免疫纳米金催化氯金酸-盐酸羟胺光度法检测免疫球蛋白G

在pH 2.27的柠檬酸钠-盐酸缓冲溶液中，纳米金对氯金酸-盐酸羟胺生成较大粒径金颗粒这一慢反应具有较强的催化作用。较大粒径金颗粒在600～1 000 nm处有一个较宽的吸收峰。将纳米金标记羊抗人IgG获得免疫纳米金，免疫纳米金也具有相同催化效果。在一定条件下，金标记羊抗人IgG与IgG发生特异性结合生成纳米金免疫复合物。以16 000 rpm速度离心分离获得未反应的纳米金标抗上层溶液。以它作为催化剂催化氯金酸-盐酸羟胺微粒反应，700 nm处的吸光度A_{700 nm}线性降低。其降低值ΔA_{700 nm}与IgG在0.1～10 ng·mL-1范围内呈良好线性关系, 检出限为0.06 ng·mL-1。本法具有灵敏、快速和较高的特异性，用于定量分析人血清IgG，结果满意。

Immunonanogold Catalytic Spectrophotometric Determination of Trace IgG

Gold nanoparticles the size of about 10 nm were prepared by improved trisodium citrate reduction procedure, and were used to label goat anti-human IgG to obtain a sensitive spectral probe for IgG in the condition of pH 6.5. The immune reaction of nanogold-labeled IgG antibody (anti-IgG) with the antigen IgG took place to form the nanogold immune complex in pH 7.0 Na₂HPO₄-C₆H₈O₇ buffer solution and in the presence of polyethylene (PEG). The optimal immunoreaction conditions were pH 7.0, 10.76 μg·mL-1 nanogold-labeled anti-IgG, 8.0% PEG 6000 and incubation time of 30 min under the ultrasonic irradiation. After centrifuging for 15 min at 16 000 rpm, the excess nanogold-labeled anti-IgG in the upper solutions was obtained, and was used to catalyze the colored particle reaction of HAuCl₄ with NH₂OH·HCl to produce gold particles with bigger size. The influence of pH value, HAuCl₄ and NH₂OH·HCl concentration, and reaction temperature and time on the immunonanogold catalytic reaction was considered spectrophotometrically. A pH 2.27 Na₃C₆H₅O₇-HCl buffer solution, 0.094 mmol·L HAuCl₄, 1.92 mmol·L NH₂OH·HCl, and reaction time of 6 min at 30 ℃ water bath were chosen for use. Results demonstrated that with increasing IgG, the concentration of gold labeled anti-IgG in the upper solution decreased, and the absorbance decreased linearly. Linear relationships between the decreased absorbance at 700 nm and the IgG concentration CIgG in the range of 0.10-10 ng·mL-1 were obtained. Its regress equation was ΔA_{760 nm}＝0.014 4ｃ_IgG+0.004 2, the related coefficient was 0.992 6, and the detection limit reached 0.06 ng·mL-1 IgG. The influence of foreign substances on the determination of 3 ng·mL-1 IgG was examined, with the relative error ±10%. Results showed that the following coexistent substances had no impact on the assay: 6 000 ng·mL-1 HSA, 6 000 ng·mL-1 gluocose, 6 000 ng·mL-1 Zn(Ⅱ)，3 000 ng·mL-1 IgA, 3 000 ng·mL-1 Ca(Ⅱ), 3 000 ng·mL-1 L-arginine, 3 000 ng·mL-1 β-phenylalanine，2 400 ng·mL-1 Cu(Ⅱ)，2 400 ng·mL-1 EDTA, 2 400 ng·mL-1 L-cystinol etc. This showed that the assay has high selectivity. The sensitive, rapid and highly specific assay was applied to the quantification of IgG in human sera, with satisfactory results.