Antibody Microarrays. Merrill Birkner ph296~December 1, 2003. Antibody (Ab) Microarray. A complete microarray-based system for profiling protein expression in biological samples; used to compare two biological samples to measure the relative differences in protein expression.
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ph296~December 1, 2003
Enzyme linked immusorbent assay (ELISA) immobilized antibodies are used to capture fluorescently labeled antigens.
Ab/Ag interaction in ELISA wells immobilized antibodies are used to capture fluorescently labeled antigens.
Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers.Proteomics 2003, 3, 56-63.
Miller, J., Zhou, H., Kwekel, J., Cavallo, R., Burke, J., Butler, E.B., Teh, B., Haab, B.
1. Compare two surfaces and antibody immobilization schemes: poly-L-lysine coated glass with a second photoreactive cross linking layer, & polyacrylamide-based hydrogels on glass.
2. Establish an efficient method to screen antibodies for those that are functional in the microarray assay.
3. Demonstrate the use of this technology to screen serum samples for potential biomarkers, by analyzing the relative protein abundances in serum samples from prostate cancer patients and controls.
N= (SIgG / μIgG)/RIgG
SIgG = the ELISA-measured IgG concentration of the serum sample on that array.
μIgG= the mean ELISA-measured IgG concentration of all of the samples.
RIgG= the average ratio of the replicate anti-IgG antibody spots on a particular array.
4 slides per sample were created (label sample A with Cy3 and Cy5 and sample B with Cy3 and Cy5). Combine one of each of these samples and scan; determine the signal ratios of the 2 slides (Cy5/Cy3). With this method potential variability is eliminated because each protein sample labeled with each dye.