| Summary: | 碩士 === 國立宜蘭大學 === 生物技術與動物科學系 === 103 === Colloidal gold is a red color nano particle with surface negatively charged. Molecules labeled with colloidal gold can be observed with the naked eye. Consequently, the color developing step in traditional enzyme immunoassay can be omitted when the antibody is labeled with colloidal gold. In addition, colloidal gold labeled antibody is more stable and sensitive compared with that labed with other dyes. Therefore, colloidal gold antiboy is wildly used in fast detection. However, the high content of Tris-HCl and variable pH resulted from proteing G column commonly used to pufify monoclonal antibodies may affect the stability of colloidal gold and its binding with antibody. This rseearch used self generated colloidal gold and monoclonal antibody against proline-rich peptide(PRP) to study the optimal binding conditions.
Protein G purified monoclonal antibodies were reacted with colloidal gold at different pH. The optimum condition was shown to be pH 9.5 ± 0.5. In high ionic strength, colloid gold may aggregate and precipitate. At pH 9.5, the stability of the colloidal gold in different concentrations of Tris-HCl were tested. The result showed that higher than 160mM Tris-HCl could cause precipitation of colloidal gold. The optimal concentration of PRP monoclonal antibody to react with colloidal gold was shown to be 185μg / ml. Using the above conditions to make rapid test strip for PRP, the control line was loaded with 125μg / ml goat anti-mouse IgG, test line was 500μg / ml PRP, and the lowest detection limit was 7.18μg / ml PRP in sample. This rapid test strip can semi-quantitative maesure the concentration of PRP, as a quick reference in production process.
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