發(fā)布時(shí)間：2018-01-13 04:07

  本文關(guān)鍵詞：免疫規(guī)劃疫苗抗體檢測(cè)蛋白芯片的研制　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蛋白芯片 免疫規(guī)劃 疫苗 保護(hù)性抗原 抗體

【摘要】：目的:接種疫苗是最經(jīng)濟(jì)、有效、方便的預(yù)防和控制疾病傳播的手段,目前疫苗技術(shù)迅速發(fā)展,已有許多疫苗用于傳染病的預(yù)防。2008年2月,衛(wèi)生部宣布我國(guó)免疫規(guī)劃病種擴(kuò)至15種,相應(yīng)疫苗也納入免疫規(guī)劃。但是,目前疫苗的免疫效果、人群的抗體水平等指標(biāo)均無(wú)法進(jìn)行大規(guī)模有效評(píng)價(jià),常規(guī)的免疫檢測(cè)方法存在的主要問(wèn)題是靈敏度不高,平行檢測(cè)抗體的種類有限等,無(wú)法滿足新形勢(shì)的需求。而蛋白芯片技術(shù)具有高通量、可平行檢測(cè)、高靈敏度、高特異性、微型化自動(dòng)化等優(yōu)點(diǎn),已成功地應(yīng)用于病原微生物抗原和抗體的檢測(cè),越來(lái)越多的引起檢驗(yàn)領(lǐng)域的關(guān)注。本研究將免疫規(guī)劃各病種病原體的保護(hù)性抗原集成在一起,研制了免疫規(guī)劃疫苗抗體檢測(cè)蛋白芯片,以實(shí)現(xiàn)高通量、高靈敏度、多元化地對(duì)疫苗抗體進(jìn)行篩查,通過(guò)一次實(shí)驗(yàn)操作能夠?qū)Χ喾N疫苗抗體進(jìn)行檢測(cè),在縮短檢測(cè)周期的同時(shí),大大提高檢測(cè)效率。 方法: 1.分別用飽和碳酸鈉溶液、重鉻酸鉀與稀鹽酸混合液、80%乙醇處理A群腦膜炎球菌多糖疫苗,分別用飽和碳酸鈉溶液、重鉻酸鉀與稀鹽酸混合液處理吸附白破聯(lián)合疫苗、麻疹減毒活疫苗、A群C群腦膜炎球菌多糖疫苗、甲型肝炎滅活疫苗、卡介苗、麻腮風(fēng)聯(lián)合減毒活疫苗,以75%乙醇處理脊髓灰質(zhì)炎減毒活疫苗糖丸,PAGE純化炭疽桿菌疫苗株裂解液中的保護(hù)性抗原(PA),并以蛋白芯片比較各處理后探針的抗原性。 2.對(duì)篩選出的檢測(cè)各疫苗抗體的抗原進(jìn)行梯度稀釋,本著既保證檢測(cè)靈敏度又節(jié)約的原則確定各抗原探針的最適點(diǎn)樣濃度。 3.以優(yōu)化好的體系制備免疫規(guī)劃疫苗抗體檢測(cè)蛋白芯片,檢測(cè)大量血清樣本以確定各探針的Cutoff值;用蛋白芯片檢測(cè)兒童血清樣本并與ELISA試劑盒的檢測(cè)結(jié)果進(jìn)行對(duì)比,觀察兩種檢測(cè)方法的結(jié)果有無(wú)統(tǒng)計(jì)學(xué)差異。 4.運(yùn)用熒光法、一步金標(biāo)銀染可視化法、兩步金標(biāo)銀染可視化法三種不同的方法檢測(cè)血清樣本,并比較其檢測(cè)效果與靈敏度。 結(jié)果: 1.用飽和碳酸鈉溶液處理A群腦膜炎球菌多糖疫苗和吸附白喉破傷風(fēng)聯(lián)合疫苗,用重鉻酸鉀與稀鹽酸混合液處理麻疹減毒活疫苗和A群C群腦膜炎球菌多糖疫苗,以75%乙醇預(yù)理脊髓灰質(zhì)炎疫苗糖丸,對(duì)炭疽桿菌疫苗株裂解液中的PA蛋白進(jìn)行純化后其抗原性都好于未處理或其它方法處理的抗原,而甲型肝炎滅活疫苗、卡介苗、麻腮風(fēng)聯(lián)合減毒活疫苗預(yù)處理前后沒(méi)有明顯變化。 2.確定了各抗原探針的點(diǎn)樣濃度:麻疹病毒H蛋白4000μg/mL,風(fēng)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,乙腦病毒E蛋白1000μg/mL,脊灰疫苗糖丸處理液1:10稀釋,百日咳FHA 125μg/mL,白喉毒素50μg/mL,破傷風(fēng)毒素250μg/mL,A群C群流腦疫苗處理液原濃度,炭疽桿菌PA蛋白250μg/mL,出血熱病毒NP蛋白330μg/mL,鉤端螺旋體抗原500μg/mL。 3.對(duì)大量血清樣本檢測(cè)結(jié)果進(jìn)行分析,確定了各探針的Cutoff值:麻疹病毒H蛋白1.41,風(fēng)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,乙腦病毒E蛋白1.87,脊灰抗原4.88,百日咳FHA 1.77,白喉毒素1.34,破傷風(fēng)毒素1.41,腦膜炎球菌多糖抗原2.95,出血熱病毒NP蛋白3.82,炭疽PA蛋白3.10,鉤端螺旋體抗原2.77。 4.蛋白芯片與ELISA兩種方法檢測(cè)血樣中各抗體的累計(jì)總符合率為91.59%,運(yùn)用統(tǒng)計(jì)學(xué)的配對(duì)計(jì)數(shù)資料檢驗(yàn)得出:χ~2=0.17,P0.05,說(shuō)明兩種方法的檢測(cè)結(jié)果無(wú)統(tǒng)計(jì)學(xué)差異。 5.對(duì)比了熒光法、一步金標(biāo)銀染可視化法、兩步金標(biāo)銀染可視化法的效果檢測(cè)與靈敏度,結(jié)果表明三種方法具有很好的一致性,且兩步金標(biāo)銀染可視化法的檢測(cè)靈敏度比熒光法提高10倍,比一步金標(biāo)銀染可視化法提高2倍。 結(jié)論:本研究比較了不同方法預(yù)處理疫苗的效果,并驗(yàn)證了所處理的疫苗具有良好的抗原性,為以疫苗作為探針檢測(cè)相應(yīng)抗體提供了實(shí)驗(yàn)依據(jù);本研究制備的免疫規(guī)劃疫苗抗體檢測(cè)蛋白芯片可同時(shí)對(duì)14種免疫規(guī)劃疫苗(除卡介苗)抗體進(jìn)行快速、準(zhǔn)確、高通量、高靈敏度、多元化地檢測(cè),并運(yùn)用高靈敏度新型納米金標(biāo)底物信號(hào)放大技術(shù)實(shí)現(xiàn)了芯片的可視化,進(jìn)一步提高檢測(cè)靈敏度,降低檢測(cè)成本,能滿足現(xiàn)場(chǎng)檢驗(yàn)和基層單位的需要,為國(guó)家和部隊(duì)順利實(shí)施免疫規(guī)劃提供了可靠的評(píng)價(jià)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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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本文編號(hào)：1417367