Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection
Fig 3
Using NP-PK and NP-Luc for rapid detection of NSE in a rat model for stroke.
A) Fluoro Jade-C staining for measurement of damaged brain tissue volume. The presence and absence of FJC-labeled degenerative neurons were imaged with epifluorescence under 20x magnification using filters for FITC. (a) Region with abundant FJC staining (bright cells) on lesioned side. (b) Region at the edge of FJC staining. (c) Region that is contralateral to (a) that did not show any FJC staining. Dotted line in B and low magnification inset indicates manually-mapped border between FJC positive and negative areas. B) FJC staining in control brain section. (a) and (b) are in area of craniectomy. C-D) Individual rat data (C-stroke and D-control) for NSE measurements at each time point (grayscale lines indicate data points for individual animals, line with symbol indicates the mean±standard deviation) as performed by tethered PK and Luc assay, and normalized to time point -1 hour. Plasma samples were collected from stroke induced or control rats pre (-1 hr), and post occlusion (0, 1, 3 and 6 hr). The mean slope of the trend (dashed line) was calculated averaging individual slopes in each group (stroke = 0.26 with 0.95 confidence interval, control = -0.02 with 0.38 confidence interval). E) Summary of rat stroke model experiments showing a statistically significant increase in NSE plasma levels in stroke (blue bars) vs. control (red bars) rats as soon as 1 hour post occlusion. F) Measurements of NSE in plasma from rats using ELISA showed elevated levels in stroke compared to control rats at the last time point (6 hr post-occlusion). P values– 0.05<*<0.1, 0.01<**<0.05. Data from 10 rats were included in our analysis, with the exclusion of 3 hour and 6 hour time points for one control animal that died (at 2 hour mark), and a 6 hour time point for a stroke animal which died and was found to have a large hemorrhage at the base of the brain.