Autoantigen_microarray_Protocol_-_AMBIS_1747972.docx

Autoantigen microarray Protocol:

Autoantigen array super panel I contains 128 autoantigens and various internal controls. The antuantigens in this panel include most of nuclear antigens, cytoplasmic antigens, membrane antigen,phospholipid antigens, as well as some novel autoantigen identified from serum and tissues. The autoantigens are printed on 16-pad FAST slide. Each chip contains 16 identical arrays and can process 15 samples and one PBS control.

The whole process for profiling IgG and IgM autoantibodies from serum or other body fluids will be done using 128-plex autoantigen arrays. Minimum 10ul of serum is required for each sample and each chip can process up to 15 samples. The samples will be treated with DNAse I, diluted 1:50, and incubated with autoantigen array. The autoantibodies binding to the antigens on the array will be detected with Cy3 labeled anti-IgG and cy5 labeled anti-IgM and the arrays will be scanned with GenePix® 4400A Microarray Scanner. The images will be analyzed using GenePix 7.0 software to generate GPR files. The averaged net fluorescent intensity (NFI) of each autoantigen will be normalized to internal controls (IgG or IgM). The final data output will be Excel spreadsheet containing non-normalized and normalized signal intensity as well as the Signal to Noise Ratio (SNR) for all antigens.