抗白念珠菌芽管单抗MAb03.2C1-C2特异性及靶抗原性质的初步研究

发布时间：2018-06-18 03:45

本文选题：MAb03.2C1-C2 + 白念珠菌　；参考：《第三军医大学》2006年硕士论文

【摘要】： 目的:观察抗白念珠菌芽管胞壁外膜单克隆抗体MAb03.2C1-C2的特异性,并初步应用于实验室口腔念珠菌病真菌涂片菌丝阳性标本中白念珠菌的检测,以探讨其用于临床真菌检测可行性及其意义,并用于白念珠菌与都柏林念珠菌的实验室鉴别。分析该单抗靶抗原表位的性质,对其抗原性质进行分析。方法:复苏冻存的杂交瘤细胞株03.2C1-C2,传代培养20代,检测培养液中单抗滴度,观察杂交瘤细胞持续分泌抗体的稳定性;用杂交瘤技术获得小鼠腹水单抗MAb03.2C1-C2。用不同念珠菌的孢子和菌丝、其他常见酵母菌、细菌做抗原,用间接免疫荧光法(IIF)检测抗体特异性。收集临床口腔念珠菌病患者口腔真菌涂片中菌丝阳性标本,用MAb03.2C1-C2通过IIF方法检测白念珠菌。用不同的液体培养基在不同的温度条件下诱导白念珠菌和都柏林念珠菌的菌丝形成,用该单抗通过IIF试验鉴别两者。用生化试剂处理白念珠菌芽管后,观察其与单抗结合情况,通过IIF试验分析抗原表位性质。结果: 1. 125株临床分离白念珠菌芽管或菌丝100%可与MAb03.2C1-C2结合,孢子均不与单抗结合。都柏林念珠菌、类星形念珠菌、热带念珠菌、克柔氏念珠菌、季也蒙念珠菌、近平滑念珠菌、高里念珠菌、葡萄牙念珠菌、光滑念珠菌、产膜念珠菌、软念珠菌、副秃发念珠菌、产阮念珠菌、木蓝念珠菌的孢子、菌丝表面没有可以与MAb03.2C1-C2相结合的靶抗原。新型隐球菌、无名酵母、粘红酵母、光滑球拟酵母、皮状丝孢酵母、大肠杆菌、金黄色葡萄球菌、绿脓杆菌表面均不存在可与该单抗相结合的靶抗原。MAb03.2C1-C2特异性好。 2.临床口腔念珠菌病患者真菌涂片中镜检菌丝阳性标本,IIF试验检测白念珠菌结果与实验室常规培养鉴定结果一致。 3.用不完全1640液37℃诱导静止态的白念珠菌和都柏林念珠菌孢子,6h后两者菌丝形成率最高。 4.复苏冻存1年、2年的杂交瘤细胞,传代培养20代后,培养液中抗体效价通
[Abstract]:Objective: To observe the specificity of monoclonal antibody MAb03.2C1-C2 of the outer membrane of Candida albicans germ tube wall, and to apply it to the detection of Candida albicans in the positive specimens of oral candidiasis fungal smear, in order to explore the feasibility and significance of its application in clinical fungal detection and to be used in the laboratory of Candida albicans and Candida albicans in Dublin. To identify the target epitopes of the monoclonal antibody and analyze its antigenic properties.
Methods: the hybridoma cell line, 03.2C1-C2, was resuscitation and cultured for 20 generations to detect the titer of the monoclonal antibody in the culture medium, and to observe the stability of the hybridoma cells continuously secreting the antibody. The hybridoma technique was used to obtain the spores and mycelium of the mouse ascites MAb03.2C1-C2. with different Candida albicans, other common yeasts, bacteria as antigens and indirect immunofluorescence. The positive specimens of mycelium in oral fungal smears of patients with oral candidiasis were collected, and Candida albicans were detected by MAb03.2C1-C2 by IIF method. The mycelia of Candida albicans and Candida Dublin were induced by different liquid medium at different temperature conditions, and the monoclonal antibody was used for the IIF test. Both of them were treated with biochemical reagent to observe the combination of Candida albicans and McAb. The antigenic epitopes were analyzed by IIF test.
Result:
1.125 clinical isolates of Candida albicans or mycelium 100% can be combined with MAb03.2C1-C2. Spores are not combined with monoclonal antibodies. Candida albicans, Candida albicans, Candida Kurus, Monilia Monilia, Candida albicans, Candida albicans, Candida albicans, Candida albicans, Candida, Candida, Candida Candida alir, Nguyen Candida, and spores of Candida albicans, and there is no target antigen on the surface of the mycelium that can be combined with MAb03.2C1-C2. New Cryptococcus, innominant yeast, mycelium yeast, smooth yeast, hyphomycosis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa have no target antigen that can be combined with the monoclonal antibody. .MAb03.2C1-C2 is very specific.
2. microscopy positive specimens of fungus in smear of oral candidiasis patients. The results of IIF test for Candida albicans were consistent with the results of routine laboratory culture.
3.. The spores of Candida albicans and Candida albicans in Dublin were induced by incomplete 1640 fluid and 37 degrees celidius, and the mycelium formation rate was highest after 6h.
4., after 1 years of cryopreservation, 2 years of hybridoma cells were passaged for 20 generations.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R379

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