Development of a fluorescent microplate assay for determining cyanovirin-N levels in plasma.
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.
Bringans
Scott D
SD
Molecular Targets Development Program, Center for Cancer Research, National Cancer Institute-Frederick, Building 562, Room 201, Frederick 21702-1201, MD, USA.
O'Keefe
Barry R
BR
Bray
Michael
M
Whitehouse
Chris A
CA
Boyd
Michael R
MR
D000818:Animals; D019380:Anti-HIV Agents / Q000032:analysis / Q000097:blood / Q000493:pharmacokinetics; D001426:Bacterial Proteins / Q000032:analysis* / Q000097:blood* / Q000493:pharmacokinetics; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis* / Q000097:blood* / Q000493:pharmacokinetics; D007163:Immunosorbent Techniques; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; C107733:cyanovirin N