抗狂犬病病毒单克隆抗体的制备和鉴定
发布时间：2018-09-04 12:20
【摘要】： 狂犬病是由狂犬病病毒引起的人畜共患的烈性传染病,也是迄今为止人类病死率最高的急性传染病。抗狂犬病病毒单克隆抗体是研究狂犬病病毒诊断方法、治疗及预防狂犬病的有力工具。为了获得高效价、高特异性的抗狂犬病病毒的单克隆抗体,进而将其应用于开发一种方便、简单、廉价、技术要求低且适合基层使用的诊断方法,本研究用人用狂犬疫苗免疫BALB/c小鼠,采用杂交瘤技术制备得到针对狂犬病病毒的稳定的、效价高的、特异性强的单克隆抗体(mAb),并初步鉴定了杂交瘤和mAb的生物学特性,包括：杂交瘤的染色体、杂交瘤分泌抗体的稳定性、mAb的效价、亚类、纯度、蛋白浓度以及mAb的特异性。为开发简单实用的检测方法打下基础。 一、融合条件的摸索 在充分了解SP2/0细胞系的生长、增殖和遗传特性后,以聚乙二醇(PEG)诱导动物细胞融合,探索不同分子量、不同浓度的PEG作为融合剂对诱导SP2/0细胞与脾细胞融合的最适条件。结果表明分子量为4000,浓度为50%的PEG诱导的细胞融合率最高,为0.06429%,为下一步制备单克隆抗体奠定基础。 二、单克隆抗体杂交瘤细胞株的制备、筛选、克隆、稳定性及染色体 1.抗体制备与细胞融合 使用人用狂犬疫苗免疫雌性BALB/c小鼠,间隔2周免疫1次。双方阵法确定了间接ELISA法的最佳抗原包被浓度为1：80,阳性血清最适工作稀释度为1：400。间接ELISA法检测抗体效价达到104的3d后,通过PEG4000使免疫B淋巴细胞和小鼠骨髓瘤细胞SP2/0融合。融合3-5d后出现杂交瘤细胞,7-10d后观察到杂交瘤细胞克隆生长。经HAT培养基选择培养后,细胞的融合率为82.99%。 2.阳性克隆的筛选 融合后14d左右,融合的杂交瘤细胞长到大约培养板的1/2面积时,按照已建立的间接ELISA法进行筛选,阳性克隆率为15.90%(38/239),选择OD值比较高的14株阳性杂交瘤细胞株进行亚克隆。 3.阳性杂交瘤细胞的克隆化培养 用显微操作法对阳性克隆的细胞株,分别进行了亚克隆,得到17珠分泌抗狂犬病病毒单克隆抗体的杂交瘤细胞株,分别命名8F3、8F4、8G8、17A4、17A5、17A7、17B2、17B5、17B4、17C2、17C6、17D2、17D4、17D8、17E3、17E4、17F3。选择OD450值较高,生长状态良好的8F3、8F4、8G8、17B2、17E3、17E4、17F3进行扩大培养和鉴定,其它株冷冻保存。 4.抗体分泌的稳定性检测 将杂交瘤细胞连续培养传代,共传18代每隔3代检测一次细胞培养上清的抗体效价,结果显示6代以后杂交瘤进入稳定分泌抗体时期。 5.杂交瘤细胞的染色体计数 杂交瘤细胞染色体的平均数为94条,符合融合细胞染色体的特点。 三、单克隆抗体的制备及初步鉴定 1.mAb的制备 通过收集培养上清、体内诱生腹水、制备实体瘤分离血清法获得并保存了8F3、8F4、8G8、17B2、17E3、17E4和17F3分泌的抗体。 2.mAb的纯化 经抗体亚类初步鉴定8F3、8F4、8G8、17B2、17E3、17E4、17F3所分泌的抗体类型皆属于IgG亚类,采用辛酸-硫酸铵法进行纯化。 3.mAb的效价 间接ELISA法检测培养上清、纯化和未纯化的腹水和实体瘤血清抗体效价。腹水和实体瘤血清中的抗体效价均高于104,最高的达1：128000,且腹水和实体瘤血清的效价比同种单抗细胞培养上清的效价均高100倍以上。 4.mAb的亚类 Ig类及亚类的鉴定结果显示8F3和8F4属于IgG2b,8G8和17E4属于IgG3、17B2属于IgG1,17E3和17F3属于IgG2a。 5.mAb的纯度 纯化后的单抗在50kD和25kD附近出现两条带,与预期的IgG抗体重链和轻链条带大小相符,而未纯化的单抗泳道上则出现多条杂带。 6.mAb的蛋白浓度 紫外分光光度法测得17B2、17E3、17E4、17F3、8F3、8F4、8G8、浓度分别为：1.6565mg/mL、0.0162mg/mL、0.0163mg/mL、1.7120mg/mL、1.6315mg/mL、1.6834mg/mL、0.0163mg/mL。 7.mAb的特异性 7株单克隆抗体与三个厂家的狂犬疫苗反应均为阳性,与犬瘟热病毒、犬细小病毒、犬传染性肝炎病毒、犬副流感病毒、乙型肝炎病毒反应均为阴性。说明7株单克隆抗体的特异性强且与其他病毒无交叉反应。
[Abstract]:Rabies is a zoonotic infectious disease caused by rabies virus. It is also an acute infectious disease with the highest mortality of human beings. Monoclonal antibody against rabies virus is a powerful tool for the study of the diagnosis, treatment and prevention of rabies virus. In order to obtain high-cost, high-specificity anti-rabies virus monoclonal antibody. In this study, human rabies vaccine was used to immunize BALB/c mice, and hybridoma technology was used to prepare stable, potent and specific monoclonal antibodies (mAb) against rabies virus, and preliminary identification was carried out. The biological characteristics of hybridoma and mAb, including chromosome of hybridoma, stability of antibody secreted by hybridoma, titer of mAb, subclass, purity, protein concentration and specificity of mAb, were studied.
First, explore the conditions of convergence.
After fully understanding the growth, proliferation and genetic characteristics of SP2/0 cell line, PEG was used to induce the fusion of SP2/0 cells and spleen cells. The optimum conditions for inducing the fusion of SP2/0 cells and spleen cells with different molecular weights and concentrations of PEG were explored. 0.06429%, lay the foundation for further preparation of monoclonal antibodies.
Two, monoclonal antibody hybridoma cell line preparation, screening, cloning, stability and chromosomes.
1. antibody preparation and cell fusion
Female BALB/c mice were immunized with rabies vaccine at intervals of 2 weeks. The optimal antigen coating concentration of indirect ELISA was 1:80 and the optimum working dilution of positive serum was 1:400. After the titer of antibody reached 104 by indirect ELISA, SP2/C of B lymphocytes and mouse myeloma cells were immunized with PEG4000 3 days later. Hybridoma cells appeared 3-5 days after fusion and clonal growth of hybridoma cells was observed 7-10 days after fusion.
2. screening of positive clones
About 14 days after fusion, when the fused hybridoma cells grew to about 1/2 of the culture plate area, the positive cloning rate was 15.90% (38/239) by indirect ELISA method, and 14 hybridoma cell lines with high OD value were selected for subcloning.
3. cloning culture of positive hybridoma cells
Hybridoma cell lines secreting anti-rabies virus monoclonal antibodies from 17 beads were obtained by subcloning the positive clones with micromanipulation. The hybridoma cell lines were named 8F3, 8F4, 8G8, 17A4, 17A5, 17A7, 17B2, 17B5, 17B4, 17C2, 17C6, 17D2, 17D4, 17D8, 17E3, 17E4, 17F3. The selected OD450 values were high and the growth status was good. 3,17E4,17F3 was expanded and identified, and other plants were cryopreserved.
4. stability of antibody secretion
The antibody titers of the supernatant of hybridoma cells were detected every three generations in 18 passages. The results showed that the hybridoma entered a stable antibody secretion stage after 6 passages.
Chromosome count of hybridoma cells 5.
The average number of chromosomes of hybridoma cells is 94, which is consistent with the characteristics of the chromosomes of fusion cells.
Three, preparation and preliminary identification of monoclonal antibodies.
Preparation of 1.mAb
The antibodies secreted by 8F3, 8F4, 8G8, 17B2, 17E3, 17E4 and 17F3 were obtained and preserved by collecting culture supernatants and inducing ascites in vivo.
Purification of 2.mAb
The antibodies secreted by 8F3, 8F4, 8G8, 17B2, 17E3, 17E4 and 17F3 were identified as IgG subclasses and purified by octanoic acid-ammonium sulfate method.
Titer of 3.mAb
The titers of antibodies in ascites and solid tumor sera were higher than 104 and the highest was 1:128000. The titers of ascites and solid tumor sera were 100 times higher than those of monoclonal antibodies.
Subclasses of 4.mAb
The identification results of Ig class and subclass showed that 8F3 and 8F4 belonged to IgG2b, 8G8 and 17E4 belonged to IgG3, 17B2 belonged to IgG1, 17E3 belonged to IgG2a.
Purity of 5.mAb
The purified monoclonal antibody showed two bands near 50 kD and 25 kD, which were consistent with the expected IgG antibody heavy and light chain bands, while the unpurified monoclonal antibody showed multiple bands on the swimming track.
Protein concentration of 6.mAb
The concentrations of 17B2, 17E3, 17E4, 17F3, 8F3, 8F4 and 8G8 were 1.656565mg/mL, 0.0162mg/mL, 0.0163mg/mL, 1.7120mg/mL, 1.6315mg/mL, 1.6834mg/mL, 0.0163mg/mL, respectively.
Specificity of 7.mAb
All the 7 monoclonal antibodies reacted positively with rabies vaccines from three manufacturers, and were negative with canine distemper virus, canine parvovirus, canine infectious hepatitis virus, canine parainfluenza virus and hepatitis B virus.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
[Abstract]:Rabies is a zoonotic infectious disease caused by rabies virus. It is also an acute infectious disease with the highest mortality of human beings. Monoclonal antibody against rabies virus is a powerful tool for the study of the diagnosis, treatment and prevention of rabies virus. In order to obtain high-cost, high-specificity anti-rabies virus monoclonal antibody. In this study, human rabies vaccine was used to immunize BALB/c mice, and hybridoma technology was used to prepare stable, potent and specific monoclonal antibodies (mAb) against rabies virus, and preliminary identification was carried out. The biological characteristics of hybridoma and mAb, including chromosome of hybridoma, stability of antibody secreted by hybridoma, titer of mAb, subclass, purity, protein concentration and specificity of mAb, were studied.
First, explore the conditions of convergence.
After fully understanding the growth, proliferation and genetic characteristics of SP2/0 cell line, PEG was used to induce the fusion of SP2/0 cells and spleen cells. The optimum conditions for inducing the fusion of SP2/0 cells and spleen cells with different molecular weights and concentrations of PEG were explored. 0.06429%, lay the foundation for further preparation of monoclonal antibodies.
Two, monoclonal antibody hybridoma cell line preparation, screening, cloning, stability and chromosomes.
1. antibody preparation and cell fusion
Female BALB/c mice were immunized with rabies vaccine at intervals of 2 weeks. The optimal antigen coating concentration of indirect ELISA was 1:80 and the optimum working dilution of positive serum was 1:400. After the titer of antibody reached 104 by indirect ELISA, SP2/C of B lymphocytes and mouse myeloma cells were immunized with PEG4000 3 days later. Hybridoma cells appeared 3-5 days after fusion and clonal growth of hybridoma cells was observed 7-10 days after fusion.
2. screening of positive clones
About 14 days after fusion, when the fused hybridoma cells grew to about 1/2 of the culture plate area, the positive cloning rate was 15.90% (38/239) by indirect ELISA method, and 14 hybridoma cell lines with high OD value were selected for subcloning.
3. cloning culture of positive hybridoma cells
Hybridoma cell lines secreting anti-rabies virus monoclonal antibodies from 17 beads were obtained by subcloning the positive clones with micromanipulation. The hybridoma cell lines were named 8F3, 8F4, 8G8, 17A4, 17A5, 17A7, 17B2, 17B5, 17B4, 17C2, 17C6, 17D2, 17D4, 17D8, 17E3, 17E4, 17F3. The selected OD450 values were high and the growth status was good. 3,17E4,17F3 was expanded and identified, and other plants were cryopreserved.
4. stability of antibody secretion
The antibody titers of the supernatant of hybridoma cells were detected every three generations in 18 passages. The results showed that the hybridoma entered a stable antibody secretion stage after 6 passages.
Chromosome count of hybridoma cells 5.
The average number of chromosomes of hybridoma cells is 94, which is consistent with the characteristics of the chromosomes of fusion cells.
Three, preparation and preliminary identification of monoclonal antibodies.
Preparation of 1.mAb
The antibodies secreted by 8F3, 8F4, 8G8, 17B2, 17E3, 17E4 and 17F3 were obtained and preserved by collecting culture supernatants and inducing ascites in vivo.
Purification of 2.mAb
The antibodies secreted by 8F3, 8F4, 8G8, 17B2, 17E3, 17E4 and 17F3 were identified as IgG subclasses and purified by octanoic acid-ammonium sulfate method.
Titer of 3.mAb
The titers of antibodies in ascites and solid tumor sera were higher than 104 and the highest was 1:128000. The titers of ascites and solid tumor sera were 100 times higher than those of monoclonal antibodies.
Subclasses of 4.mAb
The identification results of Ig class and subclass showed that 8F3 and 8F4 belonged to IgG2b, 8G8 and 17E4 belonged to IgG3, 17B2 belonged to IgG1, 17E3 belonged to IgG2a.
Purity of 5.mAb
The purified monoclonal antibody showed two bands near 50 kD and 25 kD, which were consistent with the expected IgG antibody heavy and light chain bands, while the unpurified monoclonal antibody showed multiple bands on the swimming track.
Protein concentration of 6.mAb
The concentrations of 17B2, 17E3, 17E4, 17F3, 8F3, 8F4 and 8G8 were 1.656565mg/mL, 0.0162mg/mL, 0.0163mg/mL, 1.7120mg/mL, 1.6315mg/mL, 1.6834mg/mL, 0.0163mg/mL, respectively.
Specificity of 7.mAb
All the 7 monoclonal antibodies reacted positively with rabies vaccines from three manufacturers, and were negative with canine distemper virus, canine parvovirus, canine infectious hepatitis virus, canine parainfluenza virus and hepatitis B virus.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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