间接ELISA检测鸭疫里氏杆菌抗体的研究

本文成功地建立了一种快速,敏感,特异性强的检测鸭疫里氏杆菌１型抗体的方法－－酶联免疫吸附试验（ＥＬＩＳＡ）,探讨和确定了ＥＬＩＳＡ的最适条件,通过对３２份阳性血清效价的测定,发现血清１：１００稀释时的ＯＤ值与血清效价倒数的以２为底的对数之间存在直线相关性,相关系数为０．９７,并由此建立了标准曲线和回归方程。对用不同疫苗免疫鸭的抗体消长规律测定发现,油佐剂疫苗效果最好,甲醛灭活苗两次免疫次之,甲醛灭     

间接ELISA检测鸭疫里氏杆菌抗体的研究

本文成功地建立了一种快速、敏感、特异性强的检测鸭疫里氏杆菌１型抗体的方法—酶联免疫吸附试验（ＥＬＩＳＡ）。探讨和确定了ＥＬＩＳＡ的最适条件。通过对３２份阳性血清效价的测定,发现血清１∶１００稀释时的ＯＤ值与血清效价倒数的以２为底的对数之间存在直线相关性,相关系数为０．９７,并由此建立了标准曲线和回归方程。对用不同疫苗免疫鸭的抗体消长规律测定发现,油佐剂疫苗效果最好,甲醛灭活苗两次免疫次之,甲醛灭活苗一次免疫效果最差。从抗体水平角度推断,雏鸭１０日龄左右用苗最好,每只鸭注射０．５ｍｌ是可行的。     

1型鸭疫里氏杆菌OmpA蛋白间接ELISA方法的建立

为建立检测1型鸭疫里氏杆菌(R.anatipestifer)的间接ELISA方法,本研究根据已发表的R.anatipestifer外膜蛋白A(ompA)基因序列(AF104937)设计引物,扩增1型R.anatipestifer HLG1株的ompA基因,构建重组质粒pHtb-ompA,转化大肠杆菌BL21(DE3),并利用IPTG进行诱导表达。SDS-PAGE和western blot结果表明,表达蛋白约为55 ku,具有良好的抗原活性。以纯化的OmpA为包被抗原建立间接ELISA并对条件进行优化。建立的ELISA具有良好的特异性、敏感性;与HLG1株菌体裂解蛋白为抗原的间接ELISA比较,符合率为91.3%。本研究建立的ELISA方法为R.anatipestifer的流行病学调查和SPF鸭的监测提供了快速、特异的血清学诊断方法。     

1型鸭疫里氏杆菌抗体间接ELISA检测方法的建立

以1型鸭疫里氏杆菌(RA)全基因组保守区域ompA基因的重组表达产物为包被抗原,建立了检测RA血清抗体的间接ELISA方法。原核表达的重组ompA蛋白经纯化后作为包被物,以方阵滴定法确定抗原最佳包被浓度为1.5μg/mL,待检血清最佳稀释度为1∶100。与普通的微量凝集试验相比较,该方法灵敏度高于凝集试验约16倍～128倍。与鸭源大肠埃希菌、鸭源多杀性巴氏杆菌、鸭链球菌、鸭源呼肠病毒、鸭肝炎病毒和鸭瘟病毒感染鸭血清均无交叉反应,表明该方法特异性好。利用该方法检测了雏鸭免疫RA灭活油乳剂疫苗之后血清抗体水平。     

鸭疫里氏杆菌保存条件探讨

鸭疫里氏杆菌(Riemerella anatipestifer,简称RA)感染,又称为鸭疫传染性浆膜炎(Infectious serostis of duck),曾命名为鸭疫巴氏杆菌病,其病变特征为纤维素性心包炎、肝周炎、气囊炎及脑膜充血、出血,主要感染1～8周龄雏鸭，2～3周龄雏鸭最易感染,死亡率差异较大。RA对各品种的鸭都有致病性,对鹅、鸡及其他水禽也具有致病性,发病率和死亡率都很高,已成     

应用间接免疫荧光抗体试验检测鸭疫里氏杆菌

建立间接免疫荧光抗体试验对鸭疫里氏杆菌进行特异性检测，从而与鸭大肠杆菌病、鸭霍乱等进行医分鉴别，该技术具有简便、快速、敏感和特异性强等特点，对发病小鸭的检测结果表明，鸭脑组织、鼻窦及肝脏等细菌检出率较高。     

鸭疫里氏杆菌优势血清型变化的研究

近年来，从浙江省及其周边地区鸭场患有传染性浆膜炎的病死鸭中分离鉴定出的77株鸭疫里氏杆菌，用标准定型血清作凝集试验，鉴定出：RAⅠ型33株，RAⅡ型33株，二者共占总分离茵数的85．7％，而未定型茵11株，占14．3％。结果表明：RA的优势血清型在不同的时间出现不同的变化：1998年以前为单一的RAⅠ型：1999～2000年以RAⅠ为主，占总分离茵数的51．9％；2001年以RAⅡ型占绝对优势，占总分离菌数的89．5％；而到了2002年，未定型的菌株数增加到了33．3％。同时也证明：该地区的优势血清型为RAⅠ、Ⅱ型。     

间接ELISA检测鸭疫里氏杆菌抗体的研究

以提取的血清１型鸭疫里氏杆菌（Ｒ．ａ）Ｊｂ２株的脂多糖作为包被抗原,成功地建立了一种检测１型Ｒ．ａ抗体的间接ＥＬＩＳＡ方法,特异性试验和重复性试验证明此方法的特异性高等重复性好,并比较了间接ＥＬＩＳＡ方法,间接血凝试验,玻片凝集试验和琼脂扩散试验对抗体检出敏感性,结果表明间接ＥＬＩＳＡ方法最敏感,人工感染后鸭第７２小时即可检出血清抗体为阳性（３／４）其次是间接血凝试验,第９６小时即可检出５０％（２     

鸭疫里氏杆菌外膜蛋白免疫原性研究

本研究采用超速离心法提取鸭疫里氏杆菌的外膜蛋白,在电镜下呈典型的双层泡状结构,ＳＤＳ－ＰＡＧＥ电泳分析结果细菌的外膜蛋白的分子量在２１ＫＤ和１５１ＫＤ之间。经Ｗｅｓｔｅｒｎ ｂｌｏｔ检测表明,４４ＫＤ外膜蛋白与免疫鸭血清和自然感染后康复鸭血清出现较强的阳性反应,而感染发病的濒死鸭血清反应则很微弱。     

间接荧光抗体技术检测鸭疫里氏杆菌

鸭疫里氏杆菌是危害养鸭业的重要的传染病之一,主要侵害１～８周龄肉鸭。自然感染情况下主要表现为心包炎、肝周炎,发病和死亡随着饲养环境条件有所差异,平均死亡率在２０％左右。该病在我国养鸭地区普遍存在,由于该细菌的分离和鉴定方法未能够被广大的兽医工作人员所...     

Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and gastro and extra-intestinal infections in humans. Monoclonal antibodies (MAbs) were produced against outer membrane proteins (OMPs) of E. tarda ET-7, isolated from diseased snakehead (Ophiocephalus punctatus). Two stable hybridoma clones, designated as 3F10 and 2C3 MAbs were found to be potentially specific for E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These MAbs recognized major immunogenic OMP band at 44kDa in Western blotting. Both MAbs belonged to the IgG1 isotype and recognized different epitopes of OMP as seen by competitive ELISA. These MAbs strongly reacted with all 17 isolates of E. tarda used in our study by indirect ELISA and Western blotting. Interestingly, no reaction was observed with the reference strain of E. tarda (MTCC 2400). The sensitivity of 3F10 MAb to detect whole cells of E. tarda was up to a level of 1x10(4)CFU/ml in indirect ELISA. No cross-reactivity of MAbs were seen with Escherichia coli, Salmonella arizonae, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae, Flavobacterium ferrugineum and Mycobacterium tuberculosis. These MAbs could be used for specific detection of E. tarda infection in fish by immunoassays.     

鸭疫里默氏菌研究进展

鸭传染性浆膜炎是由鸭疫里默氏菌引起的一种重要的鸭细菌病。由于该病分布的广泛性以及引起鸭的高死亡率、发育迟缓和淘汰率增加,给养鸭业造成巨大的经济损失。所以,对鸭疫里默氏菌进行深入研究,以便预防和控制、乃至最终消灭本病,有着重要的经济意义。论文从血清型、诊断方法、疫苗等方面概述了近年来鸭疫里默氏菌研究进展。     

Antigenic analysis of avian Chlamydia psittaci using monoclonal antibodies to the major outer membrane protein.

Monoclonal antibodies to the major outer membrane protein (MOMP) of Chlamydia psittaci derived from a parrot were established for antigenic analysis of avian C. psittaci. With 17 monoclonal antibodies to MOMP, 17 reactivity patterns were identified on 112 strains of C. psittaci, C. pneumoniae and C. trachomatis, which were isolated from birds, mammals and humans in Japan, U.S.A., Canada and Taiwan, from 1938 to 1987. Immunological reactivity of budgerigar-derived strains to the monoclonal antibodies was different from that of pigeon-derived strains. Imported bird-derived strains were distinguishable from domestic bird-derived strains by the reactivity to the monoclonal antibodies. A close relationship between the subtypes and geographic origins was indicated on budgerigar-derived strains. On the contrary, various reactivity patterns were shown in pigeon-derived strains isolated in a narrow area. The monoclonal antibodies established in the present work may be useful probes for ecological study of avian C. psittaci.     

抗猪传染性胃肠炎病毒重组S蛋白单克隆抗体的制备与鉴定

以纯化的猪传染性胃肠炎病毒(TGEV)重组S蛋白免疫BALB/c小鼠,通过细胞融合技术,间接ELISA方法进行筛选和有限稀释法3次克隆,获得2株稳定分泌抗TGEV重组S蛋白单克隆抗体的杂交瘤细胞株,分别为C7C882和B5G8G7,其染色体平均计数均为88±10对,制备腹水抗体效价分别为1:2×105和1:104,分泌抗体亚类均为IgM型.2株杂交瘤细胞上清与猪流行性腹泻病毒(PEDV)、猪轮状病毒(PRV)、猪伪狂犬病病毒(PrV)均无交叉反应,与TGEV感染细胞经间接免疫荧光检测均呈黄绿色荧光.     

Vaccination of ducks with recombinant outer membrane protein (OmpA) and a 41 kDa partial protein (P45N') of Riemerella anatipestifer.

The generation of protective immunity against Riemerella anatipestifer infection in ducks were investigated by immunizations with recombinant glutathione sulfatransferase (GST) fusion's proteins of OmpA, a 42kDa major outer membrane protein, and P45N', a 41kDa N-terminal fragment of a newly identified 45kDa potential surface protein from R. anatipestifer. The DNA encoding OmpA and P45N' were isolated from R. anatipestifer serotype 15 (field strain 110/89) and serotype 19 (reference strain 30/90), respectively. Immunoblotting and ELISA results showed that the purified recombinant proteins induced the production of antibodies in immunized ducks. However, neither was protective against subsequent challenge with the virulent serotype 15 strain, 34/90. All the five ducks immunized with formalinized R. anatipestifer strain 34/90 survived the challenge with the homologous strain whereas six out of seven ducks in the non-immunized control group died within a week following the challenge.     

SMD单克隆抗体的制备及测定方法的建立

用戊二醛作为偶联剂 ,将磺胺对甲氧嘧啶 (SMD )与牛血清白蛋白 (BSA )或卵清蛋白 (OVA)偶联形成完全抗原 ,经紫外分光光度计扫描鉴定。以人工抗原免疫BALB/c小鼠 ,取脾细胞与骨髓瘤细胞 (SP2 /0 -Ag1 4)融合 ,用间接ELISA联合竞争ELISA法筛选出产生针对SMD抗体的杂交瘤细胞。经克隆 ,得到 3株特异性好的阳性杂交瘤细胞 ,注入小鼠腹腔产生腹水。建立了测定SMD的ELISA法 ,其检测下限小于5ng/mL。     

Use of monoclonal antibodies to identify outer membrane antigens of Actinobacillus species

Three monoclonal antibodies (LG17, LG30, and LG33) were used to identify outer membrane antigens of Actinobacillus sp (As8C isolate) cultured from the epididymides of an infected ram lamb. Specificity of the 3 antibodies to As8C antigens was determined by use of bacterial agglutination, the enzyme-linked immunosorbent assay, and the indirect fluorescent antibody test. Results of immunoelectron microscopy confirmed that each antibody was specific for epitopes on As8C outer membrane antigens. Evaluation by use of enzyme-linked immunoelectrotransfer blot indicated that target antigens for LG17 and LG33 antibodies had molecular weights of 10 kilodaltons and 43 kilodaltons, respectively. Multiple-band staining was observed with the LG33 antibody. The target antigen for the LG30 antibody could not be discerned by use of enzyme-linked immunoelectrotransfer blot. For each of the 3 monoclonal antibodies, enzyme-linked immunosorbent assay titers were obtained for Actinobacillus seminis, A actinomycetemcomitans, and 10 field isolates of Actinobacillus spp. Target antigens for LG17 and LG30 antibodies occurred infrequently or were absent on these bacteria. However, the target antigen for the LG33 antibody was shared by Actinobacillus seminis, A actinomycetemcomitans, and the 10 field isolates of Actinobacillus spp, indicating some diversity of outer membrane antigens between isolates.     

猪细小病毒核衣壳VP2蛋白单克隆抗体的制备及部分特性鉴定

以纯化的PPV重组VP2蛋白免疫BALB/c小鼠,通过细胞融合技术,间接ELISA筛选和3次以上细胞克隆,获得了5株稳定分泌抗PPV VP2蛋白单克隆抗体杂交瘤细胞株,分别命名为2D5、1F11、2B4、3C8、1 H3。其染色体平均数均为87～102条,分泌抗体亚类4株为Ig M类,1株为IgG类,Western blot检测表明,5株单抗均识别猪细小病毒VP2蛋白;间接免疫荧光鉴定表明,5株单抗均与PPV全病毒发生反应;间接ELISA鉴定与其他相关病毒的交叉反应性表明,制备的5株单抗不与TEGV、PRV和PEDV反应,表明所制备的抗体与猪细小病毒具有较强的特异性反应。     

狂犬病病毒HepFlury株P蛋白单克隆抗体的制备及间接免疫荧光检测方法的建立

本研究以原核表达的融合型的重组蛋白pET32a(+)-P(狂犬病病毒HepFlury株P蛋白)免疫6~8周龄的BALB/c小鼠,取脾细胞和SP2/0按常规杂交瘤技术进行细胞融合,经间接免疫荧光方法(IFA)筛选及对mAb进行初步鉴定。经3次亚克隆后获得1株稳定分泌表达的杂交瘤细胞株5G8。