Validation of targets (CHAF1b and NuSAP1) by Western Blot and ELISA. Using protein microarrays, CHAF1b and NuSAP1 were identified as target proteins recognized by new antibodies that first developed 1 year after transplantation and were absent in the donor plasma or before transplantation in the patient, corroborating protein microarray results. CHAF1b and NuSAP1 were made using the E coli expression system as well as baculovirus-infected insect cells. (A) 0.5 μg protein was loaded in each well. Each lane was cut and incubated with the patient's pretransplantation (Pre) and posttransplantation plasma samples, along with the patient's donor plasma (Dnr). The probed lanes were realigned and detected by anti–human IgG Ab. Vertical lines have been inserted to indicate a repositioned gel lane. Positive controls were anti-GST mAb and anti-CHAF1b mAb. (B) Affinity purified NuSAP1 and CHAF1b obtained from E coli– and baculovirus-infected insect cells were quantitatively detected by patient plasma using IgG ELISA. There were no antibodies detected in the sera of donor, pretransplantation, or 1 month or 2 months posttransplantation, but antibodies against NuSAP1 and CHAF1b were detected 11 months after transplantation and later. (C) Antibodies against infectious antigens influenza, pneumococcus, tetanus, EBV, and VZV were detected in the donor, pretransplantation, and 1, 2, 11, 12, 14, 16, and 18 months after transplantation using IgG ELISA. Total IgG was also measured in triplicate for the same time points and is plotted as milligrams per deciliter. Error bars indicate SE of measurement.