Label-free and quantitative analysis of C-reactive protein in human sera by tagged-internal standard assay on antibody arrays

摘要

We have developed a new, high-throughput, competition-based tagged-internal standard (TIS) assay to measure the levels of blood proteins in human serum. In this assay, target proteins in the sample serum compete with tagged-internal standard proteins for binding to an antibody array. Antibody arrays are fabricated by immobilizing a target protein-specific antibody on the carboxylate-modified latex bead surface of well-type arrays. A solution of Alexa 546-conjugated target protein is added to a sample of human serum and applied to thewell-type antibody array. The array is then analyzed with a fluorescence scanner and the level of unlabeled target protein in the human sera is inferred fromthe amount of tagged protein bound to the array.We successfully applied this assay to measure the level of C-reactive protein (CRP) in 92 unlabeled human sera. The TIS assaywas found to be specific and reproducible for the quantitative analysis of CRP. The antibody array data fromthe TIS assay correlatewell with clinical laboratory data obtained using the commercialized latex-enhanced turbidimetry immunoassay (n=3, r = 0.967, CV = 0.32%). Thus, the antibody array-based TIS assay system is high-throughput, quantitative, and label-free and may be useful in the rapid serodiagnosis of human disease.