鱼类致病性豚鼠气单胞菌单克隆抗体—胶体金检测方法的建立
|
摘要
目的豚鼠气单胞菌是一种条件性致病菌：当鱼类抵抗力下降时或鱼体表有损伤时,容易被该菌侵入,反复感染后会导致其致病力增强,可形成很强的传染性。近年来,在我国养殖的南方鲇中发生了一种由豚鼠气单胞菌引起的以体表溃疡为特征的疾病,流行广、发病快、死亡率高,对水产养殖动物危害极大,导致巨大经济损失,严重危害我国渔业及水产业的发展。目前对豚鼠气单胞菌的鉴定主要依赖临床观察、解剖及一般细菌学检查法,操作过程既繁琐又费时,而且准确性不够,因此,建立快速、特异性强的诊断方法对更好防控豚鼠气单胞菌感染对水生养殖动物的危害具有重要的意义。
本研究拟利用鱼类致病性豚鼠气单胞菌为抗原,制备抗该菌的单克隆抗体,并对其生物学特性进行了鉴定,在此基础上利用胶体金技术和所制备的单克隆抗体研制能快速检测豚鼠气单胞菌感染的胶体金试纸条,并进行性能测试,从而建立鱼类致病性豚鼠气单胞菌单克隆抗体-胶体金快速检测方法,为水产上豚鼠气单胞菌病的流行监测提供一种便捷的方法,同时也为该细菌引起的疾病检测方法研究积累一些资料。
方法以福尔马林灭活的豚鼠气单胞菌按25μg/只、50μg/只分成两个剂量组,皮下多点注射免疫Balb/c小鼠,通过杂交瘤技术制备针对豚鼠气单胞菌的单克隆抗体(McAb),用间接ELISA法对所需的杂交瘤细胞株进行特异性筛选,对筛选到的高特异性的单抗进行分离、纯化及鉴定：先通过饱和硫酸铵法粗提抗体,再用Protein A亲和层析法进一步纯化,然后对单抗进行了包括亚类鉴定、效价、相对亲和力的测定,并通过单克隆抗体的位点相加实验、与豚鼠气单胞菌脂多糖(LPS)的间接ELISA实验对单抗识别的抗原表位进行了初步研究。然后采用胶体金标记技术,通过双抗体夹心法(单克隆抗体—抗原—单克隆抗体),以所得单抗作为包被及标记抗体,制备胶体金-抗体结合物和反应膜,研制豚鼠气单胞菌胶体金快速检测卡,并通过特异性、灵敏度、重复性、稳定性和临床样本的检测进行方法学评价。
结果获得了2株可分泌特异性McAb的杂交瘤细胞,并分别命名为3F3和2C9C3。经过鉴定,这2株McAb能够特异性的针对豚鼠气单胞菌,其抗体亚类分别为IgG1型和IgM型；腹水效价分别为10-6和10-5；相对亲和力较高；3F3针对豚鼠气单胞菌脂多糖表位,而2C9C3针对非脂多糖抗原位点。利用本实验制备的McAb建立了以McAb为基础的双抗夹心法膜式胶体金快速检测方法,所研制的豚鼠气单胞菌胶体金快速检测卡具有灵敏度好,最低检测量为1.71×104cfu/ml,特异性高,重复性100%,稳定性好(室温保存一年也不失活),检测时间快(5分钟可判定结果)等优点,对临床样本的检测准确率高,操作简便。
结论成功地制备出抗豚鼠气单胞菌的单克隆抗体,并研制了能快速特异检测鱼类豚鼠气单胞菌的胶体金试纸条,为水产上豚鼠气单胞菌的快速鉴定和诊断以及该菌流行的监测提供有力的工具。
Objective: Aeromonas caviae (A. caviae) is one of the most prominent pathogenic bacteria which in China has been reported that is harmful to our fresh water aquaculture. As a major causative agent of infections in fish, A. caviae becomes highly pathogenic and infective when the resistance of fish decreases or the fish is injured. In recent years, the outbreak of epizootic ulcerative syndrome associated with A. caviae was reported among the southern catfish. The diseases caused by A. caviae are characterized by rapid invasion, wide prevalence and high mortality, which can cause serious economic loss in fish keeping. At present, the commonly used diagnostic methods for A. caviae depends on clinical observation, dissection and common bacteriology tests that are tedious, time-consuming and have poor accuracy. It is obvious that the establishment of a rapid method with higher specificity is one of the better approaches to prevent and control the infection caused by A. caviae.
This research is to prepare A. caviae hybridoma cell lines, analyze the biological characteristics and develop colloidal gold test strips to establish a rapid and accurate diagnostic method for A. caviae in aquaculture
Methods: Balb/c mice were immunized with A. caviae killed by formalin as the dosage of 25μg per mouse and 50μg per mouse respectively. The hybridoma cell lines which consistently secreted monoclonal antibody (McAb) against A. caviae were obtained through cell fusion. The specificity of the McAb was analyzed by indirect ELISA. Then the subtypes were identified, the titer and relative affinity constant were measured. Tests were made to identify the antigen epitope of the McAbs by combined experimental site and indirect ELISA with LPS of A. caviae. The McAb conjugating to colloidal gold against A. caviae was established, and its specificity, sensitivity, repeatability, stability and clinic sample test were estimated respectively.
Results: From many positive hybridomas which secreted anti-Aeromonas caviae McAbs, two strains of hybridomas were screened out, and designated with 3F3 and 2C9C3. The subtypes of the McAb were IgG1 and IgM. The titer of 3F3 and 2C9C3 McAb produced by ascites fluid were 10-6 and10-5. The McAbs had high relative affinity. The two strains of McAbs targeted to different antigen epitope:3F3 targeted to the LPS of A. caviae, while 2C9C3 targeted to the non-LPS sites of A. caviae. Basing on the McAbs, the rapid detection McAbs conjugating to colloidal gold against A. caviae was developed to detect the thalli antigen of A. caviae specifically. The results demonstrated that this method had high specificity and good repeatability (100%). The minimum detection dose of strips was 1.71×104 cfu/ml. The test results by the strips could be observed within 5 minutes. The strips could be used easily and exactly in clinical test.
Conclusion: The results demonstrated that this method had high specificity, sensitivity, stability and repeatability. It could serve as an effective detection measure for clinic, and as the method of the rapid identification for A. caviae in aquaculture and monitoring the epidemic of A. caviae.
本研究拟利用鱼类致病性豚鼠气单胞菌为抗原,制备抗该菌的单克隆抗体,并对其生物学特性进行了鉴定,在此基础上利用胶体金技术和所制备的单克隆抗体研制能快速检测豚鼠气单胞菌感染的胶体金试纸条,并进行性能测试,从而建立鱼类致病性豚鼠气单胞菌单克隆抗体-胶体金快速检测方法,为水产上豚鼠气单胞菌病的流行监测提供一种便捷的方法,同时也为该细菌引起的疾病检测方法研究积累一些资料。
方法以福尔马林灭活的豚鼠气单胞菌按25μg/只、50μg/只分成两个剂量组,皮下多点注射免疫Balb/c小鼠,通过杂交瘤技术制备针对豚鼠气单胞菌的单克隆抗体(McAb),用间接ELISA法对所需的杂交瘤细胞株进行特异性筛选,对筛选到的高特异性的单抗进行分离、纯化及鉴定：先通过饱和硫酸铵法粗提抗体,再用Protein A亲和层析法进一步纯化,然后对单抗进行了包括亚类鉴定、效价、相对亲和力的测定,并通过单克隆抗体的位点相加实验、与豚鼠气单胞菌脂多糖(LPS)的间接ELISA实验对单抗识别的抗原表位进行了初步研究。然后采用胶体金标记技术,通过双抗体夹心法(单克隆抗体—抗原—单克隆抗体),以所得单抗作为包被及标记抗体,制备胶体金-抗体结合物和反应膜,研制豚鼠气单胞菌胶体金快速检测卡,并通过特异性、灵敏度、重复性、稳定性和临床样本的检测进行方法学评价。
结果获得了2株可分泌特异性McAb的杂交瘤细胞,并分别命名为3F3和2C9C3。经过鉴定,这2株McAb能够特异性的针对豚鼠气单胞菌,其抗体亚类分别为IgG1型和IgM型；腹水效价分别为10-6和10-5；相对亲和力较高；3F3针对豚鼠气单胞菌脂多糖表位,而2C9C3针对非脂多糖抗原位点。利用本实验制备的McAb建立了以McAb为基础的双抗夹心法膜式胶体金快速检测方法,所研制的豚鼠气单胞菌胶体金快速检测卡具有灵敏度好,最低检测量为1.71×104cfu/ml,特异性高,重复性100%,稳定性好(室温保存一年也不失活),检测时间快(5分钟可判定结果)等优点,对临床样本的检测准确率高,操作简便。
结论成功地制备出抗豚鼠气单胞菌的单克隆抗体,并研制了能快速特异检测鱼类豚鼠气单胞菌的胶体金试纸条,为水产上豚鼠气单胞菌的快速鉴定和诊断以及该菌流行的监测提供有力的工具。
Objective: Aeromonas caviae (A. caviae) is one of the most prominent pathogenic bacteria which in China has been reported that is harmful to our fresh water aquaculture. As a major causative agent of infections in fish, A. caviae becomes highly pathogenic and infective when the resistance of fish decreases or the fish is injured. In recent years, the outbreak of epizootic ulcerative syndrome associated with A. caviae was reported among the southern catfish. The diseases caused by A. caviae are characterized by rapid invasion, wide prevalence and high mortality, which can cause serious economic loss in fish keeping. At present, the commonly used diagnostic methods for A. caviae depends on clinical observation, dissection and common bacteriology tests that are tedious, time-consuming and have poor accuracy. It is obvious that the establishment of a rapid method with higher specificity is one of the better approaches to prevent and control the infection caused by A. caviae.
This research is to prepare A. caviae hybridoma cell lines, analyze the biological characteristics and develop colloidal gold test strips to establish a rapid and accurate diagnostic method for A. caviae in aquaculture
Methods: Balb/c mice were immunized with A. caviae killed by formalin as the dosage of 25μg per mouse and 50μg per mouse respectively. The hybridoma cell lines which consistently secreted monoclonal antibody (McAb) against A. caviae were obtained through cell fusion. The specificity of the McAb was analyzed by indirect ELISA. Then the subtypes were identified, the titer and relative affinity constant were measured. Tests were made to identify the antigen epitope of the McAbs by combined experimental site and indirect ELISA with LPS of A. caviae. The McAb conjugating to colloidal gold against A. caviae was established, and its specificity, sensitivity, repeatability, stability and clinic sample test were estimated respectively.
Results: From many positive hybridomas which secreted anti-Aeromonas caviae McAbs, two strains of hybridomas were screened out, and designated with 3F3 and 2C9C3. The subtypes of the McAb were IgG1 and IgM. The titer of 3F3 and 2C9C3 McAb produced by ascites fluid were 10-6 and10-5. The McAbs had high relative affinity. The two strains of McAbs targeted to different antigen epitope:3F3 targeted to the LPS of A. caviae, while 2C9C3 targeted to the non-LPS sites of A. caviae. Basing on the McAbs, the rapid detection McAbs conjugating to colloidal gold against A. caviae was developed to detect the thalli antigen of A. caviae specifically. The results demonstrated that this method had high specificity and good repeatability (100%). The minimum detection dose of strips was 1.71×104 cfu/ml. The test results by the strips could be observed within 5 minutes. The strips could be used easily and exactly in clinical test.
Conclusion: The results demonstrated that this method had high specificity, sensitivity, stability and repeatability. It could serve as an effective detection measure for clinic, and as the method of the rapid identification for A. caviae in aquaculture and monitoring the epidemic of A. caviae.
引文
[1]Ji L L, Wang K Y, Xiao D, et al. Isolation and identification of pathogenic bacteria causing ulcer disease of silurus meriordinalis Chen[J]. Freshwater fisheries (in Chinese).2008,38(3): 68-72.
[2]李爱华.气单胞菌(Aeromonads)分类与命名的最新进展1[J].微生物分类学.2001,23(2)：25-32.
[3]阳涛.南方鲇源豚鼠气单胞菌胞外产物活性与致病性研究[D].雅安：四川农业大学,2008.
[4]Gryllos L, Shaw J G, Gavin R E A. Role of flm Locus in MesopHilic Aeromonas Species Adherence[J]. Infect Immun.2001,69(1):65-74.
[5]陈金燕,王洪霞.一起水源性腹泻暴发的调查[J].实用预防医学.2003,10(6)：1012-1013.
[6]徐景野,傅小红,于梅.宁波市贝(甲)霓类海产品易损气单胞菌检出情况[J].实用预防医学.2001,8(6)：441442.
[7]Dwivedi M, Mishra A, Prasad A, et al. Aeromonas caviae septicemia in immunocompetent gastrointestinal carriers[J]. Braz J Infect Dis.2008,12(6):547-548.
[8]Al-Benwan K, Abbott S, Janda J M, et al. Cystitis caused by Aeromonas caviae [J]. J. Clin. Microbiol.2007,45:2348-2350.
[9]Teira R, Hernandez J L, Rojo P, et al. Bacteremia of biliary origin caused by Aeromonas caviae in a healthy patient[J]. J. Enferm Infecc Microbiol Clin.1991,9:388-389.
[10]李翠英,王平,陈玉霞.豚鼠气单胞菌引起化脓性脑膜炎一例[J].上海医学检验杂志.1994,9(3)：255.
[11]Pinna A, Sechi L A, Zanetti S, et al. Aeromonas caviae keratitis associated with contact lens wear[J]. Ophthalmology.2004,111(2):348-351.
[12]吉莉莉,汪开毓,肖丹,等.南方鲇溃疡综合症病原菌的分离与鉴定[J].淡水渔业.2008,38(3)：68-72.
[13]阳涛,汪开毓,肖丹,等.南方鲇源豚鼠气单胞菌胞外产物活性与致病性研究[J].淡水渔业.2008,38：4046.
[14]丁雷,岳永生,宋憬愚.虹鳟皮肤溃烂病的病原菌研究[J].淡水渔业.2002,32(3)：28-30.
[15]孙红祥,舒妙安.中华鳖几种常见疾病病原的分离鉴定及药敏试验[J].中国兽医报.2002, 22(2)：140-142.
[16]安利国,邢维贤,傅荣恕.鲤鱼竖鳞病的流行病学和病原学的研究[J].山东师大学报(自然科学版).1996,11(3)：68-72.
[17]樊海平,曾占壮,余培建.由豚鼠气单胞菌引起的欧洲鳗鲡败血症[J].水产学报.1999,23(3)：313-318.
[18]许信刚,伊岚,王高学.观赏金鱼豚鼠气单胞菌的分离鉴定及防治[J].西北农业学报.2006,15(1)：40-42.
[19]李小波,黄文芳.丰产鲫细菌性败血病的研究I-病原分离与鉴定[J].微生物学报.2003,30(5)：56-60.
[20]陈翠珍,张晓君,房海,等.鲢鳙打印病豚鼠气单胞茵主要特性及系统发育学分析[J].中国农学通报.2006,22(5)：455-459.
[21]徐海圣,黄立峰,王淑霞.中华绒螫蟹豚鼠气单胞菌的分离和鉴定[J].浙江大学学报.2001,27(6)：677-681.
[22]黄伟坚,黄均,秦爱珍.野生胡子鲇“腹水病”病原分离和鉴定[J].中国兽医科技.2000,30(4)：30-33.
[23]Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature.1975,256(5517):495-497.
[24]Chinchar V G, Metzger D W, A Granoff E A. Location of frog virus 3 proteins using monoclonal antibodies[J]. Virology.1984,137(1):211-216.
[25]汤仲明.生物技术药物的最新进展和展望[J].国外医药.2002,21(2)：115-117.
[26]Austin B. Monoclonal antibody-based enzyme-linked immunosorbent assays for the rapid diagnosis of clinical cases of entric red-mouth furunculosis in fish farms[J]. J. Fish Dis.1986,9: 469-474.
[28]黄钧,梁明龙,朱芸.温和气单胞菌单克隆抗体的制备及特性分析[J].河南农业科学.2006,2：108-110.
[29]Ueki N, Kayano Y, Muroga K. Pasteurella piscicida in juvenile red grouper[J]. Fish Pathology.1990,25:43-44.
[30]Balebona M C, Morinigo M A, Sedano J, et al. Isolation of Pasteurella piscicida from sea bass in south-western Spain[J]. Bulletin of the European Association of Fish Pathologists. 1992,12:168-170.
[31]Maniatis K, Morris D J, Adams A, et al. Detection of Photobacterium damsela subspecies piscicida in fixed tissue sections using immunohistochemistry and antigen retrival immunohistochemistry[J]. Journal of Fish Diseases.2000,23:343-347.
[32]Bakopoulos V, Adam A, Richards R H. Production and characterization of monoclonal antibodies against Pasteurella picicida, the causative agent of fish patearellosis[J]. Journal of Fish Diseases.1997,20:307-315.
[33]金晓航,黄威权,夏永娟,等.迟缓爱德华菌抗独特型单克隆抗体的制备及鉴定[J].水生生物学报.2005,29(3)：340-343.
[34]秦红,黄威权,杨向民,等.抗迟缓爱德华菌独特型单克隆抗体重链可变区基因的克隆及序列分析[J].细胞与分子免疫学杂志.2007,23(2)：155-156.
[35]夏永娟,黄威权,黄宝成,等.抗鳗弧菌独特型单克隆抗体的制备及鉴定[J].微生物学报.2001,41(4)：447-451.
[36]夏永娟,黄宝成,温伟红,等.抗鳗弧菌独特型单克隆抗体重链可变区基因的克隆及序列测定[J].细胞与分子免疫学杂志.2003,19(2)：176-177.
[37]陈瑞,唐旭,于辉,等.抗温和气单胞菌单克隆抗体的制备及初步鉴定[J].细胞与分子免疫学杂志.2007,23(2)：146-147.
[38]郭闯,方苹,郭立新,等.嗜水气单胞菌气溶素单克隆抗体的制备及初步应用[J].水产科学.2007,26(3)：167-170.
[39]Klesius P, Evans J, C Shoemaker E A. Rapid detection and identification of streptococcus iniae using a monoclonal antibody-based indirect fluorescent antibody technique[J]. Aquaculture.2006,258:180-186.
[40]池信才,王军,鄢庆枇,等.大黄鱼病原副溶血弧菌单克隆抗体制备及其应用[J].海洋科学.2007,31(8)：1-5.
[41]郝贵杰,沈锦玉,曹铮,等.抗哈维氏弧菌单克隆抗体杂交瘤细胞系的建立及其特性鉴定[J].23.2007,9(838-840).
[42]Caswell-Reno P, Reno P W, Nicholson B L. Monoclonal antibodies to infectious pancreatic necrosis virus (IPNV):analysis of viral epitopes and comparison of different isolates[J]. J. Gen. Virol.1986,67:2193-2205.
[43]Wolski S C, Roberson B S, Hetrick F M. Monoclonal antibodies to the Sp strain of infectious pancreatic necrosis virus[J]. Vet. Immunol. Immunopathol.1986,12:373-381.
[44]Lipipun V, Caswell-Reno P, Hsu Y L, et al. Antigenic analysis of Asian birnavirus isolates using monoclonal antibodies[J]. Fish Pathol.1989,24:155-160.
[45]Schultz C L, Lidgerding B C, Mcallister P E, et al. Production and characterization of monoclonal antibody against infectious hematopoietic necrosis virus[J]. Fish Pathol.1985,20: 339-341.
[46]Ristow S S, Arnzen J M. Development of monoclonal antibodies that recognize a type-2 specific and a common epitope on the nucleoprotein of infectious hematopoietic necrosis virus[J]. J. Aquat. Anim. Health.1989,1:119-125.
[47]Kasornchandra J, Caswell-Reno P, Lannan C N, et al. Development and characterization of monoclonal antibodies against the snakehead rhabdovirus[J]. J. Aquat. Anim. Health.1992, 4:286-291.
[48]Mourton C, de Kinkelin P, Legouvello R, et al. Diagnostic potential of monoclonal antibodies specific for the envelope glycoprotein or the nucleocapsid of the viral hemorrhagic septicaemia virus[Z]. Budapest, Hungary:1991189.
[49]Kamei Y, Potey J L, Yoshimizu M, et al. Antigenic analysis of fish viruses with monoclonal antibodies[M]. New York:VCH Publishers,1990:201-204.
[50]Arkush K D, Mcneill C, Hedrick R. Production and initial characterization of monoclonal antibodies against channel catdish virus, the causative agent of channel catfish virus disease[J]. J. Aquat. Anim. Health.1992,4:81-89.
[51]Todd D, Jewhurst V A, Welsh M D, et al. Production and characterization of monoclonal antibodies to salmon pancreas disease virus[J]. Dis. Aquat. Organ.2001,46:101-108.
[52]Nakajima K, Sorimachi M. Producion of monoclonal antibodies against red sea bream iridovirus[J]. Fish Pathol.1995,30:47-52.
[53]邴国霞,张利峰,张鹤晓,等.鲤春病毒单克隆抗体的制备[J].中国兽医杂志.2007,43(3)：56-58.
[54]Azad A A, Jagadish M N, M A Brown E A. Deletion mapping and expression in Escherichia coli of the large genomic segment of a bimavirus[J]. Virology.1987,16(1):145-152.
[55]Novoa B, Blake S, B L Nicholson E A. Application of 3 techniques for diagnosing bimavirus infection in turbot[J]. Vet Res.1995,26(5-6):493-498.
[56]贺路,杜先林,左文功,等.鱼呼肠孤病毒单克隆抗体杂交瘤细胞系的建立及鉴定[J].淡水渔业.1990,5：3-5.
[57]Falk K, Namork E, Dannevig B H. characterization and applications of a monoclonal antibody against infectious salmon anemia virus[J]. Dis Aquat Organ.1998,34(2):77-85.
[58]Krossoy B, Devold M, L Sanders E A. Cloning and identification of the infectious salmon anemia virus haemagglutinin[J]. J Gen Vifol.2001,82:1757-1765.
[59]黄倢,于佳,王秀华,等.单克隆抗体酶联免疫技术检测对虾皮下及造血组织坏死病的病原及其传播途径[J].海洋水产研究.1995,16(1)：40-50.
[60]Lorenzo G, Estepa A, Coll J M. Fast neutralization/immunoperoxidase assay for viral haemorrhagic septicaemia with anti-nucleoprotein monoclonal antibody[J]. J Virol Methods. 1996,58(1-2):1-6.
[61]Fernandez-Alonso M, Lorenzo G, L Perez E A. Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus[J]. Dis Aquat Organ.1998,34(3):167-176.
[62]Lorenzen N. Affinity purification of the structural proteins of a fish Rhabdovirus by the use of monoclonal antibodies[J]. J virol Methods.1992,38(3):297-303.
[63]Noonan B, Enzman P J, Trust T J. Recombinant infectious hematopoietic necrosis virus hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout[J]. Environ Microbiol.1995,61:3586-3591.
[64]Bearotti M, Delmas B, A Lamoureux E A. Fish rhabdovirus cell entry is mediated by fibronectin[J]. J Virol.1999,73(9):7703-7709.
[65]Nakajima K, Maeno Y, M Fukudome E A. Immunofluorescence test for the rapid diagnosis of red sea bream iridovirus infection using monoclonal antibody[J]. Fish Pathol.1995,30(b): 15-119.
[66]Alim S R, Hossain M A, Chpwdhury E K, et al. G1 antigen:a cell-surface immunoprotactive 96 kDa glycoprotein from the virulent fish pathogen Enterococcus seriolicida, its purification and characterization[J]. Letters in Applied Microbiology.2001,32:357-361.
[67]Chase J C, Booy M H, Dawson-Coates J A, et al. Immunological detection of Kudoa thyrsites spores in muscle tissues of farmed Atlantic salmon, Salmo salar L[J]. Journal of Fish Diseases.2003,26:427-431.
[68]Chase J C, Dawson-Coates J A, Haddow J D, et al. Analysis of Kudoa thyrsites(Myxozoa: Myxosporea) spore antigens using monoclonal antibodies[J]. Diseases of Aquatic Organisms. 2001,45:121-129.
[69]冯娟,胡超群.抗紫红笛鲷血清免疫球蛋白单克隆抗体的制备[J].热带海洋学报.2002,21(4)：14-21.
[70]Costa J Z, Adams A, Bron J E, et al. Identification of B-cell epitopes on the betanodavirus capsid protein[J]. Journal of Fish Diseases.2007,30:419-426.
[71]Tokuda Y, Toyohara H, Ikemoto M, et al. Distribution of immunoglobulin-positive cells in the spleen and kidney of Japanese flounder Paralichthys olivaceus[J].66.2000(1082-1086).
[72]沈红,吴国娟,李焕荣,等.磺胺甲恶唑单克隆抗体的研制[J].中国兽医杂志.2006,42(3)：12-14.
[73]龚鹏飞,王权,陈永军.孔雀石绿单克隆抗体的制备及其鉴定[J].中国兽医科学.2007,37(4)：359-362.
[74]王自良,张改平,杨艳艳,等.氯霉素单克隆抗体免疫金标快速检测试纸的研制及性能测定[J].华北农学报.2006,21(5)：127-131.
[75]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry.1976,72: 248-254.
[76]司徒镇强,吴军正.细胞培养[M].西安：世界图书出版社,1996：232-242.
[77]陈慰峰,金伯泉.医学免疫学[M].北京：人民卫生出版社,2005：32.
[78]董志伟,王琰.抗体工程[M].北京：北京医科大学出版社,2002：209-214.
[79]Davis J M. A single-step technique for selecting and cloning hybridomas for monoclonal antibody production[J]. Methods Enzymol.1986,121:307-322.
[80]汪家政,范明.蛋白质技术手册[M].北京：科学出版社,2002：238-243.
[81]苏谨,张亚莉,尤长宣,等.抗人NGF单克隆抗体的制备及初步鉴定[J].细胞与分子免疫学杂志.2002,18(2)：168-170.
[82]Friguet B, Djavadi-Ohaniance L, Pages J, et al. A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site and application to hybridomes specific for the beta 2-subunit of Escherichia coli tryptophan synthase[J]. J Immunol Methods.1983,60(3):351-358.
[83]甘露,张雅萍,肖光夏.抗脆弱类杆菌单克隆抗体的研制和鉴定[J].第三军医大学学报.1999,21(7)：485-487.
[84]Westphal O, Jann K. Bacterial lipopolysaccharide extraction with phenol-water and further applications of procedure[J]. Methods in carbohydrates chemistry.1965,5:83-91.
[85]Carlson R W, Kalembasa S, Turowski D, et al. Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.[J]. J Bacteriol.1987,169:4923-4928.
[86]Carrion M, Bhat U R, Reuhs B, et al. Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum[J]. J Bacteriol.1990,172:1725-1731.
[87]中国生物制品标准委员会.中国生物制品规程(2000版)[M].北京：化学工业出版社,2000：238-239.
[88]沈关心,周汝麟.现代免疫学实验技术[M].武汉：湖北科学技术出版社,1998：32-34.
[89]罗玉芳,范春荣,黄劲松.亲和层析一步纯化IgM类单克隆抗体[J].单克隆抗体通讯.1989,26：67-71.
[90]Deshpande S S. Enzyme immunoassay from concept to product development[M]. New York:Chapman and Hall,1996:52.
[91]Wang L F, Yu M. Epitope identification and discovery using phage display libraries: applications in vaccine development and diagnostics[J]. Current Drug Targets.2004,5(1): 1-15.
[92]Bendayan M. A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology(Review)[J]. Biotechnic & Histochemistry.2000,75:203-242.
[93]Post G. Fish Health[M]. New Jersey:T.F.H. Publications, Inc.,1987:37-41.
[94]Agnew W, Barnes A C. Streptococcus iniae:An aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination[J]. Veterinary Microbiology. 2007,122(1-2):1-15.
[95]Furones M D, Rodgers C J, Munn C B. Yersinia ruckeri, the causal agent of enteric redmouth disease (ERM) in fish[J]. Annual Review of Fish Diseases.1993,3:105-125.
[96]Ye S G, Li H, Qiao G, et al. First case of Edwardsiella ictaluri infection in China farmed yellow catfish Pelteobagrus fulvidraco[J]. Aquaculture.2009,292(1-2):6-10.
[97]耿毅,汪开毓,陈德芳,等.斑点叉尾(?)一株致病菌的分离鉴定及系统发育分析[J].微生物 学报.2006,46(4)：649-652.
[98]Ayulo A M R, Machado R A, Scussel V M. Enterotoxigenic Escherichia coli and Staphylococcus aureus in fish and seafood from the Southern region of Brazil[J]. International Journal of Food Microbiology.1994,24(1-2):171-178.
[99]Simon S S, Sanjeev S. Prevalence of enterotoxigenic Staphylococcus aureus in fishery products and fish processing factory workers[J]. Food Control.2007,18(12):1565-1568.
[2]李爱华.气单胞菌(Aeromonads)分类与命名的最新进展1[J].微生物分类学.2001,23(2)：25-32.
[3]阳涛.南方鲇源豚鼠气单胞菌胞外产物活性与致病性研究[D].雅安：四川农业大学,2008.
[4]Gryllos L, Shaw J G, Gavin R E A. Role of flm Locus in MesopHilic Aeromonas Species Adherence[J]. Infect Immun.2001,69(1):65-74.
[5]陈金燕,王洪霞.一起水源性腹泻暴发的调查[J].实用预防医学.2003,10(6)：1012-1013.
[6]徐景野,傅小红,于梅.宁波市贝(甲)霓类海产品易损气单胞菌检出情况[J].实用预防医学.2001,8(6)：441442.
[7]Dwivedi M, Mishra A, Prasad A, et al. Aeromonas caviae septicemia in immunocompetent gastrointestinal carriers[J]. Braz J Infect Dis.2008,12(6):547-548.
[8]Al-Benwan K, Abbott S, Janda J M, et al. Cystitis caused by Aeromonas caviae [J]. J. Clin. Microbiol.2007,45:2348-2350.
[9]Teira R, Hernandez J L, Rojo P, et al. Bacteremia of biliary origin caused by Aeromonas caviae in a healthy patient[J]. J. Enferm Infecc Microbiol Clin.1991,9:388-389.
[10]李翠英,王平,陈玉霞.豚鼠气单胞菌引起化脓性脑膜炎一例[J].上海医学检验杂志.1994,9(3)：255.
[11]Pinna A, Sechi L A, Zanetti S, et al. Aeromonas caviae keratitis associated with contact lens wear[J]. Ophthalmology.2004,111(2):348-351.
[12]吉莉莉,汪开毓,肖丹,等.南方鲇溃疡综合症病原菌的分离与鉴定[J].淡水渔业.2008,38(3)：68-72.
[13]阳涛,汪开毓,肖丹,等.南方鲇源豚鼠气单胞菌胞外产物活性与致病性研究[J].淡水渔业.2008,38：4046.
[14]丁雷,岳永生,宋憬愚.虹鳟皮肤溃烂病的病原菌研究[J].淡水渔业.2002,32(3)：28-30.
[15]孙红祥,舒妙安.中华鳖几种常见疾病病原的分离鉴定及药敏试验[J].中国兽医报.2002, 22(2)：140-142.
[16]安利国,邢维贤,傅荣恕.鲤鱼竖鳞病的流行病学和病原学的研究[J].山东师大学报(自然科学版).1996,11(3)：68-72.
[17]樊海平,曾占壮,余培建.由豚鼠气单胞菌引起的欧洲鳗鲡败血症[J].水产学报.1999,23(3)：313-318.
[18]许信刚,伊岚,王高学.观赏金鱼豚鼠气单胞菌的分离鉴定及防治[J].西北农业学报.2006,15(1)：40-42.
[19]李小波,黄文芳.丰产鲫细菌性败血病的研究I-病原分离与鉴定[J].微生物学报.2003,30(5)：56-60.
[20]陈翠珍,张晓君,房海,等.鲢鳙打印病豚鼠气单胞茵主要特性及系统发育学分析[J].中国农学通报.2006,22(5)：455-459.
[21]徐海圣,黄立峰,王淑霞.中华绒螫蟹豚鼠气单胞菌的分离和鉴定[J].浙江大学学报.2001,27(6)：677-681.
[22]黄伟坚,黄均,秦爱珍.野生胡子鲇“腹水病”病原分离和鉴定[J].中国兽医科技.2000,30(4)：30-33.
[23]Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature.1975,256(5517):495-497.
[24]Chinchar V G, Metzger D W, A Granoff E A. Location of frog virus 3 proteins using monoclonal antibodies[J]. Virology.1984,137(1):211-216.
[25]汤仲明.生物技术药物的最新进展和展望[J].国外医药.2002,21(2)：115-117.
[26]Austin B. Monoclonal antibody-based enzyme-linked immunosorbent assays for the rapid diagnosis of clinical cases of entric red-mouth furunculosis in fish farms[J]. J. Fish Dis.1986,9: 469-474.
[28]黄钧,梁明龙,朱芸.温和气单胞菌单克隆抗体的制备及特性分析[J].河南农业科学.2006,2：108-110.
[29]Ueki N, Kayano Y, Muroga K. Pasteurella piscicida in juvenile red grouper[J]. Fish Pathology.1990,25:43-44.
[30]Balebona M C, Morinigo M A, Sedano J, et al. Isolation of Pasteurella piscicida from sea bass in south-western Spain[J]. Bulletin of the European Association of Fish Pathologists. 1992,12:168-170.
[31]Maniatis K, Morris D J, Adams A, et al. Detection of Photobacterium damsela subspecies piscicida in fixed tissue sections using immunohistochemistry and antigen retrival immunohistochemistry[J]. Journal of Fish Diseases.2000,23:343-347.
[32]Bakopoulos V, Adam A, Richards R H. Production and characterization of monoclonal antibodies against Pasteurella picicida, the causative agent of fish patearellosis[J]. Journal of Fish Diseases.1997,20:307-315.
[33]金晓航,黄威权,夏永娟,等.迟缓爱德华菌抗独特型单克隆抗体的制备及鉴定[J].水生生物学报.2005,29(3)：340-343.
[34]秦红,黄威权,杨向民,等.抗迟缓爱德华菌独特型单克隆抗体重链可变区基因的克隆及序列分析[J].细胞与分子免疫学杂志.2007,23(2)：155-156.
[35]夏永娟,黄威权,黄宝成,等.抗鳗弧菌独特型单克隆抗体的制备及鉴定[J].微生物学报.2001,41(4)：447-451.
[36]夏永娟,黄宝成,温伟红,等.抗鳗弧菌独特型单克隆抗体重链可变区基因的克隆及序列测定[J].细胞与分子免疫学杂志.2003,19(2)：176-177.
[37]陈瑞,唐旭,于辉,等.抗温和气单胞菌单克隆抗体的制备及初步鉴定[J].细胞与分子免疫学杂志.2007,23(2)：146-147.
[38]郭闯,方苹,郭立新,等.嗜水气单胞菌气溶素单克隆抗体的制备及初步应用[J].水产科学.2007,26(3)：167-170.
[39]Klesius P, Evans J, C Shoemaker E A. Rapid detection and identification of streptococcus iniae using a monoclonal antibody-based indirect fluorescent antibody technique[J]. Aquaculture.2006,258:180-186.
[40]池信才,王军,鄢庆枇,等.大黄鱼病原副溶血弧菌单克隆抗体制备及其应用[J].海洋科学.2007,31(8)：1-5.
[41]郝贵杰,沈锦玉,曹铮,等.抗哈维氏弧菌单克隆抗体杂交瘤细胞系的建立及其特性鉴定[J].23.2007,9(838-840).
[42]Caswell-Reno P, Reno P W, Nicholson B L. Monoclonal antibodies to infectious pancreatic necrosis virus (IPNV):analysis of viral epitopes and comparison of different isolates[J]. J. Gen. Virol.1986,67:2193-2205.
[43]Wolski S C, Roberson B S, Hetrick F M. Monoclonal antibodies to the Sp strain of infectious pancreatic necrosis virus[J]. Vet. Immunol. Immunopathol.1986,12:373-381.
[44]Lipipun V, Caswell-Reno P, Hsu Y L, et al. Antigenic analysis of Asian birnavirus isolates using monoclonal antibodies[J]. Fish Pathol.1989,24:155-160.
[45]Schultz C L, Lidgerding B C, Mcallister P E, et al. Production and characterization of monoclonal antibody against infectious hematopoietic necrosis virus[J]. Fish Pathol.1985,20: 339-341.
[46]Ristow S S, Arnzen J M. Development of monoclonal antibodies that recognize a type-2 specific and a common epitope on the nucleoprotein of infectious hematopoietic necrosis virus[J]. J. Aquat. Anim. Health.1989,1:119-125.
[47]Kasornchandra J, Caswell-Reno P, Lannan C N, et al. Development and characterization of monoclonal antibodies against the snakehead rhabdovirus[J]. J. Aquat. Anim. Health.1992, 4:286-291.
[48]Mourton C, de Kinkelin P, Legouvello R, et al. Diagnostic potential of monoclonal antibodies specific for the envelope glycoprotein or the nucleocapsid of the viral hemorrhagic septicaemia virus[Z]. Budapest, Hungary:1991189.
[49]Kamei Y, Potey J L, Yoshimizu M, et al. Antigenic analysis of fish viruses with monoclonal antibodies[M]. New York:VCH Publishers,1990:201-204.
[50]Arkush K D, Mcneill C, Hedrick R. Production and initial characterization of monoclonal antibodies against channel catdish virus, the causative agent of channel catfish virus disease[J]. J. Aquat. Anim. Health.1992,4:81-89.
[51]Todd D, Jewhurst V A, Welsh M D, et al. Production and characterization of monoclonal antibodies to salmon pancreas disease virus[J]. Dis. Aquat. Organ.2001,46:101-108.
[52]Nakajima K, Sorimachi M. Producion of monoclonal antibodies against red sea bream iridovirus[J]. Fish Pathol.1995,30:47-52.
[53]邴国霞,张利峰,张鹤晓,等.鲤春病毒单克隆抗体的制备[J].中国兽医杂志.2007,43(3)：56-58.
[54]Azad A A, Jagadish M N, M A Brown E A. Deletion mapping and expression in Escherichia coli of the large genomic segment of a bimavirus[J]. Virology.1987,16(1):145-152.
[55]Novoa B, Blake S, B L Nicholson E A. Application of 3 techniques for diagnosing bimavirus infection in turbot[J]. Vet Res.1995,26(5-6):493-498.
[56]贺路,杜先林,左文功,等.鱼呼肠孤病毒单克隆抗体杂交瘤细胞系的建立及鉴定[J].淡水渔业.1990,5：3-5.
[57]Falk K, Namork E, Dannevig B H. characterization and applications of a monoclonal antibody against infectious salmon anemia virus[J]. Dis Aquat Organ.1998,34(2):77-85.
[58]Krossoy B, Devold M, L Sanders E A. Cloning and identification of the infectious salmon anemia virus haemagglutinin[J]. J Gen Vifol.2001,82:1757-1765.
[59]黄倢,于佳,王秀华,等.单克隆抗体酶联免疫技术检测对虾皮下及造血组织坏死病的病原及其传播途径[J].海洋水产研究.1995,16(1)：40-50.
[60]Lorenzo G, Estepa A, Coll J M. Fast neutralization/immunoperoxidase assay for viral haemorrhagic septicaemia with anti-nucleoprotein monoclonal antibody[J]. J Virol Methods. 1996,58(1-2):1-6.
[61]Fernandez-Alonso M, Lorenzo G, L Perez E A. Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus[J]. Dis Aquat Organ.1998,34(3):167-176.
[62]Lorenzen N. Affinity purification of the structural proteins of a fish Rhabdovirus by the use of monoclonal antibodies[J]. J virol Methods.1992,38(3):297-303.
[63]Noonan B, Enzman P J, Trust T J. Recombinant infectious hematopoietic necrosis virus hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout[J]. Environ Microbiol.1995,61:3586-3591.
[64]Bearotti M, Delmas B, A Lamoureux E A. Fish rhabdovirus cell entry is mediated by fibronectin[J]. J Virol.1999,73(9):7703-7709.
[65]Nakajima K, Maeno Y, M Fukudome E A. Immunofluorescence test for the rapid diagnosis of red sea bream iridovirus infection using monoclonal antibody[J]. Fish Pathol.1995,30(b): 15-119.
[66]Alim S R, Hossain M A, Chpwdhury E K, et al. G1 antigen:a cell-surface immunoprotactive 96 kDa glycoprotein from the virulent fish pathogen Enterococcus seriolicida, its purification and characterization[J]. Letters in Applied Microbiology.2001,32:357-361.
[67]Chase J C, Booy M H, Dawson-Coates J A, et al. Immunological detection of Kudoa thyrsites spores in muscle tissues of farmed Atlantic salmon, Salmo salar L[J]. Journal of Fish Diseases.2003,26:427-431.
[68]Chase J C, Dawson-Coates J A, Haddow J D, et al. Analysis of Kudoa thyrsites(Myxozoa: Myxosporea) spore antigens using monoclonal antibodies[J]. Diseases of Aquatic Organisms. 2001,45:121-129.
[69]冯娟,胡超群.抗紫红笛鲷血清免疫球蛋白单克隆抗体的制备[J].热带海洋学报.2002,21(4)：14-21.
[70]Costa J Z, Adams A, Bron J E, et al. Identification of B-cell epitopes on the betanodavirus capsid protein[J]. Journal of Fish Diseases.2007,30:419-426.
[71]Tokuda Y, Toyohara H, Ikemoto M, et al. Distribution of immunoglobulin-positive cells in the spleen and kidney of Japanese flounder Paralichthys olivaceus[J].66.2000(1082-1086).
[72]沈红,吴国娟,李焕荣,等.磺胺甲恶唑单克隆抗体的研制[J].中国兽医杂志.2006,42(3)：12-14.
[73]龚鹏飞,王权,陈永军.孔雀石绿单克隆抗体的制备及其鉴定[J].中国兽医科学.2007,37(4)：359-362.
[74]王自良,张改平,杨艳艳,等.氯霉素单克隆抗体免疫金标快速检测试纸的研制及性能测定[J].华北农学报.2006,21(5)：127-131.
[75]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry.1976,72: 248-254.
[76]司徒镇强,吴军正.细胞培养[M].西安：世界图书出版社,1996：232-242.
[77]陈慰峰,金伯泉.医学免疫学[M].北京：人民卫生出版社,2005：32.
[78]董志伟,王琰.抗体工程[M].北京：北京医科大学出版社,2002：209-214.
[79]Davis J M. A single-step technique for selecting and cloning hybridomas for monoclonal antibody production[J]. Methods Enzymol.1986,121:307-322.
[80]汪家政,范明.蛋白质技术手册[M].北京：科学出版社,2002：238-243.
[81]苏谨,张亚莉,尤长宣,等.抗人NGF单克隆抗体的制备及初步鉴定[J].细胞与分子免疫学杂志.2002,18(2)：168-170.
[82]Friguet B, Djavadi-Ohaniance L, Pages J, et al. A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site and application to hybridomes specific for the beta 2-subunit of Escherichia coli tryptophan synthase[J]. J Immunol Methods.1983,60(3):351-358.
[83]甘露,张雅萍,肖光夏.抗脆弱类杆菌单克隆抗体的研制和鉴定[J].第三军医大学学报.1999,21(7)：485-487.
[84]Westphal O, Jann K. Bacterial lipopolysaccharide extraction with phenol-water and further applications of procedure[J]. Methods in carbohydrates chemistry.1965,5:83-91.
[85]Carlson R W, Kalembasa S, Turowski D, et al. Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.[J]. J Bacteriol.1987,169:4923-4928.
[86]Carrion M, Bhat U R, Reuhs B, et al. Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum[J]. J Bacteriol.1990,172:1725-1731.
[87]中国生物制品标准委员会.中国生物制品规程(2000版)[M].北京：化学工业出版社,2000：238-239.
[88]沈关心,周汝麟.现代免疫学实验技术[M].武汉：湖北科学技术出版社,1998：32-34.
[89]罗玉芳,范春荣,黄劲松.亲和层析一步纯化IgM类单克隆抗体[J].单克隆抗体通讯.1989,26：67-71.
[90]Deshpande S S. Enzyme immunoassay from concept to product development[M]. New York:Chapman and Hall,1996:52.
[91]Wang L F, Yu M. Epitope identification and discovery using phage display libraries: applications in vaccine development and diagnostics[J]. Current Drug Targets.2004,5(1): 1-15.
[92]Bendayan M. A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology(Review)[J]. Biotechnic & Histochemistry.2000,75:203-242.
[93]Post G. Fish Health[M]. New Jersey:T.F.H. Publications, Inc.,1987:37-41.
[94]Agnew W, Barnes A C. Streptococcus iniae:An aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination[J]. Veterinary Microbiology. 2007,122(1-2):1-15.
[95]Furones M D, Rodgers C J, Munn C B. Yersinia ruckeri, the causal agent of enteric redmouth disease (ERM) in fish[J]. Annual Review of Fish Diseases.1993,3:105-125.
[96]Ye S G, Li H, Qiao G, et al. First case of Edwardsiella ictaluri infection in China farmed yellow catfish Pelteobagrus fulvidraco[J]. Aquaculture.2009,292(1-2):6-10.
[97]耿毅,汪开毓,陈德芳,等.斑点叉尾(?)一株致病菌的分离鉴定及系统发育分析[J].微生物 学报.2006,46(4)：649-652.
[98]Ayulo A M R, Machado R A, Scussel V M. Enterotoxigenic Escherichia coli and Staphylococcus aureus in fish and seafood from the Southern region of Brazil[J]. International Journal of Food Microbiology.1994,24(1-2):171-178.
[99]Simon S S, Sanjeev S. Prevalence of enterotoxigenic Staphylococcus aureus in fishery products and fish processing factory workers[J]. Food Control.2007,18(12):1565-1568.