Low-volume, high-throughput sandwich immunoassays for profiling plasma proteins in mice: Identification of early-stage systemic inflammation in a mouse model of intestinal cancer

An approach to high-throughput sample processing. (a) Wax is imprinted onto a microscope slide to form borders around multiple arrays. Wax is melted by the hotplate under the bath, and a slide is inserted upside-down into the holder. Bringing the lever forward raises a stamp out of the wax bath to touch the slide, imprinting the design onto the slide. Two stamps are shown in front of the device (left image). The arrays are spaced by 4.5mm, which is compatible with the 9mm spacing of standard multi-channel pipettes (middle). Samples loaded onto slides containing 12, 48, and 192 (96 samples loaded) arrays per slide are shown (right image). (b) A plan for incubating 40 different samples and eight standards on one slide, with detection by a single detection antibody. (c) Schematic illustration of a sandwich assay with fluorescence detection. Two different antibodies on an array are illustrated, and the detection antibody binds only its targeted protein bound by the corresponding capture antibody.

Standard curves of selected microarray analytes. Calibration curves were created by plotting the raw fluorescence signal (arbitrary units) against the concentration of purified antigens (pg/ml) or against the dilution factor of pooled mouse plasma sample. The zero-concentration data point is not included because of the log-scale on the x-axis.

Comparisons of levels in mutant and wildtype mice for selected analytes. (a) Distributions of concentrations. The concentrations of the indicated analytes for individual samples are indicated by each point, and the box in each plot defines the upper and lower quartiles of the distributions, with the line in each box indicating the median value. The dashed line in each plot represents the 80% specificity level (5/25 wildtype samples above the threshold), and the sensitivity at that threshold is given in each plot. (b) Scatter plot comparison of results obtained on larger arrays (48 arrays/slide) and smaller arrays (192 arrays/slide). The same set of samples was processed in parallel on two microscope slides printed with the two different array formats.

Protein expression profiles. The samples are arranged in order of increasing tumor burden for the mutant mice. The assays are arranged in order of increasing p-values from two-sample T-tests comparing the mutant and wild-type mice. Each colored square indicates the fold differences from the median of all samples for a particular analyte, according to the color bar at left.

Sample classification. (a) Scores from leave-one-out cross-validation of diagonal linear discriminant analysis (DLDA) classifiers, using seven proteins. (b) The DLDA score of each sample is plotted with respect to the number of tumors in the given sample. The Spearman's rho correlation between the two parameters was 0.19 (p=0.20).