甲型流感病毒胶乳免疫层析试剂的研制
发布时间：2019-03-03 14:27
【摘要】：目的：筛选抗甲型流感病毒核蛋白(NP)的单克隆抗体,建立简便有效的快速检测甲型流感病毒的胶乳免疫层析方法(LICA)。 方法：分别利用BCA法和SDS-PAGE法测定抗甲型流感病毒的6个单克隆抗体的蛋白浓度以及纯度,然后将间接酶联免疫法和胶体金免疫层析法相结合对6个单克隆抗体进行抗体的亲和力和特异性的鉴定,筛选获得一对抗体用于建立检测甲型流感病毒的胶乳免疫层析试剂,并对试纸条的灵敏度,特异性,稳定性,重复性进行鉴定,并用其检测349份临床样本,同时与病毒培养法和PCR法进行比较,验证LICA法检测甲型流感病毒抗原的灵敏度、特异性和可靠性。 结果：成功筛选出最佳抗体配对—AF2和AF4,建立了胶乳免疫层析的检测方法,其检测灵敏度可达胶体金试剂的5倍,检测的349份临床样本中,相对于PCR确证的91份阳性样本,试剂盒的阳性符合率可达59.34%,阴性符合率达100%,与病毒培养法相比较,阳性符合率为67.44%,阴性符合率为91.83%。 结论：利用筛选获得的抗甲型流感病毒NP蛋白的单克隆抗体成功建立了胶乳免疫层析的检测方法,操作简单、方便、快速,特异性、敏感性均较高,在临床患者流感病毒感染的检测中具有较好的应用价值。
[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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：2433785
[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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
【引证文献】
相关硕士学位论文 前1条
1 俞思明;免疫胶乳的合成表征及应用[D];华南理工大学;2012年
，本文编号：2433785
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