【摘要】： 自身紅細(xì)胞凝集試驗(yàn)(Autologous red cell agglutination assay,AGEN)是一種簡(jiǎn)便、特異又不需要任何特殊儀器的快速診斷方法。其靈敏性和特異性不亞于ELISA,且可同時(shí)檢測(cè)IgG和IgM,具有安全、快速、準(zhǔn)確、價(jià)廉的特點(diǎn),檢測(cè)時(shí)無(wú)需分離血清,無(wú)需儀器設(shè)備,僅需一滴全血,在2 min內(nèi)可得到檢測(cè)結(jié)果,非常適合條件簡(jiǎn)陋的基層醫(yī)院、義務(wù)獻(xiàn)血前以及緊急輸血前的使用,因而具有良好的應(yīng)用前景。該方法依賴于一種雙功能試劑,該試劑具有結(jié)合人紅細(xì)胞和病人血液中的抗原或抗體的特性。當(dāng)病人血液中有針對(duì)雙功能試劑的抗原或抗體存在時(shí),由于與雙功能試劑的分子間交聯(lián)而導(dǎo)致紅細(xì)胞的凝集。因此本研究的目的是制備人紅細(xì)胞非凝集型單克隆抗體,進(jìn)而形成雙功能抗體試劑,用于臨床診斷。 本研究用三種免疫原免疫BALB/c小鼠來(lái)制備人紅細(xì)胞非凝集型單克隆抗體,通過(guò)化學(xué)偶聯(lián)法制備出雙功能抗體,進(jìn)而形成全血凝集檢測(cè)試劑,并對(duì)血凝試驗(yàn)方法進(jìn)行了初步探討。主要研究?jī)?nèi)容如下: 1、分別用人O型紅細(xì)胞、紅細(xì)胞全膜和紅細(xì)胞膜蛋白作為抗原,采用腹腔加尾部靜脈注射免疫BALB/c小鼠,取免疫脾細(xì)胞和小鼠骨髓瘤細(xì)胞SP2/0進(jìn)行細(xì)胞融合,細(xì)胞比例為5∶1,經(jīng)間接ELISA、紅細(xì)胞直接凝集試驗(yàn)和間接凝集試驗(yàn)檢測(cè),采用有限稀釋法進(jìn)行克隆,經(jīng)過(guò)3次克隆后獲得1株人紅細(xì)胞非凝集型單克隆抗體雜交瘤細(xì)胞,命名為1C3。經(jīng)過(guò)多次傳代、凍存、復(fù)蘇,雜交瘤細(xì)胞分泌抗體能力仍然穩(wěn)定,其抗體亞類類型為IgG1。常規(guī)方法誘生腹水,辛酸-硫酸銨沉淀法和DEAE纖維素層析法對(duì)腹水進(jìn)行純化。腹水單抗ELISA效價(jià)在1:10~4～1:10~5之間,并與其它種屬無(wú)交叉反應(yīng),親和力高。 2、分別用胃蛋白酶消化人紅細(xì)胞非凝集型單克隆抗體和乙肝表面抗體,經(jīng)純化得到人紅細(xì)胞單抗和乙肝表面抗體的F(ab~')_2片段。通過(guò)戊二醛一步法和戊二醛兩步法將非凝集型單抗和乙肝表面抗體的F(ab~')_2片段進(jìn)行偶聯(lián)來(lái)制備雙功能抗體。經(jīng)SDS-PAGE電泳鑒定,通過(guò)戊二醛一步法成功制備了雙功能抗體,進(jìn)而形成雙功能檢測(cè)試劑,對(duì)該試劑的活性進(jìn)行了初步的鑒定,同時(shí)初步建立了一種檢測(cè)血液中乙肝表面抗原的方法。
[Abstract]:Autologous hemagglutination test (Autologous red cell agglutination assay,AGEN) is a simple, specific and rapid diagnostic method without any special instrument. Its sensitivity and specificity are no less than that of ELISA, and it can detect IgG and IgM, simultaneously with the characteristics of safety, rapidity, accuracy and low price. The detection results can be obtained in 2 min without the need of separating serum, apparatus and equipment, and only a drop of whole blood. It is very suitable for basic hospitals with poor conditions. It is used before voluntary blood donation and before emergency blood transfusion, so it has a good application prospect. The method relies on a bifunctional reagent that binds human erythrocytes to antigens or antibodies in the patient's blood. When antigen or antibody against bifunctional reagents exists in the blood of patients, the agglutination of erythrocytes is caused by intermolecular crosslinking with bifunctional reagents. The aim of this study is to prepare non-agglutination monoclonal antibody of human red blood cell and to form bifunctional antibody reagent for clinical diagnosis. In this study, BALB/c mice were immunized with three kinds of immunogens to prepare non-agglutination monoclonal antibodies against human erythrocytes. Bifunctional antibodies were prepared by chemical coupling method, and then the whole blood agglutination assay was formed. The method of hemagglutination test was discussed. The main contents are as follows: 1. BALB/c mice were immunized with human type O erythrocytes, erythrocyte membrane proteins and erythrocyte membrane proteins respectively by intraperitoneal and tail intravenous injection. The immune spleen cells and mouse myeloma cells (SP2/0) were selected for cell fusion. The proportion of cells was 5: 1. Indirect ELISA, erythrocyte agglutination test and indirect agglutination test were used to clone the cells. After three clones, a human erythrocyte non-agglutinating monoclonal antibody hybridoma cell was obtained and named as 1C3. After repeated passage, cryopreservation and resuscitation, the ability of hybridoma cells to secrete antibodies is still stable, and its antibody subtype is IgG1.. Ascites were induced by routine methods. Ascites were purified by octanoic acid-ammonium sulfate precipitation and DEAE cellulose chromatography. The ELISA titer of ascitic monoclonal antibody was between 1: 10 and 4: 10 5, and had no cross reaction with other species and had high affinity. 2. Human erythrocyte non-agglutinating monoclonal antibody and hepatitis B surface antibody were digested with pepsin, and the F (ab~') _ 2 fragment of human erythrocyte monoclonal antibody and hepatitis B surface antibody were purified. The F (ab~') _ 2 fragment of non-agglutinating monoclonal antibody and hepatitis B surface antibody was coupled with glutaraldehyde one-step method and glutaraldehyde two-step method to prepare the bifunctional antibody. The bifunctional antibody was successfully prepared by one step method of glutaraldehyde by SDS-PAGE electrophoresis, and then the bifunctional detection reagent was formed. The activity of the reagent was preliminarily identified. At the same time, a method for the detection of hepatitis B surface antigen in blood was established.
【學(xué)位授予單位】：河南工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【參考文獻(xiàn)】

中國(guó)期刊全文數(shù)據(jù)庫(kù) 前10條

1 高新,逯好英,徐東剛,楊軍,李素波,章?lián)P培;抗Glycophorin A單鏈抗體基因的構(gòu)建及酵母表達(dá)研究[J];中國(guó)輸血雜志;2003年04期

2 張嘉敏;陳和平;王芬;朱自嚴(yán);;抗人紅細(xì)胞血型糖蛋白A非多態(tài)性表位單克隆抗體的制備和鑒定[J];中國(guó)輸血雜志;2008年03期

3 劉成剛,陳志南,邊惠潔,米力,仇凱,曲萍;單克隆抗體F(ab’)_2片段2種純化方法的建立及比較[J];第四軍醫(yī)大學(xué)學(xué)報(bào);1998年04期

4 孔令宇,劉曉波,朱振宇,顧鴻飛;抗體F(ab′)_2片段制備的新方法—木瓜蛋白酶酶切法[J];臨床檢驗(yàn)雜志;2004年02期

5 邵長(zhǎng)利;史利軍;姚站馨;王宏業(yè);呂茂民;劉明;章金剛;;抗人紅細(xì)胞H抗原單鏈抗體基因克隆和表達(dá)[J];免疫學(xué)雜志;2008年02期

6 操敏,唐家琪,李光富,王長(zhǎng)軍,潘秀珍,李先富;抗人紅細(xì)胞單鏈抗體基因的構(gòu)建及表達(dá)[J];南京師大學(xué)報(bào)(自然科學(xué)版);2000年02期

7 王長(zhǎng)軍,唐家琪,李先富,潘秀珍,操敏;抗人紅細(xì)胞血型B抗原單鏈抗體基因的構(gòu)建表達(dá)[J];上海免疫學(xué)雜志;2000年01期

8 潘秀珍，，李先富，唐家琪;人紅細(xì)胞非凝集型單抗的研制及鑒定[J];上海免疫學(xué)雜志;1995年01期

9 葉群瑞,陳伯權(quán),吳美英;一種簡(jiǎn)單、快速、量大的辛酸純化單克隆抗體法[J];生物化學(xué)與生物物理進(jìn)展;1991年01期

10 米力,陳志南,余曉玲,馮強(qiáng),邊惠潔,徐力青;單克隆抗體F(ab’)_2片段的制備與質(zhì)量控制[J];中國(guó)生物制品學(xué)雜志;1999年04期

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