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Development of a fluorescent microplate assay for determining cyanovirin-N levels in plasma

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Abstract

A sensitive immunosorbent competition assay was developed for quantitation of the anti-HIV protein cyanovirin-N (CV-N) in plasma using a 96-well plate format and a fluorescent endpoint. The assay is based on the binding of CV-N in plasma to plate-bound anti-CV-N antibodies, followed by removal of the plasma and addition of europium-labeled CV-N (Eu3+-CV-N) to compete for the remaining antibody sites. Detection by addition of a dissociative fluorescence enhancement solution and time-resolved fluorescence measurements allowed correlation to the concentration of the native CV-N in plasma. A linear detection range of 1–100 nM (r2>0.99) was obtained for CV-N in mouse plasma. This assay was then utilized for analysis of plasma levels of CV-N samples following subcutaneous injection of CV-N into mice. The results of these studies confirmed the reliability and sensitivity of this assay and the feasibility of its use for pharmacokinetic studies in a variety of species.

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Acknowledgements

We thank Dr. Jim McMahon for supplying rabbit polyclonal anti-CV-N antibodies and Joshua Shamblin for excellent technical help. We also thank Dr. John Beutler for supplying Eu3+-CV-N (labeled by Perkin Elmer), Dr. Kirk Gustafson for supplying CV-N, and both of them for their critical review of this manuscript.

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  1. Michael Bray

    Present address: Biodefense Clinical Research Branch, Office of Clinical Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD, 20892, USA

Authors and Affiliations

  1. Molecular Targets Development Program, Center for Cancer Research, National Cancer Institute-Frederick, Building 562, Room 201, Frederick, MD, 21702-1201, USA

    Scott D. Bringans & Barry R. O’Keefe

  2. Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD, 21702, USA

    Michael Bray & Chris A. Whitehouse

  3. USA Cancer Research Institute, College of Medicine, University of South Alabama, Mobile, AL, 36688-002, USA

    Michael R. Boyd

Authors
  1. Scott D. Bringans
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  2. Barry R. O’Keefe
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  3. Michael Bray
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  4. Chris A. Whitehouse
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  5. Michael R. Boyd
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Corresponding author

Correspondence to Barry R. O’Keefe.

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Bringans, S.D., O’Keefe, B.R., Bray, M. et al. Development of a fluorescent microplate assay for determining cyanovirin-N levels in plasma. Anal Bioanal Chem 380, 269–274 (2004). https://doi.org/10.1007/s00216-004-2786-8

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  • DOI: https://doi.org/10.1007/s00216-004-2786-8

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