            1. Ball JM, Rushlow KE, Issel CJ, Montelaro RC. Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.. J Virol 1992 Feb;66(2):732-42.
              pmc: PMC240772pubmed: 1370556doi: 10.1128/jvi.66.2.732-742.1992google scholar: lookup
            2. Chong YH, Ball JM, Issel CJ, Montelaro RC, Rushlow KE. Analysis of equine humoral immune responses to the transmembrane envelope glycoprotein (gp45) of equine infectious anemia virus.. J Virol 1991 Feb;65(2):1013-8.
              pmc: PMC239850pubmed: 1846180doi: 10.1128/jvi.65.2.1013-1018.1991google scholar: lookup
            3. Chong YH, Payne SL, Issel CJ, Montelaro RC, Rushlow KE. Characterization of the antigenic domains of the major core protein (p26) of equine infectious anemia virus.. J Virol 1991 Feb;65(2):1007-12.
              pmc: PMC239849pubmed: 1702839doi: 10.1128/jvi.65.2.1007-1012.1991google scholar: lookup
            4. Coggins L, Norcross NL. Immunodiffusion reaction in equine infectious anemia.. Cornell Vet 1970 Apr;60(2):330-5.
              pubmed: 4986043
            5. Cordes T, Issel C. Equine infectious anemia: a status report on its control, 91-55-032. Washington, D.C.: Animal and Plant Health Inspection Service, U.S. Department of Agriculture; 1996.
            6. Fontenot JD, Ball JM, Miller MA, David CM, Montelaro RC. A survey of potential problems and quality control in peptide synthesis by the fluorenylmethoxycarbonyl procedure.. Pept Res 1991 Jan-Feb;4(1):19-25.
              pubmed: 1802234
            7. Hammond SA, Cook SJ, Lichtenstein DL, Issel CJ, Montelaro RC. Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process.. J Virol 1997 May;71(5):3840-52.
              pmc: PMC191535pubmed: 9094660doi: 10.1128/jvi.71.5.3840-3852.1997google scholar: lookup
            8. Issel CJ. EIA-hotzone project testing results. Lexington: University of Kentucky; 1997.
            9. Jolley ME. Fluorescence polarization assays for the detection of proteases and their inhibitors. J Biomol Screening 1996;1:33–38.
            10. Jolley ME. Fluorescence polarization immunoassay for the determination of therapeutic drug levels in human plasma.. J Anal Toxicol 1981 Sep-Oct;5(5):236-40.
              pubmed: 7321548doi: 10.1093/jat/5.5.236google scholar: lookup
            11. Leroux C, Issel CJ, Montelaro RC. Novel and dynamic evolution of equine infectious anemia virus genomic quasispecies associated with sequential disease cycles in an experimentally infected pony.. J Virol 1997 Dec;71(12):9627-39.
              pmc: PMC230271pubmed: 9371627doi: 10.1128/jvi.71.12.9627-9639.1997google scholar: lookup
            12. Lin M, Sugden EA, Jolley ME, Stilwell K. Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization.. Clin Diagn Lab Immunol 1996 Jul;3(4):438-43.
              pmc: PMC170364pubmed: 8807210doi: 10.1128/cdli.3.4.438-443.1996google scholar: lookup
            13. Montelaro RC, Ball JM, Rushlow KE. Equine retroviruses. The Retroviridae Vol. 2. New York, N.Y: Plenum Press; 1993. pp. 257–360.
            14. Nasir MS, Jolley ME. Fluorescence polarization: an analytical tool for immunoassay and drug discovery.. Comb Chem High Throughput Screen 1999 Aug;2(4):177-90.
              pubmed: 10469879
            15. Nielsen K, Gall D, Jolley M, Leishman G, Balsevicius S, Smith P, Nicoletti P, Thomas F. A homogeneous fluorescence polarization assay for detection of antibody to Brucella abortus.. J Immunol Methods 1996 Sep 9;195(1-2):161-8.
              pubmed: 8814332doi: 10.1016/0022-1759(96)00116-0google scholar: lookup
            16. Payne SL, Rushlow K, Dhruva BR, Issel CJ, Montelaro RC. Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli.. Virology 1989 Oct;172(2):609-15.
              pubmed: 2552661doi: 10.1016/0042-6822(89)90203-1google scholar: lookup
            17. Rushlow K, Olsen K, Stiegler G, Payne SL, Montelaro RC, Issel CJ. Lentivirus genomic organization: the complete nucleotide sequence of the env gene region of equine infectious anemia virus.. Virology 1986 Dec;155(2):309-21.
              pubmed: 2431539doi: 10.1016/0042-6822(86)90195-9google scholar: lookup
            18. Shipchandler MT, Moore EG. Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer.. Clin Chem 1995 Jul;41(7):991-4.
              pubmed: 7600701

            Citations

            This article has been cited 0 times.
            Subscribe to our newsletter
            Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.