分别分泌抗H9N2亚型禽流感病毒和新城疫病毒单克隆抗体杂交瘤细胞株的建立及初步应用
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摘要
本试验用H9N2亚型禽流感病毒(AIV)、LaSota弱毒疫苗株新城疫病毒(NDV)加入免疫增强剂作为混合免疫原,免疫8-12周龄Balb/c小鼠,采用一次融合、分别筛选的方法,取小鼠脾细胞与SP2/0骨髓瘤细胞融合,经自行建立的间接ELISA方法检测筛选,分别获得2株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞株,依次命名为C5、D5;4株稳定分泌抗NDV单克隆抗体的杂交瘤细胞株,依次命名为4B4、4B8、4C4、4E8。同时,用纯化的H9N2亚型AIV和LaSota弱毒疫苗株新城疫病毒作为单一免疫原,各自分别免疫小鼠,采用传统杂交瘤细胞技术,最后从单一AIV免疫组获得3株稳定分泌抗H9N2亚型AIV单克隆抗体的杂交瘤细胞株,依次命名为C8、D7、E8;从单一NDV免疫组获得2株稳定分泌抗NDV单克隆抗体的杂交瘤细胞株,依次命名为7D2、8C10。
经鉴定,抗H9N2亚型AIV单抗的杂交瘤细胞培养上清及小鼠腹水单抗的ELISA效价最高可达1:1024和1:102400,抗LaSota株NDV单抗的杂交瘤细胞培养上清及小鼠腹水单抗的ELISA效价最高可达1:512和1:51200。单抗亚型均为IgG类抗体分子。各株杂交瘤细胞的平均染色体数目在96-106之间,符合SP2/0细胞与正常小鼠脾细胞的染色体数目之和。连续传代25次和三次冻存复苏后,各杂交瘤细胞抗体分泌能力有所下降,但仍能稳定分泌抗体。针对H9N2亚型AIV的腹水单抗C8和针对NDV的腹水单抗7D2、4B4稳定性均较高,抗酸、碱、热的能力较强。特异性血凝抑制(HI)反应证明6株NDV单抗均显示良好的特异性。阻断试验表明7D2、C8单抗对相应的病毒具有较好的特异性。H9N2亚型AIV血凝抑制特异性腹水单抗在鸡胚中和试验中不表现中和活性,可能与AIV中具有血凝活性的抗原位点与中和位点不完全等同有关。
利用C8腹水单抗建立了检测H9N2亚型AIV的抗原捕获单抗介导双夹心间接ELISA(ACELISA)方法。经测定,鸡抗AIV高免血清的最佳包被稀释度为1:1600,C8腹水单抗的最佳稀释比例是1:1600,检测灵敏度为843.75ng/mL,且该方法具有良好的稳定性。
本试验应用混合免疫、一次融合、分别筛选的新方法,获得的杂交瘤细胞株分泌的单克隆抗体,与传统方法制备的杂交瘤细胞株分泌的单克隆抗体相比,效价相似,同样特异且稳定。与用单一纯化抗原免疫小鼠的传统方法相比,本研究建立的混合免疫、分别筛选方法,仅一次免疫、一次融合就可筛选出分别稳定分泌针对两种甚至更多种不同抗原的单克隆抗体的杂交瘤细胞株,不仅节约了实验材料、降低了成本,而且节省了时间和人力,大大提高了建立杂交瘤细胞株的工作效率,是一种建立杂交瘤细胞株的高效方法,对杂交瘤技术的学术研究和实际应用均具有重要意义。
Several hybridoma cell strains were developed by fusion of SP2/0 mouse myeloma cells with splenocytes isolated from a Balb/c mouse immunized with the mix immunity antigen constituted by the inactive purified H9N2 subtype Avian Influenza virus(AIV),Newcastle Disease virus(NDV)LaSota strain and immunity intensifier Bursin,and the technique which fused together,filtrated separately was adopted.Two indirect enzyme-linked immunosorbent assay(ELISA)methods were established to respectively detect monoclonal antibodies(McAbs)against H9N2 subtype AIV and NDV secreted by hybridoma cell strains.Two hybridoma cell strains secreting the monoclonal antibodies against H9N2 subtype AIV were named C5,D5 and four hybridoma cell strains secreting the monoclonal antibodies against NDV were named 4B4,4B8,4C4,4E8.
Three hybridoma cell strains secreting McAbs against H9N2 subtype AIV, named C8,D7,E8 were obtained after fusion with the single immunity antigen H9N2 subtype AIV,two hybridoma cell strains secreting McAbs against NDV,named 7D2, 8C10 were obtained after fusion with the single immunity antigen NDV by traditional monoclonal antibody technique.
The maximal ELISA titers of the McAbs against H9N2 subtype AIV in cell supernatants and ascites were 1:1024 and 1:102400,respectively.The maximal ELISA titers of the McAbs against NDV in cell supernatants and ascites were 1:512 and 1:51200,respectively.All McAbs were categorized into IgG subtype.Average chromosome number of each hybridoma cell strain was between 96 and 106.These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing and anabiosis three times in six months.The hybridoma cell strains C8、7D2、4B4 showed good stability to acid、alkali and heat.The result of cross-reaction in HI demonstrated that the McAbs against NDV were subtype-specific.The result of the hybridoma cell strain C8 and 7D2 by interdicted ELISA indicated that the two McAbs had good specificity to relevant viruses.The McAbs against H9N2 subtype AIV had high HI titer to H9N2 subtype AIV,but failed to neutralize the H9N2 subtype AIV in chicken egg embryo.These conflicting results of HI cross-reaction and neutralization test may be concluded that the HA epitopes is not same as the sites of neutralization in AIVs.
Antigen capture enzyme-linked immunosorbent assay(AC-ELISA)was developed by the McAbs in ascite of the cell strain C8.The optimum conditions in some critical steps of the ELISA were set as follows:the optimal concentrations of the coating antigen and the McAbs against H9N2 subtype AIV were 1:1600 and 1:1600. The limit of quantitative detection in H9N2 subtype AIV was 843.75ng/mL.The AC-ELISA established in the present study should be further consummated to suit producing rapid diagnostic slip by combining gold-labeled chromatography to be applied in practice.
经鉴定,抗H9N2亚型AIV单抗的杂交瘤细胞培养上清及小鼠腹水单抗的ELISA效价最高可达1:1024和1:102400,抗LaSota株NDV单抗的杂交瘤细胞培养上清及小鼠腹水单抗的ELISA效价最高可达1:512和1:51200。单抗亚型均为IgG类抗体分子。各株杂交瘤细胞的平均染色体数目在96-106之间,符合SP2/0细胞与正常小鼠脾细胞的染色体数目之和。连续传代25次和三次冻存复苏后,各杂交瘤细胞抗体分泌能力有所下降,但仍能稳定分泌抗体。针对H9N2亚型AIV的腹水单抗C8和针对NDV的腹水单抗7D2、4B4稳定性均较高,抗酸、碱、热的能力较强。特异性血凝抑制(HI)反应证明6株NDV单抗均显示良好的特异性。阻断试验表明7D2、C8单抗对相应的病毒具有较好的特异性。H9N2亚型AIV血凝抑制特异性腹水单抗在鸡胚中和试验中不表现中和活性,可能与AIV中具有血凝活性的抗原位点与中和位点不完全等同有关。
利用C8腹水单抗建立了检测H9N2亚型AIV的抗原捕获单抗介导双夹心间接ELISA(ACELISA)方法。经测定,鸡抗AIV高免血清的最佳包被稀释度为1:1600,C8腹水单抗的最佳稀释比例是1:1600,检测灵敏度为843.75ng/mL,且该方法具有良好的稳定性。
本试验应用混合免疫、一次融合、分别筛选的新方法,获得的杂交瘤细胞株分泌的单克隆抗体,与传统方法制备的杂交瘤细胞株分泌的单克隆抗体相比,效价相似,同样特异且稳定。与用单一纯化抗原免疫小鼠的传统方法相比,本研究建立的混合免疫、分别筛选方法,仅一次免疫、一次融合就可筛选出分别稳定分泌针对两种甚至更多种不同抗原的单克隆抗体的杂交瘤细胞株,不仅节约了实验材料、降低了成本,而且节省了时间和人力,大大提高了建立杂交瘤细胞株的工作效率,是一种建立杂交瘤细胞株的高效方法,对杂交瘤技术的学术研究和实际应用均具有重要意义。
Several hybridoma cell strains were developed by fusion of SP2/0 mouse myeloma cells with splenocytes isolated from a Balb/c mouse immunized with the mix immunity antigen constituted by the inactive purified H9N2 subtype Avian Influenza virus(AIV),Newcastle Disease virus(NDV)LaSota strain and immunity intensifier Bursin,and the technique which fused together,filtrated separately was adopted.Two indirect enzyme-linked immunosorbent assay(ELISA)methods were established to respectively detect monoclonal antibodies(McAbs)against H9N2 subtype AIV and NDV secreted by hybridoma cell strains.Two hybridoma cell strains secreting the monoclonal antibodies against H9N2 subtype AIV were named C5,D5 and four hybridoma cell strains secreting the monoclonal antibodies against NDV were named 4B4,4B8,4C4,4E8.
Three hybridoma cell strains secreting McAbs against H9N2 subtype AIV, named C8,D7,E8 were obtained after fusion with the single immunity antigen H9N2 subtype AIV,two hybridoma cell strains secreting McAbs against NDV,named 7D2, 8C10 were obtained after fusion with the single immunity antigen NDV by traditional monoclonal antibody technique.
The maximal ELISA titers of the McAbs against H9N2 subtype AIV in cell supernatants and ascites were 1:1024 and 1:102400,respectively.The maximal ELISA titers of the McAbs against NDV in cell supernatants and ascites were 1:512 and 1:51200,respectively.All McAbs were categorized into IgG subtype.Average chromosome number of each hybridoma cell strain was between 96 and 106.These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing and anabiosis three times in six months.The hybridoma cell strains C8、7D2、4B4 showed good stability to acid、alkali and heat.The result of cross-reaction in HI demonstrated that the McAbs against NDV were subtype-specific.The result of the hybridoma cell strain C8 and 7D2 by interdicted ELISA indicated that the two McAbs had good specificity to relevant viruses.The McAbs against H9N2 subtype AIV had high HI titer to H9N2 subtype AIV,but failed to neutralize the H9N2 subtype AIV in chicken egg embryo.These conflicting results of HI cross-reaction and neutralization test may be concluded that the HA epitopes is not same as the sites of neutralization in AIVs.
Antigen capture enzyme-linked immunosorbent assay(AC-ELISA)was developed by the McAbs in ascite of the cell strain C8.The optimum conditions in some critical steps of the ELISA were set as follows:the optimal concentrations of the coating antigen and the McAbs against H9N2 subtype AIV were 1:1600 and 1:1600. The limit of quantitative detection in H9N2 subtype AIV was 843.75ng/mL.The AC-ELISA established in the present study should be further consummated to suit producing rapid diagnostic slip by combining gold-labeled chromatography to be applied in practice.
引文
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