Serologic screening identifies high-titer Ab responses against DAPK2, PDGFRB, PIM1, and PRKCB1 developing after syngeneic HSCT. (A) Plasma samples collected from patient A at serial time points before and after HSCT were screened by ProtoArray protein microarray. Subarrays demonstrating DAPK2 reactivity are highlighted in the gray boxes. Spots seen in the bottom right corner of all subarray images are control spots. (B) Arrays were analyzed using 2 methods described previously.48 Six Ags, DAPK2, PDGFRB, C1orf116, RELA, PIM1, and PRKCB1, elicited significantly greater reactivity after HSCT compared with before HSCT. Significance (Z-scores) scores of Ab reactivity at time points are shown. (C) Ag binding was confirmed by immunoprecipitation of biotinylated target Ags expressed in vitro in rabbit reticulocyte lysate by patient plasma at serial time points. Immunoprecipitated Ags were detected using streptavidin-conjugated secondary Ab. Representative Western blots are shown. The first lane is whole rabbit reticulocyte lysate expressing target Ags that was not subjected to immunoprecipitation. (D) Plasma samples (1:200 dilution) collected from patient A at serial time points before and after HSCT were tested by ELISA (plates coated with 5 μg/mL of recombinant protein). Ab IgG responses against DAPK2 and PIM1 arose and peaked at 3 months after HSCT. (E) Candidate Ags elicit high-titer Ab responses, as indicated by titration studies of serum using ELISA assays. Reactivity remained detectable at dilutions of 1:10 000 (DAPK2) and 1:5000 (PIM1).
