稳定分泌抗羊种布鲁菌脂多糖单克隆抗体杂交瘤细胞株的建立

发布时间：2018-12-31 19:02

【摘要】：本研究将热酚水法提纯羊布鲁菌(16M菌株)的脂多糖(LPS)和灭活羊种布鲁菌(16M)作为免疫抗原,交替免疫6～8周龄Balb/c雌鼠。第一次免疫用羊种布鲁菌标准菌16M全菌加等量弗氏完全佐剂;第二次免疫用脂多糖加等量弗氏不完全佐剂。第三次免疫用羊种布鲁菌标准菌16M全菌(本次不加佐剂);第四次用脂多糖(本次不加佐剂),每次免疫间隔十天。四免后,测其ELISA效价达到1:10000以上的,用脂多糖(LPS)抗原腹腔超强免疫一次,三天后取小鼠的脾细胞与SP2/0骨髓瘤细胞进行细胞融合。用热酚水法提纯羊布鲁菌(16M菌株)的脂多糖作为包被抗原建立间接ELISA方法,筛选抗羊种布鲁菌(16M菌株)脂多糖的单克隆抗体杂交瘤细胞株。采用有限稀释法,经过3～4次克隆,最终获得12株分泌抗羊种布鲁菌脂多糖单克隆抗体杂交瘤细胞株,分别命名为:4A11、6B8、3H7、4D4、3E3、3F9、6E3、6F2、2C3、5D11、4C3、5H3。经过亚类鉴定,其中3E3和6E3是IgG1亚类,3F9、2C3、5D11、3H7和4D4是IgG3亚类,4C3为IgG2a亚类,而6F2、5H3、6B8、4A11是IgM亚类。选择其中两株杂交瘤细胞(4D4和3H7)进行染色体分析,试验结果所得平均染色体数目在92～108之间,符合SP2/0细胞的染色体数目与正常小鼠脾细胞的染色体数目之和,表明所获得的抗体分泌细胞为杂交瘤细胞。对其中3株杂交瘤细胞(6B8、5H3和3H7)进行特异性检测,结果显示该3株单抗只与灭活的羊种布鲁菌16M裂解抗原发生反应,呈现阳性;而不与大肠杆菌O157裂解抗原、鸡白痢沙门菌裂解抗原、鸭源鸡杆菌1-S脂多糖抗原以及福氏志贺菌解抗原反应。虎红凝集试验和试管凝集试验进一步证实此3株单抗具有很强的特异性。经检测,所得12株抗羊种布鲁菌单克隆抗体杂交瘤细胞培养上清ELISA效价在1:1000～1:10000之间,小鼠腹水单克隆抗体ELISA效价为1:160000。对其中5株杂交瘤细胞(6B8、3E3、3F9、6E3、6F2)进行连续传10、20、30代和3次冻融复苏后,杂交瘤细胞生长旺盛,虽然有部分杂交瘤细胞分泌抗体的能力有些轻微下降,但分泌抗体能力并没有消失,仍能稳定分泌高效价抗体,说明所获得的杂交瘤细胞株分泌抗体能力稳定。利用所建立的单克隆抗体细胞株,建立了一种检测布鲁菌的双夹心间接ELISA方法,并进行了特异性和敏感性检测。对模拟样品水样两份(S1、S2)、奶样两份(S3、S4)、土样两份(S5、S6)进行了检测,准确性均很高。
[Abstract]:In this study, lipopolysaccharide (LPS) and inactivated sheep strain Brucella (16M) were purified by thermophenol water method as immune antigens, and Balb/c female mice aged 6 to 8 weeks were immunized alternately. The standard strain of Brucella sheep for the first immunization was 16m and the same amount of Freund's complete adjuvant was added to the whole bacterium, and the lipopolysaccharide was added with the same amount of Freund's incomplete adjuvant for the second immunization. The standard strain of Brucella was 16m (this time without adjuvant) and lipopolysaccharide (no adjuvant was added) for the third immunization, the interval of immunization was ten days. After four immunizations, the ELISA titers of the mice whose ELISA titers were more than 1: 10 000 were tested. The lipopolysaccharide (LPS) antigen was used to immunize the spleen cells of mice with SP2/0 myeloma cells once, and three days later, the spleen cells of mice were fused with SP2/0 myeloma cells. The indirect ELISA method was established by purification of lipopolysaccharide from Brucella sheep (16M strain) by thermophenol water method. The monoclonal antibody hybridoma cell lines against lipopolysaccharide of Brucella suis (16M strain) were screened. 12 hybridoma cell lines secreting lipopolysaccharide against Brucella sibiricus were obtained by limited dilution method. The hybridoma cell lines were named 4A116B8H7H7H7D4D4E3E3E3E3F9C3F2C3D114C3C3H3H3, respectively. The results showed that 3E3 and 6E3 were IgG1 subclasses, 3F9C3D113H7 and 4D4 were IgG3 subclasses, 4C3 were IgG2a subclasses, and 6F2H3H3B8A11 subclasses were IgM subclasses. Two of the hybridoma cells (4D4 and 3H7) were selected for chromosome analysis. The results showed that the average number of chromosomes was between 922 and 108, which corresponded to the sum of the chromosome number of SP2/0 cells and that of normal mouse spleen cells. The results showed that the antibody secreting cells were hybridoma cells. Three of the hybridoma cells (6B8H3 and 3H7) were detected specifically. The results showed that the McAbs reacted with 16M lytic antigen of inactivated brucellosis. It did not react with Escherichia coli O157 lytic antigen, Salmonella pullorum lytic antigen, 1-S lipopolysaccharide antigen and Shigella flexneri lytic antigen. Tiger red agglutination test and test tube agglutination test further confirmed that the three McAbs have strong specificity. The results showed that the ELISA titer of the supernatant of 12 hybridoma cells was 1: 1000: 110000, and the ELISA titer of mouse ascites monoclonal antibody was 1: 160000. Five of the hybridoma cells (6B8O3E3E3F9C3E3O6F2) were transferred continuously for 1020 / 30 passages and 3 times of freeze-thaw resuscitation. Although some hybridoma cells had a slight decrease in the ability to secrete antibodies, the ability of secreting antibodies did not disappear. The high titer antibody can still be secreted stably, which indicates that the hybridoma cell line is stable in secreting antibody. A double sandwich indirect ELISA method for the detection of brucellosis was established by using the established monoclonal antibody cell line. The specificity and sensitivity of the method were tested. Two water samples (S _ 1 / S _ 2), two milk samples (S _ 3 / S _ 4) and two soil samples (S _ 5 / S _ 6) were tested.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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