Figure 4
Multiplex ATPS-ELISA for GVHD biomarkers.
(a) A 3D rendering showing the custom plate design consisting of 4 antibody insets within a common shallow sample well. (b) Representative images of the chemiluminescent standards. The concentrations listed above each image are the same for each biomarker in the panel. The images correspond to every third point on the quarter-logarithmic dilution curve from 10,000 pg/mL to zero. In each of the images the spot at the top corresponds to HGF, the spot on the right corresponds to elafin, the spot at the bottom corresponds to ST2 and the spot on the left corresponds to TNFR1. It is apparent from these images that elafin has the highest limit of detection due to its high background (elafin can be detected in healthy plasma as well as in GVHD+ plasma). Since the biomarker standards contained a 10% healthy pooled plasma to adjust for matrix affects (see “Plasma samples” in the Methods section), this background can be attributed to baselines levels of elafin and should not be confused as an example of a false positive signal. (c–e) Standard curves for all four GVHD biomarkers in PBS generated by densitometric quantification of chemiluminescence images compared to individual sandwich ELISAs (dashed lines). Error bars represent standard error of the mean.
