PMC

Examples of IgG and IgM profiles obtained with serum samples of SARS‐CoV‐infected patients. Sample FP B0352 was collected immediately following detection of fever in a patient in Beijing; Sample DP C08 was collected from a recovered SARS patient in Toronto. Signals in the upper panel and the BSA control were detected with anti‐human IgGs, while signals in the lower panel of the two patient samples were detected with anti‐human IgMs.

Principle of serum profiling assays performed on a functional protein microarray. A functional protein microarray, composed of hundreds of thousands of individually purified proteins, is first blocked with BSA in PBS buffer. Then, a diluted serum sample is incubated on the microarray typically at RT for 1 h. After extensive washes, bound antibodies (e.g. human IgG, IgA, or IgM) can be detected with anti‐human immunoglobulin antibodies, followed by a signal amplification step with fluorescently labeled secondary antibodies. Detection of immunoglobulin isotypes can be multiplexed with different fluorophores as illustrated.

Scheme of the two‐phase strategy for biomarker identification in human autoimmune diseases. In Phase I, a small cohort is used to rapidly identify a group of candidate biomarkers via serum profiling assays on a human protein microarray of high cost. Because a small number of microarrays are needed, cost of the experiments is relatively low. In Phase II, a focused protein microarray of low cost is fabricated by spotting down purified candidate proteins. A much larger cohort is then assayed on these arrays in a double blind fashion to validate the candidates identified in Phase I.

Identification of most relevant candidate for in‐depth in vivo studies. Assisted by GO analysis, 110 shared substrates of conserved herpesvirus kinases were plugged into the STRING database. A highly connected cluster of 15 proteins was revealed, all of which are known to play a role in DDR. Based on the literature and topology of the cluster, TIP60 emerged as the most promising candidate. Protein nodes are color coded by functional class, with proteins involved in DDR colored red. Small blue circles adjacent to protein nodes indicate that the protein is herpesvirus associated, while small pink circles indicate the proteins are associated with other viruses. Edges between the proteins represent known or predicted connections, such as protein–protein interactions, catalytic reactions, and enzyme–substrate relationships.