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  本文關(guān)鍵詞：膠體金免疫層析法快速檢測C反應(yīng)蛋白的研究　出處：《新鄉(xiāng)醫(yī)學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 比色卡 膠體金免疫層析 C反應(yīng)蛋白 細(xì)菌感染

【摘要】：背景C反應(yīng)蛋白(C reactiveprotein, CRP)自從在急性感染者血液中被發(fā)現(xiàn)后,因能夠反應(yīng)感染性疾病的動態(tài)變化,已被臨床上廣泛應(yīng)用于炎癥的輔助診斷[1],相關(guān)的檢測方法、產(chǎn)品不斷涌現(xiàn),但目前獲批的CRP定量檢測相關(guān)產(chǎn)品均需要借助設(shè)備來完成,不適合現(xiàn)場、社區(qū)醫(yī)院、鄉(xiāng)村等基層醫(yī)療機(jī)構(gòu)使用。因此,研究開發(fā)不需要借助任何設(shè)備即可快速檢測CRP含量的膠體金免疫層析試紙條具有重要的臨床價值和社會意義。目的建立基于自制比色卡半定量檢測CRP含量的雙抗體夾心膠體金免疫層析法方法1.CRP比色卡定值點的確定：根據(jù)臨床診斷標(biāo)準(zhǔn),確定比色卡三個定值點。2.CRP室內(nèi)質(zhì)控品的制備：利用CRP低值血清作為基質(zhì)血清稀釋CRP高值血清,另外添加類風(fēng)濕因子、三酰甘油等干擾物質(zhì)制備一系列室內(nèi)質(zhì)控品,并采用羅氏試劑進(jìn)行復(fù)核。3.CRP試紙條的制備及工藝優(yōu)化：采用檸檬酸三鈉還原法制備不同粒徑的膠體金,采用功能性實驗確定膠體金最優(yōu)粒徑、最佳pH和蛋白標(biāo)記量、最佳蛋白包被量；采用正交實驗確定樣品墊處理液的成分及含量；通過全血、血漿、血清的稀釋倍數(shù)研究確定不同標(biāo)本的稀釋倍數(shù),制備比色卡,并在實驗基礎(chǔ)上將反應(yīng)體積放大100倍,驗證工藝的穩(wěn)定性。4.試紙條性能評價：選用室內(nèi)質(zhì)控品對試紙條的靈敏度、精密性、批間差、特異性、鉤狀(HOOK)效應(yīng)、穩(wěn)定性、檢測環(huán)境條件等進(jìn)行研究。5.實驗室臨床標(biāo)本檢測：從中心標(biāo)本庫選200份臨床標(biāo)本進(jìn)行檢測并與對照試劑對照,確認(rèn)一致性。結(jié)果1.確定CRP比色卡三個定值點由淺到深分別為10 μg/mL、50 μg/ml、10。2.配制CRP室內(nèi)質(zhì)控品14份：含類風(fēng)濕因子(150 IU/mL)、三酰甘油(16mmol/L)和不含干擾因素的CRP終濃度分別為5μg/mL、10 μg/mL、50 μg/mL、100μg/mL。樣本共12份,HOOK效應(yīng)樣本(CRP 1046 μg/mL)及低值樣本(CRP 0.57 μg/mL)各1份。3.初步建立了比色法檢測CRP的試紙條制備方法,選用1.6的膠體金加入0.05%疊氮鈉存放,每毫升膠體金加入0.02 M K2CO3 20 μL、標(biāo)記抗體6μg。包被抗體1.2mg/mL。組條后對樣本稀釋倍數(shù)研究發(fā)現(xiàn)血清、血漿稀釋200倍、全血稀釋120倍后加樣75μL檢測結(jié)果一致。試紙條制備工藝放大實驗用室內(nèi)質(zhì)控品檢測合格。4.試紙條性能檢測顯示：CRP濃度為5μg/mL時檢測線不顯色,10μg/mL時顯色。采用P1、P2、P3連續(xù)3次,顯色無差別,且與比色卡相應(yīng)條帶顯色一致。類風(fēng)濕因子(150 IU/mL)、三酰甘油(16 mmol/L)對結(jié)果無干擾,CRP濃度1046 μg/mL時無HOOK效應(yīng)。5.檢測200份臨床標(biāo)本并與萬孚產(chǎn)品對照,全血、血清、血漿的符合率分別為：50μg/mL以下為100%; 50μg/mL-100 μg/mL之間為93.8%、99%、95.6%；100 μg/mL以上為92.6%、99%、96%。結(jié)論建立了可不借助儀器對人血中CRP半定量檢測的雙抗體夾心膠體金免疫層析方法。該方法通過自制比色卡比色可實現(xiàn)對人全血、血漿、血清中CRP的閾值區(qū)間定量。.
[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...
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.6

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