基于量子点荧光探针的H5N1亚型禽流感病毒快速检测方法的研究
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摘要
H5N1亚型高致病性禽流感(highly pathogenic avian influenza,HPAI)的频繁爆发不仅给养禽业带来巨大的经济损失,也威胁着人类的生命健康。目前,针对H5亚型禽流感病毒的检测方法主要有VI、ELISA、RT-PCR、RRT-PCR等,但这些方法存在耗时耗力、敏感性低、需要昂贵的仪器且操作要求较高等缺点。因此,建立一种简单、快速、灵敏、高通量检测H5亚型禽流感病毒(AⅣ)的新方法对于尽早地对禽流感做出初诊和进行治疗、防止病毒的进一步传播具有重要意义。本实验共分为以下三部分:
1.兔抗H5亚型禽流感病毒血凝素特异性多克隆抗体的制备及纯化。
根据设计好的免疫程序,用原核表达的H5亚型AⅣ血凝素(HA)蛋白免疫健康家兔,当抗体滴度达到所需水平时,获得抗H5亚型AⅣ的高免血清。所得高免血清分别采用辛酸—硫酸铵盐法粗提IgG和G—200凝胶柱过滤纯化,成功制备了高纯度的兔抗H5亚型AⅣ血凝素蛋白多克隆抗体。
2.量子点—兔抗H5亚型AⅣIgG荧光探针的制备
采用共价偶联的方法,利用交联剂EDC和NHS使水溶性量子点与兔抗H5亚型AⅣIgG偶联,并对偶联条件进行了摸索,成功确立了量子点与兔抗H5亚型AⅣIgG偶联的最适条件;对量子点—兔抗AⅣIgG偶联物的生物学活性进行检测后发现该标记是十分有效的,偶联物保持了抗体原有的生物学活性,适合下一步的免疫反应分析。
3.H5N1亚型禽流感病毒夹心荧光免疫法的建立
本研究以抗H5亚型禽流感病毒血凝素单克隆抗体为捕捉抗体,以量子点—兔抗H5亚型AⅣ血凝素特异性多克隆抗体荧光探针为检测抗体,采用双抗体夹心法,成功地建立了一种基于量子点荧光探针的夹心荧光免疫法(sFLISA),为临床早期快速检测H5亚型AⅣ提供了行之有效的方法。
用所建立的方法对H5N1亚型AⅣ进行定量检测,研究结果表明该方法检测H5N1亚型AⅣ的灵敏度可达0.15 ng/mL,标准曲线8 ng/mL～510 ng/mL范围内线性良好。通过与双抗体夹心ELISA进行比较,所建立的sFLISA表现出更高的灵敏度,且耗时较短。初步运用于临床检测,与Ⅵ试验同时检测103份临床鸡组织样品,相对Ⅵ试验而言,sFLISA的敏感性为93.6%,特异性为91.1%,两者的符合率为92.2%。实验结果表明,本方法特异性强,灵敏度高,可以运用于H5N1亚型AⅣ的快速检测。
本实验所建立的H5N1亚型禽流感病毒夹心荧光免疫检测方法有良好的应用价值,也为量子点标记新技术用于动物重大传染病的研究提供了重要的参考依据。
The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry industry and human health worldwide. Currently, there are several analytical methods for the detection of H5 virus were proposed, such as virus isolation (VI), standard reverse transcription-PCR (RT-PCR), real-time RT-PCR, enzyme-linked immunosorbent assay (ELISA), and so on. However, these assays have their limitations, such as time-consuming, low sensitivity, technically demanding and so on. Therefore, a simple, rapid and sensitive detection assay for the early diagnosis of H5 is required to lower the chances of spread and reduce the risk of development into an epidemic. This research include the following three parts:
1. Preparation of polyclonal antibody angainst HA protein of AIV subtype H5 and IgG extraction
According to the designed immunization schedule, two SPF rabbits were chosen and immunized with the E.coli expressed HA protein. When the antibody titer of agarose-gel precipitation(AGP) get to the level that we want, we can obtain the rabbits'serum. After these sera were purified by n-Caprylic acid-ammonium sulphate and gel column G-200 respectively, we successfully got the highly depurated rabbit anti-AIV IgG.
2. Preparation of QDs-rabbit anti-AIV IgG probe
The water-soluble CdTe QDs were linked to the rabbit anti-AIV IgG using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The couple condition was optimized, and the molar QD:IgG ratio in the reaction was discussed. The resonance light scattering (RLS) was used for detection of antigen-antibody recognition reactions, and results showed that the probe retained the activity of antibody and was suitable for immunoassay.
3. Development of sFLISA for the H5N1 subtype AIV
In this research, a novel sandwich fluorescence-linked immunosorbent assay (sFLISA) for the determination of H5 subtype AIV was developed basd on the McAb and QDs-rabbit anti-AIV IgG, which both against HA of H5 subtype AIV. The developed sFLISA supplied an effective approach to early diagnosis of AIV subtype H5N1 in fields.
Under the optimal conditions, the sFLISA allowed for H5N1 viral antigen determination in a linear range of 8 ng/mL～510 ng/mL with the limit of detection (LOD) of 0.15 ng/mL, which was lower than commercial ELISA. Moreover, in comparison with virus isolation (VI) for 103 clinic samples, the sFLISA was correlated with VI for 92.2%, the sensitivity and specificity of sFLISA were found to be 93.6% and 91.1% respectively. Results showed that the sFLISA was specific and sensitive for the H5N1 sbtype AIV.
The developed sFLISA supplied a novel approach to rapid and sensitive detection of AIV subtype H5N1, and provided significant reference for QDs in the application of animal-borne diseases.
1.兔抗H5亚型禽流感病毒血凝素特异性多克隆抗体的制备及纯化。
根据设计好的免疫程序,用原核表达的H5亚型AⅣ血凝素(HA)蛋白免疫健康家兔,当抗体滴度达到所需水平时,获得抗H5亚型AⅣ的高免血清。所得高免血清分别采用辛酸—硫酸铵盐法粗提IgG和G—200凝胶柱过滤纯化,成功制备了高纯度的兔抗H5亚型AⅣ血凝素蛋白多克隆抗体。
2.量子点—兔抗H5亚型AⅣIgG荧光探针的制备
采用共价偶联的方法,利用交联剂EDC和NHS使水溶性量子点与兔抗H5亚型AⅣIgG偶联,并对偶联条件进行了摸索,成功确立了量子点与兔抗H5亚型AⅣIgG偶联的最适条件;对量子点—兔抗AⅣIgG偶联物的生物学活性进行检测后发现该标记是十分有效的,偶联物保持了抗体原有的生物学活性,适合下一步的免疫反应分析。
3.H5N1亚型禽流感病毒夹心荧光免疫法的建立
本研究以抗H5亚型禽流感病毒血凝素单克隆抗体为捕捉抗体,以量子点—兔抗H5亚型AⅣ血凝素特异性多克隆抗体荧光探针为检测抗体,采用双抗体夹心法,成功地建立了一种基于量子点荧光探针的夹心荧光免疫法(sFLISA),为临床早期快速检测H5亚型AⅣ提供了行之有效的方法。
用所建立的方法对H5N1亚型AⅣ进行定量检测,研究结果表明该方法检测H5N1亚型AⅣ的灵敏度可达0.15 ng/mL,标准曲线8 ng/mL～510 ng/mL范围内线性良好。通过与双抗体夹心ELISA进行比较,所建立的sFLISA表现出更高的灵敏度,且耗时较短。初步运用于临床检测,与Ⅵ试验同时检测103份临床鸡组织样品,相对Ⅵ试验而言,sFLISA的敏感性为93.6%,特异性为91.1%,两者的符合率为92.2%。实验结果表明,本方法特异性强,灵敏度高,可以运用于H5N1亚型AⅣ的快速检测。
本实验所建立的H5N1亚型禽流感病毒夹心荧光免疫检测方法有良好的应用价值,也为量子点标记新技术用于动物重大传染病的研究提供了重要的参考依据。
The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry industry and human health worldwide. Currently, there are several analytical methods for the detection of H5 virus were proposed, such as virus isolation (VI), standard reverse transcription-PCR (RT-PCR), real-time RT-PCR, enzyme-linked immunosorbent assay (ELISA), and so on. However, these assays have their limitations, such as time-consuming, low sensitivity, technically demanding and so on. Therefore, a simple, rapid and sensitive detection assay for the early diagnosis of H5 is required to lower the chances of spread and reduce the risk of development into an epidemic. This research include the following three parts:
1. Preparation of polyclonal antibody angainst HA protein of AIV subtype H5 and IgG extraction
According to the designed immunization schedule, two SPF rabbits were chosen and immunized with the E.coli expressed HA protein. When the antibody titer of agarose-gel precipitation(AGP) get to the level that we want, we can obtain the rabbits'serum. After these sera were purified by n-Caprylic acid-ammonium sulphate and gel column G-200 respectively, we successfully got the highly depurated rabbit anti-AIV IgG.
2. Preparation of QDs-rabbit anti-AIV IgG probe
The water-soluble CdTe QDs were linked to the rabbit anti-AIV IgG using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The couple condition was optimized, and the molar QD:IgG ratio in the reaction was discussed. The resonance light scattering (RLS) was used for detection of antigen-antibody recognition reactions, and results showed that the probe retained the activity of antibody and was suitable for immunoassay.
3. Development of sFLISA for the H5N1 subtype AIV
In this research, a novel sandwich fluorescence-linked immunosorbent assay (sFLISA) for the determination of H5 subtype AIV was developed basd on the McAb and QDs-rabbit anti-AIV IgG, which both against HA of H5 subtype AIV. The developed sFLISA supplied an effective approach to early diagnosis of AIV subtype H5N1 in fields.
Under the optimal conditions, the sFLISA allowed for H5N1 viral antigen determination in a linear range of 8 ng/mL～510 ng/mL with the limit of detection (LOD) of 0.15 ng/mL, which was lower than commercial ELISA. Moreover, in comparison with virus isolation (VI) for 103 clinic samples, the sFLISA was correlated with VI for 92.2%, the sensitivity and specificity of sFLISA were found to be 93.6% and 91.1% respectively. Results showed that the sFLISA was specific and sensitive for the H5N1 sbtype AIV.
The developed sFLISA supplied a novel approach to rapid and sensitive detection of AIV subtype H5N1, and provided significant reference for QDs in the application of animal-borne diseases.
引文
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