Multiplexed Protein Assay

Multiplexed Protein Assay by Glass Chip-based Arrays

In this method, target protein is first immobilized to a solid support. The immobilized protein is then complexed with an antibody that is linked to an enzyme. Detection of the enzyme-complex can then be visualized through the use of a substrate that produces a detectable signal. While the traditional method works well for a single protein, the overall procedure is time consuming and requires a large sample volume. With little sample to work with, conservation of small quantities becomes a risky task. With the advancement of microarray technology over the last decade; more and more choices are available to the scientists today. This glass-chip-based multiplexed sandwich ELISA system enables researchers to accurately determine the concentration of 10 cytokines simultaneously. The system is relatively rapid and simple compared to the traditional ELISAs, which requires large sample volumes and significant processing time. Furthermore, with this system, 48 times more data can be obtained in four hours and with as little as 50 ul of samples. Each of the 10 cytokine specific capture antibody is arrayed in quadruplicate, together with positive and negative controls. The serial dilution of the cytokine standard will be used to generate a five-point standard curve. Standard cytokines and samples are assayed in each well simultaneously through a sandwich like ELISA procedure. The signals will be detected using fluorescence-based detection method for consistency and reliability. By comparing signals from unknown samples to the standard curve generated for each of the 10 cytokines, the unknown cytokine concentration in the samples will be determined. The standard curve requires six wells (including a blank), leaving 10 wells for experimental samples.