【作者】 陈继峰；

【导师】 李绍华； 李季伦；

【作者基本信息】 中国农业大学 ， 果树学， 2005， 博士

【摘要】 趋磁性细菌磁小体（BMPs）应用于免疫检测时具有很高的检测灵敏度,但许多方面仍然处于未知阶段,如影响抗体连接到BMPs的环境条件、抗体连接效率（LRA）对检测灵敏度的影响及与常用的酶联免疫吸附测定法（Enzyme linked immunosorbent assay,ELISA）检测抗原的灵敏度相比如何?有待于研究。本文以羊抗兔抗体为例,进行了抗体连接到BMPs影响因子研究,并建立了以BMPs为抗体载体的荧光检测抗原的体系:应用所建立的检测体系对果树病毒-葡萄扇叶病毒（GFLV）和李属坏死环斑病毒（PNRSV）进行了检测;并与ELISA检测方法作了对比研究。 研究表明:影响抗体连接到BMPs上的因子是多方面的,并且抗体的连接效率与检测灵敏度有关;应用BMPs的荧光检测方法比ELISA方法灵敏10⁵～10⁶倍。 由实验结果得知,许多缓冲液可以作为羊抗兔抗体连接到BMPs反应体系的缓冲液,从实验所采用的12种常用缓冲液,在通常所使用的浓度与pH值条件下对LRA的影响研究得知,LRA变化范围为35～95 μg/mg。进一步对两种有机缓冲液（Tris-Cl,HEPES）和一种无机缓冲液（Na₃PO₄）的pH值和浓度研究表明,不同的缓冲液使LRA达到最高时的pH值和溶液浓度不同。当pH 5.6时10～20 mM的Tris-Cl,pH 3.5时20 mM的HEPES,pH 5.6时1～5 mM的Na₃PO₄,其LRA明显高于其他pH值和浓度条件下的。由此得知,偏酸的环境有利于抗体连接到BMPs,而且有机溶液的浓度较无机的高。温度对LRA也有影响,不同的连接温度使LRA达到最高的时间不同。在4-37℃之间连接抗体时,较高的连接温度使LRA达到最高所用的时间较短,4℃时需要12小时,室温（24℃）下需要4小时,37℃时需要2小时。在1 ml的反应体系中抗体用量越多,BMPs用量越少,获得的LRA越高。在1ml的连接体系中,当抗体的用量为300μg,BMPs用量为0.1 mg时,LRA可达762.6μg/mg。并且BMPs的质量与LRA有关,高质量的BMPs可以获得高的LRA。冷冻干燥BMPs对抗体连接效率有影响,使LRA降低;并且冷冻干燥前预处理方式使LRA有异,在液氮中预处理的BMPs的LRA明显高于在-70^C和-20℃预处理的。超声波清洗器悬浮和摇床晃动有利于抗体的连接,缩短LRA达到最大值的时间。 抗体的连接效率与抗原检测的灵敏度有很大的关系,连接效率越高,抗原检测的灵敏度也越高。在对ELISA和以BMPs为抗体载体的荧光检测方法比较研究表明,对兔IgG检测时,后者的灵敏度比前者提高10⁶倍;以羊抗兔IgG-兔IgG的抗体抗原关系所建立的应用BMPs的荧光检测体系对果树病毒（GFLV,PNRSV）的检测表明,其灵敏度是ELISA的10⁶倍。由实验得出结论:以BMPs为抗体载体的荧光检测方法检测抗原时具有很高的检测灵敏度。更多还原

【Abstract】 The effect of incubation factors on immobilization efficiency of antibodies, using goat anti-rabbit IgG as an illustration, onto bacterial magnetic particles (BMPs), focusing on the concentration of immunoglobulin G (IgG) and BMPs, buffer selection and its suitable conditions for the conjugation, and the other incubation factors such as, temperature and duration, shaking or stationary condition, BMPs quality, freeze-dry treatment were carried out in this study. New antigen detection method was established by using BMPs while rabbit IgG was applied as a sample, and later on the established detection method used for determination fruit tree virus- Prunus necrotic ring spot virus (PNRSV) and grape fanleaf virus.The resultant data showed that, many factors could influence goat anti rabbit IgG (antibody) immobilized onto BMPs; different linkage rate of antibody (LRA) had varied antigen (rabbit IgG) detection sensitivity; high LRA can obtained high detection sensitivity; and the BMPs based fluorescence assay had much higher detection sensitivity than the method of ELISA in the detection of rabbit IgG and fruit tree virus.Many buffers can be the conjugation buffers for goat anti-rabbit IgG onto BMPs. 12 buffers tested under the normal buffer concentration and pH, the LRAs obtained in the range of 35-95 μg/mg. The selected three buffers, 2 organic (Tris-Cl and HEPES) and 1 inorganic (Na3PO4) were used to investigate the effect of buffer pH and concentration on immobilization efficiency. The pHs and concentrations were different when the LRAs reached the peak value. The suitable pH and concentrations were pH 5.6, 1-5 mM for buffer Na3PO4; pH 5.6, 10-20 mM for Tris-Cl; and pH3.5, 20 mM for HEPES. Therefore, the lower pH benefits LRA, and the concentration of organic buffer was higher than inorganic buffer. Temperature was also affect on LRA, which could reach the peak point faster with higher temperature when 4℃ to 37℃ were used. High quality with appropriate amount of BMPs and enough amount of antibody quantity would lead high LRA. When 0.1 mg of BMPs and 300 μg of antibody mixed into 1 ml of conjugation buffer, it resulted the LRA of 762.6 μg /mg. Freeze-dry BMPs caused LRA decreased, and different pre-treatment of freeze-dry BMPs had significant difference of LRA, which was much higher of pre-treatment in liquor nitrogen than that in -70℃ and -20℃. Resuspending or shaking the conjugation reactants could accelerate the LRA peak appeared, and much higher than that in a stationary stage.Comparing to the different detection methods of ELISA and BMPs based fluorescence assay, the later one had much higher detection sensitivity. The detection sensitivity of the later method was 10 times higher than the former one for rabbit IgG detection and fruit tree virus determination.更多还原