發(fā)布時(shí)間：2019-01-11 08:54

【摘要】：抗人紅細(xì)胞膜抗原非凝集型單克隆抗體(mAb)是與自身全血凝集試驗(yàn)相關(guān)的各類雙特異性抗體分子的重要組成部分。以非凝集型單抗為基礎(chǔ)的雙特異性抗體分子可快速、準(zhǔn)確地檢測(cè)人體血液中感染病原體的抗原或抗體成分。 本研究應(yīng)用雜交瘤技術(shù),將人O型紅細(xì)胞膜免疫BALB/c小鼠,取免疫小鼠的脾細(xì)胞與SP2/0骨髓瘤細(xì)胞融合,聚凝胺試管法篩選出識(shí)別紅細(xì)胞表面共同抗原的抗體,玻片法剔除凝集型抗體(IgM、IgA),再將分泌非凝集型抗體(IgG)的雜交瘤細(xì)胞株有限稀釋法克隆3次,對(duì)雜交瘤細(xì)胞的穩(wěn)定性和mAb的特性進(jìn)行鑒定。 本研究獲得1株可穩(wěn)定分泌IgG1類mAb的雜交瘤細(xì)胞,命名為2E8。mAb2E8針對(duì)紅細(xì)胞膜H抗原,沒(méi)有血型交叉反應(yīng)和種屬交叉反應(yīng),細(xì)胞培養(yǎng)上清與人的A、B、AB和O型紅細(xì)胞均能形成很強(qiáng)的凝集,凝集效價(jià)為1:1024,腹水凝集效價(jià)達(dá)到1:64×10~6。mAb2E8與紅細(xì)胞結(jié)合后能誘導(dǎo)白細(xì)胞或激活補(bǔ)體,有效地破壞、溶解紅細(xì)胞。此外,本研究建立了篩選針對(duì)紅細(xì)胞表面抗原mAb的新方法—聚凝胺試驗(yàn),為制備與紅細(xì)胞相關(guān)的各種mAb的篩選提供一個(gè)簡(jiǎn)單快捷的途徑。 本研究通過(guò)雜交瘤技術(shù),成功地制備了針對(duì)紅細(xì)胞膜H抗原的非凝集性mAb,并對(duì)抗體的各項(xiàng)指標(biāo)進(jìn)行了較全面的鑒定。該mAb的凝集效價(jià)、相對(duì)親和力及特異性均良好,且具有較強(qiáng)的體外生物學(xué)活性,為構(gòu)建與自身全血凝集試驗(yàn)相關(guān)的雙特異性抗體分子和單抗異聚體(HP)奠定了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Anti-human erythrocyte membrane antigen non-agglutination monoclonal antibody (mAb) is an important component of all kinds of bispecific antibody molecules associated with autohemagglutination test. Bispecific antibody molecules based on non-agglutinating monoclonal antibodies can be used to detect the antigen or antibody components of infected pathogens in human blood quickly and accurately. In this study, BALB/c mice were immunized with human O type erythrocyte membrane by hybridoma technique. The spleen cells of immunized mice were fused with SP2/0 myeloma cells. The agglutination antibody (IgM,IgA) was eliminated by slide method. The hybridoma cell line which secreted non-agglutinative antibody (IgG) was cloned for 3 times by limited dilution method. The stability of the hybridoma cells and the characteristics of mAb were identified. In this study, we obtained a hybridoma cell line which can secrete IgG1 mAb stably, named 2E8.mAb2E8 for erythrocyte membrane H antigen, no blood group cross reaction and species cross reaction, cell culture supernatant and human Agna B, The agglutination titer of AB and O-type red blood cells was 1: 1024, and the agglutination titer of ascites reached 1:64 脳 10~6.mAb2E8, which could induce white blood cells or activate complement, effectively destroy and dissolve red blood cells. In addition, a new method for screening erythrocyte surface antigen (mAb) was established, which provides a simple and rapid way for the preparation of various mAb related to erythrocyte. In this study, nonagglutinating mAb, against erythrocyte membrane H antigen was successfully prepared by hybridoma technique, and various indexes of antibody were comprehensively identified. The agglutination titer, relative affinity and specificity of the mAb were good, and had strong biological activity in vitro, which laid a solid foundation for the construction of the bispecific antibody molecule and the monoantibody heteropolymer (HP) related to the whole blood agglutination test.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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本文編號(hào)：2406931