Development Of High-sensitivity Immunochromatography Assay By Using A Novel Sensitive Gold Nano-particle Enlargement Method

Understanding biological processes at the molecular level with accurate-quantification needs advanced bio-analysis. The rapid, sensitive, accurate methods were widely produced for the detection of bacteria, virus, pesticide and antibiotic residues in the veterinary fields. Gold-immunochromatographic assay (GICA) is based on the use of an antibody conjugated with gold nanoparticles that reacts specifically with an antigen to be tested. It is rapid, sensitive, low cost, and suitable for the on-site detection of antibodies or antigens. The method has been used for the surveillance and diagnosis of poultry infectious diseases, such as ND, AIV and bovine viral diarrhea virus (BVDV). But GICA has lower sensitivity than fluorescent, radioactive, and enzyme-colorometric method. If the Au nanoparticles increased, the observation will be beneficial and enhanced by naked eye. In this paper, a new technique that based on colloidal gold immunochromatography principle was introduced, which enabled enhancement of detection sensitivity by enlarging immunogold particles immobilized on the nitrocellulose strip. The conjugated nanoparticle provides an extremely high megascopic signal for analysis.First, colloidal gold nanoparticles (AuNPs) were synthesized and conjugated with monoclonal antibody by self assembling technique. Then, another monoclonal antibody was utilized to coat the Nitrocellulose (NC) membrane as a test line (T line), while sheep anti-mouse antibody was sprayed as a quality control line (C line). The identification was made by the presence of color in grid sections captured with gold particles and amplified by hydroxylamine chloride on the surface of NC membrane and the absence of color was assumed to be negative by comparing its color with negative control. To improve the sensitivity, we use a mixture of HAuCl4 and L-Ascorbic Acid(C6H8O6) or NH2OH·HCl to enlarge gold nanoparticles. The detecting limit is significantly increased by 10～100 times. Validation was performed by several methods:UV-vis absorption, transmission electron microscopy (TEM), cross reactivity and stability measurement.