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.     

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.     

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.     

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。     

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.     

酶联免疫法检测动物源性食品中甲羟孕酮的残留

本试验旨在利用酶联免疫技术对鸡肉和鱼肉中的甲羟孕酮药物残留进行检测。经过测试,该试剂盒对鸡肉和鱼肉样本的检测限均为ug／kg;当药物添加浓度为1、2、4ug／kg时,其平均回收率范围是65．5％～94．6％,批内、批间相对标准偏差均小于10％;交叉反应率试验表明该试剂盒的特异性良好：酶联免疫法与仪器法的验证结果一致。     

Validation of the BioPRYN enzyme‐linked immunosorbent assay for detection of pregnancy‐specific protein‐B (PSPB) and diagnosis of pregnancy in American bison (Bison bison)

This study assessed the accuracy of the commercial BioPRYN^® ELISA for the detection of pregnancy‐specific protein‐B (PSPB) using a single blood sample to determine pregnancy status in American bison (Bison bison). A total of 49 bison cows were used in the study, and sampled at two time‐points during the gestation period, fall and spring, correlating with early‐ to mid‐term gestation (average 62.9 days post‐mating) and mid‐ to late‐term gestation (average 229.2 days post‐mating), respectively. Sensitivity of the test during early‐ to mid‐term gestation sampling period (fall) was 87.1%, while specificity was 100%; sensitivity of the test during late‐term gestation sampling period (spring) was 96.3%, while specificity remained at 100%. In total, the test showed a total sensitivity of 91.4%, specificity of 100% and total accuracy of 93.8%, similar to domestic cattle. Use of the single‐sample BioPRYN^® ELISA in American Bison for pregnancy diagnosis is economical and practical, minimizing animal handling time, frequency and subsequent stress while providing accurate results for pregnancy diagnosis at 62 days post‐mating. This method should be considered over more traditional pregnancy diagnosis methods for use in managed bison herds.     

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。     

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。     

酶联免疫吸附（ELISA）法测定生猪尿液中的沙丁胺醇

将标准品、样品和和酶标记物一并加入到微孔板中孵育,样品中的游离沙丁胺醇与酶标记的沙丁胺醇竞争结合酶标板微孔中固定相化的沙丁胺醇特异性抗体,通过清洗步骤洗掉未结合的酶标记沙丁胺醇,再通过酶的专一性显色剂显色,微孔显蓝色,加入反应终止液使颜色由蓝色转为黄色。在450nm处测量吸光度（OD）值,并设置标准曲线,通过标准曲线测定样品中沙丁胺醇的含量,结果显示：OD值的大小与样品中沙丁胺醇的含量成反比。     

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。     

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.     

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%。     

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.     

现场诊断奶牛早孕与发情的酶免疫分析法的改进

报道了一种建立并经改进的现场检测乳汁孕酮诊断奶牛早 和发情的酶免疫分析法，有准、早、易、安全等优点。     

Assessment of Progesterone Concentration Using Enzymeimmunoassay, for Early Pregnancy Diagnosis in Sheep and Goats

The objective of this study was to determine a value of serum progesterone (P₄) concentration, assessed using an enzymeimmunoassay (EIA), for the early distinction between pregnant and non‐pregnant ewes and goats. Adult, non‐lactating ewes of Chios (n=53), Berrichon (n=30) and Sfakia (n=45) breeds were synchronized during the breeding season with progestagens and gonadotrophins and mated to fertile rams (Experiment I). Adult, lactating goats of Swiss breeds (Alpine and Saanen, n=104) and indigenous Greek breed (n=45) were synchronized during the transitional season with progestagens, PGF₂α and gonadotrophins. Cervical artificial insemination (AI) with fresh semen was applied once, 42–44 h after sponge removal (Experiment II). Jugular blood samples were collected on day 19 after sponge removal (ewes) or on day 21 after AI (goats) and serum P₄ concentration was determined by EIA. Progesterone concentrations ≥1.0, ≥1.5, ≥2.5 and ≥4.0 ng/ml were tested as indicative of pregnancy. Pregnancy diagnosis was verified on birth. In the case of sheep, using a discriminatory level of 2.5 ng/ml, overall accuracy of pregnancy diagnosis was 91.4% and predictive value of negative and positive diagnoses were 98.3 and 85.3%, respectively. In the case of goats, predictive value of negative diagnosis was 95.8 and 94.0% and predictive value of positive diagnosis 71.3 and 71.7%, for 1.5 and 2.5 ng/ml, respectively; overall accuracy was 79.2% using either level. The other discriminatory levels tested did not improve these results. A significant positive correlation was observed between P₄ concentration and the number of lambs or kids born, and further analysis indicated that this relationship is not a simple linear function. Based on the results of this study, P₄ concentrations of 2.5 ng/ml in the case of ewes and 1.5–2.5 ng/ml in the case of goats, determined with EIA, are proposed as discriminatory levels between pregnant and non‐pregnant animals, at an interval of one oestrous cycle after service.     

Computed Tomography and Cross‐Sectional Anatomy of the Metatarsus and Digits of the One‐humped Camel ( Camelus dromedarius) and Buffalo ( Bos bubalis)

The purpose of the present study was to provide a detailed computed tomography (CT) and cross‐sectional anatomic reference of the normal metatarsus and digits for the camel and buffalo, as well as to compare between metatarsus and digits in these animals to outstand a basis for diagnosis of their diseases. Advantages, including depiction of detailed cross‐sectional anatomy, improved contrast resolution and computer reformatting, make it a potentially valuable diagnostic technique. The hind limbs of 12 healthy adult camel and buffalo were used. Clinically relevant anatomic structures were identified and labelled at each level in the corresponding images (CT and anatomic slices). CT images were used to identify the bony and soft tissue structures of the metatarsus and digits. The knowledge of normal anatomy of the camel and buffalo metatarsus and digits would serve as initial reference to the evaluation of CT images in these species.     

Endometrial transcript profile of progesterone‐regulated genes during early pregnancy of Water Buffalo (Bubalus bubalis)

Progesterone (P₄) plays a key role in the establishment and maintenance of pregnancy in most mammals. Unravelling the expression of progesterone‐regulated genes can expand the understanding of the embryonic mortality. Accordingly, we studied the relative mRNA expression of the P₄‐regulated genes in the buffalo. Uteri were collected from the abattoir and categorized into nonpregnant late luteal phase, stage I (28–38th days of gestation) and stage II (48–56th days of gestation) of pregnancy (n = 6/group). After extraction of total RNA from the endometrial tissues, we carried out qRT‐PCR for determining the relative mRNA expression of the P₄‐regulated genes using nonpregnant late luteal phase as calibrator group. The expression of LGALS3BP (essential for maternal recognition of pregnancy) gene was found to be significantly upregulated (p < 0.05), while MUC1 (important for embryo attachment) gene was downregulated in stage I and II of pregnancy. We observed no significant change in the expression of LGALS1, LGALS9 and CTSL genes. The SLC5A11 and SLC2A1 genes (involved in the transport of glucose to endometrium) in early pregnancy were upregulated in the pregnancy stage I (p < 0.05) relative to nonpregnant late luteal phase. The CST3 gene was significantly upregulated in pregnancy stage II (p < 0.01). These results provide molecular insights into the specific pathways involved in foeto‐maternal communication during early pregnancy in buffaloes.