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Applied Microbiology and Biotechnology

, Volume 98, Issue 21, pp 9073–9081 | Cite as

Development of antigen capture ELISA for the quantification of EIAV p26 protein

  • Zhe Hu
  • Hao Chang
  • Man Ge
  • Yuezhi Lin
  • Xuefeng Wang
  • Wei Guo
  • Xiaojun WangEmail author
Methods and protocols

Abstract

An antigen capture enzyme-linked immunosorbent assay (AC-ELISA) was established based on two monoclonal antibodies (mAbs) for the quantification of equine infectious anemia virus (EIAV). Two p26-specific monoclonal antibodies were developed in mice. The mAb 9H8 was coated in microtiter plates as the capture antibody; the other mAb, 1G11, was coupled to horseradish peroxidase (HRP) and used as the detection antibody. The limit of detection for the EIAV p26 protein was 0.98 ng/ml, and the linearity range was 3.9–62.5 ng/ml. The sensitivity of p26 AC-ELISA for the detection of the virus (EIAV infectious clone, FDDVcmv3–8) was the same as that for the purified p26 protein. No cross-reaction with other equine viruses was observed by this method. The intra- and inter-assay coefficients of variation were below 8.3 and 10.3 % for testing p26 and FDDVcmv3–8, respectively. The AC-ELISA was also compared to Western blotting (WB) and reverse transcriptase (RT) assays, validating the sensitivity, accuracy, and reliability of this method. Both the AC-ELISA and RT assay showed good agreement, with a correlation coefficient of R 2 = 0.9946. Sample analysis showed that this AC-ELISA is a useful tool for quantifying EIAV p26 in cell lysates and culture medium.

Keywords

AC-ELISA EIAV p26 Capsid protein Quantitation of viral load 

Notes

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (31222054 to Xiaojun Wang and 31300713 to Zhe Hu) and grants from the State Key Laboratory of Veterinary Biotechnology (SKLVBP201426 to Zhe Hu).

Conflict of interest

The authors have no conflicts of interest to declare.

References

  1. Boldbaatar B, Bazartseren T, Koba R, Murakami H, Oguma K, Murakami K, Sentsui H (2013) Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates. J Virol Methods 189(1):41–46PubMedCrossRefGoogle Scholar
  2. Bourinbaiar AS (1991) HIV and gag. Nature 349(6305):111PubMedCrossRefGoogle Scholar
  3. Carpenter S, Chesebro B (1989) Change in host cell tropism associated with in vitro replication of equine infectious anemia virus. J Virol 63(6):2492–2496PubMedPubMedCentralGoogle Scholar
  4. Cheevers WP, McGuire TC (1985) Equine infectious anemia virus: immunopathogenesis and persistence. Rev Infect Dis 7(1):83–88PubMedCrossRefGoogle Scholar
  5. Chen C, Li F, Montelaro RC (2001) Functional roles of equine infectious anemia virus Gag p9 in viral budding and infection. J Virol 75(20):9762–9770PubMedCrossRefPubMedCentralGoogle Scholar
  6. Clabough DL, Gebhard D, Flaherty MT, Whetter LE, Perry ST, Coggins L, Fuller FJ (1991) Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus. J Virol 65(11):6242–6251PubMedPubMedCentralGoogle Scholar
  7. Cook RF, Cook SJ, Li FL, Montelaro RC, Issel CJ (2002) Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV). J Virol Methods 105(1):171–179PubMedCrossRefGoogle Scholar
  8. Craigo JK, Montelaro R (2008) Equine infectious anemia virus (Retroviridae). Encyclopedia of virology, vol 3. Elsevier, Oxford, pp 167–174Google Scholar
  9. Craigo JK, Montelaro RC (2013) Lessons in AIDS vaccine development learned from studies of equine infectious, anemia virus infection and immunity. Viruses 5(12):2963–2976PubMedCrossRefPubMedCentralGoogle Scholar
  10. Craigo JK, Ezzelarab C, Montelaro RC (2012) Development of a high throughput, semi-automated, infectious center cell-based ELISA for equine infectious anemia virus. J Virol Methods 185(2):221–227PubMedCrossRefPubMedCentralGoogle Scholar
  11. de Arruda Coutinho LC, de Jesus AL, de Paiva Fontes KF, Coimbra EC, Mariz FC, de Freitas AC, de Cassia Carvalho Maia R, de Castro RS (2013) Production of equine infectious anemia virus (EIAV) antigen in Pichia pastoris. J Virol Methods 191(2):95–100PubMedCrossRefGoogle Scholar
  12. Hu Z, Wu X, Ge J, Wang X (2013) Inhibition of virus replication and induction of human tetherin gene expression by equine IFN-alpha1. Vet Immunol Immunopathol 156(1–2):107–113PubMedCrossRefGoogle Scholar
  13. Issel CJ, Coggins L (1979) Equine infectious anemia: current knowledge. J Am Vet Med Assoc 174(7):727–733PubMedGoogle Scholar
  14. Issel CJ, Adams WV Jr, Meek L, Ochoa R (1982) Transmission of equine infectious anemia virus from horses without clinical signs of disease. J Am Vet Med Assoc 180(3):272–275PubMedGoogle Scholar
  15. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517):495–497PubMedCrossRefGoogle Scholar
  16. Layne SP, Merges MJ, Dembo M, Spouge JL, Nara PL (1990) HIV requires multiple gp120 molecules for CD4-mediated infection. Nature 346(6281):277–279PubMedCrossRefGoogle Scholar
  17. Liang H, He X, Shen RX, Shen T, Tong X, Ma Y, Xiang WH, Zhang XY, Shao YM (2006) Combined amino acid mutations occurring in the envelope closely correlate with pathogenicity of EIAV. Arch Virol 151(7):1387–1403PubMedCrossRefGoogle Scholar
  18. Lichtenstein DL, Rushlow KE, Cook RF, Raabe ML, Swardson CJ, Kociba GJ, Issel CJ, Montelaro RC (1995) Replication in vitro and in vivo of an equine infectious anemia virus mutant deficient in dUTPase activity. J Virol 69(5):2881–2888PubMedPubMedCentralGoogle Scholar
  19. Mascola JR, D’Souza P, Gilbert P, Hahn BH, Haigwood NL, Morris L, Petropoulos CJ, Polonis VR, Sarzotti M, Montefiori DC (2005) Recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines. J Virol 79(16):10103–10107PubMedCrossRefPubMedCentralGoogle Scholar
  20. McKeating JA, Moore JP (1991) HIV infectivity. Nature 349(6311):660PubMedCrossRefGoogle Scholar
  21. Montelaro RC, Ball J, Rushlow KE (1993) In: Levy JA (ed) The Retroviridae, vol 2. Plenum, New York, pp 257–360Google Scholar
  22. Piatak M Jr, Saag MS, Yang LC, Clark SJ, Kappes JC, Luk KC, Hahn BH, Shaw GM, Lifson JD (1993) High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 259(5102):1749–1754PubMedCrossRefGoogle Scholar
  23. Quinlivan M, Cook RF, Cullinane A (2007) Real-time quantitative RT-PCR and PCR assays for a novel European field isolate of equine infectious anaemia virus based on sequence determination of the gag gene. Vet Rec 160(18):611–618PubMedCrossRefGoogle Scholar
  24. Rongxian SZW (1983) Development and use of an equine infectious anemia donkey leukocyte attenuated vaccine. In: Tashijan R (ed) Equine infectious anemia virus: a national review of policies, programs, and future objectives. American Quarter Horse Association, Amarillo, TX, pp 135–148Google Scholar
  25. Salinovich O, Payne SL, Montelaro RC, Hussain KA, Issel CJ, Schnorr KL (1986) Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection. J Virol 57(1):71–80PubMedPubMedCentralGoogle Scholar
  26. Sellon DC, Fuller FJ, McGuire TC (1994) The immunopathogenesis of equine infectious anemia virus. Virus Res 32(2):111–138PubMedCrossRefGoogle Scholar
  27. Shane BS, Issel CJ, Montelaro RC (1984) Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody. J Clin Microbiol 19(3):351–355PubMedPubMedCentralGoogle Scholar
  28. Shen T, Liang H, Tong X, Fan X, He X, Ma Y, Xiang W, Shen R, Zhang X, Shao Y (2006) Amino acid mutations of the infectious clone from Chinese EIAV attenuated vaccine resulted in reversion of virulence. Vaccine 24(6):738–749PubMedCrossRefGoogle Scholar
  29. Soneoka Y, Cannon PM, Ramsdale EE, Griffiths JC, Romano G, Kingsman SM, Kingsman AJ (1995) A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res 23(4):628–633PubMedCrossRefPubMedCentralGoogle Scholar
  30. Sponseller BA, Sparks WO, Wannemuehler Y, Li Y, Antons AK, Oaks JL, Carpenter S (2007) Immune selection of equine infectious anemia virus env variants during the long-term inapparent stage of disease. Virology 363(1):156–165PubMedCrossRefGoogle Scholar
  31. Wang G, Gao Y, Huang H, Su X (2010) Multiplex immunoassays of equine virus based on fluorescent encoded magnetic composite nanoparticles. Anal Bioanal Chem 398(2):805–813PubMedCrossRefGoogle Scholar
  32. Wang X, Wang S, Lin Y, Jiang C, Ma J, Zhao L, Lv X, Wang F, Shen R, Kong X, Zhou J (2011a) Genomic comparison between attenuated Chinese equine infectious anemia virus vaccine strains and their parental virulent strains. Arch Virol 156(2):353–357PubMedCrossRefGoogle Scholar
  33. Wang X, Wang S, Lin Y, Jiang C, Ma J, Zhao L, Lv X, Wang F, Shen R, Zhou J (2011b) Unique evolution characteristics of the envelope protein of EIAV(LN(40)), a virulent strain of equine infectious anemia virus. Virus Genes 42(2):220–228PubMedCrossRefGoogle Scholar
  34. Zhang W, Auyong DB, Oaks JL, McGuire TC (1999) Natural variation of equine infectious anemia virus Gag protein cytotoxic T lymphocyte epitopes. Virology 261(2):242–252PubMedCrossRefGoogle Scholar
  35. Zielonka J, Bravo IG, Marino D, Conrad E, Perkovic M, Battenberg M, Cichutek K, Munk C (2009) Restriction of equine infectious anemia virus by equine APOBEC3 cytidine deaminases. J Virol 83(15):7547–7559PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Zhe Hu
    • 1
  • Hao Chang
    • 1
  • Man Ge
    • 1
  • Yuezhi Lin
    • 1
  • Xuefeng Wang
    • 1
  • Wei Guo
    • 1
  • Xiaojun Wang
    • 1
    Email author
  1. 1.State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research InstituteCAASHarbinPeople’s Republic of China

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