【摘要】：目的：篩選抗甲型流感病毒核蛋白(NP)的單克隆抗體,建立簡便有效的快速檢測甲型流感病毒的膠乳免疫層析方法(LICA)。 方法：分別利用BCA法和SDS-PAGE法測定抗甲型流感病毒的6個單克隆抗體的蛋白濃度以及純度,然后將間接酶聯(lián)免疫法和膠體金免疫層析法相結合對6個單克隆抗體進行抗體的親和力和特異性的鑒定,篩選獲得一對抗體用于建立檢測甲型流感病毒的膠乳免疫層析試劑,并對試紙條的靈敏度,特異性,穩(wěn)定性,重復性進行鑒定,并用其檢測349份臨床樣本,同時與病毒培養(yǎng)法和PCR法進行比較,驗證LICA法檢測甲型流感病毒抗原的靈敏度、特異性和可靠性。 結果：成功篩選出最佳抗體配對—AF2和AF4,建立了膠乳免疫層析的檢測方法,其檢測靈敏度可達膠體金試劑的5倍,檢測的349份臨床樣本中,相對于PCR確證的91份陽性樣本,試劑盒的陽性符合率可達59.34%,陰性符合率達100%,與病毒培養(yǎng)法相比較,陽性符合率為67.44%,陰性符合率為91.83%。 結論：利用篩選獲得的抗甲型流感病毒NP蛋白的單克隆抗體成功建立了膠乳免疫層析的檢測方法,操作簡單、方便、快速,特異性、敏感性均較高,在臨床患者流感病毒感染的檢測中具有較好的應用價值。
[Abstract]:Objective: to screen monoclonal antibodies against nucleoprotein (NP) of influenza A virus and establish a simple and effective latex immunochromatographic method for detection of influenza A virus (LICA). Methods: the protein concentration and purity of six monoclonal antibodies against influenza A virus were determined by BCA method and SDS-PAGE method, respectively. Then the affinity and specificity of six monoclonal antibodies were identified by indirect enzyme-linked immunosorbent assay (Elisa) and colloidal gold immunochromatography, and a pair of antibodies were selected and used to establish latex immunochromatographic reagents for detecting influenza A virus. The sensitivity, specificity, stability and repeatability of the test strip were identified and compared with the virus culture method and PCR method to verify the sensitivity of Liga method for detecting influenza A virus antigen, and using it to detect 349clinical samples, and compared it with the virus culture method and PCR method, and verified the sensitivity of Lica method for detecting influenza A virus antigen. Specificity and reliability. Results: the best antibody pairing-AF2 and AF4, were successfully screened and the latex immunochromatographic assay was established. The sensitivity was 5 times higher than that of colloidal gold reagent. Compared with 91 positive samples confirmed by PCR, the sensitivity of the assay was 5 times higher than that of colloidal gold reagent. The positive and negative coincidence rates of the kit were 59.34% and 100% respectively. Compared with the virus culture method, the positive coincidence rate was 67.44% and the negative coincidence rate was 91.83%. Conclusion: the monoclonal antibody against NP protein of influenza A virus has been successfully established for the detection of latex immune chromatography. The method is simple, convenient, rapid, specific and sensitive. It has good application value in the detection of influenza virus infection in clinical patients.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

【引證文獻】

相關碩士學位論文 前1條

1 俞思明;免疫膠乳的合成表征及應用[D];華南理工大學;2012年

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本文編號：2433785