Control of antibody orientation for sensitive protein microarray

A55 Microarray technology allows high throughput analysis of various biomolecules of interest. While DNA microarray shows the simultaneous expression level of a genome, the investigation of the change in protein levels gives us more meaningful clues to understand the cellular response to the various stimuli. Cytokines from immune cells act as messenger proteins to trigger defensive network to non-self molecules. One of immune assay, ELISA is widely used to measure the concentration of particular cytokine in serum or culture medium. The assay is based on the specific recognition between antibody and antigen. In this study, we present an advanced method to immobilize the whole IgG-type antibody or its fragments on the planar solid substrate. The chemically modified glass slide enables the capture probes to stand upright via covalent linkage to avoid the random orientation. The distance between neighboring anchorage sites for capture probes is also controlled to minimize the steric hindrance of capture probes from 3.5 nm to 6 nm. Our results show the effect of antibody orientation on detection sensitivity of cytokines using the microarray-type immunoaasay whose analytic capacity is equivalent or higher than the well-based immunoarray.