【作者】 陈汉忠；

【导师】 蒋金书；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2003， 博士

【摘要】 我们利用经典的免疫学理论和成熟的电化学技术，设计和构建了一种电化学免疫传感器，将其应用于兽医方面的抗原抗体反应检测分析，试图在兽医领域建立一种新的快速、灵敏、准确的抗原抗体反应检测、诊断方法，供生产上使用。 本研究的内容主要包括3个方面：一是筛选适当的材料，设计和构建电化学免疫传感器；二是采用适当的方法建立电化学传感器的实验条件，并测定传感器的各种性能；三是应用电化学免疫传感器检测几种常见寄生虫病、传染病的抗原抗体反应，了解研制的电化学免疫传感器的实用效果。 工作电极、辅助电极、参比电极、反应池(样品池)等都是构建电化学免疫传感器的重要部件。我们通过一系列的实验研究筛选到了适当的制作电化学免疫传感器电极基片和反应池等材料，并使用这些材料成功的制作了各种性能良好的电极和反应池，最后利用这些器件建立了一种电化学免疫传感器检测系统。 我们应用化学修饰的方法，使电极基片表面产生活泼基团，从而成功地将生物分子(抗原)固定在基片上。实验测试的结果表明，固定后的抗原，保持了良好的反应性和稳定性，可用于抗原抗体反应的测试。 传感器检测实验条件的建立是实现传感器实用化的关键步骤。我们利用成熟和操作简易的恒电法来建立了传感器检测的实验条件。传感器性能的测试研究表明，研制的电化学免疫传感器具有良好的稳定性、选择性、适用性和特异的检测效果。但目前由于受一些条件所限，传感器测定的结果，相对偏差(RSD)仍较大，因此传感器目前仍不适用于定量分析。 将电化学免疫传感器应用于兽医临床和现场的抗原抗体检测分析和诊断是我们的最终目的，因此测试其在兽医方面的应用具有特别重要的意义。在实验研究中，我们先后应用电化学免疫传感器测试了大片形吸虫，鸡新城疫(ND)、鸡传染性法氏囊病(IBD)、鸡减蛋综合征(EDS)等的抗原抗体反应，结果与我们先前初步设定的判断标准基本相符。但是我们认为提出一个更为合理的、适用于检测临床和现场样品的阳性判定标准，还需做进一步的实验研究。 本研究构建的电化学免疫传感器经初步的实验研究就显示了其许多优良特性：传感器适用于不同类型的抗原抗体反应检测，具有良好的稳定性和特异的检测效果；检测快速，操作简便；样品血清用量少；传感器微型巧小，携带方便，检测仪器价格便宜等优点，预示了其良好的应用前景，我们相信，通过进一步的设计和实验研究，就有可能将传感器应用于临床和现场抗原抗体反应快速、准确检测分析和疫病诊断。更多还原

【Abstract】 We have designed and fabricated a kind of electrochemical immunosensor by using the theory of immunology and the technology of electrochemistry and apply the sensor to detect reactions of antibody and antigen on veterinary. By this way, we surpose to set up a new quick, sensitive, specific detecting method of antibody - antigen reactions for the veterinary practice use.The contents of the research mainly include three aspects:Firstly, it is to select proper materials for designing and fabricating the electrochemical immunosensor. Secondly, using the proper method to set up the experiment conditions and judge the characters of the sensor. Thirdly for examining the result of the electrochemical immunosensor made, we use the sensor to detect antibody-antigen reactions of several common diseases of parasites and microbes.Working electrodes, auxiliary electrodes, referent electrodes, reaction pools and so on are all the important components for making the electrochemical immunosensor. It is very important to select proper materials to design and make these components.Through a series of selecting experiments, we found the proper matrix materials and utilized these materials to make the electrodes and reaction pools with good properties. Finally, we successfully made a system of electrochemical immunosensor by using these compenonts.It is the key technology to immobilize biomolecules such as antibody and antigen onto the matrix of electrodes and maintain their reactivity and stability. We use the methods of chemical modifications to create active basal molecules on the matrix surface of electrodes and successfully immobilize the biomolecules onto the matrix. The experiment results indicated the immobilized antigen still keep its good reactivity and stability and can use for detecting reactions of antibody-antigen.The key procedure for practice use of the sensor is to set up its experiment conditions. We sut up the experiment conditions by using the mature and simple voltammetry.The results of detecting the properties of our sensor indicated the sensor has good stability, selectivity, adaptability and the specific detecting effects. But as some conditions are confined the detecting RSD of the sensor is still not so good. So the sensor is not suit to quantitative analysis at present.Our final aim is to use the sensor to detect reactions of antibody-antigen in veterinary clinic and field. So it is very important to exam their applicability in veterinary. In this study we had used the sensor to detect antibody-antigen reactions of Fasciolsis,ND, IBD and EDS. It is proved the sensor is fitted to use in detecting antibody-antigen reactions of these diseases. The results of detecting antibody-antigen reactions of Fasciola gegatica, ND, IBD and EDS by using the sensor basically conform to that with standard samples. Through mis experiment we think to set up a more reasonable standard for judgingthe clinic and field samples we still want to make further experiments.Through the primary studies, our sensor has showed many excellent characters such as adaptability and specificity, quick detection, simple operation ,small amount consumption of sample serums, miniature of the sensor, convenience to carry and the detecting instrument is cheap and so on. With the many excellent and special characters the sensor has displayed its light apply perspectives. We believe through further designing and experiments it is possible to use the sensor to quick detect reactions of antibody-antigen and diagnose diseases in veterinary clinic and fields.更多还原