补体C3的免疫共振散射光谱分析
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摘要
第一部分:绪论
介绍了共振散射技术发展历史、分析应用以及发展前景。综述了金纳米微粒的制备、表征、组装和生物标记中的应用以及补体C3的分析进展。
第二部分:免疫共振散射光谱分析法简便快速测定补体C3
利用补体C3与羊抗人C3抗体在pH 6.0的Na2HPO4-C6H8O7缓冲溶液和聚乙二醇(PEG)存在下发生特异性反应生成免疫抗体抗原复合物,导致体系在340nm处的共振散射峰增强,建立了一个测定补体C3的免疫共振散射光谱法。C3的浓度在0.167-3.33μg·mL-1的范围内,与340nm处的共振散射强度呈线性,回归方程为I340nm=0.1603C+12.42,R2=0.9926,检出限为0.058μg·mL-1。该方法具有试样用量少、简单快速的特点,用于定量分析人血清中的补体C3,得到的结果较为满意。
第三部分:快速灵敏的免疫纳米金共振散射光谱探针测定补体C3
采用改良柠檬酸钠还原法制备了粒径约为10.0nm的胶体金颗粒,在pH 7.5条件下制备了金标记羊抗人C3抗体共振散射光谱探针。在pH值为5.6的Na2HPO4-C6H8O7缓冲溶液中及PEG存在下,金标记上羊抗人C3抗体与补体C3发生特异性结合,形成胶体金免疫复合物,导致体系在580nm处的共振散射强度急剧增强。补体C3浓度在0.00833-0.200μg·mL-1范围内与共振散射强度的增大值呈良好线性关系,检出限为0.0028μg·mL-1。此法用于人血清测定补体C3具有简便、快速等特点。获得较满意的结果。
第四部分:纳米金标免疫抗体催化增强反应的共振散射光谱研究及其超痕量检测补体C3
在pH 7.5条件下用金纳米颗粒标记羊抗人补体C3获得C3共振散射光谱探针。当pH值为5.6的Na2HPO4-C6H8O7缓冲溶液及PEG存在条件下,金标记羊抗人补体C3与补体C3发生特异性结合生成胶体金免疫复合物。以12000rpm速度离心分离获得未反应的金标抗上层溶液。以它作晶种,在pH 2.97柠檬酸钠-盐酸缓冲溶液-53.33μg·mL-1 HAuCl4-74.13μg·mL-1NH2OH·HCl溶液中及37℃条件下反应3分钟内,免疫金纳米微粒粒径表面积迅速增大,大大提高了585nm处金纳米微粒的共振散射强度I585nm。结果表明,随着C3浓度增大,离心上层溶液中金标抗浓度降低,I585nm线性降低。其降低值△I585nm与补体C3浓度在5.0-160.0pg·mL-1范围内呈良好线性关系,检出限为1.52pg·mL-1。本法具有灵敏、快速和高的特异性,用于定量分析人血清补体C3,结果满意。
PartⅠIntroduction
Some basic knowledge of historical investigation of light scattering, resonance scattering and the application of resonance scattering technology in studying analytical chemistry and nanoparticles were introduced. Summarized the preparation, identification and assembly of gold nanoparticles and its application in biochemical analysis in recent years. The analytical progress of C3 was also reviewed.
PartⅡRapid immune resonance scattering spectral assay of complement 3
In pH 6.0 Na2HPO4-C6H8O7 buffer solutions, goat anti-human complement 3 would combine with complement 3(C3) and produce immunocomplex particles, which resulted in resonance scattering (RS) effect at 340nm, 360nm and 470nm. By the experiment, we found in the presence of appropriate concentration polyethyleneglyco (PEG), which could enhance the RS intensities and sensitivity for analysis of C3. Maxium RS intensity was produced at 60 mg·mL-1 PEG-10000. Under the optimal conditions, the intensities△IRS were proportional to the concentration C3, linear relationship was found between intensity of RS and the concentration in the range of 0.167-3.33μg·mL-1 for C3, regression equation was△I340nm=0.1603C+12.42, correlation coefficient R2=0.9926, and the detection limit reached 0.058μg·mL-1. The method was successfully applied to quantitative determination of C3 in human sera, with satisfactory results. The method was simple, rapid and reliable for human sera assay.
PartⅢA rapid and sensitive immunonanogold resonance scattering spectral probe for C3.
An improved trisodium citrate reduction method was used to prepare about 10 nm size gold nanoparticles and it was used to label goat anti-human complement 3 (anti-C3) in the pH 7.5 to obtain a sensitive and selective immunoresonance scattering spectral probe. It is based on the immune reaction between labeled anti-C3 and C3 in the pH 5.6 Na2HPO4-C6H8O7 buffer solutions. As added C3 the gold labeled-antiC3 would form immunogold complex in the action of polyethylene (PEG), resulting in enhancing the resonance scattering (RS) intensities at 580 nm greatly. Well linear relationships between the enhanced RS intensities (△IRS) and the C3 concentration in the range of 0.00833-0.200μg·mL-1 were obtained, with a detection limit of 0.00280μg·mL-1C3. The rapid and sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.
PartⅣUltrasensitive immunoresonance spectral determination of C3 based on nano-gold labeling catalytic enhancement
In the condition of pH 7.5 goat anti-human C3 was labeled by nano-gold to obtain an immunoresonance scattering spectral probe for C3. The immune reaction between gold-labeled antibodies and the antigens took place in pH 5.6 Na2HPO4-C6H8O7 buffer solution in the presence of polyethylene (PEG). Centrifuged at 12000rpm, we obtained supernatant of unreacted gold labeled anti-C3. In pH 2.97 sodium citrate buffer solution, the immunogold in the supernatant, as a seed catalysed the immunereaction between 53.33μg·mL-1 HAuCl4 and 74.13μg·mL-1NH2OH·HCl for 3 min at 37℃, to form larger size and surface gold particles, resulting in enhancement the resonance scattering (RS) intensities at 585 nm greatly. With increasing C3, the concentration of gold labeled-antiC3 in supernatant decreased and I585nm decreased either. Linear relationships between the decreased RS intensities (△IRS) and the C3 concentration in the range of 5.0-160.0pg·mL-1 were obtained, the detection limit reached at 1.52pg·mL-1 C3. The sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.
介绍了共振散射技术发展历史、分析应用以及发展前景。综述了金纳米微粒的制备、表征、组装和生物标记中的应用以及补体C3的分析进展。
第二部分:免疫共振散射光谱分析法简便快速测定补体C3
利用补体C3与羊抗人C3抗体在pH 6.0的Na2HPO4-C6H8O7缓冲溶液和聚乙二醇(PEG)存在下发生特异性反应生成免疫抗体抗原复合物,导致体系在340nm处的共振散射峰增强,建立了一个测定补体C3的免疫共振散射光谱法。C3的浓度在0.167-3.33μg·mL-1的范围内,与340nm处的共振散射强度呈线性,回归方程为I340nm=0.1603C+12.42,R2=0.9926,检出限为0.058μg·mL-1。该方法具有试样用量少、简单快速的特点,用于定量分析人血清中的补体C3,得到的结果较为满意。
第三部分:快速灵敏的免疫纳米金共振散射光谱探针测定补体C3
采用改良柠檬酸钠还原法制备了粒径约为10.0nm的胶体金颗粒,在pH 7.5条件下制备了金标记羊抗人C3抗体共振散射光谱探针。在pH值为5.6的Na2HPO4-C6H8O7缓冲溶液中及PEG存在下,金标记上羊抗人C3抗体与补体C3发生特异性结合,形成胶体金免疫复合物,导致体系在580nm处的共振散射强度急剧增强。补体C3浓度在0.00833-0.200μg·mL-1范围内与共振散射强度的增大值呈良好线性关系,检出限为0.0028μg·mL-1。此法用于人血清测定补体C3具有简便、快速等特点。获得较满意的结果。
第四部分:纳米金标免疫抗体催化增强反应的共振散射光谱研究及其超痕量检测补体C3
在pH 7.5条件下用金纳米颗粒标记羊抗人补体C3获得C3共振散射光谱探针。当pH值为5.6的Na2HPO4-C6H8O7缓冲溶液及PEG存在条件下,金标记羊抗人补体C3与补体C3发生特异性结合生成胶体金免疫复合物。以12000rpm速度离心分离获得未反应的金标抗上层溶液。以它作晶种,在pH 2.97柠檬酸钠-盐酸缓冲溶液-53.33μg·mL-1 HAuCl4-74.13μg·mL-1NH2OH·HCl溶液中及37℃条件下反应3分钟内,免疫金纳米微粒粒径表面积迅速增大,大大提高了585nm处金纳米微粒的共振散射强度I585nm。结果表明,随着C3浓度增大,离心上层溶液中金标抗浓度降低,I585nm线性降低。其降低值△I585nm与补体C3浓度在5.0-160.0pg·mL-1范围内呈良好线性关系,检出限为1.52pg·mL-1。本法具有灵敏、快速和高的特异性,用于定量分析人血清补体C3,结果满意。
PartⅠIntroduction
Some basic knowledge of historical investigation of light scattering, resonance scattering and the application of resonance scattering technology in studying analytical chemistry and nanoparticles were introduced. Summarized the preparation, identification and assembly of gold nanoparticles and its application in biochemical analysis in recent years. The analytical progress of C3 was also reviewed.
PartⅡRapid immune resonance scattering spectral assay of complement 3
In pH 6.0 Na2HPO4-C6H8O7 buffer solutions, goat anti-human complement 3 would combine with complement 3(C3) and produce immunocomplex particles, which resulted in resonance scattering (RS) effect at 340nm, 360nm and 470nm. By the experiment, we found in the presence of appropriate concentration polyethyleneglyco (PEG), which could enhance the RS intensities and sensitivity for analysis of C3. Maxium RS intensity was produced at 60 mg·mL-1 PEG-10000. Under the optimal conditions, the intensities△IRS were proportional to the concentration C3, linear relationship was found between intensity of RS and the concentration in the range of 0.167-3.33μg·mL-1 for C3, regression equation was△I340nm=0.1603C+12.42, correlation coefficient R2=0.9926, and the detection limit reached 0.058μg·mL-1. The method was successfully applied to quantitative determination of C3 in human sera, with satisfactory results. The method was simple, rapid and reliable for human sera assay.
PartⅢA rapid and sensitive immunonanogold resonance scattering spectral probe for C3.
An improved trisodium citrate reduction method was used to prepare about 10 nm size gold nanoparticles and it was used to label goat anti-human complement 3 (anti-C3) in the pH 7.5 to obtain a sensitive and selective immunoresonance scattering spectral probe. It is based on the immune reaction between labeled anti-C3 and C3 in the pH 5.6 Na2HPO4-C6H8O7 buffer solutions. As added C3 the gold labeled-antiC3 would form immunogold complex in the action of polyethylene (PEG), resulting in enhancing the resonance scattering (RS) intensities at 580 nm greatly. Well linear relationships between the enhanced RS intensities (△IRS) and the C3 concentration in the range of 0.00833-0.200μg·mL-1 were obtained, with a detection limit of 0.00280μg·mL-1C3. The rapid and sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.
PartⅣUltrasensitive immunoresonance spectral determination of C3 based on nano-gold labeling catalytic enhancement
In the condition of pH 7.5 goat anti-human C3 was labeled by nano-gold to obtain an immunoresonance scattering spectral probe for C3. The immune reaction between gold-labeled antibodies and the antigens took place in pH 5.6 Na2HPO4-C6H8O7 buffer solution in the presence of polyethylene (PEG). Centrifuged at 12000rpm, we obtained supernatant of unreacted gold labeled anti-C3. In pH 2.97 sodium citrate buffer solution, the immunogold in the supernatant, as a seed catalysed the immunereaction between 53.33μg·mL-1 HAuCl4 and 74.13μg·mL-1NH2OH·HCl for 3 min at 37℃, to form larger size and surface gold particles, resulting in enhancement the resonance scattering (RS) intensities at 585 nm greatly. With increasing C3, the concentration of gold labeled-antiC3 in supernatant decreased and I585nm decreased either. Linear relationships between the decreased RS intensities (△IRS) and the C3 concentration in the range of 5.0-160.0pg·mL-1 were obtained, the detection limit reached at 1.52pg·mL-1 C3. The sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.
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