Figure 3. Development of an on-chip in vitro BCR-ABL kinase radio assay.

(a) Anti-ABL immunoblot of the Ba/F3 cell line with and without expression of constitutively active BCR-ABL kinase (loading control, anti-Actin immunoblotting). (b) The on-chip BCR-ABL kinase reaction was performed with 12,000 Ba/F3 + BCR-ABL cells (left column) and parental Ba/F3 cells (right column). Final results are represented by an overlay of optical and beta camera false-color images (20 min acquisition) of the lower substrate capture columns. The color bar scale (counts per second per mm²) and total detected activity (pCi) for each ROI (region of interest, yellow circle) are indicated. (c) Comparison between traditional test tube-based (off-chip) and on-chip in vitro BCR-ABL kinase radio assays. Off-chip assays were performed with 10⁷ cells and on-chip assays with 4,500 cells. Results reflect absolute quantitation, and the error bars represent the standard deviation for three assays performed on different days using aliquots of the same lysate with different chips. (d) Demonstration of linearity using BCR-ABL expressing Ba/F3 cells. In each experiment, samples were diluted with parental non-BCR-ABL-expressing cells as necessary to keep the total cell number constant at 15,000. The graph incorporates data from a total of 16 sample aliquots (8 pair-wise chip experiments performed on different chips using mixed aliquots of the same lysates). The results from each experiment were normalized such that the mean detection signal was set equal to the mean number of cells in the experiment divided by 3000. Non-specific background signal was not subtracted. Thus, the results represent relative quantitation designed to identify the linear region of the platform. The R-squared value from a linear regression fit is 0.997. Error bars represent the standard deviation.