本文選題：D-二聚體 + 甲基纖維素半固體基　； 參考：《華南理工大學》2012年碩士論文

【摘要】：D-二聚體是人體內纖維蛋白降解的特異性產物，血液中D-二聚體的含量反應了人體內血栓的形成及溶解狀態(tài)，D-二聚體的含量測定對血栓性疾病的檢測及監(jiān)控有指導性作用。D-二聚體的檢測試劑主要應用抗D-二聚體單克隆抗體與D-二聚體抗原的免疫反應，定性或定量檢測D-二聚體�？笵-二聚體單克隆抗體是各種檢測試劑的生物原材料，抗體的性質直接影響試劑檢測結果的準確性和可靠性。故特異性強的抗D-二聚體單克隆抗體的制備是開發(fā)各類D-二聚體檢測試劑的關鍵。國內外多利用傳統(tǒng)雜交瘤細胞篩選方法或基因工程方法制備抗D-二聚體單克隆抗體，主要應用于D-二聚體酶聯免疫檢測試劑（其中包括微孔板ELISA、免疫熒光ELISA、化學發(fā)光ELISA）以及用于膠體金免疫層析試劑、半定量乳膠凝集試劑、全血凝集試劑和免疫比濁試劑。 本試驗以D-二聚體為免疫原，按常規(guī)方法免疫BALB/c小鼠；首先利用甲基纖維素半固體基改進的傳統(tǒng)雜交瘤技術，篩選出了141株雜交瘤細胞株；接著應用D-二聚體、纖維蛋白原、纖維蛋白、及纖維蛋白降解物為篩選原料，利用間接ELISA篩選技術從141株雜交瘤細胞株中篩選出10株穩(wěn)定的陽性雜交瘤細胞株；最后通過單克隆抗體體外生產法，，生產10株抗D-二聚體的單克隆抗體，分別命名為1-3C、2-1B、2-1D、2-4B、2-6D、4-1D、4-3C、4-3D、4-4C、5-3B。ELISA檢測結果表明，10株單克隆抗體腹水效價可達1×10~6�？贵w類—亞類—型鑒定結果顯示，抗體型為IgG1的有2-1D，為IgG2a有1-3C、2-1B、2-4B、4-3C、4-3D，為IgG2b有2-6D、4-1D、4-4C、5-3B。Westernblot結果表明與纖維蛋白原有交叉抗體有4-1D、5-3B，與纖維蛋白原降解物有交叉的抗體有C1、C2，只與D-二聚體有反應而無交叉的抗體有1-3C、2-1B、2-6D、4-3C、4-3D、4-4C。通過雙抗夾心ELISA方法進行單克隆抗體的配對，結果顯示15對配對顯示陽性結果。最后依次采用ELISA、膠體金及熒光膠乳定量法篩選出適用于熒光定量法的2-6D與C2抗體對。 熒光膠乳免疫層析法是一種快速免疫學方法，具有簡便快捷，敏感特異的優(yōu)點。本試驗利用配對的D-二聚體單克隆抗體，制備熒光膠乳免疫層析試紙條，并將其應用于熒光定量檢測儀（廣州萬孚生物技術有限公司）。敏感性結果表明，本試驗制備的熒光膠乳試紙條可以敏銳的檢測出含量僅為100μg/L濃度的D-二聚體；在特異性試驗中沒有交叉，通過臨床試驗對醫(yī)院樣品進行檢測，結果顯示陽性符合率92.1%，陰性符合率更高達98%。 本研究結果表明，改進的雜交瘤細胞篩選方法制備出的抗D-二聚體單克隆抗體親和性和特異性好。通過雙抗夾心ELISA法-膠體金法篩選出的抗體對應用于免疫熒光膠乳定量層析法具有高的敏感性和特異性，可達到與國外同類試劑盒相同的效果。該研究成果為我國血栓類疾病的檢測、監(jiān)控和治療提供了有效的技術支持。
[Abstract]:D-dimer is a specific product of fibrin degradation in human body. The content of D- dimer in blood reflects the formation and dissolution of thrombus in human body. The determination of D- dimer content has instructive effect on the detection and monitoring of thrombotic diseases. The detection reagent of D- dimer is mainly used to resist D- dimer. Immune reaction of Monoclonal Antibody to Ddimer Antigen, Qualitative or quantitative detection of D-dimer. Anti-D-dimer monoclonal antibody is the biological raw material of various detection reagents. The nature of the antibody directly affects the accuracy and reliability of the detection results of the reagents. So the preparation of anti-D-dimer monoclonal antibody is the key to develop all kinds of D-dimer detection reagents. Traditional hybridoma cell screening methods or genetic engineering methods are used to prepare monoclonal antibodies against D- dimer at home and abroad. It is mainly used in D- dimer enzyme linked immunoassay (including microporous plate ELISA, immunofluorescence ELISA, chemiluminescence ELISA), colloidal gold immunochromatographic reagent, semi-quantitative latex agglutination reagent. BALB / c mice were immunized with D- dimer as immunogen, and 141 hybridoma cell lines were screened by methylcellulose semisolid modified hybridoma technique. Then 10 stable hybridoma cell lines were screened from 141 hybridoma cell lines by indirect Elisa using D- dimer fibrinogen fibrin and fibrin degradation as raw materials. Finally, 10 strains of monoclonal antibodies against D- dimer were produced by the method of monoclonal antibody production in vitro. The results showed that the ascites titers of 10 strains of McAbs were 1 脳 10 ~ (6). The results of antibody class-subclass-type identification showed that, The results of Western blot for IgG1, IgG2a, IgG2a and IgG2a were as follows: 1C 2a, 1-3CU 2-4BU 2-4BN 4-3D, and IgG2b, IgG2b, 2-6DX 4-4CU 4-3B Western blot. The results of Western blot showed that there were 4-1D5-3B with fibrinogen biodegradable antibody, C1C2with fibrinogen degradation, and 1-3CU 2-1B2-6B2-3C4-3D4-4C with fibrinogen biodegradable antibody. Monoclonal antibodies were paired by double antibody sandwich Elisa. The results showed that 15 pairs showed positive results. Finally, 2-6D and C2 antibodies suitable for fluorescence quantitative assay were screened by ELISA-colloidal gold and fluorescent latex quantitative method. Fluorescent latex immunochromatography was a rapid immunological method with the advantages of simplicity, rapidity, sensitivity and specificity. In this study, a fluorescent latex immunochromatographic strip was prepared by using the matched monoclonal antibody of D-dimer, and was applied to the fluorescence quantitative detector (Guangzhou Wanfu Biotechnology Co., Ltd.) The sensitivity results showed that the fluorescent latex test strip prepared in this experiment could be used to detect D- dimer with a concentration of only 100 渭 g / L, and there was no crossover in the specific test, and the hospital samples were detected by clinical trial. The results showed that the positive coincidence rate was 92.1%, and the negative coincidence rate was even higher. The results showed that the modified screening method of hybridoma cells had good affinity and specificity of anti-D-dimer monoclonal antibodies. The antibodies screened by double antibody sandwich Elisa and colloidal gold method have high sensitivity and specificity for immunofluorescence latex quantitative chromatography, and can achieve the same effect as foreign similar kits. The results provide effective technical support for the detection, monitoring and treatment of thrombotic diseases in China.
【學位授予單位】：華南理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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