Analysis with antibody arrays

Protease inhibitor (PI) cocktail effects on antibody microarray experiments using two-color rolling circle amplification (TC-RCA) detection [10] were investigated. Three different serum samples were used, and each sample had four preparations, namely: no PI added, PI added prior to sample labeling, PI added after the sample labeling, and PI added both before and after the sample labeling. Representative images from each of the four conditions are shown in Fig. 6. The background level

Fig. 4 Comparison of twenty blood samples collected into tubes having either PI cocktail and EDTA (blue circles) or EDTA alone (red squares). Samples represent "time zero" timepoints, processed from whole blood into plasma, aliquoted, and frozen, within 15 min from blood draw. Upper graph: 20data points for each tube type, showing m/z ratio of approximately 6803 and signal intensity, after normalization of each of the forty SELDI mass spectra represented. Lower graph: whisker plot of the same data. Note that the samples with PI display both higher overall signal intensities and a wider intensity distribution than the untreated plasma samples. These SELDI experiments do not provide information on the identity of the discriminatory peaks.

Fig. 4 Comparison of twenty blood samples collected into tubes having either PI cocktail and EDTA (blue circles) or EDTA alone (red squares). Samples represent "time zero" timepoints, processed from whole blood into plasma, aliquoted, and frozen, within 15 min from blood draw. Upper graph: 20data points for each tube type, showing m/z ratio of approximately 6803 and signal intensity, after normalization of each of the forty SELDI mass spectra represented. Lower graph: whisker plot of the same data. Note that the samples with PI display both higher overall signal intensities and a wider intensity distribution than the untreated plasma samples. These SELDI experiments do not provide information on the identity of the discriminatory peaks.

of nonspecific binding to the NC matrix was highest in the sample without PI, and the background appeared lowest when PI was used both before and after the labeling. Plots of the average backgrounds for each serum sample in each condition show the decrease in background with each addition of PI. This effect was observed more in the 543 nm (green) channel (Fig. 7A) than in the 633 nm (red) channel (Fig. 7B). The average signal-to-background (S/B) ratios increase with the addition of PI, both in the 543 nm channel (Fig. 7C) and the 633 nm channel (Fig. 7D), showing that the signals do not decrease at the same rate as the backgrounds.

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