应用McAb—RPHA检测囊虫病猪循环抗原

应用单克隆抗体致敏红细胞,以反向被动血凝试验(RPHA)检测囊虫病猪血清中的循环抗原.囊虫病猪血清的检出率为94.55%(104/110),健猪血清阳性率为8.5%(8/94).假阳性反应主要来自棘球蚴感染.囊虫病猪血清中的循环抗原可耐受100℃30min而仍保持其抗原活性,提示其可能是一种多糖类物质.     

应用猪囊虫匀浆抗原间接血凝试验检测猪囊虫病抗体的研究

从囊虫匀浆浸提液中分离出一种抗原性与特异循环抗原相同的成分,以这种抗原成分通过间接血凝试验检测病、健猪的血清抗体,结果病猪抗体阳性率为93.2％(96/103),健猪阴性(?)合率为100％。     

抗猪囊虫循环抗原杂交瘤细胞系的建立和鉴定

建立了8株抗猪囊虫循环抗原(CA)的杂交病细胞系。其中6F12和2E7为抗囊虫特异McAb;McAb 1C6、1C7、1B5、4D9、2B9和8D8与囊虫、(?)球蚴、细颈囊尾蚴抗原均可发生反应。这些McAb的腹水ELISA效价为105～107,细胞培养上清液效价为102,并均可与病猪血清中的囊虫CA反应形成沉淀线。用1B5、6F12和8D8分别致敏血球,以反向间接血凝试验检测98份囊虫病猪血清,检出率分别为70.41%(69/98)、6.12%(6/98)和7.14%(7/98)。本研究制备的McAb可用于猪囊虫循环抗原检测。     

囊虫病特异诊断方法的研究

应用间接ELISA对囊虫粗抗原、B抗原、用等电聚焦技术分离的抗原组分及特异McAb亲和层析抗原的特异性作了评价,结果均不理想。用McAb分析查明,在某些囊虫抗原分子上既有特异性抗原决定簇,又有非特异性决定簇。应用特异McAb以R-PIⅡ和Ⅰ-ELISA检测囊虫病人和猪的血清抗体,结果完全消除了假阳性反应;用R-PIⅡ微量法和Ⅰ-ELISA检测,病人血清的阳性率为87.36%(76/87)和89.66%(78/87),病猪血清的阳性率为89.32%(92/103)和86.41%(89/103);用R-PHI玻片法检测,病猪血清的阳性率为87.32%(90/103)。     

应用间接血凝技术诊断马脑脊髓丝虫病的研究

用DEAE-Sephadex A50柱层析法对粗抗原进行分离纯化,用琼脂凝胶双扩散法对分离的成分进行抗原性分析,查用旧含0.2M NaCl的PBS洗脱的部分(SD_(0.2)为特异抗原成分,而用含0.35M NaCl的PBS洗脱的部分(SD_(0.35))为交叉反应成分。用组抗原致敏的血球检测抗体时,健马血清的阴性符合率为22％,而用(SD_(0.2))致敏的血球检测时阴性符合率为100％。本项研究首先应用反向间接血凝技术险出病马血清中的循环抗原。用SD_(0.2)致敏的血球检测抗体,同时用反向间接血凝技术检测循环抗原,对发病马的总检出率为82.2％。对自然感染病马系列血清的检测结果表明,病马均能在发病前检出抗体;循环抗原出现在感染早期尚未检出抗体之前和感染后期抗体转阴之后。     

用微量间接血凝法诊断牛贝诺孢子虫病的研究

本试验是在1983年研制成功牛贝诺孢子虫抗原及初步应用于临床的基础上,进一步用-20℃条件下保存30个月的病牛皮肤制备牛贝诺孢子虫抗原,对应用微量间接血凝试验诊断牛贝诺孢子虫病进行了扩大复试。结果表明,15份健康牛血清全部为阴性;140头(份)带虫牛血清有133头(份)呈阳性反应,阳性符合率达95.0％;49头类症病牛血清,除1份肉孢子虫血清效价为1:40外,其余均为阴性;对疫区的496头被检牛,通过临床、组织学以及眼巩膜包囊等检查,检出阳性牛33头,检出率为6.65％,通过间接血凝试验检出阳性牛84头,检出率达16.94％,后者检出率明显高于前者。由此认为,间接血凝试验对牛贝诺孢子虫病有很高的检出率和特异性,可用于临床诊断和疫区普查。     

检测伊氏锥虫循环抗原的McAb-RPHA试验

用一株抗伊氏锥虫、马媾疫锥虫和布氏锥虫共同抗原(与泰氏锥虫等抗原无交叉反应)的单克隆抗体建立了检测伊氏锥虫循环抗原(TcA)的反向间接血凝试验。用以检测人工感染兔,于感染后8～10天TcA转阴;如不治疗直至观察期结束持续阳性;治疗后一周即转阴。用以检测疫区36份虫血症阳性水牛血清,25份阳性(69.44％);16份虫血症阴性、IHA阳性水牛血清,3份阳性;25份虫血症和IHA阴性水牛血清,全部阴性。     

抗独特型抗体检测H9亚型禽流感血清抗体

为了探讨用抗独特型抗体替代病毒或病毒亚单位检测H9亚型禽流感血清抗体的可行性,本试验分别用全病毒抗原和抗独特型抗体(Ab2)作检测原,通过琼脂免疫扩散试验(AGP)和间接酶联免疫吸附试验(ELISA)检测了鸡的血清样品。在AGP试验中,分别用Ab2和禽流感病毒(AIV)对10份血清样品检测的符合率为70%。在间接ELISA试验中,Ab2和AIV抗原对10份血清样品检测的符合率为90%。用Ab2间接ELISA试验检测出的血清阳性率(8/10)高于血凝抑制试验(6/10)和AGP试验(5/10)。结果表明,在一定的情况下Ab2可以用来代替具有污染环境风险的病毒抗原来检测抗病毒抗体。     

乌兰县柯柯镇北柯柯村绒山羊衣原体病的血清学调查

应用衣原体间接血凝试验(IHA)对青海省乌兰县柯柯镇北柯柯村的267份绒山羊的血清,进行衣原体间接血凝抗体的检测。结果检出39份阳性血清,血清阳性率为14.61%。(39/267)     

用反向间接血凝抑制试验检测传染性法氏囊病血清抗体

利用扬州大学农学院畜禽病原微生物研究室提供的传染性法氏囊病单克隆抗体致敏红细胞，用反向间接血凝抑制试验检测传染性法氏囊病血清抗体。试验表明：反向间接血凝抑制试验具有很高的特异性，能被传染性法氏囊病抗原特异性地阻断，且不与鸡马立克氏病、白血病、传染性脑脊髓炎、减蛋综合征、新城疫等血清出现交叉反应。该法敏感性比琼脂扩散试验高２８倍，检出率高约６０％。与琼脂扩散试验的相关系数为０．９４００。用此法测定雏鸡母源抗体，测得雏鸡于１３～１５日龄时母源抗体降至免疫临界线     

Prevalence of antibodies to porcine adenovirus in swine by indirect fluorescent antibody test

An indirect fluorescent antibody test was developed for routine identification of a porcine adenovirus and its specific antibody. Two specific-pathogen-free young pigs were inoculated with the viral antigen prepared in continuous porcine kidney cell cultures, and their sera were used as an antibody reagent to standardize the test. Sera of adult pigs with respiratory problems, obtained from pig farms in Quebec, were tested for antibodies to this virus; 83 of 540 sera tested (15.2%) were found to be positive.     

Pestivirus infections in Norway. Serological investigations in cattle, sheep and pigs.

Serum samples from 1,133 dairy cows (187 herds), 3,712 ewes (103 flocks) and 1,317 adult pigs (877 herds), were tested for neutralizing antibodies against the NADL strain of bovine virus diarrhoea virus. The prevalence rate of seropositive animals was 18.5% in cattle, 4.5% in sheep and 2.2% in pigs, such seroreactors being found in 28% of the cattle herds and 18% of the sheep flocks. In all three species the rate showed considerable herd and geographical variation. In cattle the seroreactor rate was similar in herds with normal reproduction and in 62 herds with problems of repeat breeding. Of 31 pig sera containing antibodies against the NADL strain, 27 were also positive in a neutralization test for antibodies against swine fever virus (Baker strain). However, all sera showed a higher titre of antibodies against the bovine strain than against the swine fever virus. It was concluded that the immune response of the pigs had been induced by ruminant pestivirus, and not by swine fever virus.     

Indirect fluorescence antibody studies of porcine cytomegalo virus infections in the Netherlands

Summary Sera from 683 pigs of 41 swine herds with clinical atrophic rhinitis (A R), from 477 pigs of 37 herds with no A R history, from 267 breeding sows and breeding boars for slaughtering, from 22 boars at an artifical insemination centre, and from 103 SPF pigs were tested for the presence of antibodies to porcine cytomegalo virus (PCMV). The herds examined were spread all over the Netherlands. For the presence of antibodies to PCM V the indirect fluorescence antibody test was used. To obtain the antigen, the PCMV had been grown in pig lung macrophage cultures in Petri dishes for 10-12 days. These macrophages were dropped into the wells of slides. The serum dilution 1:20 of all the 103 sera from SPF pigs were negative, but 93 per cent of the other sera were positive. No marked differences were found between swine herds with clinical atrophic rhinitis and herds with no A R history. The FA titres in both types of herds seem to be at a comparable level.     

副猪嗜血杆菌Apd-ELISA抗体检测试剂盒的制备和初步应用

旨在对副猪嗜血杆菌（Haemophilus parasuis,HPS）的黏附素蛋白（autotransporter passenger domain,Apd）ELISA抗体检测方法进行参数优化、临界值确定和应用评价,获得性能稳定的副猪嗜血杆菌病抗体检测试剂盒。首先通过猪的免疫攻毒试验获得HPS阴、阳性对照血清,优化Apd-ELISA的反应参数;然后用背景确认的阴、阳性血清确定其临界值,并对封闭液及酶标板的包装方式进行选择;最后用Apd-ELISA试剂盒对临床样品进行检测并与荷兰Biocheck的OppA-ELISA试剂盒进行比较。结果表明,抗原包被质量浓度为0.5 μg·mL^-1、被检血清以1：200倍稀释后反应45 min以及二抗稀释度为1：15 000时,Apd-ELISA的反应背景下降,区分度提高。根据背景确认的27份阳性和40份阴性血清的OD_{630 nm}值以及临界值计算公式（S-N）/（P-N）,得到Apd-ELISA的阴阳性临界值是0.33。证实用封闭液K封闭和真空包装的酶标板可以在50℃保存4 d（相当于在4℃保存22个月）。用Apd-ELISA试剂盒检测1 179份临床血清,表明母猪、后备和育肥猪以及仔猪的HPS血清阳性率分别为83.76%、61.09%和27.48%。与荷兰的Biocheck试剂盒进行比较,发现Apd-ELISA与Biocheck试剂盒（OppA-ELISA）对HPS临床阴性血清和疫苗免疫血清检测的符合率为100%,对HPS临床阳性血清的符合率仅为4.76%（6/126）。然而,Apd-ELISA与OppA Western blot的阳性符合率可达到73.02%（92/126）。本研究获得了能有效区分HPS阴、阳性血清和稳定保存的Apd-ELISA抗体检测试剂盒,可用于HPS灭活疫苗免疫后的抗体检测和副猪嗜血杆菌病的血清抗体检测。     

Development and Preliminary Application of Apd-ELISA Kit for Antibody Detection of Haemophilus parasuis

The present study aimed to develop an ELISA for detecting antibodies of Haemophilus parasuis by optimization of reaction conditions, determination of its cutoff value and evaluation of its application with autotransporter passenger domain (Apd) as the coating antigen. Firstly, the porcine sera from immunization and challenge experiments were used as HPS-positive and -negative control sera to optimize the Apd-ELISA reaction parameters and conditions. Next, the cutoff value was determined based on testing of the serum samples with confirmed background, and then the blocking solutions and the packing methods of plates were selected. Finally, Apd-ELISA was used to detect clinical serum samples and then compared with the Biocheck OppA-ELISA kit from Netherland. With the coating antigen at 0.5 μg·mL^-1, the tested sera being diluted at 1:200 and incubation for 45 min and HRP-labeled goat anti-pig second antibody being diluted at 1:15 000, Apd-ELISA displayed the improved discriminative capability and the reduced background signal. The cutoff value was determined to be 0.33 by calculation formula (S-N)/(P-N) and the OD_{630 nm} value of the 27 positive porcine sera and 40 negative porcine sera with the confirmed background. The blocking solution K and the vaccum package can preserve ELISA plates for 4 days at 50 ℃ (comparable to 22 months at 4 ℃). Detection of 1 179 clinical sera with Apd-ELISA indicated that the positive rates were 83.76%, 61.09% and 27.48% in sows, fattening pigs and piglets, respectively. Compared with the OppA-ELISA kit from Biocheck (OppA-ELISA), Apd-ELISA showed 100% identity to the results of OppA-ELISA kit in clinically negative sera and experimentally positive sera from the pigs immunized with the inactivated vaccine, but 4.76%(6/126)identity to the results in the sera from clinically positive sera. However, Apd-ELISA displayed 73.02%(92/126)identity to the results of OppA-Western blot in the sera from clinically and experimentally infected pigs. The present study obtained the Apd-ELISA kit that can effectively distinguish HPS-positive and -negative sera and can be stably preserved, which would be applied for antibody monitoring among pigs immunized with HPS-inactivated vaccine and subjected to infection of H. parasuis.     

Evaluation of an in-house TgSAG1 (P30) IgG ELISA for diagnosis of naturally acquired Toxoplasma gondii infection in pigs

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect (κ=0.9124, p≥0.05) and between IFAT and IB was moderate (κ=0.53, p≥0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC=0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.     

Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% has positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5 IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of ASFV-infected pigs.     

猪圆环病毒2型免疫过氧化物酶单层细胞试验抗体检测试剂盒的研制及应用

本研究建立了一种免疫过氧化物酶单层细胞试验（Immunoperoxidase monolayer assay,IPMA）,用于猪圆环病毒2型血清抗体检测,通过对IPMA反应条件的优化,组装了诊断试剂盒。研究结果表明,用IPMA检测猪圆环病毒2型人工感染猪血清,于感染后3周抗体阳转,第3周～10周抗体阳性检出率为92.8%（52/56）,对照组猪血清抗体检测均为阴性（33/33）。试剂盒在-20℃稳定保存18个月与其他几种猪病毒参考血清无交叉反应,与用重组蛋白抗原建立的rcELISA符合率为89.2%。对来自黑龙江、吉林、河北、上海、内蒙古、云南、江西等地猪场健康成年猪血清480份和发病猪血清424份进行了检测,抗体检出率分别为91.7%和79.2%,表明我国猪群中猪圆环病毒2型污染相当严重。该试剂盒的研制为我国PCV2流行病学调查和疫苗免疫效果的评价提供了技术手段。     

猪肺炎支原体乳酸脱氢酶基因的克隆、表达、纯化及其间接ELISA检测方法的建立

根据GenBank中的猪肺炎支原体乳酸脱氢酶（LDH）基因序列设计1对引物，PCR扩增LDH基因，首先将扩增片段连接到克隆载体pMD18-T上，然后连接到表达载体pGEX—KG上，经测序正确后诱导表达。重组质粒在大肠杆菌中表达的目的蛋白以可溶性蛋白和包涵体2种形式存在。将超声波破碎的诱导菌液高速离心，其上清用Glutathione Sepharose4Bbead亲和层析纯化。用猪肺炎支原体的标准阳性血清对纯化蛋白作Western—blot检测，出现目的条带；以纯化蛋白为抗原建立了检测猪肺炎支原体抗体的间接ELISA方法，该方法具有较好的稳定性和重复性，较高的特异性与敏感性。用建立的ELISA方法与中国兽医药品监察所研制的IHA试荆盒同时检测120份临床血清，二者总符合率为92．5％。用建立的ELISA方法检测了671份临床送检不同年龄阶段的猪血清，结果显示断奶前仔猪猪肺炎支原体（Mycoplasma hyopnenmoniae，MHP）感染率为44．3％，保育猪为3．0％，育肥猪为17．44％，种猪为73．41％，这初步反映了猪喘气病在各个年龄阶段的感染率。