Antibody microarrays of plasma from bone marrow transplant patients to find markers of graft versus host disease

A non-invasive diagnostic test does not exist for acute graft versus host disease (aGVHD). We therefore sought to identify biomarkers for aGVHD using antibody microarrays (Schleicher and Schuell Serum Biomarker Chips, Whatman) that simultaneously assayed 120 plasma proteins. We measured these proteins in a set of 42 patient plasma samples following an allogeneic bone marrow transplant (BMT): 21 patients with a diagnosis of aGVHD grade II-IV (+GVHD) and 21 patients without aGVHD (–GVHD) at similar times after transplant. We excluded data from 2 hybridizations that had very bright dots and appeared as outliers in preliminary principal components analysis, so that we finally compared 20 +GVHD to 20 -GVHD samples. Keywords: disease state analysis, antibody microarray The arrays are printed on nitrocellulose-coated glass slides, with two identical arrays on each slide, so that two hybridizations can be preformed per slide. We formed a reference pool by combining equal amounts plasma from the 21 +GVHD and 21 -GVHD samples, which was labled with biotin-ULS. Each individual sample was labled with flourescein-ULS, combined with an aliqout of the reference pool and hybridized to a single array (no replicates). One +GVHD sample and one -GVHD sample were assayed on the two arrays of each glass slides, with the placement of the two samples alternating so that +GVHD samples used the top array on approximately half of the slides. We performed the assays in 3 batches, each batch having 7 +GVHD and 7 -GVHD samples. In the second batch we used an additional slide for control experiments in which one or both of the reference pool aliquots hybridized to the arrays were unlabeled.