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

Analytical and Bioanalytical Chemistry
Scott D BringansMichael R Boyd

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.

References

Mar 1, 1984·Analytical Biochemistry·I HemmiläT Lövgren
Jul 17, 1998·Nature Structural Biology·C A BewleyA M Gronenborn
Apr 30, 2003·Antiviral Research·Laura G BarrientosMichael R Boyd
Jul 25, 2003·Antimicrobial Agents and Chemotherapy·Barry R O'KeefeMichael R Boyd
Aug 19, 2003·AIDS Research and Human Retroviruses·Che-Chung TsaiMichael R Boyd
Mar 6, 2004·AIDS Research and Human Retroviruses·Che-Chung TsaiMichael R Boyd

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