本文選題：熒光微球　切入點(diǎn)：免疫層析　出處：《鄭州大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景前列腺特異性抗原(Prostate specific antigen,PSA)由前列腺上皮組織分泌,分子量為34KD,已廣泛應(yīng)用于前列腺癌的輔助診斷,免疫學(xué)方法檢測(cè)形式有兩種,即游離前列腺特異性抗原(free-PSA,f PSA)、與α1-抗糜蛋白結(jié)合的前列腺特異性抗原(PSA-ACT),通常所說(shuō)的總前列腺抗原(t PSA)是f PSA和PSA-ACT的總和。國(guó)際上認(rèn)為,PSA濃度小于4ng/m L是正常水平,前列腺癌風(fēng)險(xiǎn)隨PSA濃度升高而增加,當(dāng)PSA濃度在4~10ng/m L之間時(shí)被認(rèn)為是診斷灰區(qū),t PSA和f PSA占t PSA百分比(%f PSA)常用來(lái)提高診斷灰區(qū)的前列腺癌診斷效率。定量檢測(cè)t PSA和f PSA的方法主要有:化學(xué)發(fā)光法、時(shí)間熒光免疫分辨法、電化學(xué)發(fā)光法等,這些方法雖然具備靈敏度高、定量準(zhǔn)確、重復(fù)性和穩(wěn)定性較好等優(yōu)點(diǎn),但是需要大型儀器,適合樣本量多的大中型醫(yī)院,不適合單個(gè)標(biāo)本測(cè)定,從而限制了t PSA和f PSA檢測(cè)項(xiàng)目在社區(qū)醫(yī)院的開(kāi)展。目前國(guó)內(nèi)檢測(cè)t PSA和f PSA的試劑盒中,缺少一種可以準(zhǔn)確、快速定量,并且操作簡(jiǎn)便可實(shí)現(xiàn)床旁檢測(cè)方法,熒光微球免疫層析技術(shù)可實(shí)現(xiàn)床旁檢測(cè),如能研究成功,可彌補(bǔ)國(guó)內(nèi)t PSA、f PSA檢測(cè)方法的缺憾。目的建立一種性能優(yōu)良的定量檢測(cè)t PSA及f PSA的方法,為t PSA及f PSA的檢測(cè)提供一種新的技術(shù)和方法。方法應(yīng)用t PSA及f PSA蛋白制備室內(nèi)參考品;用熒光微球與抗體偶聯(lián),并利用中心提供的熒光免疫層析實(shí)驗(yàn)技術(shù)和平臺(tái)對(duì)本實(shí)驗(yàn)條件進(jìn)行多方面的研究和優(yōu)化。通過(guò)分析試紙條的最低檢出限、特異性、準(zhǔn)確度、線性、精密性以及穩(wěn)定性,對(duì)其性能進(jìn)行研究。取已經(jīng)過(guò)羅氏試劑盒(電化學(xué)發(fā)光法)定值的血清87份,用本實(shí)驗(yàn)建立的檢測(cè)方法進(jìn)行測(cè)定,比較兩種檢測(cè)方法的相關(guān)性。結(jié)果研究結(jié)果顯示,本實(shí)驗(yàn)制備的t PSA及f PSA室內(nèi)參考品在≤-20℃環(huán)境下至少能夠穩(wěn)定保存12個(gè)月,2-8℃環(huán)境下至少能夠穩(wěn)定保存4周;本試驗(yàn)制備的試紙條t PSA的最低檢出限為0.08ng/m L,f PSA的最低檢出限為0.06ng/m L;與CEA、AFP、CA125均無(wú)交叉反應(yīng);試紙條批內(nèi)變異系數(shù)均小于10%,批間變異系數(shù)均小于15%,回收率在97.06%-103.11%之間;試紙條在2-8℃儲(chǔ)存條件下至少可穩(wěn)定存放6個(gè)月,37℃儲(chǔ)存條件下可穩(wěn)定存放10天;用本實(shí)驗(yàn)建立的熒光免疫層析試紙條與羅氏試劑盒(電化學(xué)發(fā)光法)進(jìn)行相關(guān)性分析,檢測(cè)t PSA的R2為0.9727,回歸方程為Y=0.9372X+0.2626,檢測(cè)f PSA的R2為0.9888,回歸方程為Y=0.9981X-0.0537,兩種檢測(cè)方法具有良好的相關(guān)性。結(jié)論(1)制備出穩(wěn)定的t PSA、f PSA室內(nèi)參考品;(2)初步建立了同時(shí)檢測(cè)t PSA、f PSA熒光層析定量檢測(cè)方法。(3)制備了性能良好的t PSA、f PSA熒光層析定量檢測(cè)試紙條。
[Abstract]:Background Prostate specific antigenase (PSAs) is secreted from the epithelium of the prostate with a molecular weight of 34kD. It has been widely used in the auxiliary diagnosis of prostate cancer. There are two kinds of immunological methods for detection of prostate cancer. That is, free prostate specific antigen free-PSAF PSAA, a prostate-specific antigen (PSA-ACTA) binding to 偽 1-antichyme protein, is the sum of f PSA and PSA-ACT. The risk of prostate cancer increases with the increase of PSA concentration. When the concentration of PSA is between 4 ~ 10 ng / mL, it is considered to be a diagnostic gray area t PSA and f PSA as a percentage of t PSA) is often used to improve the diagnostic efficiency of prostate cancer in the gray area. The main methods for quantitative detection of t PSA and f PSA are chemiluminescence method. Although these methods have the advantages of high sensitivity, accurate quantification, reproducibility and stability, they need large instruments and are suitable for large and medium-sized hospitals with a large sample size. T PSA and f PSA are not suitable for single specimen determination, which limits the development of t PSA and f PSA detection in community hospitals. At present, there is a lack of a kit for detecting t PSA and f PSA in China, which can be accurately and rapidly quantified. It is easy to operate and can be used for bedside detection. Fluorescence microsphere immunochromatography can be used to detect bedside, if it can be studied successfully, This method can make up for the shortcoming of domestic t PSA and f PSA detection methods. Objective to establish a method for quantitative detection of t PSA and f PSA with good performance. Methods the t PSA and f PSA proteins were used to prepare indoor reference materials, and fluorescent microspheres were used to couple with antibodies, and a new technique and method for the detection of t PSA and f PSA were provided. The experimental conditions of this experiment were studied and optimized by using the fluorescence immunochromatographic technique and platform provided by the center. The minimum detection limit, specificity, accuracy, linearity, precision and stability of the test strip were analyzed. 87 samples of serum which had passed the value of Roche kit (electrochemiluminescence method) were used to determine the correlation between the two methods. T PSA and f PSA reference materials prepared in this experiment can be kept stably for at least 12 months and 2 to 8 鈩，

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