单抗捕捉ELISA检测猪附红细胞体血清抗体方法的建立

猪附红细胞体病是由附红细胞体(Eperythrozoon)寄生于多种动物和人红细胞表面、血浆和骨髓而引起的一种人畜共患病[1-2].本病自1928年发现以来被人们长期忽视,近年来随着人附红细胞体病病例的增多,家畜附红细胞体病的暴发流行,引起国内外学者的关注.本试验首次应用自主研制的猪附红细胞体单克隆抗体,初步建立了一种简便、经济、特异的单抗捕捉ELISA方法,用于检测猪附红细胞体的血清抗体,为诊断试剂盒的研制奠定了基础.     

猪附红细胞体双抗体夹心ELISA检测方法的建立和应用

为建立猪附红细胞体双抗体夹心ELISA(DAS-ELISA)检测方法,本研究以抗猪附红细胞体单克隆抗体为捕获抗体,兔抗猪附红细胞体多克隆抗体为检测抗体,优化工作条件,建立猪附红细胞体DAS-ELISA检测方法.结果显示:该方法的最佳工作条件为:捕获抗体包被浓度为5.42 g/mL,检测抗体浓度为6.84 g/mL,酶标...     

单抗捕捉ELISA检测猪附红细胞体血清抗体方法的建立

在制备针对猪附红细胞体病的特异性单克隆抗体的基础上,建立了单抗捕捉快速检测猪附红细胞体病抗体的ELISA方法,并对影响ELISA结果的各项反应条件进行了优化.结果表明,猪附红细胞体腹水单抗的最佳稀释度为1:20 000,猪附红细胞体病抗原工作质量浓度为4.088 mg/L,血清稀释度为1:40,HRP标记的兔抗猪IgG的工作浓度为1:5 000.待检血清和酶标二抗的反应时间均为37℃1 h;底物在室温显色时间为10 min.已建立的ELISA方法与PCR平行检测50份样本,总符合率为92%,表明该方法特异性强、快速、敏感性高.用该法检测了各地采集的171份血清,阳性率为40.4%,与镜检的符合率为59.2%;阴性率为47.4%,与镜检的符合率为66.7%,总符合率为67.3%.     

应用间接血凝试验诊断猪附红细胞体病

附红细胞体病 (Eperythrozoonosis)是由附红细胞体 (Eperythrozoon)寄生于红细胞表面、血浆及骨髓内而引起的一种以红细胞压积降低、血红蛋白下降、白细胞增多、贫血、黄疸、发热为主要临床特征的人兽共患病 [1,2 ]。近年来随着人附红细胞体病例的增多 ,家畜附红细胞体病的暴发流行 ,已引起国内外学者的广泛关注 [3 ,4]。本试验探索了将用 IHA于猪附红细胞体病的诊断 ,以便为该病的临床诊断提供一种较为敏感、特异、实用和快速的血清学诊断方法。1　材料与方法1.1　血清样品　猪附红细胞体病阳性血清和阴性血清均由本教研室提供 ,被检血清…     

猪附红细胞体病间接ELISA诊断试剂盒的研制及初步应用

以纯化的猪附红细胞体特异性抗原为包被抗原,在建立猪附红细胞体病间接ELISA检测方法的基础上对各反应参数进行了优化,并初步组装成试剂盒。组装的试剂盒抗原最佳包被质量浓度为30 mg/L,被检血清稀释度为1∶100,酶标二抗的最适工作滴度为1∶4 000,底物TMB最适反应条件为室温15 min;检测血清的判定标准为D450值≥0.13定为阳性反应,D450值≤0.11定为阴性反应,0.11~0.13定为疑似反应;该试剂盒不与猪大肠杆菌病、猪弓形虫病及猪瘟等阳性血清发生交叉反应,具有较好的特异性;在不同时间对阴阳性样品重复检测8次,阴阳性血清的D450值变异系数均未超过10%,说明该试剂盒具有良好的重复性;通过对组装试剂盒中各组分的稳定性和保存时间的测定结果证实,该试剂盒的保质期暂定为6个月;应用保存6个月的试剂盒通过对吉林省延边地区的60份猪血清样本检测,并与间接ELISA比较,其阳性符合率达100%,说明该试剂盒具有较好的稳定性。     

利用IFAT检测猪附红细胞体的研究

利用猪附红细胞体抗原玻片，通过IFAT方法，对96份疫区血清样本进行检测，并与血涂片镜检方法进行比较。结果显示IFAT法比血涂片法敏感性要高出40个百分点：用猪弓形体阳性血清、猪蛔虫阳性血清、猪瘟阳性血清进行对照，结果均阴性，表明IFAT法特异性强，敏感性高。     

猪附红细胞体病

猪附红细胞体病通常发生在哺乳猪、怀孕的母猪以及受到高度应激的肥育猪身上；病原是一种寄生在猪红血球上的立克次氏体，其可造成红血球的改变而容易为体内的网状内皮系统或是被吞噬细胞所破坏，因而造成红血球数量的减少，导致贫血、发育减慢、料肉比提高。传染途径主要经由节肢动物叮咬以及器械的机械性传播。     

猪附红细胞体病

猪附红细胞体病是由寄生于猪红细胞和血浆中的附红细胞体而引起的传染性疾病.经口感染或媒介昆虫(如猪虱)吸血而感染,一般在温度、湿度较高的夏秋季节多发,主要引起猪高热、贫血、黄疸和全身发红,因此又称红皮病.尤其是1月龄左右的仔猪发病更严重,可引起大批死亡.     

单克隆抗体间接免疫荧光(IFA)检测猪附红细胞体

为建立利用单克隆抗体检测猪附红细胞体病的间接免疫荧光(IFA)方法,用抗猪附红细胞体的单克隆抗体,对48份疑似猪附红细胞体感染的病猪猪血进行免疫荧光检测,并与血涂片镜检及PCR检测方法进行比较,结果显示IFA检测的阳性率为68.75%,与PCR检测阳性率为77.08%符合率较高,比血涂片的瑞氏和姬姆萨染色镜检阳性率高出近30个百分点;而用猪肺炎支原体、猪链球菌和猪巴氏杆菌作对照,结果均呈阴性。建立的用抗猪附红细胞体的单克隆抗体检测猪附红细胞体病的IFA特异性强,敏感性高;检测临床样品的结果准确可靠。     

猪附红细胞体病的诊治报告

1993年6月,地处江汉平原的我公司所属养猪场发生了一种以无名高热、贫血或黄疸、胸腹水肿以及鼻腔有脓性分泌物为特征的疾病。经过流行病学、临床病理和实验室诊断,诊断为猪附红细胞体病。一、流行病学该场1993年3月从省畜科所引进后备母猪48头;5月初,又从外地购进育肥仔猪158头,引进第三     

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

An indirect hemagglutination test for the diagnosis of Eperythrozoon suis infection in swine.

An indirect hemagglutination test was developed to detect naturally occurring eperythrozoonosis in swine and to follow experimentally induced infections serologically. The antigen used was prepared from the plasma of acutely infected, splenectomized pigs. The test was specific and reliable in detecting swine latently infected with the disease.     

Serologic progression studies using CF and ELISA for the detection of antibodies against Eperythrozoon suis infection of swine

Serological studies by means of a CF-Test and Elisa were undertaken for evaluation of antibody titers against Eperythrozoon suis infection in swine. The animals were partly splenectomized and underwent artificial infection. It could be shown that the splenectomized animals showed higher and longer lasting antibody titers than the non splenectomized swine. It is stated, that the above mentioned tests can be used in diagnosis on a herd basis but not on a single animal basis.     

Indirect hemagglutination for Eperythrozoon suis detection in experimentally and spontaneously infected swine

Ten splenectomized and non-splenectomized pigs were experimentally infected with E. suis bearing red blood cells in order to determine the antibody response. All animals were monitored for antibody titer by indirect hemagglutination over a period of 80-290 days postinfection. Latent E. suis infection only yielded a detectable antibody titer in one pig. Acutely infected pigs had a titer ranging up to 1:640. Maximum antibody response lasted only 2 months and dropped below the level of detection of our assay within 2 to 3 months. At this time, the clinical symptoms could reappear and antibodies were again detectable. However, no booster effect was observed with this second outbreak. We also determined the antibody frequency in 138 pigs from 16 herds in Southern Germany. Pigs from only 4 out of 6 clinically positive herds had antibody titer against E. suis. 20 out of 78 pigs of the clinical positive herds demonstrated a detectable E. suis antibody titer. In 10 herds that were asymptomatic and presumed uninfected all 80 pigs were serologically negative for E. suis.     

间接竞争ELISA检测TGEV方法的建立

猪传染性胃肠炎(transmissible gastroenteri-tis,TGE)是由冠状病毒科猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)引起的以仔猪呕吐、腹泻、严重脱水为特征的消化道传染病。病毒粒子随粪便大量排出,是造成病毒扩大传播、仔猪大批死亡的重要原因。采取粪便样品进行病原检测,对该病早期诊断具有重要意义。目前针对TGEV感染有多种快速诊断方法,概括起来可分为针对病毒结构蛋白进行的各种免疫学诊断技术和近些年发展的以病毒核酸为基础的分子生物学检测方法。其中免疫学方法以其特异性强、易操作、不需要昂贵仪器等特点…     

猪化脓隐秘杆菌抗体间接ELISA检测方法的建立

为建立检测化脓隐秘杆菌(T.pyogenes)血清抗体的间接ELISA方法,本研究以超声破碎处理T.pyo-genes分离株TP-2849(NZ_CP029004)的全菌蛋白作为包被抗原,经反应条件优化建立了T.pyogenes抗体间接ELISA检测方法。该方法与7种常见病原阳性血清均无交叉反应,特异性较强;敏感性试验显示阳性血清的检测下限为1∶128,敏感性较高;组内、组间变异系数均小于9.5%,重复性较好。对吉林省某养殖场100份猪血清检测结果显示,该方法与PCR检测方法总符合率为94%。本研究建立的间接ELISA方法为T.pyogenes抗体检测提供了一种新手段。     

猪流感抗体间接ELISA检测方法的建立

猪流感病毒A／Swine／Fujian／668／2001（H3N2）株感染的鸡胚尿囊液，经差速离心后，再经蔗糖密度梯度离心，提纯、纯化的猪流感病毒经NP-40处理并反复冻融，作为猪流感间接ELISA抗原，确立了间接ELISA检测方法。对29份HI试验猪流感为阴性的血清进行了检测，经统计学分析，确定间接ELISA判定标准，被检血清OD490nm值≥0．20判定为阳性。该方法对猪瘟等11种猪疫病阳性血清无交叉反应，批内和批间重复试验的吸收变异系数分别在3．34％～8．12％和6．2％～9．04％之间。与HI的符合率达到92．8％，经卡方检验（P〈0．01）比HI试验敏感。为猪流感抗体检测提供了快速、准确、简便的方法。     

Establishment of an efficient enzyme-linked immunosorbent assay for the detection of Eperythrozoon sius antibody in swine

The Eperythrozoon suis (E. suis) antigen was purified using a Sephadex G-200 chromatograph, and thereby, a high-affinity, specific E. suis antigen was collected and confirmed with Western blotting. Using this antigen, an enzyme-linked immunosorbent assay (ELISA) system to detect the antibody against E. suis in swine was established. There was no cross-reaction with swine sera, which were affected with Mycoplasmal pneumonia, swine fever, swine colibacillosis, or toxoplasmosis. A comparison of this ELISA system with an indirect hemagglutination (IHA) test using 78 swine samples revealed that the ELISA system significantly improved the sensitivity, specificity, and stability for the serodiagnosis of swine E. suis.