用于诊断阿根廷奶牛牛奶和血清样本中牛布鲁氏菌病的间接酶联免疫吸附测定法评估

Evaluation of an indirect ELISA for the diagnosis of bovine brucellosis in milk and serum samples in dairy cattle in Argentina.

作者信息

Vanzini V R, Aguirre N, Lugaresi C I, de Echaide S T, de Canavesio V G, Guglielmone A A, Marchesino M D, Nielsen K

机构信息

Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Agropecuaria Rafaela, Santa Fe, Argentina.

出版信息

Prev Vet Med. 1998 Sep 1;36(3):211-7. doi: 10.1016/s0167-5877(98)00080-4.

DOI:10.1016/s0167-5877(98)00080-4

PMID:9785376

Abstract

An indirect enzyme-linked immunosorbent assay (ELISA) for Brucella abortus antibodies detection in bovine milk and serum samples was validated. The assay use B. abortus smooth lipopolysaccharide as antigen, immobilized on a polystyrene matrix; milk diluted 1:2 or serum diluted 1:50, in a buffer containing divalent cation chelating agents EDTA and EGTA (ethyleneglycol-bis-aminoether-N,N,N',N'-tetraacetic acid) to reduce non-specific reactions; and a mouse monoclonal antibody specific for an epitope of bovine IgG1, conjugated with horseradish peroxidase. A total of 2646 sera and 2119 milk samples from cows older than 24 months were obtained from 12 brucellosis-free herds for at least the previous 5 years. Milk samples were obtained in parallel with serum samples. The remaining 527 serum samples were from dry cows. All cattle were vaccinated with B. abortus strain 19 between 3-10 months of age. Five hundred and fifty-two milk samples and 562 serum samples were obtained from 6 infected herds with abortions where B. abortus was isolated at least once no more than 6 months before sampling. The complement-fixation test (CFT) on serum samples was considered the gold standard. Serum samples were also tested with the official screening test: the buffered plate antigen (BPA) test. The cut-off point was determined using receiver-operating characteristic (ROC) analysis. For milk samples, it was fixed at 36 percent positivity (PP) giving a sensitivity of 99.6% with a 95% confidence interval (CI) of 98.6-99.9%. The specificity was 99.1% (CI 98.9-99.4%). For serum samples, the cut-off was fixed at 53 PP giving a sensitivity of 99.6% (CI 98.6-99.9%) and a specificity 98.6% (CI 98-99%). The BPA test showed a relative sensitivity of 99.6% (CI 98.6-99.9%) and a relative specificity of 98.6% (CI 98.1-99%). Our results indicate that the indirect ELISA is a highly sensitive and specific test and can be adapted to process a large number of samples.

摘要

一种用于检测牛乳和血清样本中布鲁氏菌流产亚种抗体的间接酶联免疫吸附测定（ELISA）方法得到了验证。该测定使用固定在聚苯乙烯基质上的布鲁氏菌流产亚种光滑脂多糖作为抗原；牛奶用含二价阳离子螯合剂乙二胺四乙酸（EDTA）和乙二醇双氨基醚-N,N,N',N'-四乙酸（EGTA）的缓冲液稀释1:2，血清稀释1:50，以减少非特异性反应；以及一种针对牛IgG1表位的小鼠单克隆抗体，与辣根过氧化物酶偶联。从至少在过去5年无布鲁氏菌病的12个牛群中获取了2646份血清样本和2119份来自24个月以上奶牛的乳样。乳样与血清样本同时采集。其余527份血清样本来自干奶牛。所有牛在3至10月龄时均接种了布鲁氏菌流产亚种19号菌株。从6个有流产且在采样前不超过6个月至少一次分离出布鲁氏菌流产亚种的感染牛群中获取了552份乳样和562份血清样本。血清样本的补体结合试验（CFT）被视为金标准。血清样本也用官方筛查试验：缓冲板抗原（BPA）试验进行检测。通过受试者操作特征（ROC）分析确定临界值。对于乳样，临界值固定为36%的阳性率（PP），灵敏度为99.（此处原文有误，应为99.6%）%，95%置信区间（CI）为98.6 - 99.9%。特异性为99.1%（CI 98.9 - 99.4%）。对于血清样本，临界值固定为53 PP，灵敏度为99.6%（CI 98.6 - 99.9%），特异性为98.6%（CI 98 - 99%）。BPA试验显示相对灵敏度为99.6%（CI 98.6 - 99.9%），相对特异性为98.6%（CI 98.1 - 99%）。我们的结果表明，间接ELISA是一种高度灵敏且特异的检测方法，可适用于处理大量样本。