胶体金免疫层析法快速检测C反应蛋白的研究

发布时间：2018-01-11 11:19

本文关键词：胶体金免疫层析法快速检测C反应蛋白的研究　出处：《新乡医学院》2015年硕士论文　论文类型：学位论文

【摘要】：背景C反应蛋白(C reactiveprotein, CRP)自从在急性感染者血液中被发现后,因能够反应感染性疾病的动态变化,已被临床上广泛应用于炎症的辅助诊断[1],相关的检测方法、产品不断涌现,但目前获批的CRP定量检测相关产品均需要借助设备来完成,不适合现场、社区医院、乡村等基层医疗机构使用。因此,研究开发不需要借助任何设备即可快速检测CRP含量的胶体金免疫层析试纸条具有重要的临床价值和社会意义。目的建立基于自制比色卡半定量检测CRP含量的双抗体夹心胶体金免疫层析法方法1.CRP比色卡定值点的确定：根据临床诊断标准,确定比色卡三个定值点。2.CRP室内质控品的制备：利用CRP低值血清作为基质血清稀释CRP高值血清,另外添加类风湿因子、三酰甘油等干扰物质制备一系列室内质控品,并采用罗氏试剂进行复核。3.CRP试纸条的制备及工艺优化：采用柠檬酸三钠还原法制备不同粒径的胶体金,采用功能性实验确定胶体金最优粒径、最佳pH和蛋白标记量、最佳蛋白包被量；采用正交实验确定样品垫处理液的成分及含量；通过全血、血浆、血清的稀释倍数研究确定不同标本的稀释倍数,制备比色卡,并在实验基础上将反应体积放大100倍,验证工艺的稳定性。4.试纸条性能评价：选用室内质控品对试纸条的灵敏度、精密性、批间差、特异性、钩状(HOOK)效应、稳定性、检测环境条件等进行研究。5.实验室临床标本检测：从中心标本库选200份临床标本进行检测并与对照试剂对照,确认一致性。结果1.确定CRP比色卡三个定值点由浅到深分别为10 μg/mL、50 μg/ml、10。2.配制CRP室内质控品14份：含类风湿因子(150 IU/mL)、三酰甘油(16mmol/L)和不含干扰因素的CRP终浓度分别为5μg/mL、10 μg/mL、50 μg/mL、100μg/mL。样本共12份,HOOK效应样本(CRP 1046 μg/mL)及低值样本(CRP 0.57 μg/mL)各1份。3.初步建立了比色法检测CRP的试纸条制备方法,选用1.6的胶体金加入0.05%叠氮钠存放,每毫升胶体金加入0.02 M K2CO3 20 μL、标记抗体6μg。包被抗体1.2mg/mL。组条后对样本稀释倍数研究发现血清、血浆稀释200倍、全血稀释120倍后加样75μL检测结果一致。试纸条制备工艺放大实验用室内质控品检测合格。4.试纸条性能检测显示：CRP浓度为5μg/mL时检测线不显色,10μg/mL时显色。采用P1、P2、P3连续3次,显色无差别,且与比色卡相应条带显色一致。类风湿因子(150 IU/mL)、三酰甘油(16 mmol/L)对结果无干扰,CRP浓度1046 μg/mL时无HOOK效应。5.检测200份临床标本并与万孚产品对照,全血、血清、血浆的符合率分别为：50μg/mL以下为100%; 50μg/mL-100 μg/mL之间为93.8%、99%、95.6%；100 μg/mL以上为92.6%、99%、96%。结论建立了可不借助仪器对人血中CRP半定量检测的双抗体夹心胶体金免疫层析方法。该方法通过自制比色卡比色可实现对人全血、血浆、血清中CRP的阈值区间定量。.
[Abstract]:Background C reactive protein (CRP) has been found in the blood of patients with acute infection because of its ability to respond to the dynamic changes of infectious diseases. It has been widely used in assistant diagnosis of inflammation. [1], related testing methods, products continue to emerge, but the current approved CRP quantitative testing products need to be completed with equipment, is not suitable for field, community hospitals. Used in primary medical institutions such as villages. It is of great clinical value and social significance to develop a colloidal gold immunochromatographic strip for rapid detection of CRP content without any equipment. Objective to establish a semi-quantitative detection method for CRP content based on self-made colorimetric card. Double antibody sandwich colloidal gold immunochromatographic method 1. Determination of CRP colorimetric calorimetric point:. According to the clinical diagnostic criteria. To determine the three fixed value points of colorimetric cards. 2. Preparation of indoor quality control products: low value serum of CRP was used as matrix serum to dilute high value serum of CRP, and rheumatoid factor was added. Triacylglycerol and other interfering substances were used to prepare a series of indoor quality control products, and the preparation and process optimization of the test strip of .3.CRP were carried out by using Roche reagent: the colloidal gold with different particle size was prepared by tri-sodium citrate reduction method. The optimum particle size, optimal pH, protein labeling amount and protein encapsulation amount were determined by functional experiments. The composition and content of the sample pad solution were determined by orthogonal experiment. Through the study of dilution times of whole blood, plasma and serum, the dilution times of different samples were determined, the colorimetric cards were prepared, and the reaction volume was enlarged 100 times on the basis of experiments. Test strip performance evaluation: the sensitivity, precision, batch difference, specificity, hookk effect and stability of the test strip were selected. Detection of environmental conditions. 5. Laboratory clinical specimen detection: 200 clinical specimens were selected from the central specimen bank for detection and compared with the control reagent. Confirm consistency. Result 1. Determine the three fixed points of CRP colorimetric card from shallow to deep to 10 渭 g / mL 50 渭 g / ml respectively. 10.2.The preparation of 14 CRP indoor quality control products: containing rheumatoid factor (150 IUU / mL). The final concentrations of triacylglycerol (16mmol / L) and CRP without interference factor were 5 渭 g / mL and 10 渭 g / mL, 50 渭 g / mL, respectively. There were 12 samples of 100 渭 g / mL. HOOK effect sample (1046 渭 g / mL) and low value sample (0.57 渭 g / mL). A method for the preparation of test strip for the detection of CRP by colorimetric method was established. 0.05% sodium azide was added to 1.6 colloidal gold, and 0.02 M K _ 2CO _ 3 20 渭 L per milliliter of colloidal gold was added. After labeling antibody 6 渭 g. Coated with 1.2 mg / mL. group, the dilution times of the sample were studied and the serum and plasma diluted 200 times. After 120 times of whole blood dilution, the test results of 75 渭 L were the same. The test strip was qualified for laboratory quality control. 4. The test strip performance test showed that:. When the concentration of CRP was 5 渭 g / mL, the detection line did not show color. At 10 渭 g / mL, using P1P2P2P3 for 3 consecutive times, there was no difference in color development, which was consistent with the corresponding band of colorimetric card. The rheumatoid factor was 150 IUmL). There was no HOOK effect at 1046 渭 g / mL triacylglycerol / L. 200 clinical samples were detected and compared with Wanfu products, whole blood. The coincidence rates of serum and plasma were below 50 渭 g / mL and 100 渭 g / mL, respectively. The range of 50 渭 g / mL-100 渭 g / mL was 93.8% and 95.6%; More than 100 渭 g / mL was 92.6%. Conclusion A double antibody sandwich colloidal gold immunochromatographic method was established without the aid of instrument for the detection of CRP in human blood. This method can be used to detect human whole blood and plasma by self-made colorimetric Kabi colorimetry. The threshold interval of CRP in serum...
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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