利用Asia1型口蹄疫病毒VP1蛋白的单克隆抗体建立单抗竞争ELISA方法

Agricultural Science＆Technology，2009，10(3)：104—107 Copyright⑥2009，Information Institute of HAAS All rights resewed AnimaI Science Establ ish ment of Monoclonal Anti body Competitive ELISA Using Protein of Asia Monoclonal Anti body Agai nst VP1 1 Type Foot-and- Mouth Disease Vi rus UN Tong，SHAO Jun-lun，CONG Guo·zheng，DU Jun-zheng，GAO Shan-dian，CHANG Hui。yun ，XIE Qing’ge Key Laboratory of Animal Virology，Ministry of Agriculture／State Key Laboratory of Veterinary Etiological Biology／Lanzhou Veterinary Research Institute，Chinese Academy of Agricultural Sciences，Lanzhou 730046 Abstract Using the purified VP1 protein of Asia 1 type foct-and—mouth disease virus as the antigen．the purified monoclonal antibody was la- beled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study．Ten positive porcine foct。 and—mouth disease serums and more than two hundreds negative serum were tested，and the results were the same as the background of sam。 pies．The sensitivity test and replicate test indicated that this method was stable and sensitive，which was suitable for monitoring Asia 1 type por- cine foot— and——mouth disease virus antibody． Key words Asia 1 FMDV：VP1 monoclonal antibody：Competitive ELlSA Foot．and．mouth disease is a kind of infectious disease of artiodactyls caused by foot-and mouth disease virus．which is pyretic．acute and high—tangent⋯ ．The disease distributes in the worldwide and leads to people’s Iire and social economic lnstability due to its strong harmfulness．SO it js also known as “politicaI economic disease“1 2j．Thus ． this disease has been Iisted as class A fulminating infectious disease by Office Inter· nationaf des Epizooties f OIE)and Food and Agriculture O卜 ganization f FAO)．Foot-and-mouth disease has seven serum types such as O，A and C(European type)，Asia 1(Asian type)，SAT1，SAT2 and SAT3 (African type)，and there is no cross—protection among different types of serum ．How． ever，the antigenicity are various among subtypes in the same types of serum．and the serologicaI cross—reactivity is also dif— ferent，which causes serious problems for diagnosis and pre． vention of foot—and—mouth disease． Foot·and-mouth disease virus belongs to the family Picor．． navidae，genus Aphthovirus．which shows sphericaI structure with regular dodecahedron．while its diameter is 20 —30 nm and molecular weight is 6．9 x 1 O Pa with sedimentation coeffi． cient of 140s一146s ．The complete virus panicles are com． posed of capsid protein，positive·strand RNA virus，some non-structural protein with non—trace value and actin in host cells，which has no envelope and is sensitive to pH value． The fulI length RNA of foot—and-mouth disease virus is about 8．5 kb，while VP4．VP2，VP3 and VP1 are four types of structuraI proteins，which constitutes the virus capsid protein with 60 molecular weight for each onel ．The structuraI pro． tein VP1 3 js constituted by 20o一3oo amino acids．while VP1 is the only virus protein which is resistant to protein hydrolase and neutralizing antibody or protection in animals．140—160 amino acids of VP1 are exposed to the surface of virus parti． cles．which is known as foot-and-mouth disease virus．circle and constitutes the major antigen site together with C end of Received：April 23，2009 Accepted：June 2．2009 SuppoSed by National High—tech R&D Program (863)Subsidized Project(2006AA1 0A204)；Special Fund for Basic Scientific Re． search-related Subsidy of State—leveI and Public．welfare Scientific Research lnstitutes． Corresponding author．E-maiI：changhuiyun@ 126．com VP1．The peptide synthesized by the separated foot—and— mouth disease virus—circle can produce neutralizing protective antibody with high Jeve1．GH circle of VP1 including 134—16O residues not only conStitutes the major antigen site，but also has the consewed RGD sequence(Arg-Gly—Asp)that is es- sentiaI for virus absorbing cellsl ． Recently，Asia 1 lvpe foot—and-mouth disease has 0(3- curred in many provinces or cities in China．which severely in— fluences livestock product trade and people’s Iire．Therefore． it seems especially important for preventing from the sprea— ding of epidemic and enhancing the monitoring of antibody fev· els，which is also necessary to establish the cOrrespOnding antibody testing method．The monoclonaI antibody has higher homogeneity and specificity，which has been widely used in biologicaI and medicaI fields．A testing method with strong sensitivity and good specificity was established by the mono- clonal antibody against VP1 protein of Asia 1 type to monitor the antibody Ievels of aniodactyIs in this study．which provided effective data for lmmune protection and references for im— mune controI． Materials and Methods Materials Newborn bovine serum was purchased frclm GIBICO Big— logical Company． penicillin and streptomycin frOm Harbin Pharmaceutical Group Sixth Pharm Factory．glycerine frOm Sigma Company，sodium periodate from Shanghai Biological Reagent Company，skim milk fr0m Feihe Dairy Co．．Ltd and Babl／c mouse frOm Lanzhou lnstitute of Biological Products． Hybridoma cell recovery。mouse rehydration preparation and test for titers and subtypes The frozen hybridoma ceils were recovered by experi． mentaI guide for cell culture．and the cultivated hybddoma cells were injected into mouse peritoneal treated bv the steri． 1ized glycerine．After severaI days．mouse rehydration was collected and precipitated with the saturated ammonium sul fate to remove lipid，then purified with octanoic acid．ammOn_． um sulfate to obtain the monoclonaI antibody．The titers of monoclonal antibody were measured by the indirect ELlSA method．while the subtype was determined with SIGMA mon． LIN Tong et a1．Establishment of Monoclonal Antibody Competitive ELISA Using Monoclonal Antibody Against VP]Protein of Asia 1 Type Foot—and—Mouth Disease Virus 1 05 oclonaI antibody subtype kit and its steps were described in the instruction． Preparation for the enzyme-labeled antibody Preparation for the enzyme—labeled antibody was refer．． enced to the modified sodium periodates enzyme．．1abeled anti．． body method． Competitive ELISA reaction procedure The expression protein was diluted to the optimum coat． ing concentration by 0．05％ PBS with pH value of 9．4．while each hole of the coating enzyme-labeled board was 1 oo uI， and there was a blank in each board as the zero adjustment for the reading data of enzyme·labeled instrument．The en— zyme-labeled board was slightly vibrated and cultivated for 2 h al 37℃．and then sealed for overnight at 4 oC after the coat． ing solution was discharged． Next day，the sealed solution was cleared and washed for four times．and then enzyme—la— beled monoclonal antibody was added after being dried．Next． the reaction continued for 30 min at 37 oC ． and the substrate showed color in dark for 1 0 min after being washed for five times． Finally． the reaction was terminated with 2 mol／L H2 SO4 to measure its DD跚 value． Determination for antigen and enzyme-labeled antibody concentration The working concentration of coating antigen and en— zyme-labeled antibody was determined with method of square titration． Analysis of the optimaI dilution of serum or reaction time and positive or negative judgment Ten positive serums referenced to the Iaboratory standard and ten standard negative serums were selected and diluted to twofold，fourfold，eightfold and tenfoId respectively，then the inhibitory rate of positive or negative serum was deter- mined for the optimal cI¨ution of serum．The inhibitory rate was referenced with the inhibitory rate test of 3D monoclonal antibody byYang M ．Inhibitory rate of serum ( )=(0 value of negative control一0D锄 value of test serum)／(o‰ value of negative control一 o value of positive contro1)x 1 00％． Thus，o value was measured by the competitive ELISA steps and the inhibitory rate of serum was calculated for the optimal reaction time． Fifty porcine foot—and-mouth disease positive serums and two hundreds porcine negative serum were tested by the competitive ELlSA method．and the inhibi- tory rate of serum was also calculated．sO the positive or neg· ative threshold value was iudged based on the distribution of Table 2 The optimal dilution of serum inhibitory rate of positive or negative serum． Sealed solution and determination for sealing time The samples were sealed with PBST diluted solution in． cluding 0．5％ ，1％ BSA，5％ skim milk and 5％ ．1O％ new． born bovine serum．Next．the sealed solution was incubated for 15 min，30 min．45 min，1 h．1．5 h and 2 h at 37 oC or sealed at 4 oC overnight(12—14 h)to determine the optimaI sealing concentration and time． Specificity and repeatability identification The positive serum of classical swine fever virus，porcine blue-ear disease serum and positive serum of foot．-and-mouth disease A．O．C and Asia 1 type or porcine negative serum were tested for three times by the established monoclonal antj． body competitive ELISA method to calculate the inhibitory ． ． rate Results and Analysis Determination for titers of monoclonaI antibody rehydraUon The monoclonaI antibody subtype was identified with the SIG- MA monoclonaI antibody subtype kit．and the results showed that two monoclonal antibodies of 1 B8 and 5 E1 were all classi- fled as IgG1，which could proliferated indefinitely after being recovered and its titers were 1 O f Table 1)． Table 1 Titers of monoclonal antibody The data in the Table were the average of three repeats Markers and titers of enzyme-labeled antibody The purified monoc JonaI antibody 1 B8 was Iabeled with HRP by the modified sodium periodates enzyme-．1abeled anti-- body method L ． and its titers were tested after being Iabeled for severaI times．The Iabeled monoclonal antibody with the best titer was the optimaI dilution of protein and the optimal enzyme-labeled concentration．Therefore．the dilution of mon— oclonal antibody was 2．76 ug／mI and the optimal enzyme-la- beled concentration was 1：1 2 8oo． Dilution of serum and the optimaI competitive time From the results of twofold，fourfold．eightfold and tenfold dilution comprehensively，the optimaI dilution was twofold． and there was gradient sign for the jnhibitory rate of serum． Meanwhile．the optimal competitive time was 60 min at 37℃ based on the inhibitory rate test of serum． Positive or negative judgment The inhibitory rate of fifty positive serums f≥50％ ，while thal of one hundred eighty—seven negative serums f≤40％ and thal of the other thirteen serums 40％ ≤ f≤6o％ ．Thus． the inhibitory rate，≥6o％ was classified as positive，while 4O％ as negative．and 40％ ≤f≤60％ as suspicious． Selection for se aled solution The results suggested Ihat the optimaI sealed condition was 1％ BSAfor2 h at 37℃ or overnight at 4 oC。 Specificity and repe atability The D value of classicaI swine fever virus，porcine blue．ear disease and A，O，C and Asia 1 type FMD serum was tested，and the inhibitory rate except for Asia 1 type，≤ 50％ was classified as negative based on the results de- scribed in“1．7“．However．the inhibitory rate of Asia 1 type serum 』≥60％ was classified as positive。which was in ac- 106 cordance with the samples． The above serums were tested for three times and the re- suits were the same，which {ndicated that the established monoclonal antibody competitive ELISA method had g0ocl sta。 bility and repeatability． DISCUSSlonS As one of the fulminating infectious disease．foot-and— mouth disease is paid attention internationally．but some de- veloped countries declare their countries have no foot--and·- mouth disease． which sets internationaI trade barriers for manv developing countries and severely influences animal or Iivestock product trade ．Recently．conventionaIinactivated vaccines have been used for controlling the epidemic and out- break of foot—and-mouth disease In many developing coun— tries．However．the common international ways to test the an- tibod y Ievel of immune animals are virus neutralization test and Iiquid block ELISA．which are also the specified detection method in the international trade． The competitive ELlSA method is established by using monoclonaI antibody against Asia 1 type FMD with higher specificity and sensitivity in this study．which can avoid the influence of unknown factors in polyclonal antibodies to enhance the monitoring of antibody Jevels．According to the competitive ELISA method ．the opti— maI results are used in each step frOm coating．sealing and the optimaI competitive time to color reaction．which reduces the influence of non—specific background．Moreover．the posi- tive or negative iudgment is also determined based on the re- suits．However．the above results are alI obtained in the Iabo- ratory．while lts practicaI application stilI needs a lot of data and further study． Agricultural Science＆Technotogy Vo1．10，No．3，2009 Helerences 『1]BANER J，GYARMATI P，YA∞ UB A，ef a1．Microarray-based molecular detection of foot—and·mouth disease．vesicular stomatitis and swine vesicular disease viruses，using padlock probes I J『． Journal of Virological Methods．2007．143：2O0—206． f2]SANYAL A．VENKATAR MANAN R，PA丌NAlK B．Antigenic lea- tures of foot and mo uth disease viruser O type AsiaI as revealed by mo noclonal antibod ies and Reutralization escapemutants i J 1． Virus Res．19g7。4：168—173． 『3]AGGARWAL N．BARNET-r PV．Antigenic sites of foot-and—mouth disease virus(FMDV)：an analysis of the specificities of anti．FM- DV antibodies after vaccination of naturally susceptible host spe- cies『J]．J Gen Vi巾l。2002．83：775—782． f41 YU XL，XIAO SB，FANG LR，ef a High expression of the foot— and-mouth disease structuraI protein P1 in Escherichia coil and analysis of its biology activity[J]．Chin J Biotsch，2OO5，21(1)： 163—166． 『5 1 ARMSTRONG RM．The detection of antibodies agains foot-and- mouth disease virus in sheep milkfJ]．J VIr0 Methods，1997，69 (1—2)：45-51． 『61 KITSON JD．Sequence analysis of mo nocJonaI antibody resistant- mutant of type O FMDV：evidence for the involvemo nt of the three surface exposed capsid proteins in four antigenic sites『J] Virolo— gy，1990，179：26—34． 『7]、限NG M．CU lJ0 A，Ll M．et a1．Identification of a major anti- bOdv binding epitope in the non-structuraI protein 3D of foot-and一- mo uth disease virus in cattle and the development of a monoclonal antib0dy wilh cIiagnostic applications『J1．JournaI of Immunological Method s．2OD7，321：174 —181． 『8]ARMSTRONG RM，COX sJ．AGGARWAL N，ef a1．Detection of antibody to the foot-and·mouth disease virus(FMDV)non-struc- tural polyprotein 3ABC in sheep by ELISA[J]．Journal of Virologi· caJ Method s．制 7，125：153—163． 『9]BARNARD AL．ARRIENS A．COX S。ef Immune response characteristics foIlowing emergency vaccination of pigs against foot-and-mouth disease『J]．Vaccine．2005．23：10G7—1047． Responsible editor-CHEN Juan Responslble translator：LI Jing—wei Responsible proofreader：WU Xiao-yan 利用Asia l型口蹄疫病毒vP1蛋白的单克隆抗体建立单抗竞争 ELISA方法 林彤，邵军军，丛国正，独军政，高闪电，常惠芸 ，谢庆阁 (中国农业科学院兰州兽医研究所，家畜疫病病原生物学国家重 点实验室，农业部畜禽病毒学重点实验室，甘肃兰州 730046) I 材料与方法 1．1 材料 新生牛血清购自GIBICO生物公司；青霉素、链霉素 购自哈尔滨制药六厂；降脂烷购 自sigma公司；过碘酸钠购 自上 海生物试剂公司；脱脂乳购自飞鹤乳业公司；Babl／c小鼠购自兰 州生物制品研究所。 1．2 杂交瘤细胞的复苏、小鼠复水制备、效价及亚型测定 按 照细胞培养实验指导复苏冻存杂交瘤细胞，将培养好的杂交瘤 细胞注射人经灭菌降脂烷处理的小鼠腹腔，几天后收集小鼠腹 水，用饱和硫酸铵沉淀去脂，再用辛酸 一硫酸铵进一步纯化，获 得单克隆抗体。单抗的效价采用间接 ELISA方法测定；亚型测 定用SIGMA单抗亚型试剂盒测定，具体步骤按照说明书操作。 1．3 酶标抗体的制备 酶标抗体的制备参照该实验室改良后 的碘酸钠酶标抗体法进行。 1．4 竞争ELISA反应程序 将表达蛋白用 0．05％、pH值 9．4 的PBS稀释至最适包被浓度，包被酶标板 ，每孑L 100 l，每板均 设空白孔，作为酶标仪读数调零用，轻振酶标板，37℃温育 2 h， 弃包被液，封闭，4℃过夜。次El，控干封闭液，洗涤4次，尽量减 少背景值，加入被检血清5O l，反应 1 h，洗涤4次，拍干后加酶 标单抗，37℃反应30 min，洗涤5次，加入底物避光显色 lO min， 用2 mol／L H：SO 终止反应，测 OD 值。 1．5 抗原浓度及酶标抗体浓度的确定 采用方阵滴定来确定 包被抗原和酶标抗体的工作浓度。 1．6 检测血清最佳稀释度、最佳反应时间及阴阳性的界定 选 取参考实验室阳性血清 10份，标准阴性血清 1O份，分别稀 释2、4、8、16倍 ，测定阴阳血清的抑制率，从而确定最适稀释倍 数。抑制率参照 Yang M等关于3D单克隆抗体所做的抑制率实 验。血清抑制率(，％)=(阴性对照 OD 值 一被检血清 0D 值)／(阴性对照 OD6，。一阳性对照 OD 。)x 100％。按照竞争 ELISA操作步骤，测 OD 。值，计算血清抑制率，确定最佳反应时 间。用竞争 ELISA检测 5O份猪口蹄疫感染阳性血清、200份猪 阴性血清，计算血清抑制率，并根据阴阳性血清抑制率分布界定 阳性和阴性临界值。 1．7 封闭液及封闭时间的确定 用含有0．5％、1％的 BSA．5％ 脱脂奶粉，以及 5％、10％新生牛血清 PBST稀释液封闭，按照37 ℃孵育 15 min、30 min、45 min、1 h、1．5 h、2 h或 4℃封闭过夜 (12—14 h)，确定最佳封闭浓度及封闭时间。 LIN Tong et aL Establishment of Monoclonal Antibody Competitive ELISA Using Monoclonal Antibody Against VP!Protein of Asia 1 Type Foot-and-Mouth Disease Virus 1 07 1．8 特异性及重复-眭鉴定 用建立的单抗竞争 ELISA检测猪 瘟阳性血清，猪蓝耳病血清，口蹄疫 A、O、C、Asial型血清阳性样 品，以及猪阴性血清。将上述样品测定 3次，计算抑制率。 2 结果与 2．1 单抗腹水效价测定 利用 SIGMA亚类鉴定 KIT鉴定单克 隆抗体的亚类，结果表明1B8、5E1这 2株单抗都属于IgG1，经复 苏后可以持续传代，而且2株单抗的效价都稳定在 1O (表 1)。 2．2 酶标抗体的标记及效价 将纯化后的一株单抗 1B8经改 良后的过碘酸钠法标记 HRP，多次标记后测其效价。取效价最 好的一次标记单抗作为蛋白最佳稀释度和酶标记最佳稀释度。 最后确定单 抗稀 释浓度 为 2．76 g／ml，最佳标 记浓 度为 1：12 800。 2．3 血清稀释度及最佳竞争时间确定 综合比较稀释 2、4、8、 16倍的试验结果，确定 2倍稀释为最佳稀释度，此时血清抑制率 有梯度出现(表 2)。同时，根据血清抑制率试验结果测得最佳 竞争时间为 37℃温育 60 min。 表1 单克隆抗体效价 注：表中数据为3次试验数据的平均值。 表2 血清最适稀释度 2．4 阴阳性的界定 供试 5O份阳性血清 1／>50％；200份阴性 血清，其中 187份，≤40％，13份血清 40％≤，≤60％。根据试验 结果，将抑制率1>t60％界定为阳性，抑制率40％≤，≤60％界定为 可疑，抑制率 ，≤40％界定为阴性。 2．5 封闭液的选择 试验结果表明，1％的 BSA 37 oC 2 h或者 4℃过夜后封闭效果最佳。 2．6 特异性及重复-陛 将猪瘟，蓝耳病，A、C、O、Asia1型 FMD 血清测 DD 值，根据方法“1．7”所得试验结果将抑制率 ，≤50％ 判为阴性，而将Asial血清抑制率11>60％判为阳性，结果与样品 相符。将上述血清测定 3次，结果均相同，说明该竞争 ELISA方 法具有良好的稳定性和可重复性。 3 讨论 该试验主要应用特异性和敏感性较高的抗 Asial型FMD单 (上接第 103页) 表1 美凤蝶不同阶段核酸含量 p,g／mg 注 ：1 2为非滞育蛹，其中1为非滞育蛹前期，2为非滞育蛹后期；3 8为越冬蛹，其中3—7为从 1O月下旬至翌年 2月下旬，8为3月中 旬 ；9为越冬蛹羽化成虫初期。 含量呈下降趋势。1～3月含量变化不大，差异不显著。滞育 解除后羽化成虫初期含量较高，达 1．727 0 g／mg。与 RNA变 化一样，不同时期 DNA含量变化与该时期生命活动特征相对 抗建立竞争 ELISA，该方法可以避免多抗中不明因子的影响，从 而提高抗体水平的监测。根据竞争 ELISA的试验步骤，尽量每 一 步都采用最佳试验结果，从包被、封闭、最佳竞争时间以及显 色等，从而减低非特异性背景的影响，阴阳性的界定也是根据试 验结果来判定。但是，上述结果均为在实验室取得，具体应用于 实践还需大量数据来充实，有待进一步研究。 基金项目 国家高技术研究发展(863)资助项目(2006AA10A204)； 中央级公益性科研院所基本科研业务费专项资金项目资助。 作者简介 林彤(1977一)，男，四川中江人，硕士，助理研究员，从事动 物病毒学研究。 通讯作者。 收稿日期 2009-04-23 修回日期 2009-06．-02 应，在非滞育蛹初期可能细胞分裂较为活跃 ；滞育期间变化表 明，在 12月下旬的大幅度上升可能与抗冻物质的合成有关；滞 育蛹羽化成虫 DNA含量较高，超过滞育期间最高含量的2倍， 可能与生殖系统的快速发育有关。 3 结论与讨论 美凤蝶非滞育蛹 RNA含量为 4．614 0—7．946 3 n1g，滞育 蛹为4．326 0～5．885 3 mg，羽化成虫初期为2o．779 3 g／n1g。 非滞育蛹DNA含量为0．448 7—0．535 0峭／n1g，滞育蛹为0．452 0 ～ 0．828 3 oeVmg，羽化成虫初期为 1．727 0 oe／mg。 美凤蝶在11月下旬，RNA和RNA／DNA值增加，而从l1月 下旬至 1月下旬水分含量也持续上升，据此推断美凤蝶越冬蛹 在 l 1月下旬滞育开始解除，进入滞育发育结束期。从水分含量 比值变化推断，3月中旬左右滞育已完全解除，进入无滞育发育 阶段。 基金项目 国家林业局引进国际先进林业科学技术计划项 目(2005-4-59 和2008-4-68)。 作者简介 易传辉(1970一)，男，四川开江人，博士，副教授，从事昆虫生 态、观赏昆虫培育与利用研究。 通讯作者。 收稿日期 2009-02-11 修回日期 2009-06-02