用于检测牛抗边缘无形体抗体的快速侧向流动分析方法的验证与现场评估。

Nielsen K, Yu W L, Kelly L, Williams J, Dajer A, Gutierrez E, Ramirez Cruz G, Renteria T, Bermudez R, Algire J

Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield), Ottawa, Ontario, Canada.

J Immunoassay Immunochem. 2009;30(3):313-21. doi: 10.1080/15321810903084749.

The lateral flow assay (LFA) is a rapid diagnostic test which may be performed under most conditions and is especially useful for field applications. This type of assay was applied to the detection of antibody to bovine Anaplasma marginale using sera from endemic areas and from areas which have been free from infection for more than 25 years. Briefly, the test uses recombinant A. marginale major surface protein 5 peptide (Msp5), immobilized on a cellulose acetate membrane. A serum sample is added to a pad containing a monoclonal antibody specific for bovine IgG(1), conjugated with colloidal gold, located at one end of the strip. The sample and gold conjugate are wicked along the membrane and if antibody is present in the serum, a visible line will form between the Msp5-antibody-conjugate immune complex in minutes. An additional band of recombinant protein A/G was added to the membrane as a positive control reaction of the monoclonal antibody conjugate. For comparison, direct examination of blood smears and a nested polymerase chain reaction (PCR) were performed on some of the samples. Using samples from herds in one endemic area, the PCR gave a sensitivity value of 9.2% while a commercial competitive enzyme immunoassay (CELISA) gave a sensitivity value of 17.2% and the LFA values of 20.5%. In a second endemic area, selected samples, all positive by direct examination gave a 71.7% sensitivity values with the PCR, 94.5% with the CELISA and 95.5% with the LFA. Using sera from a disease-free area, the specificity values were 100% for the PCR (testing a proportion of randomly selected samples), 99.5% for the CELISA and 98.0% for the LFA. It is envisaged that the validated LFA will be a useful tool for screening cattle moving from an area with infection to a disease-free area.

侧向流动分析（LFA）是一种快速诊断测试，可在大多数条件下进行，尤其适用于现场应用。这种分析方法被用于使用来自流行地区和已超过25年无感染地区的血清检测牛边缘无浆体抗体。简而言之，该测试使用固定在醋酸纤维素膜上的重组边缘无浆体主要表面蛋白5肽（Msp5）。将血清样本添加到试纸条一端的一个垫上，该垫含有与胶体金结合的针对牛IgG(1)的单克隆抗体。样本和金标记物沿膜向上迁移，如果血清中存在抗体，几分钟内Msp5-抗体-标记物免疫复合物之间会形成一条可见线。在膜上添加了一条额外的重组蛋白A/G带作为单克隆抗体标记物的阳性对照反应。为作比较，对部分样本进行了血涂片直接检查和巢式聚合酶链反应（PCR）。使用来自一个流行地区牛群的样本，PCR的灵敏度值为9.2%，而商业竞争酶免疫分析（CELISA）的灵敏度值为17.2%，LFA的灵敏度值为20.5%。在第二个流行地区，所有经直接检查呈阳性的选定样本，PCR的灵敏度值为71.7%，CELISA为94.5%，LFA为95.5%。使用来自无病地区的血清，PCR（检测部分随机选择的样本）的特异性值为100%，CELISA为99.5%，LFA为98.0%。预计经过验证的LFA将成为筛查从感染地区转移到无病地区的牛的有用工具。