抗Asia1型口蹄疫病毒单克隆抗体的制备及其金标试纸条的研制
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摘要
利用杂交瘤细胞技术，以表达的Asial型口蹄疫病毒VP1蛋白免疫BALB／c小鼠，无菌条件下取小鼠脾细胞与SP2／0细胞进行细胞融合。经多次克隆和间接ELISA筛选，获得三株能分泌抗Asial型口蹄疫的单克隆杂交瘤细胞株，并对其特性作了鉴定。在此基础上，利用胶体金免疫层析技术制备了Asial型口蹄疫病毒单抗胶体金诊断试纸条。
经单克隆抗体亚类鉴定，三株单克隆抗体依次为IgG1、IgG2a和IgG2b。杂交瘤细胞株的染色体计数为104条，在杂交瘤细胞染色体范围内。用间接ELISA对腹水和细胞上清液中单抗效价进行测定，三株单抗的腹水中单抗效价≥1∶10~6，而细胞上清中单抗效价≥1∶10~4。用叠加实验对3株单抗的抗原位点进行了分析，发现三株单抗针对2个不同的抗原位点，其中2株单抗作用于同一抗原表位。对所获得的三株单抗经SDS-PAGE电泳，结果显示，所有单抗重链的分子量均为45kD左右，轻链的分子量为25kD左右。三株杂交瘤细胞和注射小鼠腹腔内的杂交瘤细胞经过5次连续传代后，用间接ELISA对每代次的细胞上清中的单抗效价进行了检测，传代细胞每代的单抗效价稳定在1∶10~4左右，腹水的抗体效价稳定于1∶10~5～10~6。用Asial单抗分别与O、A、C、Asial型病毒抗原和Asial重组抗原进行反应，结果三株单抗均与Asial型口蹄疫病毒和表达VP1蛋白有特异的反应，而与C、O和A型口蹄疫抗原不发生交叉反应。
利用获得的单抗成功研制出了检测Asial型口蹄疫病毒抗原的金标快速诊断试纸条。用该试纸条分别对A、O、C和Asial型口蹄疫病毒抗原以及猪水泡病病毒抗原等87份田间样品进行了检测，发现检测Asial型口蹄疫病毒抗原的试纸条出现2条明显的条带，而A、O、C型口蹄疫病毒抗原以及猪水泡病病毒抗原只出现对照线，检测线没有条带，说明该试纸条不口蹄疫病毒A、O、C型以及猪水泡病病毒抗原发生反应，特异性强。用该试纸条对口蹄疫细胞毒液(10~(-7)TCID50)的10倍系列稀释液进行了检测，最低可以检测到大约10~(-4)TCID_(50)。初步实验确定该试纸条在4℃、37℃和室温下的有效期为4个月。对于Asial型口蹄疫病毒金标试纸条的特性和应用Asial型口蹄疫病毒单克隆抗体的诊断价值还有待进一步的研究。
The monoclonal antibody(McAb) has strong specific and repeat characters, and it can be make into productions of detection and be used in research fields. The purposes of this paper were for obtain monoclonal antibodies to Asial type Foot-and-Mouth Disease Virus(FMDV), and for make them into detective productions of FMDV antigen with a rapid, specific, sensitive and convenient mean.
The BALB/C mice were immunized 4 times with Asial type FMDV. VP1 protein。The myeloma cell line SP2/0 was fused with the spleen of immunized BALB/C by PEG-1000, and by several limiting dilution, three specific monoclonal antibodies were screened by indirect ELISA. The three specific monoclonal antibodies cell strains named 1B8, 5E1 and 5E2, respectively.
Among the three cell strains, 1B8 belonged to IgG_1 subclass of Ig, 5E1 and 5E2 belonged to IgG2b and IgG2a subclass of Ig,. The results of specificity cross test indicated that the three cell strains' monoclonal antibodies were not specific reaction with the A, C or O type of FMDV. Their titers in cell culture supematant were 1∶10~4, Their titers of abdomen liquor were 1∶4×10~6, respectively.
The purified protein of monoclon by SDS-PAGE showed the molecular weights of the heavy chain and light chain were about 45.0 kD and 25.0 kD. The number of chromosomal to SP2/0 cell strains were 50, to hybridoma cell strains were 104。
By mean of indirect ELISA, the antigenic epitopes was detected and recognized by1B8, 5E1 and 5E2. The results indicated that the epitopes recognized 1B8 overlapped partly that of 5E2, and the epitopes recognized 1B8 is different to that of 5E1.
In the appliance research test, lemon acid was made to colloidal gold and marked it with monoclonal antibody. The strips can recognize Asial type FMDV and VP1 protein, can detective 10~(-4) TCID_(50) virus particle, can store three months in different temperature condition。through experiment we find these strips have highly specificity、sensitivity and stability。
In this whole experiment, we obtained monoclonal antibodies to FMDV, and had a significance trial for make them into detective productions of FMDV antigen in the future.
经单克隆抗体亚类鉴定，三株单克隆抗体依次为IgG1、IgG2a和IgG2b。杂交瘤细胞株的染色体计数为104条，在杂交瘤细胞染色体范围内。用间接ELISA对腹水和细胞上清液中单抗效价进行测定，三株单抗的腹水中单抗效价≥1∶10~6，而细胞上清中单抗效价≥1∶10~4。用叠加实验对3株单抗的抗原位点进行了分析，发现三株单抗针对2个不同的抗原位点，其中2株单抗作用于同一抗原表位。对所获得的三株单抗经SDS-PAGE电泳，结果显示，所有单抗重链的分子量均为45kD左右，轻链的分子量为25kD左右。三株杂交瘤细胞和注射小鼠腹腔内的杂交瘤细胞经过5次连续传代后，用间接ELISA对每代次的细胞上清中的单抗效价进行了检测，传代细胞每代的单抗效价稳定在1∶10~4左右，腹水的抗体效价稳定于1∶10~5～10~6。用Asial单抗分别与O、A、C、Asial型病毒抗原和Asial重组抗原进行反应，结果三株单抗均与Asial型口蹄疫病毒和表达VP1蛋白有特异的反应，而与C、O和A型口蹄疫抗原不发生交叉反应。
利用获得的单抗成功研制出了检测Asial型口蹄疫病毒抗原的金标快速诊断试纸条。用该试纸条分别对A、O、C和Asial型口蹄疫病毒抗原以及猪水泡病病毒抗原等87份田间样品进行了检测，发现检测Asial型口蹄疫病毒抗原的试纸条出现2条明显的条带，而A、O、C型口蹄疫病毒抗原以及猪水泡病病毒抗原只出现对照线，检测线没有条带，说明该试纸条不口蹄疫病毒A、O、C型以及猪水泡病病毒抗原发生反应，特异性强。用该试纸条对口蹄疫细胞毒液(10~(-7)TCID50)的10倍系列稀释液进行了检测，最低可以检测到大约10~(-4)TCID_(50)。初步实验确定该试纸条在4℃、37℃和室温下的有效期为4个月。对于Asial型口蹄疫病毒金标试纸条的特性和应用Asial型口蹄疫病毒单克隆抗体的诊断价值还有待进一步的研究。
The monoclonal antibody(McAb) has strong specific and repeat characters, and it can be make into productions of detection and be used in research fields. The purposes of this paper were for obtain monoclonal antibodies to Asial type Foot-and-Mouth Disease Virus(FMDV), and for make them into detective productions of FMDV antigen with a rapid, specific, sensitive and convenient mean.
The BALB/C mice were immunized 4 times with Asial type FMDV. VP1 protein。The myeloma cell line SP2/0 was fused with the spleen of immunized BALB/C by PEG-1000, and by several limiting dilution, three specific monoclonal antibodies were screened by indirect ELISA. The three specific monoclonal antibodies cell strains named 1B8, 5E1 and 5E2, respectively.
Among the three cell strains, 1B8 belonged to IgG_1 subclass of Ig, 5E1 and 5E2 belonged to IgG2b and IgG2a subclass of Ig,. The results of specificity cross test indicated that the three cell strains' monoclonal antibodies were not specific reaction with the A, C or O type of FMDV. Their titers in cell culture supematant were 1∶10~4, Their titers of abdomen liquor were 1∶4×10~6, respectively.
The purified protein of monoclon by SDS-PAGE showed the molecular weights of the heavy chain and light chain were about 45.0 kD and 25.0 kD. The number of chromosomal to SP2/0 cell strains were 50, to hybridoma cell strains were 104。
By mean of indirect ELISA, the antigenic epitopes was detected and recognized by1B8, 5E1 and 5E2. The results indicated that the epitopes recognized 1B8 overlapped partly that of 5E2, and the epitopes recognized 1B8 is different to that of 5E1.
In the appliance research test, lemon acid was made to colloidal gold and marked it with monoclonal antibody. The strips can recognize Asial type FMDV and VP1 protein, can detective 10~(-4) TCID_(50) virus particle, can store three months in different temperature condition。through experiment we find these strips have highly specificity、sensitivity and stability。
In this whole experiment, we obtained monoclonal antibodies to FMDV, and had a significance trial for make them into detective productions of FMDV antigen in the future.
引文
[1] Sobrino F, Saiz M, J imenez Clavero M A, at al. Foot and mouth disease virus: a long known virus, but a current threat [J]. Vet Res, 2001, 32 (1): 1-30.
[2] Kohler G. Milsten. continuous cultures of fused cell secreting antibody of predefined specificity[J] Nuture, 1975, 256: 495-772.
[3] Chen D, Shou C. Molecular Cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11[J]. Biochem Cell Biol, 1999, 11: 177-173
[4] 叶敏．基因工程抗体．上海科学技术文献出版社，2000，46-62
[5] Sharon J, Gefer M L, Morrison, et al. Expression of a VHCκchimaeric protein in mouse myeloma cells [J]. Nature, 1984, 309 (24): 364-367.
[6] Bouliance G L, Hozumi N, Shulwan M J, et al. Production of functional chimeric mouse/human antibody [J]. Nature, 1984, 312 (13): 643-646.
[7] BarbasC F, KangA S, LernerR S, et al. Assembly of combinatorial antibody libraries on phage surfaces:the geneⅢ site[J]. ProcNatI Acad Sci USA, 1991 88 (18): 7978-7982
[8] Villen J, Borras E, Schaaper W M, et al. Synthetic peptides as functional mimics of a viral discontinuous antigenic site[J]. Biologicals, 2001, 29(3-4): 265-269.
[9] Vaughan T J,Williams A J, Pritchard K, et al. Human antibodies with subnanomolar affinities isolated from alargenon-immunized phage display library[J]. Nature Biotechnol, 1996, 14: 309-314.
[10] Winter G, Griffiths A D. Making antibody by phage display technology [J]. Ann Rev Immunol, 1994, 12: 433-448
[11] Pluck A. Mono and bivalent antibody f ragment produced in E. coli: engineering, folding, and antigen binding [J]. Immunol Rev, 1992, 130 (2): 51-58
[12] Hanes J, Pluckthun A. Invitro selection and evolution of functional proteins by using ribosome display[J]. Proc Natl Acad Sci USA, 1997, 94: 4937-4942.
[13] Schaffitzel C, Hanes J, Jermutus L, et al. Ribosome display: aninvitro method for election and evolution of antibodies from libraries[J]. J Immunol Methods, 1999, 231: 119-135.
[14] Mann M J, Gibbons G H, Hutchinson H, et al. Pressure-mediated oligo nucleotide transfection of rat and human cardiovascular tissues[J]. Proe Natl Acad Sci USA, 1999, 96(11): 6411-6416
[15] Rizzuto G, Cappelletti M, Maione D, et al. Efficient and regulated erythroppoietin production by naked DNA injection and muscle electroporation [J]. Proc Natl Acad Sci USA, 1999, 96(11): 6417-6422.
[16] Spiller O B, Harris C L, Morgan B P, et al. Efficient generation of monoclonal antibodies against surface-expressed proteins by hyper expression in rodent cells[J]. J Immunol Meth, 1999, 224; 51-60.
[17] Kilpatrick K E, Wring S A, Walker D H, et al, Rapid development of affinity matured monoclonal antibodies using RIMMS[J]. Hybridoma, 1997, 16(4): 381-389.
[18] Alligood K J, Milla M, Rhodes N, et al. Monoclonal antibodies generated against recombinant ATM support kinase activity[J]. Hybridoma, 2000, 19(4): 317-320.
[19] Kilpatrick K E, Danger D P, Hull-ryde E A, et al. High-affinity monoclonal antibodies to PED/PEA-15 generated using 5ug DNA[J]. Hybridoma, 2000, 19(4): 297-302.
[20] Baxt B, Vakharia V, Moore D M, et al. Analysis of Neutralizing Antigenic Sites on the Surface of Type A12 Foot-and-Mouth Disease Virus[J]. J Virol, 1989, 63: 2143-2151.
[21] 谢庆阁主编 口蹄疫[M] 北京：中国农业出版社，2004
[22] 徐春河等 口蹄疫和猪水泡病诊断技术[M] 兰州：中国农业科学院兰州兽医研究所编，1984．
[23] 谢庆阁主编 口蹄疫[M] 北京：中国农业出版社，2004
[24] Alzahrani A J, Vallely P J and McMahon E T. Development of a noval nested in situ PCR-ISH methods for detection of hepatitis C virus RNA in liver tissue. J Virol Meth, 2002, 99: 53-60.
[25] 吴乃虎编著 基因工程原理(上)[M]．北京：科学出版社，1999
[26] Grubman M J, Baxt B. Foot and mouth disease [J]. Clin M icrobiol Rev, 2004, 17(2): 465-493
[27] 殷震，刘景华主编．动物病毒学[M]．(第二版)．北京：科学出版社，1997．
[28] 胡大利，张培因，尹晓光，等．抗O型口蹄疫病毒VP1单克隆抗体的制备与鉴定[J]．细胞与分子免疫学杂志．2006，22(6)：811～815
[29] Wissink H J, van Wijk. The major envelope protein, GP5, of a European porcine reproductive andrespiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain [J]. J Gen Virol, 2003, 84: 1535-1543.
[30] Sanyal A, Gurumurthy C B, Venkataramanan R, et al. Antigenic characterization of foot-and-mouth disease virus serotype Asial field isolates using polyclonal and monoclonal antibodies[J]. Vet Microbiol, 2003, 93: 1-11.
[31] 李海燕，辛晓光，田国斌，等．间接酶联免疫吸附试验检测禽流感抗体的最佳工作条件．[J]．中国畜禽传染病，1998，20(4)：233-235
[32] 细胞和分子免疫学实验技术[M]．金伯泉主编．西安：第二军医大学出版社，2002．
[33] 李金明．临床酶免疫测定技术[M]．北京：人民军医出版社．2005．
[34] 细胞培养[M]．司徒镇强，吴军正编．西安：世界图书出版西安公司，2004．
[35] 马刚，李作生，余兴龙，等．猪瘟病毒保护性抗原E2蛋白单抗的制备及其抗原表位的初步分析[J]．中国兽医学报，2002，22(2)：121-123．
[36] 陈士友，陈溥言，蔡宝祥，等．禽呼肠孤病毒单克隆抗体杂交瘤细胞株的建立[J]．病毒学报，1991，7(3)：209．
[37] 钟发刚，沈敏，王新华，等．抗牛病毒性腹泻／粘膜病病毒单克隆抗体的抗原结合位点分析[J]．中国畜禽传染病，1997，19(6)：48-49．
[38] 胡北侠，吴艳涛，刘秀梵，等．新城疫病毒融合蛋白单克隆抗体的研制[J]．畜牧兽医学报，2003，34(5)：498-501．
[39] 钱琨，秦爱建，邵红霞，等．抗禽流感病毒H9亚型血凝素特异性单克隆抗体的研制[J]．扬州大学学报(农业与生命科学版)，2003，24(3)：5-7．
[40] 武志强，杨奇伟，付朝阳，等．单抗ELISA在鸡传染性支气管炎病毒分型中的 应用[J]．中国畜禽传染病，1997，3：48-50．
[41] 金山，王永坤，董国雄．单克隆抗体亲和层析纯化传染性法氏囊病病毒方法的建立[J]．中国畜禽传染病，1997，4：59-61．
[42] 何永强，杜青云，盛祖恬，等．猪口蹄疫和水泡病病毒免疫鉴别诊断方法的比较研究[J]．中国预防兽医学报，1999，21(3)：228-230．
[43] 王川庆，梁宏德，郭军庆，等．不同方法对鸡胚和法氏囊中囊病病毒抗原含量的检测比较[J]．中国预防兽医学报，2001，23(6)：458-460．
[44] 李学伍，张改平，肖治军，等．IBD试纸条对中强毒力苗毒及分离野毒的检测研究[J]．中国预防兽医学报，2003，25(4)：298-301．
[45] 田克恭．犬细小病毒单克隆抗体研究进展[J]．中国兽医杂志，1999，25(2)：54-57．
[46] 王中力，宋晓晖，陈西召，等．犬细小病毒病免疫胶体金诊断技术的研究[J]．中国预防兽医学报，2004，26(1)：62-66．
[47] 苗得园，杨兵，李富强，等．单抗夹心LAB-ELISA检测猪伪狂犬病病毒的研究[J]．华北农学报，2001，16(1)：127-131．
[48] 黄红亮，陈焕春，覃雅丽，等．抗伪狂犬病病毒gG蛋白单克隆抗体的制备及鉴定[J]．中国兽医学报，2004，24(3)：243-245．
[49] 张国中，赖志，赵继勋．禽副粘病毒2型(PMV-2)群特异性单克隆抗体的研究[J]．畜牧兽医学报，2003，34(2)，188-190．
[50] 李玉峰，曹福敏，李峰，等．单抗ELISA试剂盒检测肉鸽新城疫病毒初步研究[J]．山东家禽，2004，6：7-8．
[51] 陆芹章，罗廷荣，温和心，等．猪瘟单克隆抗体的制备及ACI-ELISA检测猪瘟病毒的研究[J]．中国预防兽医学报，2004，26(5)：368-372．
[52] 周维松，刘秀梵．鸡新城疫病毒单克隆抗体的中和作用[J]．病毒学报，1991，7(1)：23-29．
[53] 陈万荣，付少才，刘树玲．抗犬瘟热病毒单克隆抗体的制备及初步应用[J]．中国兽医学报，2002，22(5)：455-456．
[54] 谷红，张中直，杨汉春，等．猪繁殖与呼吸综合征病毒单克隆抗体的研究[J]．中国兽医科技，2000，30(12)：9-11．
[55] 张改平，李学伍，杨艳艳，等．鸡传染性法氏囊病快速诊断试纸条的研制及特性测定[J]．畜牧兽医学报，2004，35(1)：89-92．
[56] 鲁会军，金宁一，韩松等．O型口蹄疫泛亚毒株单克隆抗体的制备及抗原表位差异分析[J]．中国兽医学报，2005，25(5)：494-496．
[57] 何永强，盛祖恬，杜青云．猪口蹄疫病毒位点及其抗原性差异分析[J]．中国兽医学报，2004，24(5)：451-453．
[58] Zamorano P, Wigdorovitz A, Chaher M T, et al. Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VPl FMDV 01 Campos[J]. Virology, 1994, 201(2): 383-387.
[59] Zhang Q, Yang Y Q, Zhang Z Y, et al. Immunogenicity of a recombinant fusion protein of tandem repeat epitopes of foot-and-mouth disease virus type Asia 1 for guinea pigs[J]. Acta Virol, 2002, 46(1): 1-9.
[60] Aggarwal N, Barnett P V. Antigenic sites of foot-and-mouth disease virus(FMDV): an analysis of the specificities of anti-FMDV antibodies after vaccination of naturally susceptible host species[J]. J Gen Virol, 2002, 83: 775-782.
[61] Baxt B, Vakharia V, Moore D M, et al. Analysis of Neutralizing Antigenic Sites on the Surface of Type A12 Foot-and-Mouth Disease Virus[J]. J Virol, 1989, 63: 2143-2151.
[62] 李一经，李海滨，贾永清等．聚乙二醇硫酸铵沉淀法对不同亚类单克隆抗体活性的影响[J]．黑龙江畜牧兽医，1998，3：6-7．
[63] Rimmelzwaan G F, Groen J, Juntti N, et al. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies[J]. J Virol methods, 1987, 15(4): 313-322.
[64] Butchaiah G, Morgan D O. Neutralization antigenic sites on type Asia-1 foot-and-mouth disease virus defined by monoclonal antibody-resistant variants[J]. Virus Res, 1997, 52(2): 183-194.
[65] Blanco E, Garcia-Briones M, Sanz-Parra A, et al. Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus[J]. J Virol, 2001, 75(7): 3164-3174.
[66] Thomas A M, Woortmeijer R J, Puijk W, et al. Antigenic Sites on Foot-and-Mouth Disease Virus Type A[J]. J Virol, 1988, 62: 2782-2789
[67] Beggs M, Novotny M, Sampedro S, et al. A selfperforming chromatographic immunoassay for the qualitative determination of human chorionic gonadotrophin (HCG) in urine and serum [J]. Clin Chem, 1990, 36: 1084-1085.
[68] 分子克隆实验指南[M] (第二版)．J萨姆布鲁克，E F弗里奇，T曼尼阿蒂斯(金冬雁，黎孟枫译)．北京：科学出版社，1996．
[69] Curry S, Fry E, Blakemore W, et al. Perturbations in the surface structure of A22Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity[J]. Structure, 1996, 4: 135-145.
[70] Chambers M A, Dougan G, Newman J, et al. Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions[J]. J Virol, 1996, 70(6): 4045-4052.
[71] 王玉金，王玉珠，杨书豪，等．胶体金免疫层析法检测血清癌胚抗原[J]．标记免疫分析与临床．2003，(3)：167～170．
[72] Domingo E, Verdaguer N, Ochoa WF, et al. Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus[J]. Virus Res, 1999, 62(20): 169-175.
[73] 金颜辉，黄印尧，张长弓等．应用金免疫层析技术检测猪瘟抗体的研究[J]．中国动物检疫，2004，21(5)：26-27．
[74] 赵启祖，谢庆阁．口蹄疫病毒抗原位点研究及应用，畜禽重大疫病免疫防制研究进展[M]．谢庆阁，翟中和主编．北京：中国农业出版社，1996．
[75] Wensvoort G, Terpstra C, Boonstra J, et al. Production ofmonoclonal antibodies against swine fever virus and their use in laboratory diagnosis[J]. Vet Microbiol, 1986, 12: 101-108.
[76] MeDonald C A. A Rapid One-Step Colored Particle Lateral-Flow Immunoassay for the Detection of Group 1 Streptococcal Antigen Extracted Directly from Throat Swabs[C]. In Proceedings of the 93rd General Meeting of American Society of Microbiology. 1999.
[77] Cuzzubbo A J, Chenthamarakshan V, Vadivelu J, et al. Evaluation of a New Commercially Available Immunoglobulin M and Immunoglobulin G Immunochromatographic Test for Diagnosis of Melioidosis Infection[J]. Clin Microbiol. 2000, 38: 1670～1671.
[78] Lou S C, Patel C, Ching S, et al. One-step competitive immunochromatographic assay for semiquantitative determination of lipoprotein(a) in plasma[J]. Clin Chem. 1993, 39: 619～624.
[79] Hyeong-soon Shin, Chang-kyu Kim, Kwang-soon Shin, et al. Pretreatment of Whole Blood for Use in Immunochromatographic Assays for Hepatitis B Virus Surface Antigen[J]. Clin Diagn Lab Immunol. 2001, 8: 9～13.
[80] 刘宏伟，张彬，葛洪，等．全血肌红蛋白快速免疫层析分析[J]．标记免疫分析与临床．2001，8(4)：220～223．
[81] Esfandiari J, Klingeborn B. A comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats and mink[J]. Vet Med B Infect Dis Vet Public Health. 2000, 47(2): 145～153.
[82] 徐葛林，胡巧玲，吴杰，等．检测狂犬病毒的胶体金免疫层析法的建立及应用[J]．中国人兽共患病杂志．2004，20(3)：236～240．
[83] 李学伍，张改平．邓瑞广．猪旋毛虫病快速检测试纸条的研制及特性测定[J]．畜牧兽医学报，2004，35(3)：343-346．
[84] 邓艳琴，严延生，何似，等．应用快速免疫层析法检测恙虫病特异性IgM、IgG及总抗体[J]．中国人兽共患病杂志，2003，19(6)，52-54．
[85] 催尚金，姜建宏，周艳君，等．猪繁殖和呼吸综合征胶体金抗体检测技术的建立和初步应用[J]．中国预防兽医学报，2005，27(3)：211-216．
[86] 江静，刘一兵，周棱，等．甲基苯丙胺免疫层析试纸条的研制[J]．同位素．2004，17(2)：79～83
[87] 唐雨德，周东明，陆承平．快速检测猪囊虫抗体的金免疫层析法的建立及应用[J]．中国人兽共患病杂志，2003，19(5)，77-79．
[2] Kohler G. Milsten. continuous cultures of fused cell secreting antibody of predefined specificity[J] Nuture, 1975, 256: 495-772.
[3] Chen D, Shou C. Molecular Cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11[J]. Biochem Cell Biol, 1999, 11: 177-173
[4] 叶敏．基因工程抗体．上海科学技术文献出版社，2000，46-62
[5] Sharon J, Gefer M L, Morrison, et al. Expression of a VHCκchimaeric protein in mouse myeloma cells [J]. Nature, 1984, 309 (24): 364-367.
[6] Bouliance G L, Hozumi N, Shulwan M J, et al. Production of functional chimeric mouse/human antibody [J]. Nature, 1984, 312 (13): 643-646.
[7] BarbasC F, KangA S, LernerR S, et al. Assembly of combinatorial antibody libraries on phage surfaces:the geneⅢ site[J]. ProcNatI Acad Sci USA, 1991 88 (18): 7978-7982
[8] Villen J, Borras E, Schaaper W M, et al. Synthetic peptides as functional mimics of a viral discontinuous antigenic site[J]. Biologicals, 2001, 29(3-4): 265-269.
[9] Vaughan T J,Williams A J, Pritchard K, et al. Human antibodies with subnanomolar affinities isolated from alargenon-immunized phage display library[J]. Nature Biotechnol, 1996, 14: 309-314.
[10] Winter G, Griffiths A D. Making antibody by phage display technology [J]. Ann Rev Immunol, 1994, 12: 433-448
[11] Pluck A. Mono and bivalent antibody f ragment produced in E. coli: engineering, folding, and antigen binding [J]. Immunol Rev, 1992, 130 (2): 51-58
[12] Hanes J, Pluckthun A. Invitro selection and evolution of functional proteins by using ribosome display[J]. Proc Natl Acad Sci USA, 1997, 94: 4937-4942.
[13] Schaffitzel C, Hanes J, Jermutus L, et al. Ribosome display: aninvitro method for election and evolution of antibodies from libraries[J]. J Immunol Methods, 1999, 231: 119-135.
[14] Mann M J, Gibbons G H, Hutchinson H, et al. Pressure-mediated oligo nucleotide transfection of rat and human cardiovascular tissues[J]. Proe Natl Acad Sci USA, 1999, 96(11): 6411-6416
[15] Rizzuto G, Cappelletti M, Maione D, et al. Efficient and regulated erythroppoietin production by naked DNA injection and muscle electroporation [J]. Proc Natl Acad Sci USA, 1999, 96(11): 6417-6422.
[16] Spiller O B, Harris C L, Morgan B P, et al. Efficient generation of monoclonal antibodies against surface-expressed proteins by hyper expression in rodent cells[J]. J Immunol Meth, 1999, 224; 51-60.
[17] Kilpatrick K E, Wring S A, Walker D H, et al, Rapid development of affinity matured monoclonal antibodies using RIMMS[J]. Hybridoma, 1997, 16(4): 381-389.
[18] Alligood K J, Milla M, Rhodes N, et al. Monoclonal antibodies generated against recombinant ATM support kinase activity[J]. Hybridoma, 2000, 19(4): 317-320.
[19] Kilpatrick K E, Danger D P, Hull-ryde E A, et al. High-affinity monoclonal antibodies to PED/PEA-15 generated using 5ug DNA[J]. Hybridoma, 2000, 19(4): 297-302.
[20] Baxt B, Vakharia V, Moore D M, et al. Analysis of Neutralizing Antigenic Sites on the Surface of Type A12 Foot-and-Mouth Disease Virus[J]. J Virol, 1989, 63: 2143-2151.
[21] 谢庆阁主编 口蹄疫[M] 北京：中国农业出版社，2004
[22] 徐春河等 口蹄疫和猪水泡病诊断技术[M] 兰州：中国农业科学院兰州兽医研究所编，1984．
[23] 谢庆阁主编 口蹄疫[M] 北京：中国农业出版社，2004
[24] Alzahrani A J, Vallely P J and McMahon E T. Development of a noval nested in situ PCR-ISH methods for detection of hepatitis C virus RNA in liver tissue. J Virol Meth, 2002, 99: 53-60.
[25] 吴乃虎编著 基因工程原理(上)[M]．北京：科学出版社，1999
[26] Grubman M J, Baxt B. Foot and mouth disease [J]. Clin M icrobiol Rev, 2004, 17(2): 465-493
[27] 殷震，刘景华主编．动物病毒学[M]．(第二版)．北京：科学出版社，1997．
[28] 胡大利，张培因，尹晓光，等．抗O型口蹄疫病毒VP1单克隆抗体的制备与鉴定[J]．细胞与分子免疫学杂志．2006，22(6)：811～815
[29] Wissink H J, van Wijk. The major envelope protein, GP5, of a European porcine reproductive andrespiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain [J]. J Gen Virol, 2003, 84: 1535-1543.
[30] Sanyal A, Gurumurthy C B, Venkataramanan R, et al. Antigenic characterization of foot-and-mouth disease virus serotype Asial field isolates using polyclonal and monoclonal antibodies[J]. Vet Microbiol, 2003, 93: 1-11.
[31] 李海燕，辛晓光，田国斌，等．间接酶联免疫吸附试验检测禽流感抗体的最佳工作条件．[J]．中国畜禽传染病，1998，20(4)：233-235
[32] 细胞和分子免疫学实验技术[M]．金伯泉主编．西安：第二军医大学出版社，2002．
[33] 李金明．临床酶免疫测定技术[M]．北京：人民军医出版社．2005．
[34] 细胞培养[M]．司徒镇强，吴军正编．西安：世界图书出版西安公司，2004．
[35] 马刚，李作生，余兴龙，等．猪瘟病毒保护性抗原E2蛋白单抗的制备及其抗原表位的初步分析[J]．中国兽医学报，2002，22(2)：121-123．
[36] 陈士友，陈溥言，蔡宝祥，等．禽呼肠孤病毒单克隆抗体杂交瘤细胞株的建立[J]．病毒学报，1991，7(3)：209．
[37] 钟发刚，沈敏，王新华，等．抗牛病毒性腹泻／粘膜病病毒单克隆抗体的抗原结合位点分析[J]．中国畜禽传染病，1997，19(6)：48-49．
[38] 胡北侠，吴艳涛，刘秀梵，等．新城疫病毒融合蛋白单克隆抗体的研制[J]．畜牧兽医学报，2003，34(5)：498-501．
[39] 钱琨，秦爱建，邵红霞，等．抗禽流感病毒H9亚型血凝素特异性单克隆抗体的研制[J]．扬州大学学报(农业与生命科学版)，2003，24(3)：5-7．
[40] 武志强，杨奇伟，付朝阳，等．单抗ELISA在鸡传染性支气管炎病毒分型中的 应用[J]．中国畜禽传染病，1997，3：48-50．
[41] 金山，王永坤，董国雄．单克隆抗体亲和层析纯化传染性法氏囊病病毒方法的建立[J]．中国畜禽传染病，1997，4：59-61．
[42] 何永强，杜青云，盛祖恬，等．猪口蹄疫和水泡病病毒免疫鉴别诊断方法的比较研究[J]．中国预防兽医学报，1999，21(3)：228-230．
[43] 王川庆，梁宏德，郭军庆，等．不同方法对鸡胚和法氏囊中囊病病毒抗原含量的检测比较[J]．中国预防兽医学报，2001，23(6)：458-460．
[44] 李学伍，张改平，肖治军，等．IBD试纸条对中强毒力苗毒及分离野毒的检测研究[J]．中国预防兽医学报，2003，25(4)：298-301．
[45] 田克恭．犬细小病毒单克隆抗体研究进展[J]．中国兽医杂志，1999，25(2)：54-57．
[46] 王中力，宋晓晖，陈西召，等．犬细小病毒病免疫胶体金诊断技术的研究[J]．中国预防兽医学报，2004，26(1)：62-66．
[47] 苗得园，杨兵，李富强，等．单抗夹心LAB-ELISA检测猪伪狂犬病病毒的研究[J]．华北农学报，2001，16(1)：127-131．
[48] 黄红亮，陈焕春，覃雅丽，等．抗伪狂犬病病毒gG蛋白单克隆抗体的制备及鉴定[J]．中国兽医学报，2004，24(3)：243-245．
[49] 张国中，赖志，赵继勋．禽副粘病毒2型(PMV-2)群特异性单克隆抗体的研究[J]．畜牧兽医学报，2003，34(2)，188-190．
[50] 李玉峰，曹福敏，李峰，等．单抗ELISA试剂盒检测肉鸽新城疫病毒初步研究[J]．山东家禽，2004，6：7-8．
[51] 陆芹章，罗廷荣，温和心，等．猪瘟单克隆抗体的制备及ACI-ELISA检测猪瘟病毒的研究[J]．中国预防兽医学报，2004，26(5)：368-372．
[52] 周维松，刘秀梵．鸡新城疫病毒单克隆抗体的中和作用[J]．病毒学报，1991，7(1)：23-29．
[53] 陈万荣，付少才，刘树玲．抗犬瘟热病毒单克隆抗体的制备及初步应用[J]．中国兽医学报，2002，22(5)：455-456．
[54] 谷红，张中直，杨汉春，等．猪繁殖与呼吸综合征病毒单克隆抗体的研究[J]．中国兽医科技，2000，30(12)：9-11．
[55] 张改平，李学伍，杨艳艳，等．鸡传染性法氏囊病快速诊断试纸条的研制及特性测定[J]．畜牧兽医学报，2004，35(1)：89-92．
[56] 鲁会军，金宁一，韩松等．O型口蹄疫泛亚毒株单克隆抗体的制备及抗原表位差异分析[J]．中国兽医学报，2005，25(5)：494-496．
[57] 何永强，盛祖恬，杜青云．猪口蹄疫病毒位点及其抗原性差异分析[J]．中国兽医学报，2004，24(5)：451-453．
[58] Zamorano P, Wigdorovitz A, Chaher M T, et al. Recognition of B and T cell epitopes by cattle immunized with a synthetic peptide containing the major immunogenic site of VPl FMDV 01 Campos[J]. Virology, 1994, 201(2): 383-387.
[59] Zhang Q, Yang Y Q, Zhang Z Y, et al. Immunogenicity of a recombinant fusion protein of tandem repeat epitopes of foot-and-mouth disease virus type Asia 1 for guinea pigs[J]. Acta Virol, 2002, 46(1): 1-9.
[60] Aggarwal N, Barnett P V. Antigenic sites of foot-and-mouth disease virus(FMDV): an analysis of the specificities of anti-FMDV antibodies after vaccination of naturally susceptible host species[J]. J Gen Virol, 2002, 83: 775-782.
[61] Baxt B, Vakharia V, Moore D M, et al. Analysis of Neutralizing Antigenic Sites on the Surface of Type A12 Foot-and-Mouth Disease Virus[J]. J Virol, 1989, 63: 2143-2151.
[62] 李一经，李海滨，贾永清等．聚乙二醇硫酸铵沉淀法对不同亚类单克隆抗体活性的影响[J]．黑龙江畜牧兽医，1998，3：6-7．
[63] Rimmelzwaan G F, Groen J, Juntti N, et al. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies[J]. J Virol methods, 1987, 15(4): 313-322.
[64] Butchaiah G, Morgan D O. Neutralization antigenic sites on type Asia-1 foot-and-mouth disease virus defined by monoclonal antibody-resistant variants[J]. Virus Res, 1997, 52(2): 183-194.
[65] Blanco E, Garcia-Briones M, Sanz-Parra A, et al. Identification of T-cell epitopes in nonstructural proteins of foot-and-mouth disease virus[J]. J Virol, 2001, 75(7): 3164-3174.
[66] Thomas A M, Woortmeijer R J, Puijk W, et al. Antigenic Sites on Foot-and-Mouth Disease Virus Type A[J]. J Virol, 1988, 62: 2782-2789
[67] Beggs M, Novotny M, Sampedro S, et al. A selfperforming chromatographic immunoassay for the qualitative determination of human chorionic gonadotrophin (HCG) in urine and serum [J]. Clin Chem, 1990, 36: 1084-1085.
[68] 分子克隆实验指南[M] (第二版)．J萨姆布鲁克，E F弗里奇，T曼尼阿蒂斯(金冬雁，黎孟枫译)．北京：科学出版社，1996．
[69] Curry S, Fry E, Blakemore W, et al. Perturbations in the surface structure of A22Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity[J]. Structure, 1996, 4: 135-145.
[70] Chambers M A, Dougan G, Newman J, et al. Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions[J]. J Virol, 1996, 70(6): 4045-4052.
[71] 王玉金，王玉珠，杨书豪，等．胶体金免疫层析法检测血清癌胚抗原[J]．标记免疫分析与临床．2003，(3)：167～170．
[72] Domingo E, Verdaguer N, Ochoa WF, et al. Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus[J]. Virus Res, 1999, 62(20): 169-175.
[73] 金颜辉，黄印尧，张长弓等．应用金免疫层析技术检测猪瘟抗体的研究[J]．中国动物检疫，2004，21(5)：26-27．
[74] 赵启祖，谢庆阁．口蹄疫病毒抗原位点研究及应用，畜禽重大疫病免疫防制研究进展[M]．谢庆阁，翟中和主编．北京：中国农业出版社，1996．
[75] Wensvoort G, Terpstra C, Boonstra J, et al. Production ofmonoclonal antibodies against swine fever virus and their use in laboratory diagnosis[J]. Vet Microbiol, 1986, 12: 101-108.
[76] MeDonald C A. A Rapid One-Step Colored Particle Lateral-Flow Immunoassay for the Detection of Group 1 Streptococcal Antigen Extracted Directly from Throat Swabs[C]. In Proceedings of the 93rd General Meeting of American Society of Microbiology. 1999.
[77] Cuzzubbo A J, Chenthamarakshan V, Vadivelu J, et al. Evaluation of a New Commercially Available Immunoglobulin M and Immunoglobulin G Immunochromatographic Test for Diagnosis of Melioidosis Infection[J]. Clin Microbiol. 2000, 38: 1670～1671.
[78] Lou S C, Patel C, Ching S, et al. One-step competitive immunochromatographic assay for semiquantitative determination of lipoprotein(a) in plasma[J]. Clin Chem. 1993, 39: 619～624.
[79] Hyeong-soon Shin, Chang-kyu Kim, Kwang-soon Shin, et al. Pretreatment of Whole Blood for Use in Immunochromatographic Assays for Hepatitis B Virus Surface Antigen[J]. Clin Diagn Lab Immunol. 2001, 8: 9～13.
[80] 刘宏伟，张彬，葛洪，等．全血肌红蛋白快速免疫层析分析[J]．标记免疫分析与临床．2001，8(4)：220～223．
[81] Esfandiari J, Klingeborn B. A comparative study of a new rapid and one-step test for the detection of parvovirus in faeces from dogs, cats and mink[J]. Vet Med B Infect Dis Vet Public Health. 2000, 47(2): 145～153.
[82] 徐葛林，胡巧玲，吴杰，等．检测狂犬病毒的胶体金免疫层析法的建立及应用[J]．中国人兽共患病杂志．2004，20(3)：236～240．
[83] 李学伍，张改平．邓瑞广．猪旋毛虫病快速检测试纸条的研制及特性测定[J]．畜牧兽医学报，2004，35(3)：343-346．
[84] 邓艳琴，严延生，何似，等．应用快速免疫层析法检测恙虫病特异性IgM、IgG及总抗体[J]．中国人兽共患病杂志，2003，19(6)，52-54．
[85] 催尚金，姜建宏，周艳君，等．猪繁殖和呼吸综合征胶体金抗体检测技术的建立和初步应用[J]．中国预防兽医学报，2005，27(3)：211-216．
[86] 江静，刘一兵，周棱，等．甲基苯丙胺免疫层析试纸条的研制[J]．同位素．2004，17(2)：79～83
[87] 唐雨德，周东明，陆承平．快速检测猪囊虫抗体的金免疫层析法的建立及应用[J]．中国人兽共患病杂志，2003，19(5)，77-79．
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