摘要
用甲醛灭活哈维氏弧菌(Vibrio harveyi)GYC1108-1制备免疫原免疫8周龄雌性BALB/c小鼠,利用淋巴细胞杂交瘤技术,用间接酶联免疫吸附试验(ELISA)对杂交瘤进行筛选,阳性克隆经2—3次亚克隆后,共获得5株针对GYC1108-1的单克隆抗体。选取1B7制备小鼠腹水抗体,其细胞上清及腹水效价分别为1∶3200和1∶32000。同样用灭活的哈维氏弧菌免疫新西兰大白兔,5次免疫后颈动脉采血,离心取血清,并用饱和硫酸铵法进行纯化,制备了兔抗哈维氏弧菌多克隆抗体,其ELISA效价为1∶51200。利用1B7单克隆抗体和兔抗哈维氏弧菌多克隆抗体以及山羊抗小鼠HRP酶标二抗,建立了检测哈维氏弧菌的三抗体夹心酶联免疫吸附试验(TAS-ELISA)方法。该方法对哈维氏弧菌的最小检出浓度为1×104个/mL。用该TAS-ELISA方法检测大黄鱼(Pseudosciaena crocea)样品,54尾患病大黄鱼中有45尾检出哈维氏弧菌,而14尾健康大黄鱼都没有检出哈维氏弧菌。由此可见,本试验建立的TAS-ELISA方法,可以用于患病大黄鱼哈维氏弧菌的快速诊断。
Vibrio harveyi is a kind of important pathogenic bacteria for seawater animals, such as prawn, crab, calm and ormer. Along with the development of seawater fish cage-culture in China, Vibrio harveyi also becomes a main cause of disease of cage-culture fish, including Lutianus erythopterus, Seriola dumerili, Epinephelus coioides and Pseudosciaena crocea. One pathogenic V. harveyi strain, GYC1108-1, which was isolated from diseased Pseudosciaena crocea in Zhejiang Province, 2003, was identified after morphological observation, biochemical characteristic analysis, 16sRNA and HSP60 gene sequence detection. V. harveyi GYC1108-1 was inactivated by formaldehyde to prepare antigen for the immunizing of 8-week-old female BALB/c mice. Spleen cells collected from immunized mice after the fifth immunization were fused with SP2/0-Ag-14 myeloma cells. Indirect Enzyme-Linked Immunosorbent Assay （ELISA） was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones. After three cycles of subcloning, five McAbs against the GYC1108-1 were selected and designated as 1B7, 1D7, 2G8, 3G6 and 3H7 respectively. The five McAbs in culture liquid were proved to have high ELISA titers between 1：400--1：3200. By using the immunoglobulin subtypes kit, 1B7 and 2G8 were identified to be IgG1 ; 1D7 and 3G6 to be IgG3; 3H7 to be IgG2a, respectively. The 1 B7 was chose to prepare ascites and the ELISA titers of 1B7 in culture liquid and ascites were 1 ： 3200 and 1：32000, respectively. One rabbit was immunized with V. harveyi GYC1108-1. The immune sera of rabbit were collected after five immunizations. Antibodies （IgG） obtained from the immune sera were purified by ammonium sulfate fractionation. The ELISA titer of the multi-clone antibody was 1：51200. A triple antibody sandwich enzyme-linked immunosorbent assay（TAS-ELISA） was developed with the plates pre-coated with rabbit polyclonal antibodies against V. harveyi for bacteria capturing, followed by samples incubation, McAb（ 1 B7） and goat-anti-mnuse-IgG antibodies labeled with HRP. The lowest detectable concentration of V. harveyi detected by the TAS-ELISA was 1 × 10^4cells/mL. The TAS-ELISA was used for detecting V. harveyi in Pseudosciaena crocea, V. harveyi was detected from 45 fish among 54 diseased P. crocea, and no V. harveyi was detected from 14 healthy P. crocea. The results indicated that the TAS-ELISA specially against V. harveyi could be used for the rapid diagnosis of diseased P. crncea.
出处
《水生生物学报》
CAS
CSCD
北大核心
2009年第3期413-417,共5页
Acta Hydrobiologica Sinica
基金
浙江省科技厅重点项目(2005F12005)资助
