Informative Value of an Indirect Enzyme‐Linked Immunosorbent Assay (ELISA) for the Detection of Bovine Viral Diarrhoea Virus (BVDV) Antibodies in Milk

Bulk and individual milk samples from 117 herds located in Brittany (west France) were used to assess: (i) the performance characteristics of an indirect enzyme‐linked immunosorbent assay (ELISA) applied to individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV); and (ii) the relationship between the bulk milk result obtained from this test and the within‐herd prevalence of antibody‐positive lactating cows. This ELISA test was based on a monoclonal antibody directed against non‐structural protein NS2‐3 of pestiviruses. At the individual level, based on 1113 matched milk/serum samples, the sensitivity and specificity of this test applied to milk, compared with the virus neutralization test on serum, were 95.0 and 97.7%, respectively. At the herd level, the relationship between the optical density percentage (OD%) of bulk milk and the within‐herd prevalence of antibody‐positive lactating cows was assessed using the receiver operating characteristics (ROC) analysis. Classes of OD% of bulk milk were determined so that they were associated with minimum intraclass and maximum between‐class variances of within‐herd prevalence of antibody‐positive cows. The ROC analysis resulted in two classes of bulk milk results corresponding to different expected levels of within‐herd prevalence. Herds with an OD% of bulk milk <75% and ≥75% had a mean observed prevalence of antibody‐positive cows of 8.9 and 60.6%, respectively. Herds with a bulk milk result <75% were expected to be BVDV free, whereas large variations in prevalence of antibody‐positive cows existed in the herds with OD% ≥75%. The test described in this study is suitable to identify herds likely to have a low prevalence of BVDV antibody‐positive cows.     

Early Pregnancy Detection of Iraqi Riverine Buffalo (Bubalus bubalis) Using the BioPRYN Enzyme‐Linked Immunosorbent Assay for PSPB and the Progesterone Assay

This study was undertaken to detect pregnancy in Iraqi riverine buffalo (Bubalus bubalis) using three different methods (rectal palpation, plasma progesterone concentration and detection of the presence of pregnancy‐specific protein B (PSPB) with the BioPRYN^® enzyme‐linked immunosorbent assay (ELISA) test. The aim of the study was to identify the most sensitive, early and accurate method for detecting pregnancy. Twenty‐two female riverine buffalo that were 6.0 ± 0.93 years old were used. Four blood samples per buffalo were taken via jugular venipuncture at days 22–24, 32–34, 42–44 and 58–61 post‐mating (PM) to measure the progesterone concentration (ng/ml) and to detect the presence of plasma PSPB. The rectal palpation method was employed to evaluate all buffalo on days 42–44 and 58–61 PM. The BioPRYN^® test differed (p < 0.01) from the other tests with earlier accuracy for detecting pregnant and non‐pregnant buffalo. Eighty‐eight percent of pregnant and 76.9% of non‐pregnant buffalo were distinguished early (days 22–24 PM) using BioPRYN^® and plasma PSPB‐ELISA level (2.09 ± 0.12 ng/ml) in relation to 66.7% and 53.9% detected using the progesterone assay at similar days (4.30 ± 0.40 ng/ml). In conclusion, these results described, for the first time, the early and accurate pregnancy detection of water riverine buffalo using BioPRYN^® technology and provided the plasma levels of PSPB using an ELISA test. These findings will improve the reproductive and productive efficiency of Iraqi riverine buffalo by adapting the recent management and reproductive strategies in Iraq and in the world.     

Detection of Enterohemorrhagic Escherichia coli in Meat Foods using DNA Probes,Enzyme‐Linked Immunosorbent Assay and Polymerase Chain Reaction

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhoea with blood and haemolytic uremic syndrome (HUS) in children and elderly people. Infections with EHEC are a world‐wide public health problem, related to consumption of contaminated ground beef. The aim of this study was to establish whether different meat foods sold in Santiago, Chile pose an infection risk by EHEC and to evaluate three different diagnostic techniques in foods, to determine which is most applicable for use in Chile. A parallel analysis was performed on 64 samples of meat foods (23 refrigerated ground meat, 23 refrigerated long pork sausages and 18 frozen hamburgers) sold in Santiago, Chile using DNA probes, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Twenty‐four samples (24 of 64=37.5 %) were positive by DNA probes, ELISA or PCR. The positive and negative predictive values, sensitivity and specificity of ELISA were 26.7, 81.6, 30.8 and 78.4 %, respectively. The positive and negative predictive values, sensitivity and specificity of PCR were 91.7, 96.2, 84.6 and 98 %, respectively. The EHEC serogroups most frequently isolated were O158, O157, O119, O125 and O114. These results show that, although molecular techniques such as enzyme immunoassays are useful for EHEC detection in meat foods, PCR has advantages in terms of sensitivity, specificity, cost and ease of implementation in Chile.     

Variance Components of an Enzyme‐linked Immunosorbent Assay for Detection of IgG Antibodies in Milk Samples to Mycobacterium avium subspecies paratuberculosis in Dairy Cattle

Milk samples from 120 cows were tested up to 10 times in an enzyme‐linked immunosorbent assay (ELISA) for detection of antibodies to Mycobacterium avium ssp. paratuberculosis. The purpose of the study was to estimate variance components of the assay attributable to laboratory factors using mixed model theory. Because of significant interaction between the between‐run, between‐day and between‐plate variables, the ELISA‐plate variable was nested in run‐number and run‐number was nested in day‐number. The nested variable accounted for 68% of the laboratory variability (P<0.001), whereas the intra‐plate variability accounted for only 0.04% of the laboratory variability (P>0.9). Therefore, it was concluded that the intra‐plate variability could be ignored whereas the variability from the combined run‐day‐plate variable should be considered in any analyses based on the ELISA.     

The Ability of the Enzyme‐Linked Immunosorbent Assay to Detect Antibody against Staphylococcus aureus in Milk following Experimental Intramammary Infection

Changes in the milk antibody levels against Staphylococcus aureus were measured at the start of an experimental intramammary instillation of either S. aureus (Study I) or Staphylococcus hyicus (Study II). A commercial enzyme‐linked immunosorbent assay system was used. Twenty‐one Holstein cows were enrolled in Study I and 15 Holstein cows were used in Study II. Pathogen instillation began 21 days before the start of the non‐lactating period. Cows received intramammary antibiotic treatment in all quarters immediately after the last milking, the start of the non‐lactating period. Lacteal secretions were collected before the start of the non‐lactating period, and during the immediate postpartum period in both studies, and during the non‐lactating period in Study I. Milk was cultured for mastitis pathogens and S. aureus antibody levels and somatic cell counts were determined from all samples. There was an approximate 2‐week delay in the elevation in antibody levels in response to the instillation of S. aureus. Antibody levels remained elevated in cows with S. aureus intramammary infections postpartum, but were below threshold in cows where intramammary infections were cured during the non‐lactating period. Antibody levels were elevated by S. hyicus intramammary infections, remained elevated for the first 12 days postpartum, but were below threshold by day 21 postpartum. Cows with incipient intramammary S. aureus infections might be misclassified as false negatives by the antibody test. However, results suggest that cows with S. hyicus intramammary infections that were not cured would not be misclassified if milk is withheld from test for the first 30 days postpartum, as recommended by the manufacturer of the test.     

抗氧氟沙星单克隆抗体的制备及ELISA快速试剂盒的初步研制

本试验通过碳二亚胺法,将半抗原氧氟沙星与载体牛血清白蛋白（BSA）和卵清白蛋白（OVA）偶联,制备得到氧氟沙星免疫原和包被原,并制备抗氧氟沙星的单克隆抗体,初步研制测定氧氟沙星的ELISA试剂盒,经过测试,对鱼肉样本中氧氟沙星的检测限为1 μg/kg,添加回收率在62.8%～97.7%之间,批内、批间试验的相对标准偏差均小于10%。     

间接ELISA检测鸭肝炎病毒抗体的研究

以蔗糖密度梯度离心法纯化的病毒作为包被抗原，建立了检测鸭肝炎病毒（ＤＨＶ）抗体的间接ＥＬＩＳＡ方法。经特异性及重复性试验，效果良好。ＥＬＩＳＡ效价与琼扩、中和效价存在平行关系。经ＥＬＩＳＡ检测，１日龄雏鸭免疫后，４日龄可检出ＥＬＩＳＡ抗体，１０日龄达到峰值。ＤＨＶ高免血清在雏鸭体内作用维持时间为１０ｄ左右。攻毒保护试验表明，攻毒前雏鸭的血清抗体水平与攻毒后雏鸭存活率具直接相关性。     

Screening for the Fish Disease Agent Aeromonas salmonicida with an Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract

Serological detection of bacterial pathogens in fish tissue is an important tool for surveying epidemiological situations. Whenever antibacterial treatment of fish is recommended, it becomes necessary, however, to culture the pathogen for sensitivity testing. An enzyme-linked immunosorbent assay (ELISA) was developed to identify Aeromonas salmonicida in culture. This serological identification can partly substitute for biochemical characterization of the organism and thus decrease the time between isolation and sensitivity testing by at least 3 d. The ELISA works with only one bacterial colony and yields results within 4 h. During this time, a bacterial suspension can be prepared for the resistance test. The specificity of an antiserum, raised in rabbits, against whole cells of A. salmonicida can be increased by adsorption with strains of cross-reacting species. However, difficulties arise when serologically heterogeneous species (e.g., A. hydrophila) are used as the cross-reacting bacterium. In the present study, severalfold adsorption with four isolates did not totally rule out cross-reactivity against additional strains. Therefore, the strain in question should also be checked for colony morphology, production of pigment, or presence of cytochrome oxidase to validate the serologically obtained result.     

用酶联免疫吸附法检测细胞培养物中兔出血症病毒

用本实验室提纯的兔出血症病毒抗体（IgG）,采用改良过碘酸钠（NaIO4）交联法,将辣根过氧化物酶（HRP）标记在抗体上,以4×10聚苯乙烯微量酶标板作为载体,用酶联免疫吸附法双抗体夹心法检测了兔肾上皮细胞（RK）培养的RHDV16代、18代细胞毒和羊睾丸细胞（ST）培养的RHDV19代、24代、26代细胞毒,均呈特异性阳性反应,P/N值〉2．1,正常细胞培养物标本为阴性结果,P／N值〈2．1,试验结果与免疫荧光试验相吻合。     

补体结合酶联免疫吸附试验方法的建立

为改进免疫学诊断技术的准确性,研究了一种基于补体结合的免疫学检测新技术———补体结合酶联免疫吸附试验（CF-ELISA）。CF-ELISA技术采用酶标记抗菊糖纯化豚鼠补体C3抗体及其酶显色系统作为补体参与反应的指示系统,用ELISA方法进行补体结合试验。经对布氏菌病抗体检测的初步试验结果显示,CF-ELISA技术可检测到0.01 IU的布氏菌病抗体,灵敏度与间接酶联免疫吸附试验（iELISA）相当,是虎红平板凝集试验（RBPT）试管凝集试验（SAT）的5 000倍、补体结合试验（CFT）的10 000倍。对349份确诊布氏菌病感染群牛、羊血清的检测结果显示,CF-ELISAi、ELISA、CFT、SAT、RBPT的阳性率分别为35.82%3、6.39%、31.81%、30.09%、36.1%,CF-ELISA与iELISA、CFT、SAT、RBPT的阳性符合率分别为：98.4%、88.8%、80.0%、90.6%。CF-ELISAi、ELISA、CFT、SAT、RBPT对490份布氏菌病阴性群牛、羊血清的阴性率分别为100%、99.6%、100%、99.4%、99.8%,CF-ELISA与iELISA、CFT、SAT、RBPT的阴性符合率分别为：99.6%1、00%、99.4%、99.8%。研究表明,CF-ELISA是具有高特异性和高敏感性的布氏菌病免疫学检测技术。     

Enzyme-Linked Immunosorbent Assay (Elisa) for Detection of Antibodies to Mycobacterium Para-Tuberculosis in Cattle

Paratuberculosis may be diagnosed by clinical, bacteriological and immunological methods, but so far only the demonstration of M. paratuberculosis is considered a definite proof of the infection. World-wide use is being made of the complement fixation (CF) test as a valuable immunological test for diagnosis of clinical cases, but its low specificity and sensitivity makes its value problematic in non-clinical cases.     

检测鸡肉中甲羟孕酮酶联免疫试剂盒的研制

试验旨在利用酶联免疫技术研制一种快速检测鸡肉中甲羟孕酮的试剂盒。经测试,该试剂盒对鸡肉样本的检测限为1 μg/kg,批内、批间相对标准偏差均小于10%;稳定性测试结果表明,试剂盒能在4℃保存12个月;交叉反应率结果表明,单克隆抗体特异性良好。该试剂盒的操作时间仅需45 min,适合现场大量样本的快速检测。     

牛奶及奶粉中三聚氰胺残留酶联免疫检测方法的研究

通过4,6-二氨基-2-氯-1,3,5-三嗪与对氨基苯丁酸反应获得三聚氰胺半抗原,再以活性酯法与载体蛋白偶联制备三聚氰胺免疫原或包被原。免疫BALB/c小鼠,利用杂交瘤技术制备出针对三聚氰胺的特异性单克隆抗体。采用间接竞争酶联免疫吸附法(ciELISA)建立检测三聚氰胺的标准曲线,线性范围是17.4～345.5ng/mL,50%抑制浓度(IC50)61.3ng/mL。牛奶和奶粉加标回收率在68.1%～91.0%,变异系数在2.4%～14.5%。结果表明,该方法可以满足牛奶和奶粉中三聚氰胺残留分析要求。     

恩诺沙星酶联免疫吸附检测方法(ELISA)的建立及其与HPLC方法的比较研究

建立了恩诺沙星残留检测的酶联免疫吸附检测方法(ELISA),并对方法的准确度和灵敏度等指标进行了评价,确定方法的最低检出限(LOD)为1ng/mL,线性范围为1ng/mL-1000ng/mL。以虾肉为样本进行的加标实验表明,在5ng/mL-200ng/mL的加标浓度下,孔间变异系数为9.8%-17.4%,批间变异系数为11.9%-23.4%,添加回收率为60.07%-120.8%。对ELISA和高效液相色谱(HPLC)的检测性能进行了比较,并通过水产品、畜禽等市售食品的同步检测进行了验证,结果表明在实验范围内,二种方法之间呈现较好的相关性,R2=0.9896,这表明建立的ELISA检测方法有较高的可信度是比较高的,可以有效地反映药物的残留情况。     

新城疫病毒抗体ELISA检测方法的建立与应用

为建立一种快速的新城疫病毒抗体检测方法,本研究以原核表达的重组HN蛋白作为诊断抗原,建立了检测新城疫病毒抗体的间接ELISA诊断方法.该方法检测AIV(H9亚型)、IBV、IBDV、EDSV 4种常见禽病病原的阳性血清均为阴性;检测灵敏度为1:12 800;批内重复性试验、批间重复性试验的变异系数分别小于5％和10％;与血凝抑制试验(HI)符合率为96.1％.本研究建立的NDV HN间接ELISA检测方法具有良好的特异性、敏感性和重复性,为NDV的抗体检测及流行病学调查等快速诊断提供一种技术手段.     

An enzyme-linked immunosorbent assay (ELISA) for detection of Marek's disease virus-specific antibodies and its application in an experimental vaccine trial

An enzyme-linked immunosorbent assay (ELISA) for the detection of Marek's disease virus (MDV)-specific antibodies was developed. Chicken embryo cells (CEC) or chicken kidney cells (CKC) were infected with MDV vaccine strain CVI988/Rispens, and infected-cell lysates were prepared at day 5 post-infection by freeze-thawing. Uninfected-cell lysates served as negative controls. Sera were used at a 1 : 100 dilution and were added in parallel to wells containing the infected and uninfected cell lysates. The optical densities at 492 nm (OD(492 nm)) were measured after detection of bound chicken antibodies with anti-chicken IgG peroxidase conjugate and colour reactions using o-phenylenediamine (OPD) as a substrate. The best results concerning the signal-to-noise ratio were obtained by using CKC cells rather than CEC for antigen preparation. The OD(492 nm) of plasma or serum samples with infected CKC was <0.02 when samples of unvaccinated and unchallenged maternal antibody-negative white leghorn chickens were tested. Sera and plasma samples of positive control birds exhibited OD(492 nm) of <0.01 when tested with uninfected CKC. The assay was used to monitor a trial that compared experimental BAC DNA vaccines and a commercial vaccine. Sustained seroconversion and antibody titers that were constantly rising until day 84 after vaccination (71 days after challenge) was observed only when chickens did not develop Marek's disease. In contrast, chickens developing the disease mounted marginal and short-lived antibody titers only. We conclude that the developed ELISA may be a valuable tool for the evaluation of the efficacy of MDV vaccination under experimental but possibly also under field conditions.