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

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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.

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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).

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The authors have no conflicts of interest to declare.

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Authors and Affiliations

  1. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS, 427 Maduan Street, Harbin, 150001, People’s Republic of China

    Zhe Hu, Hao Chang, Man Ge, Yuezhi Lin, Xuefeng Wang, Wei Guo & Xiaojun Wang

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  1. Zhe Hu
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  2. Hao Chang
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  3. Man Ge
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  4. Yuezhi Lin
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  5. Xuefeng Wang
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  6. Wei Guo
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  7. Xiaojun Wang
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Correspondence to Xiaojun Wang.

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Hu, Z., Chang, H., Ge, M. et al. Development of antigen capture ELISA for the quantification of EIAV p26 protein. Appl Microbiol Biotechnol 98, 9073–9081 (2014). https://doi.org/10.1007/s00253-014-6078-8

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  • DOI: https://doi.org/10.1007/s00253-014-6078-8

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