Image analysis quantification of the Miles assay.
The Miles assay for vascular permeability has high intra- and interassay coefficients of variation (CVs). Quantification, usually by dye extraction and spectrophotometry, is time consuming. In this study, quantification by this means was compared with image analysis using the Olympus CUE-2 Image Analyzer (version 4). The test substance was recombinant human vascular permeability factor (rhVPF). The quantification process took approximately 10 min with image analysis. Formamide extraction and spectrophotometry required 1 hr of preparation, 4-6 days of incubation, and 1 hr for filtration and spectrophotometry. Between assay CVs ranged from 0 to 30% for spectrophotometry, but were all < 10% for image analysis. The sensitivity (2SD above the negative control mean) of the image analysis approach was 64 +/- 25 ng/mL, whereas for spectrophotometry it was 65 +/- 29 ng/mL. Interanimal CVs for rhVPF at 200 and 1000 ng/mL were 15% and 26% when assessed by spectrophotometry and 7% and 22%, respectively, by image analysis. The R2 value for the correlation of image analysis with spectrophotometry was 81.4%. Test substances injected close to the spine evoked a greater permeability response than those injected laterally: at 200 ng/mL p = 0.005, at 1000 ng/mL p = 0.1 (unpaired t tests).
Monash University Department of Obstetrics and Gynaecology, Clayton, Victoria, Australia.
N
N
McClure
D M
DM
Robertson
P
P
Heyward
D L
DL
Healy
D000818:Animals; D002199:Capillary Permeability; D016228:Endothelial Growth Factors / Q000493:pharmacokinetics; D005070:Evans Blue; D006168:Guinea Pigs; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008222:Lymphokines / Q000493:pharmacokinetics; D008297:Male; D010599:Pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D005070:Evans Blue