| dc.description.abstract | The construction of an antibody microarray for one specific target, with improved methods of protein immobilization on solid matrix, is always of great interest to the field of systems biology. Unlike DNA or RNA, proteins have unique 3D structures that are critical to their functions, but have the tendency of denaturing quickly after leaving their natural chemical and physical environments, a tendency that poses a serious challenge to making antibody microarrays viable. Consequently, the selection of suitable surface chemistry is requirement unique to antibody microarrays; it is not needed for, say, DNA microarrays. For enhancing the performance, how antibodies are captured and fixed on the slide surface plays an increasingly vital role in maintaining the conformation and orientation of the antibody. For an attachment method to be effective, it is necessary that it is applicable to a wide range of antibodies and, when the antibodies are bound to the surface of solid substrates, their activities and binding capacity are preserved. Immobilization methods using efficient surface chemistry should be reliable, applicable to antibodies with universal properties, amenable to high-throughput automation, and it should fully preserve the binding capabilities of the antibodies – through maintenance of the correct orientation of antibody epitopes – and minimize nonspecific binding.
We present a novel immobilization method to capture antibodies that uses Protein G coating on aldehyde-derivatized slide. To maintain the antibody activity and enhance performance of array-based immunoassays, protein G was used to allow a shorter duration of immunoglobulin G immobilization at 4 °C, with the antibody placed in the appropriate orientation with uniform face-out epitopes. The multiplexed detection of six pain-related message molecules (PRMMs) was used as examples for the development of array-based immunoassays: substance P, calcitonin gene-related peptide, nerve growth factor, brain-derived neurotrophic factor, tumor necrosis factor-α, and β-endorphin. Compared to non-protein G immunoassays, protein G shortened the antibody immobilization time at 4 °C from overnight to 2 hours. It provides effective high-density protein immobilization without activity loss or incorrect orientation of the capture antibody. This new platform of antibody microarray is a useful tool for analysis of PRMMs as demonstrated in our experimental results with fluorescence immunoassay methods. A mechanism of protein binding to solid surface coated with Protein G that results in improved detection sensitivity, excellently low limit of detection (LOD), and remarkably high signal-to-noise (SN) ratio are provided. In our array validation, the LOD of six PRMMs were sensitive to average 100 pg/ml,
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better than other proteomic tools. Our method exhibited excellent specificity, the cross-reactivity was observed to have contrast 50-times seen typically in conventional array surface.
In conclusion, our experimental results suggested that Protein G, a novel linker molecule, was an efficient antibody immobilizer, and that Protein G-coated method was a highly sensitive antibody microarray. Key obstacles overcame and achievement obtained in this work include: prevention of antibody denaturation by shortened immobilization time; full preservation of antibody’s binding capacity and effective increase of array sensitivity by absence of extraneous modification tag on antibody; good reproducibility assured by more stable fixation of antibody on surface; increase of signal-to-noise ratio and reduction of non-specific binding by uniform arrangement of capture antibody. We demonstrated the applicability for a small-scale project, and believe it forms the foundation large-scale scale-up. We believe our method can be a useful tool for the development of a high detection sensitivity, antigen-antibody interaction, microarray-based screening system for new drug target discovery and biomarker assays. | en_US |