免疫规划疫苗抗体检测蛋白芯片的研制

发布时间：2018-01-13 04:07

本文关键词：免疫规划疫苗抗体检测蛋白芯片的研制　出处：《中国人民解放军军事医学科学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的:接种疫苗是最经济、有效、方便的预防和控制疾病传播的手段,目前疫苗技术迅速发展,已有许多疫苗用于传染病的预防。2008年2月,卫生部宣布我国免疫规划病种扩至15种,相应疫苗也纳入免疫规划。但是,目前疫苗的免疫效果、人群的抗体水平等指标均无法进行大规模有效评价,常规的免疫检测方法存在的主要问题是灵敏度不高,平行检测抗体的种类有限等,无法满足新形势的需求。而蛋白芯片技术具有高通量、可平行检测、高灵敏度、高特异性、微型化自动化等优点,已成功地应用于病原微生物抗原和抗体的检测,越来越多的引起检验领域的关注。本研究将免疫规划各病种病原体的保护性抗原集成在一起,研制了免疫规划疫苗抗体检测蛋白芯片,以实现高通量、高灵敏度、多元化地对疫苗抗体进行筛查,通过一次实验操作能够对多种疫苗抗体进行检测,在缩短检测周期的同时,大大提高检测效率。方法: 1.分别用饱和碳酸钠溶液、重铬酸钾与稀盐酸混合液、80%乙醇处理A群脑膜炎球菌多糖疫苗,分别用饱和碳酸钠溶液、重铬酸钾与稀盐酸混合液处理吸附白破联合疫苗、麻疹减毒活疫苗、A群C群脑膜炎球菌多糖疫苗、甲型肝炎灭活疫苗、卡介苗、麻腮风联合减毒活疫苗,以75%乙醇处理脊髓灰质炎减毒活疫苗糖丸,PAGE纯化炭疽杆菌疫苗株裂解液中的保护性抗原(PA),并以蛋白芯片比较各处理后探针的抗原性。 2.对筛选出的检测各疫苗抗体的抗原进行梯度稀释,本着既保证检测灵敏度又节约的原则确定各抗原探针的最适点样浓度。 3.以优化好的体系制备免疫规划疫苗抗体检测蛋白芯片,检测大量血清样本以确定各探针的Cutoff值;用蛋白芯片检测儿童血清样本并与ELISA试剂盒的检测结果进行对比,观察两种检测方法的结果有无统计学差异。 4.运用荧光法、一步金标银染可视化法、两步金标银染可视化法三种不同的方法检测血清样本,并比较其检测效果与灵敏度。结果: 1.用饱和碳酸钠溶液处理A群脑膜炎球菌多糖疫苗和吸附白喉破伤风联合疫苗,用重铬酸钾与稀盐酸混合液处理麻疹减毒活疫苗和A群C群脑膜炎球菌多糖疫苗,以75%乙醇预理脊髓灰质炎疫苗糖丸,对炭疽杆菌疫苗株裂解液中的PA蛋白进行纯化后其抗原性都好于未处理或其它方法处理的抗原,而甲型肝炎灭活疫苗、卡介苗、麻腮风联合减毒活疫苗预处理前后没有明显变化。 2.确定了各抗原探针的点样浓度:麻疹病毒H蛋白4000μg/mL,风疹病毒E1蛋白500μg/mL,腮腺炎病毒NP蛋白1000μg/mL,HAV vp1蛋白1000μg/mL,HAV vp3蛋白1000μg/mL,HBsAg 500μg/mL,乙脑病毒E蛋白1000μg/mL,脊灰疫苗糖丸处理液1:10稀释,百日咳FHA 125μg/mL,白喉毒素50μg/mL,破伤风毒素250μg/mL,A群C群流脑疫苗处理液原浓度,炭疽杆菌PA蛋白250μg/mL,出血热病毒NP蛋白330μg/mL,钩端螺旋体抗原500μg/mL。 3.对大量血清样本检测结果进行分析,确定了各探针的Cutoff值:麻疹病毒H蛋白1.41,风疹病毒E1蛋白1.76,腮腺炎病毒NP蛋白1.51,HAV vp1蛋白2.03,HAV vp3蛋白1.99,HBsAg 1.10,乙脑病毒E蛋白1.87,脊灰抗原4.88,百日咳FHA 1.77,白喉毒素1.34,破伤风毒素1.41,脑膜炎球菌多糖抗原2.95,出血热病毒NP蛋白3.82,炭疽PA蛋白3.10,钩端螺旋体抗原2.77。 4.蛋白芯片与ELISA两种方法检测血样中各抗体的累计总符合率为91.59%,运用统计学的配对计数资料检验得出:χ~2=0.17,P0.05,说明两种方法的检测结果无统计学差异。 5.对比了荧光法、一步金标银染可视化法、两步金标银染可视化法的效果检测与灵敏度,结果表明三种方法具有很好的一致性,且两步金标银染可视化法的检测灵敏度比荧光法提高10倍,比一步金标银染可视化法提高2倍。结论:本研究比较了不同方法预处理疫苗的效果,并验证了所处理的疫苗具有良好的抗原性,为以疫苗作为探针检测相应抗体提供了实验依据;本研究制备的免疫规划疫苗抗体检测蛋白芯片可同时对14种免疫规划疫苗(除卡介苗)抗体进行快速、准确、高通量、高灵敏度、多元化地检测,并运用高灵敏度新型纳米金标底物信号放大技术实现了芯片的可视化,进一步提高检测灵敏度,降低检测成本,能满足现场检验和基层单位的需要,为国家和部队顺利实施免疫规划提供了可靠的评价工具。
[Abstract]:Objective: vaccination is the most economical, effective, convenient communication of disease prevention and control measures, the current vaccine technology develops rapidly, there are many vaccines for infectious disease prevention.2008 in February, the Ministry of Health announced that China's national immunization program expanded to 15 diseases, the corresponding vaccine into the immunization program. However, the current vaccine the immune effect, the index of population antibody level were unable to carry out large-scale effective evaluation, the main problems of immune conventional detection methods is not high sensitivity type parallel detection antibody is limited, can not meet the needs of the new situation. And the protein chip technology is a high throughput, parallel detection, high sensitivity and high specificity. The miniaturization of automation, has been successfully applied to the detection of pathogen antigen and antibody, caused more and more attention in the field of test. This study will be immune planning various disease pathogens The protective antigen integrated with developed protein chip for detection of EPI vaccine antibody, to achieve high sensitivity, high flux, diversified screening of vaccine antibody, through an experimental operation can detect a variety of vaccine antibody, shorten the detection cycle and greatly improve the detection efficiency.
Method:
1. respectively with saturated solution of sodium carbonate, potassium dichromate and dilute hydrochloric acid mixed solution, 80% ethanol treatment group A meningococcal polysaccharide vaccine, respectively with a saturated solution of sodium carbonate, potassium dichromate and dilute hydrochloric acid mixed solution adsorption white broken vaccine, Measles Vaccine live, A group C group meningococcal polysaccharide vaccine, BCG, Inactivated Hepatitis A Vaccine, Ma mumps and rubella combined vaccine, with 75% ethanol for Poliomyelitis Vaccine in Dragee Candy, PAGE purified protective antigen of Bacillus anthracis vaccine strain in the lysate (PA), and to probe the antigenicity of protein chip comparison after treatment.
2., we made gradient dilution for the antigen screened for each vaccine antibody, and determined the best concentration of each antigen probe based on the principle of ensuring sensitivity and saving.
3. prepared protein chip for detection of EPI vaccine antibody detection, a large number of serum samples to determine the probe Cutoff value; protein chip to detect children serum samples detection results and ELISA kit were compared to observe the results of the two detection methods have no significant difference.
4., using fluorescence method, one-step gold labeling silver staining visualization method, two step gold standard silver staining visualization method, three different ways to detect serum samples, and compare their detection effect and sensitivity.
Result:
1. with saturated A meningococcal polysaccharide vaccine and Diphtheria and Tetanus Combined Vaccine Adsorbed with sodium carbonate solution, potassium dichromate and dilute hydrochloric acid treatment of mixture of Measles Vaccine live and A group C meningococcal polysaccharide vaccine, with 75% ethanol preconditioning polio vaccine was purified after sugar pill, its antigenicity is better than untreated or other treatment of antigen, and Inactivated Hepatitis A Vaccine anthrax vaccine strains of BCG lysate PA protein with MMR live attenuated rabies vaccine pretreatment had no obvious changes.
2. determine the sample concentration of each antigen probe: measles virus H protein 4000 g/mL, rubella virus E1 protein 500 g/mL, mumps virus NP protein VP1 protein HAV 1000 g/mL, 1000 g/mL, HAV VP3 protein 1000 g/mL, HBsAg 500 g/mL, E 1000 g/mL protein of Japanese encephalitis virus, polio vaccine pill treatment liquid diluted 1:10 pertussis FHA 125 g/mL diphtheria toxin 50 g/mL tetanus toxin, 250 g/mL, A group C meningococcal polysaccharide vaccine treatment liquid concentration, Bacillus anthracis PA protein 250 g/mL, hemorrhagic fever virus NP protein, 330 g/mL, 500 g/mL. of Leptospira antigen
3. the analysis of a large number of serum samples detection results, determine the probe Cutoff value: 1.41 H protein of measles virus, rubella virus E1 protein 1.76, mumps virus NP protein 1.51, HAV VP1 2.03 protein, HAV VP3 protein 1.99, HBsAg 1.10, Japanese encephalitis virus E protein 1.87, poliovirus antigen 4.88, pertussis FHA 1.77 1.34, diphtheria toxin, tetanus toxin 1.41, meningococcal polysaccharide antigen 2.95, hemorrhagic fever virus NP protein 3.82, anthrax PA protein 3.10, Leptospira antigen 2.77.
4. protein chip and ELISA two ways to detect the total coincidence rate of antibodies in blood samples was 91.59%. The data obtained by statistical paired count data showed that: ~2=0.17 and P0.05 showed that there was no statistical difference between two methods.
5. comparison of fluorescence method, one-step glss, two step glss effect detection and sensitivity. The results show that the three methods have good consistency, sensitivity and the two step glss 10 times higher than 2 times higher than the fluorescence method. Step glss.
Conclusion: This study compared the different methods of pretreatment effects of vaccine, it is verified that the treatment vaccine has good antigenicity and provide experimental basis for the vaccine as a probe to detect the corresponding antibodies; protein chip detection of the research on the preparation of EPI vaccine and antibody of 14 EPI vaccine (except BCG) fast, accurate and high-throughput antibody, high sensitivity, multiple detection, and the use of new high sensitivity gold nanoparticle substrate signal amplification technology to realize the visualization of the chip, further improve the detection sensitivity, low detection cost, can meet the needs of field inspection and grass-roots units, to provide a reliable evaluation tool for the state and the army the smooth implementation of immunization programs.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【参考文献】