藻红蛋白荧光标记技术及在烟草花叶病毒检测上的应用
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摘要
藻红蛋白是具有优良荧光特性的新型荧光探针,在荧光检测中有着广泛的应用前景。本文以藻红蛋白为荧光探针,探讨了藻红蛋白荧光标记技术及荧光探针在烟草花叶病毒免疫检测中的应用效果。
从海藻中分离纯化出高纯度的荧光标记物—藻红蛋白,同时用正辛酸沉淀法纯化TMV抗血清;经纯度、浓度和抗体效价的测定,获得符合荧光标记的主要材料。
测定了不同pH值、离子强度、添加剂对RPE荧光强度的影响,结果表明:在pH 5—9范围内,藻红蛋白的荧光强度能保持较好的稳定性,pH3和pH12时其荧光强度影响很大:0.15-0.2M离子强度的PB(或加0.1—0.2M NaCl溶液)缓冲溶液能保持藻红蛋白的荧光强度的稳定性;添加Tween-20和EDTA时,Tween-20浓度以0.1%为宜,EDTA则以1mmol/L浓度最合适。
筛选了异功能试剂活化组合,建立标记流程,优化交联反应条件。用异功能试剂SPDP活化藻红蛋白,2-IT巯基化抗体,通过两步交联法制备荧光探针。同时根据交联反应对藻红蛋白荧光强度和抗体的活性的影响,优化交联反应条件,结果为:试剂SPDP活化藻红蛋白,二者摩尔比100:1,反应时间在0.5—1h之间为宜;2-IT巯基化抗体,二者摩尔比200:1,反应时间2h为宜。
应用硝酸纤维素膜和Sephadex G-50葡聚糖凝胶为固相载体,建立免疫检测流程,探讨其在烟草花叶病毒(Tobacco mosaic virus,TMV)免疫检测上的应用。以ELISA检测的结果为标准,检测了55份TMV样品。结果为:阳性的检出率为100%、假阳性的检出率为75%、阴性检出率为83.3%、总体检出率为94.55%,结果表明用藻红蛋白标记的荧光抗体的免疫检测具有较好的特异性。
Phycoerythrin is a new type of labeling probe with remarkable fluorescent characteristics, which was widely used in fluorescence immunoassay. The fluorescence labeling technique of phycoerythrin and application for the immunoassay of Tobacco mosaic Virus were studied in this research using it as a labeling probe.The phycoerythrin was isolated and purified from the algae. The TMV antiserum was purifed with n-octanoic acid precipitation. The main materials suitable for fluorescence labeling were obtained through the measurement of the purity, concentration and antibody titer.The effects of different pH values、 ionic strength and additives on PE fluorescence intensity was measured in this study. The results showed that the stability of fluorescence intensity of PE was better maintained in a range of pH from 5 to 9 and ionic strength within 150~200mmol/L PBS (or 100~200mmol/L NaCl solution added), but pH3 and pH12 had an obvious effect on the fluorescence intensity of this protein. The additive of 0.1% Tween20 and lmmol/L EDTA was also favourable to the phycoerythrin's stability.Several heterobifunctional reagent combinations were screened, the labeling protocol was established, and Experiment conditions for crosslinking reaction were optimized. SPDP was used to activated the phycoerythrin, 2-IT was used to modified multiclonal antibodies, labeling probe was prepared by the two-step conjugation. The better protocol was as follows: the proper molar ratio of SPDP to PE was 100: 1, and the corresponding reaction time was 0.5-1h;, the proper molar ratio of 2-IT to multiclonal antibodies was 200: 1, and the corresponding reaction time was 2h.Phycoerythrin fluorescence immunoassay protocol was established by the use
从海藻中分离纯化出高纯度的荧光标记物—藻红蛋白,同时用正辛酸沉淀法纯化TMV抗血清;经纯度、浓度和抗体效价的测定,获得符合荧光标记的主要材料。
测定了不同pH值、离子强度、添加剂对RPE荧光强度的影响,结果表明:在pH 5—9范围内,藻红蛋白的荧光强度能保持较好的稳定性,pH3和pH12时其荧光强度影响很大:0.15-0.2M离子强度的PB(或加0.1—0.2M NaCl溶液)缓冲溶液能保持藻红蛋白的荧光强度的稳定性;添加Tween-20和EDTA时,Tween-20浓度以0.1%为宜,EDTA则以1mmol/L浓度最合适。
筛选了异功能试剂活化组合,建立标记流程,优化交联反应条件。用异功能试剂SPDP活化藻红蛋白,2-IT巯基化抗体,通过两步交联法制备荧光探针。同时根据交联反应对藻红蛋白荧光强度和抗体的活性的影响,优化交联反应条件,结果为:试剂SPDP活化藻红蛋白,二者摩尔比100:1,反应时间在0.5—1h之间为宜;2-IT巯基化抗体,二者摩尔比200:1,反应时间2h为宜。
应用硝酸纤维素膜和Sephadex G-50葡聚糖凝胶为固相载体,建立免疫检测流程,探讨其在烟草花叶病毒(Tobacco mosaic virus,TMV)免疫检测上的应用。以ELISA检测的结果为标准,检测了55份TMV样品。结果为:阳性的检出率为100%、假阳性的检出率为75%、阴性检出率为83.3%、总体检出率为94.55%,结果表明用藻红蛋白标记的荧光抗体的免疫检测具有较好的特异性。
Phycoerythrin is a new type of labeling probe with remarkable fluorescent characteristics, which was widely used in fluorescence immunoassay. The fluorescence labeling technique of phycoerythrin and application for the immunoassay of Tobacco mosaic Virus were studied in this research using it as a labeling probe.The phycoerythrin was isolated and purified from the algae. The TMV antiserum was purifed with n-octanoic acid precipitation. The main materials suitable for fluorescence labeling were obtained through the measurement of the purity, concentration and antibody titer.The effects of different pH values、 ionic strength and additives on PE fluorescence intensity was measured in this study. The results showed that the stability of fluorescence intensity of PE was better maintained in a range of pH from 5 to 9 and ionic strength within 150~200mmol/L PBS (or 100~200mmol/L NaCl solution added), but pH3 and pH12 had an obvious effect on the fluorescence intensity of this protein. The additive of 0.1% Tween20 and lmmol/L EDTA was also favourable to the phycoerythrin's stability.Several heterobifunctional reagent combinations were screened, the labeling protocol was established, and Experiment conditions for crosslinking reaction were optimized. SPDP was used to activated the phycoerythrin, 2-IT was used to modified multiclonal antibodies, labeling probe was prepared by the two-step conjugation. The better protocol was as follows: the proper molar ratio of SPDP to PE was 100: 1, and the corresponding reaction time was 0.5-1h;, the proper molar ratio of 2-IT to multiclonal antibodies was 200: 1, and the corresponding reaction time was 2h.Phycoerythrin fluorescence immunoassay protocol was established by the use
引文
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