单抗夹心ELISA检测鸡新城疫病毒抗原的研究
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摘要
鸡新城疫（Newcastle Disease ND）俗称亚洲鸡瘟，是由禽副粘病毒1型（APMV-1）引起的鸡的一种高度接触性传染病。近年来，随着非典型ND传播与流行的日渐增多，传统的诊断方法已不能完全满足临床需要。本研究应用自制单抗建立的单抗夹心ELISA试验方法，可对该病作出快速、准确诊断。
本文分为两部分。第一部分是鼠抗新城疫病毒（Newcastle Disease Virus NDV）单克隆抗体的制备及其生物学特性鉴定，第二部分是应用自制单抗建立夹心ELISA试验方法并对其反应条件进行优化。
蔗糖密度梯度超速离心法纯化NDV效果良好。接种NDV F48E9株于10～11日龄鸡胚尿囊腔，平均每枚鸡胚收获尿囊液约8mL，血凝价（HA效价）在29～212之间。尿囊液粗提后再经20～60%、梯度为20%的蔗糖密度梯度超速离心法进一步纯化，在40～60%的蔗糖梯度层有一乳白色的病毒带。透析后，测其HA效价高达215，较纯化前有明显提高。用Folin酚法测得病毒的蛋白浓度约为1mg/mL。
建立了筛选阳性杂交瘤细胞的间接ELISA试验方法并对其反应条件进行优化。当纯化的NDV抗原每孔包被47ng，酶标抗体羊抗鼠IgG-HRP的稀释度为1∶800时，阳性对照的OD492值大于1.0，而阴性对照的OD492值小于0.1。分别用鼠抗NDV抗血清和健康鼠血清作为阳性和阴性对照，对照血清的稀释度为1∶1600。
用蔗糖密度梯度超速离心法纯化的NDV作为抗原，按常规方法免疫6～8周龄雌性Balb/C小鼠。小鼠脾细胞与骨髓瘤细胞SP2/0按2∶1至5∶1的比例混合，经50%PEG 4 000融合后，HAT培养基选择培养10～12天进行阳性筛选。融合细胞分置6块96孔细胞培养板培养，共576孔，其中391孔有杂交瘤细胞生长，融合率为67.9%；经间接ELISA检测上清得阳性细胞孔数40，阳性率为6.9%。挑选出其中OD492值在1.0以上的孔共10孔进行有限稀释法克隆或亚克隆。最终得到3株能稳定分泌抗体并产生腹水的杂交瘤细胞3株，分别命名为1F9、1C6、4D5。
按5×105细胞/0.2 mL/只的量将3株杂交瘤细胞分别注入小鼠腹腔，约15
天后，小鼠腹部明显涨大，用注射器分次收集腹水，腹水呈黄色至淡血红色不等。平均每只小鼠可获4～5mL腹水。应用辛酸-硫酸铵法对腹水进行纯化，蛋白得率为28.2%～31.4%。
对3株单抗的部分生物学特性进行鉴定，结果为：三株单抗均具有较高的ELISA效价（1.5×104～1×105）；1F9、4D5两株单抗同时具有较高的HI效价（29，28），而中和效价（VN）偏低（二者都小于10）；单抗1C6株具有较高的VN效价(103)，却无血凝抑制能力（HI效价为0）。
配对试验的结果显示：1C6、 4D5分别作为包被单抗和酶标记单抗时，ELISA反应有较高的OD492值，系统的敏感性较其它组合要高。在此基础上建立了单抗夹心ELISA试验方法，并对其反应条件进行优化。试验结果表明：酶标板经紫外线照射处理，包被单抗1C6、酶标单抗4D5-HRP分别作800倍、1600倍稀释，包被液、封闭液、酶标抗体稀释液分别选用0.05mol/L pH9.6 Na2CO3-NaHCO3、l%BSA和4%NCS-PBS，酶标抗体和底物分别作用30min和15min时所测得的阳性OD492值相对较高，阳性OD492值与阴性OD492值之比最大，试验效果最好。
对单抗夹心ELISA反应系统的特异性、敏感性和重复性作了鉴定。结果表明：NDV阳性血清可特异性阻断酶标抗体与NDV之间的的反应；该系统最少可检出2.5ng/mL的NDV纯化蛋白；该系统检测阳性样品的变异系数（CV）小于5%。
Newcastle Disease(ND), commonly called as asian roup, is a highly contagious disease of birds caused by avian paramyxovirus serotype 1(APMV-1). In recent years, traditional diagnosis method would no longer effective in clinical along with the rising rate of non-typicle ND, a kind of ND which has no typical symptom and pathological changes. We prepared three lines of monoclonal antibodies(McAbs) and developed a McAbs sandwich enzyme-linked immunosorbant assay(ELISA) which could satisfy the clinical demand in this study.
The paper included two parts. The destiny of the former was to get the McAbs against Newcastle Disease Virus(NDV) which would be used in the sandwich ELISA. In the second part, we developed the sandwich ELISA based on two lines of the McAbs and optimized the terms of the assay.
It is very effective to concentrate virions by high speed centrifugation through sucrose cushions. First of all, we inoculate NDV F48E9 strain in 10 to 11-day-old embryos and harvested 8mL allantoic fluids average. Then the concentrated viral stock was overlaid on 20～60%,20% distance sucrose gradient. Last, harvested the fractions between 40～60% gradient. After purifying, the hemagglutination (HA) titers of NDV was 215, much higher than the allantoic fluids, 28～29, and the concentration was 1 mg/mL by Folin phenol method.
An indirect ELISA procedure was established and optimized to screen positive hybridoma. The optimal coating quantity was 47 ng per well and the 4D5-HRP should be diluted by 800 times. Serum samples from immunized and healthy mice were measured as positive and negative control respectively.
Through immuning Balb/C mouse with immunogen that is virulent NDV F48E9 strain purified by the method of sucrose density centrifuge, cellular fusion, clone and detection, we obtained three lines of hybridoma cells named 1F9、1C6、4D5 that could secrete McAbs. The fusion rate was 67.9%(391/576) and the positive rate was 6.9%(40/576). Hybridoma that producing McAbs anti-NDV was prepared by fusion of SP2/0 murine myeloma cells with spleen cell isolated from
the immunized mouse.
McAbs were produced in vivo by mouse ascites method and 4～5 mL ascites per mouse could be harvested. The ascites was purified by C8H16O2-(NH4)2SO4 and the recovery rate of target protein was 28.2～31.4%.
Some of the biology characteristics of the McAbs cell lines were verified and the results demonstrated that all of them had high ELISA titers(1.5×104～1×105). 1F9 and 4D5 had high haemagglutination inhibition (HI) titers(29,28) also, but the virus neutralisation(VN) tiers were lower than 10. While 1C6 had high VN tiers(103) even though the HI tiers was 0.
1C6 and 4D5 were used as coating and peroxidase-labelling McAbs respectively by antibody pairing test. Based on them, we developed and optimized the sandwich ELISA. The results shown that: (1)the plate which irradiated by X-ray could adsorb more coating protein; (2)1C6 and 4D5-HRP should diluted by 800 and 1600 times respectively; (3) 0.05mol/L pH9.6 Na2CO3-NaHCO3, l%BSA and 4%NCS-PBS were suitable to work as coating buffer, blocking buffer and peroxidase-labelling antibody diluting buffer respectively; (4)the reacting time of 4D5-HRP and OPD was 30min and 15min respectively.
The sandwich ELISA based on McAbs was experimented with specificity, sensitivity and repetition. The result demonstrated that: (1)NDV-specific antibodies could stop the reaction between 4D5-HRP and NDV; (2)the minimum detectable concentration by this assay was 2.5ng/mL; (3)CV of intra-assay was smaller than 5%(n=6).
本文分为两部分。第一部分是鼠抗新城疫病毒（Newcastle Disease Virus NDV）单克隆抗体的制备及其生物学特性鉴定，第二部分是应用自制单抗建立夹心ELISA试验方法并对其反应条件进行优化。
蔗糖密度梯度超速离心法纯化NDV效果良好。接种NDV F48E9株于10～11日龄鸡胚尿囊腔，平均每枚鸡胚收获尿囊液约8mL，血凝价（HA效价）在29～212之间。尿囊液粗提后再经20～60%、梯度为20%的蔗糖密度梯度超速离心法进一步纯化，在40～60%的蔗糖梯度层有一乳白色的病毒带。透析后，测其HA效价高达215，较纯化前有明显提高。用Folin酚法测得病毒的蛋白浓度约为1mg/mL。
建立了筛选阳性杂交瘤细胞的间接ELISA试验方法并对其反应条件进行优化。当纯化的NDV抗原每孔包被47ng，酶标抗体羊抗鼠IgG-HRP的稀释度为1∶800时，阳性对照的OD492值大于1.0，而阴性对照的OD492值小于0.1。分别用鼠抗NDV抗血清和健康鼠血清作为阳性和阴性对照，对照血清的稀释度为1∶1600。
用蔗糖密度梯度超速离心法纯化的NDV作为抗原，按常规方法免疫6～8周龄雌性Balb/C小鼠。小鼠脾细胞与骨髓瘤细胞SP2/0按2∶1至5∶1的比例混合，经50%PEG 4 000融合后，HAT培养基选择培养10～12天进行阳性筛选。融合细胞分置6块96孔细胞培养板培养，共576孔，其中391孔有杂交瘤细胞生长，融合率为67.9%；经间接ELISA检测上清得阳性细胞孔数40，阳性率为6.9%。挑选出其中OD492值在1.0以上的孔共10孔进行有限稀释法克隆或亚克隆。最终得到3株能稳定分泌抗体并产生腹水的杂交瘤细胞3株，分别命名为1F9、1C6、4D5。
按5×105细胞/0.2 mL/只的量将3株杂交瘤细胞分别注入小鼠腹腔，约15
天后，小鼠腹部明显涨大，用注射器分次收集腹水，腹水呈黄色至淡血红色不等。平均每只小鼠可获4～5mL腹水。应用辛酸-硫酸铵法对腹水进行纯化，蛋白得率为28.2%～31.4%。
对3株单抗的部分生物学特性进行鉴定，结果为：三株单抗均具有较高的ELISA效价（1.5×104～1×105）；1F9、4D5两株单抗同时具有较高的HI效价（29，28），而中和效价（VN）偏低（二者都小于10）；单抗1C6株具有较高的VN效价(103)，却无血凝抑制能力（HI效价为0）。
配对试验的结果显示：1C6、 4D5分别作为包被单抗和酶标记单抗时，ELISA反应有较高的OD492值，系统的敏感性较其它组合要高。在此基础上建立了单抗夹心ELISA试验方法，并对其反应条件进行优化。试验结果表明：酶标板经紫外线照射处理，包被单抗1C6、酶标单抗4D5-HRP分别作800倍、1600倍稀释，包被液、封闭液、酶标抗体稀释液分别选用0.05mol/L pH9.6 Na2CO3-NaHCO3、l%BSA和4%NCS-PBS，酶标抗体和底物分别作用30min和15min时所测得的阳性OD492值相对较高，阳性OD492值与阴性OD492值之比最大，试验效果最好。
对单抗夹心ELISA反应系统的特异性、敏感性和重复性作了鉴定。结果表明：NDV阳性血清可特异性阻断酶标抗体与NDV之间的的反应；该系统最少可检出2.5ng/mL的NDV纯化蛋白；该系统检测阳性样品的变异系数（CV）小于5%。
Newcastle Disease(ND), commonly called as asian roup, is a highly contagious disease of birds caused by avian paramyxovirus serotype 1(APMV-1). In recent years, traditional diagnosis method would no longer effective in clinical along with the rising rate of non-typicle ND, a kind of ND which has no typical symptom and pathological changes. We prepared three lines of monoclonal antibodies(McAbs) and developed a McAbs sandwich enzyme-linked immunosorbant assay(ELISA) which could satisfy the clinical demand in this study.
The paper included two parts. The destiny of the former was to get the McAbs against Newcastle Disease Virus(NDV) which would be used in the sandwich ELISA. In the second part, we developed the sandwich ELISA based on two lines of the McAbs and optimized the terms of the assay.
It is very effective to concentrate virions by high speed centrifugation through sucrose cushions. First of all, we inoculate NDV F48E9 strain in 10 to 11-day-old embryos and harvested 8mL allantoic fluids average. Then the concentrated viral stock was overlaid on 20～60%,20% distance sucrose gradient. Last, harvested the fractions between 40～60% gradient. After purifying, the hemagglutination (HA) titers of NDV was 215, much higher than the allantoic fluids, 28～29, and the concentration was 1 mg/mL by Folin phenol method.
An indirect ELISA procedure was established and optimized to screen positive hybridoma. The optimal coating quantity was 47 ng per well and the 4D5-HRP should be diluted by 800 times. Serum samples from immunized and healthy mice were measured as positive and negative control respectively.
Through immuning Balb/C mouse with immunogen that is virulent NDV F48E9 strain purified by the method of sucrose density centrifuge, cellular fusion, clone and detection, we obtained three lines of hybridoma cells named 1F9、1C6、4D5 that could secrete McAbs. The fusion rate was 67.9%(391/576) and the positive rate was 6.9%(40/576). Hybridoma that producing McAbs anti-NDV was prepared by fusion of SP2/0 murine myeloma cells with spleen cell isolated from
the immunized mouse.
McAbs were produced in vivo by mouse ascites method and 4～5 mL ascites per mouse could be harvested. The ascites was purified by C8H16O2-(NH4)2SO4 and the recovery rate of target protein was 28.2～31.4%.
Some of the biology characteristics of the McAbs cell lines were verified and the results demonstrated that all of them had high ELISA titers(1.5×104～1×105). 1F9 and 4D5 had high haemagglutination inhibition (HI) titers(29,28) also, but the virus neutralisation(VN) tiers were lower than 10. While 1C6 had high VN tiers(103) even though the HI tiers was 0.
1C6 and 4D5 were used as coating and peroxidase-labelling McAbs respectively by antibody pairing test. Based on them, we developed and optimized the sandwich ELISA. The results shown that: (1)the plate which irradiated by X-ray could adsorb more coating protein; (2)1C6 and 4D5-HRP should diluted by 800 and 1600 times respectively; (3) 0.05mol/L pH9.6 Na2CO3-NaHCO3, l%BSA and 4%NCS-PBS were suitable to work as coating buffer, blocking buffer and peroxidase-labelling antibody diluting buffer respectively; (4)the reacting time of 4D5-HRP and OPD was 30min and 15min respectively.
The sandwich ELISA based on McAbs was experimented with specificity, sensitivity and repetition. The result demonstrated that: (1)NDV-specific antibodies could stop the reaction between 4D5-HRP and NDV; (2)the minimum detectable concentration by this assay was 2.5ng/mL; (3)CV of intra-assay was smaller than 5%(n=6).
引文
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48罗廷荣,余克伦,韦家槐.ABC-ELISA检测鸡新城疫病毒[J].病毒学报,1990(3):262-268
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64 Alexander D.J,Manvell R.J,Lowings J.P,et al. Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies. Avian Pathology,1997(2):399-418.
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72陈伯权,吴美英,叶群瑞.几种部分纯化单克隆抗体方法的比较[J].病毒学报,1990(2):122-125
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41 赵虎,吴艳涛,文其乙等.新城疫单抗ELISA试剂盒检测鸽新城疫病毒[J]. 畜牧兽医杂志,2001(3):6-8
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43 曹洪敬,王文志,崔锦鹏等.Dot-ELISA间接法检测鸡新城疫病毒(NDV)的研究[J].中国动物检疫,1998(6):1-3
44 Swain P,Verma KC,Kataria JM,et al. Monoclonal antibody based dot-enzyme linked immunosorbent assay (Dot-ELISA) and agar gel precipitation test (AGPT) for identification of Newcastle disease virus (NDV). Indian J Exp Biol,1999(10):1037-1038
45 Cadman H.F,Kelly P.J,De Angelis,et al. Comparison of enzyme-linked immunosorbent assay and haemagglutination inhibition test for the detection of antibodies against Newcastle disease virus in ostriches (Struthio camelus). Avian Pathology,1997(2):357-363.
46 孙建宏,曹殿军,刘培欣等.检测NDV特异性IgG、IgM、IgA的间接ELISA方法的建立[J].中国预防兽医学报,2001(1):23-25
47 赵虎,张如宽,刘秀梵.新城疫单抗ELISA试剂盒监测免疫鸡群中新城疫强毒[J].中国预防兽医学报,2001(3):222-225
48罗廷荣,余克伦,韦家槐.ABC-ELISA检测鸡新城疫病毒[J].病毒学报,1990(3):262-268
49 郑世军,甘孟侯.间接法DOT-ELISA检测新城疫病毒的研究[J].畜牧兽医学报,1992(2):187-192
50 秦爱建,董国雄,沈素芳等.免疫酶斑点试验快速检测新城疫病毒[J].中国畜禽传染病,1990(4):34-35
51 曹殿军,卢景良,王莉林等.应用单克隆抗体鉴别新城疫病毒强、弱毒株的研
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52 卢建红,谈志祥,鲁杏华等.应用聚乙二醇介导的单抗夹心ELISA监测湖南新城疫病毒强毒感染[J].湖南农业大学学报(自然科学版),2001(5):391-393
53 宁官保,刘晋平,赵宇君等.用Dot-ELISA检测鸡新城疫病毒的研究[J].动物医学进展,1998(4):37-39
54 C.L.Kho,M.L.Mohd-Azmi,S.S.Arshad,et al. Performance of an RT-nested PCR ELISA for detection of Newcastle disease virus[J]. Journal of Virological Methods,2000(86):71-83
55 夏梦岩,张卓然.基因扩增产物微孔杂交法检测新城疫病毒致病毒株[J].中国动物检疫,2002(5):22-25
56谢芝勋,谢志勤,庞耀珊等.应用多重聚合酶链反应同时检测鉴别鸡新城疫病毒、鸡传染性支气管炎病毒、鸡传染性喉气管炎病毒和鸡毒支原体的研究[J]. 中国预防兽医学报,2000(6):443-446
57,刘福安.用PCR检测鸡新城疫病毒的初步试验[J].中国兽医科技, 1995(10):20-20
58东生,宋长绪.DNA-RNA杂交法检测新城疫病毒的研究[J].中国动物检疫,1997(5):10-11
59 Norbert St?uber,Katrin Brechtbühl,Lukas Bruckner,et al. Hofmann.Detection of Newcastle disease virus in poultry vaccines using the polymerase chain reaction and direct sequencing of amplified cDNA[J]. Vaccine,1995(4):360-364
60 黄庚明,辛朝安.应用逆转录套式PCR检测新城疫病毒核酸[J].中国预防兽医学报,2001(4):295-299
61 夏梦岩,张卓然,秦成等.基因扩增产物微孔杂交法检测新城疫病毒致病毒株[J].中国动物检疫,2002(5):22-25
62 贺东生,宋长绪,刘福安等.用核酸探针检测新城疫病毒的研究[J].中国畜禽传染病,1997(4):3-4
63 周维松,刘秀梵.新城疫病毒单克隆抗体的中和作用[J].病毒学报,1991(1): 23-29
64 Alexander D.J,Manvell R.J,Lowings J.P,et al. Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies. Avian Pathology,1997(2):399-418.
65 程由铨,李怡英,林天龙等.新城疫病毒单克隆抗体的特性及应用[J].病毒学报,1987(4):332-337
66 曲鲁江,曹殿军,单文鲁.单克隆抗体技术在新城疫研究中的应用[J].预防兽医学进展,2000(4):19-21
67 周维松,刘秀梵.用单克隆抗体研究鸡新城疫病毒抗原变异与流行病学的相关性[J].中国病毒学,1992(4):449-455
68 许辉.单克隆抗体的纯化[J].生物工程学报,1986(1):6-9
69 郭春祥,郭锡琼.介绍一种简单、快速、高效的辣根过氧化物酶标记抗体的过碘酸钠法[J].上海免疫学杂志,1983(2):97-100
70 黄宇峰,杨毓华,李保同.降植烷、石蜡油、不完全福氏佐剂诱导小鼠产生腹水型McAb的实验研究[J].单克隆抗体通讯,1988(3):78
71 徐修礼.广谱抗LPS-McAb的交叉反应性及内毒素定量双抗体夹心ELISA的建立.第四军医大学硕士论文,2001:42
72陈伯权,吴美英,叶群瑞.几种部分纯化单克隆抗体方法的比较[J].病毒学报,1990(2):122-125
73 梁荣,伊岚,李健强.小鼠IgG类单克隆抗体三种纯化方法的比较.细胞与分子免疫学杂志,1996(1):55-58
74 董伟,文其乙,吴艳涛等.NDV单抗针对多肽抗原的鉴定[J].江苏农业研究,1999(3):59-62
75杨书豪,马丽丽,杨艳艳等.抗人甲胎蛋白单克隆抗体的制备及鉴定.细胞与分子免疫学杂志,1997(3):45-47
76 李晨旭,赵洪阳,紫外处理聚苯乙烯微孔板的ELISA法检测抗原.白求恩医科大学学报,1998(1):103-104