Figure 1.

Protein microarray is a screening tool that reproducibly detects serums reactivities to non-HLA antigens. (A) Data normalization approach for two representative serum samples from two different patients. Raw signals are shown on the left. Unmanipulated positive control (red) and negative control (black) signals are shown in the next panel. The third panel depicts the signals for normalized positive and negative controls. The final panel on the right shows post normalization histogram for all reactivities. The vertical dotted line in the right two panels is drawn at log₂10. The numbers refer to percentages of reactivities above log₂10 threshold. (B) Assay validation with positive control sera. Normalized signal values are plotted for positive control serum (closed circles) and four healthy control serum samples (open circles). The horizontal dotted line is drawn at the significance level of log₂ 10. (C) Assay variability. Representative Spearman plots of a single serum sample tested on three separate arrays. Spearman plots comparing pairs of arrays with a mean tau of 0.90. The experiment was repeated twice with comparable results (data not shown). (D) Effects of serum dilution. The left panel shows box plots (median, 25^th and 75^th percentiles) for four different dilutions of the same serum sample tested on four separate arrays. The dashed box surrounds the dilution recommended by the manufacturer. Median reactivity for each array titrates down with serum dilution. The right panel shows signal histograms with numbers of significant reactivities >10 for each dilution.