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An in vivo Assay to Test Blood Vessel Permeability
Published on: March 16, 2013
Development of time-resolved immunofluorometric assay of vascular permeability factor
K T Yeo1, T M Sioussat, J D Faix
1Department of Pathology, Beth Israel Hospital, Boston, MA.
Insights
A new time-resolved immunofluorometric assay accurately quantifies guinea pig vascular permeability factor (VPF) in biological fluids. This sensitive method is highly correlated with the Miles assay but offers superior detection limits for VPF.
Area of Science:
- Biochemistry
- Immunology
- Assay Development
Background:
- Vascular Permeability Factor (VPF) plays a crucial role in vascular physiology.
- Accurate quantification of VPF in biological fluids is essential for research.
Purpose of the Study:
- To develop and validate a novel two-site time-resolved immunofluorometric assay for guinea pig VPF.
- To assess the sensitivity, specificity, and correlation with existing bioassays.
Main Methods:
- Immobilization of anti-VPF antibodies (C-IgG) on microtiter wells.
- Labeling of anti-VPF antibodies (N-IgG) with a Eu(3+)-chelate.
- Two-step incubation assay followed by time-resolved fluorescence measurement.
Main Results:
- The assay demonstrated analytical sensitivity of 0.35 VPF units with intra-assay CV of ~20%.
- High specificity for VPF was confirmed through peptide pre-treatment and VPF pre-extraction.
- The immunoassay showed a strong correlation (r2 = 0.94) with the Miles permeability assay.
Conclusions:
- The developed assay provides a sensitive and specific method for VPF quantification.
- This immunofluorometric assay is approximately 30-fold more sensitive than the classical Miles assay.
- The assay is suitable for quantifying VPF in various biological fluid samples.
Abstract:
We describe a two-site time-resolved immunofluorometric assay for guinea pig vascular permeability factor (VPF) for quantifying VPF in different biological fluids. Antibody against the carboxy terminus (C-IgG) is immobilized on microtiter wells, and antibody against the amino terminus (N-IgG) is labeled with Eu(3+)-chelate. Line 10 tumor culture medium, known to be rich in VPF, is assayed in a two-step incubation. Bound Eu3+ is then quantified by dissociation into a fluorescent enhancement solution, with measurement of the time-resolved fluorescence. The analytical sensitivity is 0.35 VPF unit, and the intra-assay CV is about 20%. The assay is specific for VPF, because pre-treatment with the appropriate C- or N-peptide, or pre-extraction of VPF, greatly decreases fluorescence. The VPF immunoassay is highly correlated (r2 = 0.94) with the Miles permeability assay, the classical bioassay of VPF. In addition, the immunofluorometric assay is about 30-fold more sensitive than the Miles assay.
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