A-352 Investigation of the Immunochromatographic Method using 150 nm Gold Colloids (1)-Comparison of Different Types of Gold Colloids

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Abstract Background Gold colloids, which have been extensively used in immunochromatography, are typically 40 nm in particle size. Recently, 150 nm gold colloidal nanoparticles, which are prepared by gold coating a silica nucleus up to a diameter of 150 nm, have attracted considerable attention. These nanoparticles enable high sensitivity in immunochromatography. The purpose of this study is to compare the antibodies labeled with 40 and 150 nm gold colloids using the physisorption and covalent bonding methods, which are applied to the immunochromatography method developed in our laboratory. Methods 1) For the physisorption method, 40 nm gold colloids (gold colloid AuH2, Morinaga Institute of Biochemistry) and 150 nm gold nanoshells (nanoComposix) were used to physically adsorb the antibody. The covalent gold conjugation kit (nanoComposix) was used to prepare 40 nm gold colloid-labeled antibodies by covalent bonding, and the high sensitivity gold conjugation kit was used to perform covalent bonding of the antibody and gold colloid after buffer displacement of the antibody.2) Anti-human albumin monoclonal antibody (abcam) was applied as the solid-phase antibody onto a nitrocellulose membrane (HF075, Merck Millipore) and dried. A human serum albumin sample (Fujifilm Wako Pure Chemicals) was diluted to a concentration of 1 mg/mL using phosphate-buffered saline (PBS). The sample was mixed with the gold colloid-labeled antibodies and a blocking solution (EZBlock Chemi, ATTO). Subsequently, 90 μL of the mixture was added dropwise and allowed to expand for 15 min before analysis. Results 1) Comparison of detection linesA red line was detected in the judgment zone for the 40 nm gold colloid, whereas a black line was detected in the judgment zone for the 150 nm gold colloid. The background of the detection area for the physisorption method was higher than that for the covalent bonding method. 2) Comparison of detection sensitivitiesUsing the physisorption method, the 40 nm gold colloid detected up to 10 ng/mL. In contrast, using the covalent bonding method, the 40 nm gold colloid detected up to 100 ng/mL. Using the physisorption method, the 150 nm gold colloid detected up to 3 ng/mL when tested at 100, 10, and 3 ng/mL. In contrast, using the covalent bonding method, the 150 nm gold colloid detected up to 3 ng/mL. Conclusion Currently, 40 nm gold colloids are extensively used as a labeling material in immunochromatography. However, the use of 150 nm gold colloids, which have better detection sensitivity, may increase sensitivity and lead to more accurate detection.

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