Chemiluminescent immunoassay as a microtiter system for the detection of Salmonella antibodies in the meat juice of slaughter pigs

Chemiluminescent immunoassay (CLIA) was applied in the screening of swine meat juice samples obtained from different laboratories in Germany, using the indirect enzyme linked immunosorbent assay (ELISA) as test for comparison. Out of the 1350 samples tested, 987 were found acceptable for validation of results. A good level of agreement between the two tests was obtained with a kappa value of 0.824 at 20% cut-off and 0.798 at 40% cut-off. At 20% and 40% cut-off levels, a sensitivity of 96.2% and 97.3%, respectively, and a specificity of 94.6% and 95.1%, respectively, were demonstrated between CLIA and ELISA. The detecting lipopolysaccharide (LPS) antigen was tested for specificity and a cross-reaction with two Escherichia coli and Yersinia strains was found when tested with ELISA. This reaction was not observed in CLIA, possibly because of the broader measurement spectrum of this test, which allows a more distinctive definition of immunologic reactions. The same explanation can be given for the increased number of meat juice samples which were positively detected only in ELISA but not in CLIA. Because of the wide detection range in CLIA, a normalization scheme was necessary to obtain reproducible results in this test system. The samples positively classified in screening were further tested for reciprocal titres in both test systems, and a higher correlation between screening and titration results was obtained for CLIA. Based on the results of this study, CLIA can be used as a reference method in detecting Salmonella antibodies in the meat juice of slaughter pigs.     

猪巨细胞病毒gB基因的原核表达与间接ELISA检测方法的建立

猪巨细胞病毒(PCMV)是引起新生仔猪死亡、鼻炎、肺炎和生长速度减缓的重要病原之一,我国尚无有效的诊断方法.为建立检测PCMV的ELISA方法,本研究以重组PCMV囊膜糖蛋白B(gB蛋白)为包被抗原,优化反应条件:抗原包被量0.4 μg/mL,待检血清稀释度1∶100,37℃包被2h后4℃过夜,5％脱脂乳37℃封闭2h,二抗1∶10 000稀释,37℃作用2h,抗体临界值为S/P≥0.354判为阳性,S/P≤0.295判为阴性,介于二者之间为可疑,建立了PCMV抗体间接ELISA检测方法.与猪瘟、猪伪狂犬病、猪附红细胞体病、副猪嗜血杆菌及猪圆环病毒2型等血清抗体无交叉反应,批间和批内重复性较好,对江苏省259份仔猪血清样品进行检测,抗体阳性率达56.76％,从而为PCMV检测和流行病学调查提供了有效方法.     

检测鸭呼肠孤病毒抗体间接ELISA方法的建立

RT—PCR扩增鸭呼肠孤病毒（Duckreovirus,DRV）的dB基因,克隆到pET-32a（＋）表达载体,再转化大肠杆菌Transetta（DE3）;含有重组质粒pET—σB的大肠杆菌经IPTG诱导,获得大小为55000的以包涵体形式表达的σB重组蛋白。Westernblotting显示,σB重组蛋白能够与兔抗DRV多抗血清特异性结合。以高亲和NI—NTA树脂在变性条件下纯化、梯度尿素复性的σB重组蛋白为包被抗原,建立了检测DRV抗体的间接ELSIA方法。分别以间接ELSIA方法和血清中和试验对DRV感染鸭血清和SPF鸭血清进行检测,两者的符合率为100％。该间接ELSIA方法对鸭瘟病毒、鸭病毒性肝炎病毒和禽流感病毒阳性血清均无交叉反应。     

Sampling plan for the establishment of a serologic Salmonella surveillance for slaughter pigs with meat juice ELISA

The guidelines of the German Ministery of Food, Agriculture and Forestry outlining a Salmonella surveillance programme, "Leitlinien für ein Programm zur Reduzierung des Eintrags von Salmonellen durch Schlachtschweine in die Fleischgewinnung" (February 5th, 1998), provide a staggered spot-check size depending on the annual production of slaughtery pigs. A classification of farms into three quality categories (< 20%, 20-40%, and > 40%) is performed by salmonella antibody levels detected in meat samples using ELISA. Beside a fundamental inquiry into the salmonella status, the programme ought to lead to a decreased burden on slaughtery pigs and finally to a reduced salmonella entry into meat handling and processing companies. The spot-check plan is based on an unfavourable initial position and does not consider the real situation of salmonella load in pig fattening farms. For many farms the procedure will lead to an unjustified expenditure of examinations. In simple model calculations it is shown how a significant reduction of testing amount can be reached and statistical reliability is guaranteed, too. At the same time, we attempt to find a compromise between optimal spot check size and practicability. For reasons of free enterprise, an additional category would be desirable containing farms without any positive antibody titres in the samples. The results achieved so far indicate that a large number of German slaughter pig producers would fall into this category, without the necessity of a higher examination effort.     

间接ELISA在检测巴氏杆菌抗体中封闭条件的选择

间接ELISA在检测抗体中广泛应用,而封闭作为间接ELISA中必不可少的一步可直接影响试验的准确性.利用巴氏杆菌提取的包被抗原,经方阵滴定法确定抗原和血清的最佳工作浓度为1∶400和1∶100.而且通过对几种封闭液分别37℃水浴2h后4℃24h、4℃ 24h、37℃水浴3h封闭对比找出最佳的封闭条件为含0.5%BSA 2%蔗糖的PBS封闭液在37℃水浴2h后4℃24h.     

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。     

猪胸膜肺炎放线杆菌间接ELISA检测方法的建立

【摘 要】用纯化的重组蛋白抗原作为ELISA包被抗原,通过对抗原包被浓度、血清稀释倍数、酶标二抗稀释倍数、抗原和血清反应时间、血清和酶标二抗反应时间、显色剂作用时间和中止液滴加量的优化,建立了检测胸膜肺炎放线杆菌抗体的间接ELISA方法。通过特异性实验证明该ELISA方法特异性较强。将建立的ELISA方法与IDEXX公司的标准试剂盒进行了比较,二者的符合率较高,说明建立的ELISA方法比较敏感,为ELISA检测方法的商品化奠定了基础。     

The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses.

In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.     

检测肠炎沙门氏菌ELISA方法的建立与应用研究

从已建立的抗沙门氏茵单抗中筛选出适用于肠炎沙门氏菌ELISA检验的3—47—26单抗．采用筛选强阳性杂交瘤细胞株制备了高效价的3—47—26单抗．改进用辣根过氧化物酶标记高效价单抗的方法，进行了HRP标记单抗的免疫生物学特性的鉴定。确定了包被浓度和酶标抗体的工作浓度：HRP-3—47—26为1：100；LPS的包被浓度为400ng／mL。试验中采用LPS与多聚赖氨酸的结合，解决了包被过程中的解吸附作用，增强了包被的稳定性，同时也减少了假阳性反应，优化了包被过程。建立了检测肠炎沙门氏菌的抗原竞争ELISA方法，利用样品中的LPS抑制酶标抗体与包被的LPS结合，以降低底物反应的颜色从而达到检测的目的。通过对210份肠炎沙门氏菌感染鸡泄殖腔棉拭子、羽毛和组织样品先筛选后鉴定的方法进行检测具有明显的抑制作用，通过与国标法对大量的样品检测结果比较表明，竞争ELISA方法的检出率为18．09％；检出阳性样品36份，国标法检出率为17．14％；两者符合率为97．14％。竞争ELISA敏感性和特异性分别为94．4％和97．7‰从而为肠炎沙门氏菌的检测提供了一种敏感、特异的检测方法。     

牛传染性鼻气管炎间接ELISA诊断方法的建立

以牛肾细胞系（MDBK）培养牛传染性鼻气管炎病毒（IBRV）Bartha Nu/67株,经超速离心纯化病毒,再经超声破碎处理后作为诊断抗原,建立了检测牛血清IBRV抗体的间接酶联免疫吸附试验.该ELISA的判定标准为：血清D490 nm值大于0.369的判为阳性,小于0.295的判为阴性,在0.295与0.369之间的为可疑.特异性和重复性试验结果表明,该方法特异性高、重复性好.与法国进口ELISA抗体诊断试剂盒比较,其符合率为96.3%;与中和试验比较,符合率为95.8%,且敏感性更高.应用该诊断方法调查了我国部分地区IBRV的感染情况,结果显示,这些地区的IBRV感染率为67.1%.     

Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.     

禽脑脊髓炎病毒VP1蛋白间接ELISA检测方法的建立

对AEV陕西分离株VP1基因进行原核表达,采用SDS-PAGE和Western blotting检测表达产物;以纯化的蛋白为包被抗原建立ELISA方法,并对反应条件进行优化.结果表明,VP1蛋白在大肠杆菌中成功表达,产物约为50 000的融合蛋白,具有良好的反应原性;优化的ELISA最佳工作条件为:重组抗原包被质量浓度3.8 mg/L,37℃2h后4℃过夜,1％ BSA 37℃封闭2h,待检血清1∶50稀释37℃孵育0.5h,酶标抗体1:4 000稀释,37℃作用30min,底物37℃显色5 min,临界值为0.360;建立的ELISA方法特异性强、重复性好、敏感性高;临床检测180份样品,与美国IDEXX公司AEV抗体检测试剂盒检测结果符合率为91.9％.该方法可用于临床样品的大批量检测.     

Development and preliminary application of an indirect ELISA for detecting antibodies against Avian Influenza Virus

Fragment of 759 bp DNA spanning the Matrix 1 (M1) gene of Avian Influenza Virus (AIV) was inserted into an expression vector pET28c to construct a recombinant plasmid pET28c-M1. The pET28c-M1 plasmid was transformed into the Escherichia coli BL21 (DE3) competent cell to produce a recombinant strain E. coli 21 (DE3). After being induced by Isopropyl-b-D-galactopyranoside (IPTG), E. coli 21 (DE3) expressed a 28-kDa fusion protein at a high level. This protein can bind anti-AIV (H5N1) positive serum by Western-blot analysis. After being denatured, renatured, and purified by Ni(2+)-column, the fusion protein was used as an antigen to develop Matrix 1 Enzyme-Linked Immunosorbent Assay (M1-ELISA) for detecting antibodies against AIV from chicken serum. We found that this indirect M1-ELISA was sensitive for differentiating antisera against AIV and antisera against other six kinds of avian viruses apart from AIV and this method is more sensitive than Hemagglutination Inhibition (HI) test. When compared with HI test and ELISA (IDEXX) in evaluating 581 serum samples from field vaccinated chickens, this assay showed 93.3% agreement ratio with the HI test, as well as 96.0% agreement ratio with ELISA (IDEXX). In a preliminary application, the assay successfully detected 19 AIVs from 51 nonvaccinated chicken lungs. It concludes that an indirect ELISA was successfully developed for detecting AIV. The assay is specific and sensitive. The application will greatly contribute to the long-term prevention and control of avian influenza in China.     

Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting blue-tongue virus antibodies in cattle and sheep

The performances of a competitive enzyme-linked immunosorbent assay (ELISA) using a group specific monoclonal antibody against bluetongue virus, an indirect ELISA and the standard agar gel immunodiffusion (AGID) test were compared in the detection of serum antibody against bluetongue virus. Test sera consisted of 1300 bovine, 530 ovine and 160 carpine samples from bluetongue-free areas of Canada, 605 bovine and ovine field samples from the USA and Barbados and 464 samples from 79 cattle and sheep experimentally infected with 19 South African and five USA serotypes of bluetongue virus. The diagnostic specificity of the competitive ELISA, as determined for the bluetongue virus-free cattle sera was superior (99.92 per cent) to that of the indirect ELISA (99.85 per cent) and the AGID (99.0 per cent). The specificities of the competitive ELISA for sheep (99.63 per cent) and goats (100.0 per cent) sera were also higher than those of the AGID test. The performance of the ELISA tests was similar whether a gamma-ray-irradiated (2.0 Mrad) or a non-irradiated bluetongue virus antigen preparation was used. The competitive ELISA results for bovine field sera from endemic areas demonstrated a relatively low level of agreement (92.04 per cent) with AGID test results, with 9.7 per cent false negatives. The possible presence in these sera of antibody to cross-reacting antigens or to other orbiviruses, eg, epizootic haemorrhagic disease virus, which react in the AGID but not in the competitive ELISA may account for this lack of agreement.(ABSTRACT TRUNCATED AT 250 WORDS)     

检测新城疫病毒中和抗体的间接ELISA和竞争ELISA方法的初步建立

鸡新城疫(ND)是由鸡新城疫病毒引起的急性传染病，是对养禽业危害最大的禽病之一。融合蛋白(F)是NDV的主要保护性抗原，对新城疫的免疫保护起着重要的作用。基因工程苗是防治ND的一种新型疫苗，本室在前期研究中，以282E4株鸡痘病毒为载体表达了NDV F40E8株的F基因，在SPF鸡上显示了较好的保护效力，但F蛋白激发机体产生的中和抗体用常规的HI试验无法测出抗体效价。     

Possibilities for standardization of ELISA for detection of Salmonella antibodies in sera and meat juices of pigs

Programmes for controlling salmonella infections in German piggeries are based on the meat-juice-ELISA conducted in various investigation centres by using different test-kits. A usual procedure for harmonization (standardisation) of results is the calculation of the percentage of antibody-concentration from field samples in relation to the extinctions of a set of control-sera with known antibody concentrations. Whether this system is still acceptable in case of using different test-kits seems to be questionable. In principle, difficulties arise by calculating field results from the regression curve of control-sera because the calculated percentages of antibodies do not represent the antibody concentration but, instead, the percentages of the extinctions measured, and secondly, because control-sera presently in use are directed against different salmonella serovars. In regard to the number of laboratories involved and because of a variety of test-kits used it seems to be more adequate to include only one anti-Salmonella Typhimurium standard-serum at a given antibody concentration which is to be tested repeatedly on every test-plate. Simultaneously, further controls should include another anti-Salmonella Typhimurium and one anti-Salmonella Choleraesuis serum which should provide results similar to the Danish system which is regarded as a standard. As well, a negative serum must be included in the test and a minimum difference in extinctions between this negative serum and the standard positive control-serum should be reached to prove the validity of results from the test plate.     

可溶性E2蛋白作为抗原的检测猪瘟病毒血清抗体间接ELISA方法的建立

以可溶性重组E2蛋白作为抗原,建立了猪瘟病毒(CSFV)血清抗体间接ELISA诊断方法(rE2-ELISA).将猪瘟疫苗毒株E2基因主要抗原区(A-D)基因克隆到表达栽体pGEX-6p-1上,转化E.coli,降低诱导温度至20℃,获得48 000大小E2融合蛋白,部分目的蛋白以可溶性形式表达.Western blotting试验证实,E2融合蛋白可以和CSFV阳性血清发生特异性结合.亲和层析纯化后的E2融合蛋白作为抗原,建立了检测CSFV血清抗体的间接rE2-ELISA方法.该方法的特异性试验结果表明,与PRRSV、PCV2、PPV和PRV阳性血清之间不存在交叉反应;用rE2-ELISA和国外同类试剂盒(CSF-Ab-Kit)检测142份田问血清样品,2种试剂盒的阳性检测率分别为83.81%和88.73%.因此,rE2-ELISA猪瘟抗体检测试剂盒具有良好的敏感性和特异性,适合应用在大规模的CSFV血清抗体的检测工作中.     

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.