斑点酶免疫渗滤法检测纳米细菌致兔胆结石血清学方法的建立

发布时间：2018-03-17 17:40

  本文选题：纳米细菌　切入点：纳米细菌检测　出处：《华北煤炭医学院》2008年硕士论文　论文类型：学位论文

【摘要】：纳米细菌(Nanobacteria,Nb)是由芬兰科学家Kajander于1989年发现的一种超微细菌,它们呈球形或杆状,具有细胞壁,直径在80～500nm之间,菌落常呈蔟状;能生成含有磷灰石的生物膜,具有很强的抗热、抗γ射线辐射及抵御抗生素的能力;能通过100nm的滤菌器,且具独特的生物矿化能力。近年来,有研究资料表明,纳米细菌与胆囊结石、肾结石、动脉粥样硬化等多种骨骼外钙化性或硬化性疾病有关,引起了科学界极大的关注。 采用抗纳米细菌单克隆抗体作免疫荧光及免疫组化检测,扫描电镜、透射电镜及免疫电镜等形态学观察,是目前鉴定纳米细菌的主要方法。 目的 本研究旨在通过对胆石症患者胆汁纳米细菌分离培养,免疫动物,制备多克隆抗体;并对试验条件的探讨优选,建立斑点酶免疫渗滤法检测动物血清中特异性纳米细菌抗体的初步诊断方法。 方法 1.选择无急性胆囊炎发作病史,腹腔镜手术前未接受抗生素治疗的6例胆囊结石患者,无菌操作抽取胆汁进行纳米细菌培养,同时设DMEM全培养液培养和合成羟基磷灰石培养为阴性对照。 2.用数显浊度仪测量胆石症患者胆汁分离培养物和对照组中纳米细菌生长情况并绘制生长曲线。 3.用扫描电镜对胆石症患者胆汁分离培养物进行能谱分析,并用透射电镜对纳米细菌培养物进行形态学观察。 4.用纳米细菌培养物制成纳米细菌滴片,分别按文献方法进行间接免疫荧光染色、Von KOSSA染色及Hochest 33258染色。 5.纯化抗原,通过考马斯亮蓝定量蛋白浓度为174.2μg/ml,保存备用。 6.免疫家兔 免疫家兔3只,背部多点接种含福氏完全佐剂的纳米细菌(总量200μg/ml/只)。2周后接种含福氏不完全佐剂的纳米细菌(总量50μg/ml/只)进行加强免疫(每次间隔2周,共4次),制备纳米细菌多克隆抗体,并用ELISA测定其含量。 7.建立纳米细菌多克隆抗体斑点酶免疫渗滤检测法 将兔抗纳米细菌多克隆抗体用二倍顺序稀释法稀释至1:6400～1:51200;纳米细菌抗原稀释至1?10～1:160等不同浓度;通用型抗鼠/兔酶标抗体稀释至1:10～1:40。用方阵滴定法确定所有试验点及其相应的抗原抗体反应浓度。每个试验点重复6次,以出现肉眼能判读的棕色斑点或圆圈者为阳性,不能判读者为阴性。对每个阳性反应棕色斑点,用“Motic Med 6.0数码医学图象分析系统(A)”读取光密度值(OD值),求出每个试验点的平均OD值。 分析数据,选择检测纳米细菌多抗的最佳兔抗纳米细菌抗原滴度和酶标抗体滴度的反应组合。抗原和酶标抗体滴度固定后,进一步检测不同稀释度的多克隆抗体,读取数据,分析OD值变化与纳米细菌多抗含量的关系,绘制标准曲线,求出回归方程及相关系数。 8.斑点酶免疫渗滤法的特异性检测及评价 用最佳纳米细菌抗原滴度和酶标抗体滴度的反应组合,以斑点酶免疫渗滤法对不同浓度的实验动物感染阳性血清标本和阴性血清标本进行检测,通过诊断灵敏度、诊断特异性和假阳性率等指标确定样本的最佳稀释浓度。 用最佳纳米细菌抗原滴度和酶标抗体滴度的反应组合,对纳米细菌感染实验各组家兔血清标本进行检测,并评价其诊断灵敏度、诊断特异度、阳性结果预期值、阴性结果预期值及总有效率。 结果 1. 6例胆石症患者胆汁样本在含有10%γ-FBS的DMEM、37℃和5%CO2培养条件下,纳米细菌培养均为阳性。 2.胆石症患者胆汁培养物在倒置相差显微镜下可见做布朗运动的微小的颗粒。培养过程中纳米细菌缓慢生长,培养液的pH值无明显变化;传代后的纳米细菌仍维持上述生物学特性。 3.经数显浊度仪测量,在4周内,每5天测量一次,纳米细菌培养组浊度一直呈上升趋势,其倍增时间约为3天,而DMEM和羟基磷灰石对照组无明显变化,经180℃干烤4h灭活的纳米细菌培养组也未见明显变化。 4.纳米细菌的鉴定 透射电镜下观察,纳米细菌约为80～350nm,呈椭球形或短棒状颗粒,聚集成簇状,其表面覆有细菌被膜。 扫描电镜能谱分析(EDX)显示,纳米细菌含有钙、磷、铝、硅、硫等元素,其钙/磷比值为1.62,与羟基磷灰石中钙/磷比值的1.66相近。 Von KOSSA钙染色法结果显示纳米细菌形成的矿化外壳呈黑色。 间接免疫荧光染色显示纳米细菌被荧光抗体结合,在荧光显微镜下激发绿色荧光。 纳米细菌可被Hoechst 33258染色,发出特征性的蓝色荧光,提示纳米细菌含有DNA成分。 5.免疫家兔血清经ELISA检测,多抗的浓度为56.25mg/ml。 6.斑点酶免疫渗滤法检测Nb多克隆抗体的最佳反应滴度为:Nb抗原1:20(8.71μg/ml),通用型酶标抗体浓度1:10。建立不同稀释浓度的多抗与OD值变化关系的标准曲线,求出回归方程Y=0.0832X+0.0745,相关系数r=0.876(P0.05)。确定最小Nb多克隆抗体稀释比例为1:12800,最小抗体检出浓度为68.59ng/ml。 7.用最佳纳米细菌抗原滴度和酶标抗体滴度的反应组合,以斑点酶免疫渗滤法对不同浓度的实验动物感染阳性血清标本和阴性血清标本进行检测,确定最佳血清样本工作滴度为1:6400。 8.用斑点酶免疫渗滤法检测对纳米细菌感染实验各组家兔血清标本进行检测的结果:40份阳性,56份阴性;而芬兰nanobac公司ELISA试剂盒检测结果为:38份阳性,58份阴性。检测结果经配对资料χ2检验,与纳米细菌感染家兔致胆结石结果相关(χ2=88.055, P=0.001),且两者具有高度诊断一致性(kappa值为0.957)。 结论 1.患有胆囊结石的患者胆汁中存在纳米细菌感染。纳米细菌体积微小,生长缓慢,在生理条件下其菌体表面可生成羟基磷灰石矿化外壳。 2.纳米细菌具有免疫原性,可以通过免疫动物制备抗血清。 3.初步建立斑点酶免疫渗滤法对兔血清中纳米细菌抗体检测法,其灵敏度:96.55%;特异度:82.09%;约登指数:0.7867;总有效率:86.46%;阳性预期值:70.00%;阴性预期值:98.21%。
[Abstract]:Nanobacteria (Nanobacteria, Nb) is a kind of micro bacteria discovered by scientists in Finland Kajander in 1989, they are spherical or rod-shaped, with cell wall, with a diameter of 80 ~ 500nm, colonies are usually tufted; can produce biofilm containing apatite, with strong heat resistance, anti radiation ability and resist antibiotics; through the filter of 100nm, and has the unique ability of biomineralization. In recent years, studies show that with gallstones, kidney stones, atherosclerosis and other bone calcification or sclerosis disease, have attracted great attention.
Immunofluorescence and immunohistochemistry were used for the detection of monoclonal antibodies against nanomaterials. Scanning electron microscopy, transmission electron microscopy and immunoelectron microscopy were the main methods to identify nanomaterials.
objective
The aim of this study is to prepare polyclonal antibodies from bile bacteria and nanomaterials in patients with cholelithiasis.
Method
1., 6 patients without cholecystolithiasis who had no history of acute cholecystitis before laparoscopic surgery were selected. The bacteria were cultured under sterile operation and cultured in nanomaterials. At the same time, DMEM total culture medium and hydroxyapatite culture were used as negative control.
2. the growth of bile separation culture in cholelithiasis patients and the nanoscale in the control group were measured with the digital turbidimeter, and the growth curve was plotted.
3. scanning electron microscopy was used to analyze the bile separation culture of cholelithiasis, and the morphology of the nanoscale bacteria was observed by transmission electron microscope.
4. nanoscale bacterial cultures were used to produce nanoscale bacterial drops. Indirect immunofluorescence staining, Von KOSSA staining and Hochest 33258 staining were carried out according to the literature method.
The 5. purified antigen, and preserved through the examination of quantitative protein concentration of Coomassie blue is 174.2 g/ml.
6. immunized rabbits
Immune 3 rabbits back multipoint inoculation with complete Freund's adjuvant nanobacteria (total 200 g/ml/).2 weeks after inoculation with Freund's incomplete adjuvant nanobacteria (total 50 g/ml/ only) to strengthen immunity (the time interval of 2 weeks, a total of 4 times), the preparation of nanobacteria polyclonal antibody, and its content was determined by ELISA.
7. establishment of multi clone antibody dot enzyme immunofiltration assay for nanoscale bacteria
Rabbit anti nanobacteria polyclonal antibody with two times dilution in order to 1:6400 ~ 1:51200; nanobacterial antigen diluted to 1? 10 ~ 1:160 in different concentration; universal anti mouse / rabbit HRP was diluted to 1:10 ~ 1:40. with square titration method to determine all the test points and the corresponding concentration of the antigen antibody reaction. Each test was repeated 6 times, with the naked eye can interpret brown spots or circle is positive, not negative. For each sentence readers positive brown spots, analysis system with Motic Med 6 digital medical image (A) "to read the value of optical density (OD), calculated the average OD value of each test point.
Analysis of the data, select the best detection of nanobacteria polyclonal anti rabbit nanobacterial antigen titer and the antibody titer of the enzyme labeled antigen and enzyme reaction. Antibody titer after fixation, further detection of polyclonal antibody, different dilutions of the read data, analysis of the relationship between od changes and nano bacteria antibody content, standard curve for the regression equation and correlation coefficient.
Specific detection and evaluation of 8. dot enzyme immunoassay
The reaction in combination with the best nanobacterial antigen titer and the antibody titer by ELISA, dot immuno enzyme filtration assay to detect different concentrations of experimental animal infected with positive serum samples and negative serum samples, the diagnostic sensitivity, specificity and the optimum concentrations of false positive rate and other indicators to determine the sample.
Using the best combination of nano bacterial antigen titer and enzyme antibody titer, we detected the serum samples of each group, and evaluated their diagnostic sensitivity, diagnostic specificity, positive results, expected value and total effective rate.
Result
In 1.6 cases of cholelithiasis, the bile samples were positive in the culture of DMEM, 37 and 5%CO2, with 10% gamma -FBS.
2.. Bile bacteria in cholelithiasis patients can be seen as tiny particles of Brown movement under inverted phase contrast microscope. During the culture process, nanomaterials grew slowly, and the pH value of culture solution did not change significantly.
3. by digital turbidity measurement, in 4 weeks, measured once every 5 days, nanobacteria culture group turbidity has been on the rise, the doubling time is about 3 days, but no obvious changes of DMEM and ha control group cultured groups showed no significant change at 180 dry roasted inactivated 4H nanoparticles bacteria.
Identification of 4. nanoscale bacteria
Under transmission electron microscopy, it was observed that the nanometers were about 80 ~ 350nm, with ellipsoidal or short rod like particles, which were clustered together, and the surface was covered with bacterial membrane.
Scanning electron microscopy (EDX) shows that nanomaterials contain calcium, phosphorus, aluminum, silicon, sulfur and other elements. The ratio of calcium to phosphorus is 1.62, which is close to 1.66 of the ratio of calcium to phosphorus in hydroxyapatite.
The results of Von KOSSA calcium staining showed that the mineralized shell formed by nanometers was black.
Indirect immunofluorescence staining showed that nanophores were combined with fluorescent antibodies and stimulated green fluorescence under the fluorescence microscope.
Nanometers can be stained by Hoechst 33258 and emit a characteristic blue fluorescence, suggesting that nanbacteria contain DNA components.
5.鍏嶇柅瀹跺厰琛，

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