College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China.
Biosens Bioelectron. 2011 Mar 15;26(7):3353-60. doi: 10.1016/j.bios.2011.01.018. Epub 2011 Jan 22.
Rabies, canine distemper, and canine parvovirus are common contagious viral diseases of dogs and many other carnivores, and pose a severe threat to the population dynamics of wild carnivores, as well as endangering carnivore conservation. However, clinical diagnosis of these diseases, especially canine distemper and canine parvovirus, is difficult because of the broad spectrum of symptoms that may be confused with other respiratory and enteric diseases of dogs. The most frequently used and proven techniques for diagnosing viral diseases include the conventional enzyme-linked immunosorbent assay (ELISA), rapid fluorescent focus inhibition test (RFFIT), mouse neutralisation test (MNT), and fluorescent antibody virus neutralization (FAVN) test. However, these methods still have some inherent limitations. In this study, a magnetic protein microbead-aided indirect fluoroimmunoassay was developed to detect canine virus specific antibodies, human rabies immunoglobulin, CDV McAbs, and CPV McAbs. In this assay, an avidin-biotin system was employed to combine magnetic microbeads and virus antigens (rabies virus, canine distemper virus, and canine parvovirus). Quantification of the targeted virus antibodies was analyzed through indirect fluoroimmunoassay using the specific antigen-antibody reaction, as well as their corresponding FITC-labeled detection antibodies (mouse anti-human IgG/FITC conjugate or rabbit anti-dog IgG/FITC conjugate). The results indicated that the fluorescence intensity increased when a higher concentration of the targeted analyte was used, but the control had almost no fluorescence, much like the conventional ELISA. For human rabies immunoglobulin, CDV McAbs, and CPV McAbs, the minimum detectable concentrations were 0.2 IU/mL, 0.3 ng/mL, and 0.5 ng/mL, respectively. All of these results indicate that this assay can be employed to determine the presence of canine virus specific antibodies. In addition, the method devised here can be utilized as a general protocol in other bacterial and viral marker analysis.
狂犬病、犬瘟热和犬细小病毒是犬和许多其他食肉动物常见的传染性病毒病，对野生动物种群动态构成严重威胁，同时也危及食肉动物的保护。然而，由于这些疾病的症状范围广泛，可能与犬的其他呼吸道和肠道疾病混淆，因此临床诊断这些疾病（尤其是犬瘟热和犬细小病毒）非常困难。最常用和经过验证的诊断病毒病的技术包括常规酶联免疫吸附测定（ELISA）、快速荧光焦点抑制试验（RFFIT）、鼠中和试验（MNT）和荧光抗体病毒中和（FAVN）试验。然而，这些方法仍然存在一些固有的局限性。在这项研究中，开发了一种基于磁性蛋白微球的间接荧光免疫测定法，用于检测犬病毒特异性抗体、人狂犬病免疫球蛋白、CDV McAbs 和 CPV McAbs。在该测定法中，采用亲和素-生物素系统将磁性微球与病毒抗原（狂犬病病毒、犬瘟热病毒和犬细小病毒）结合。通过间接荧光免疫测定法，利用特定的抗原-抗体反应以及相应的 FITC 标记检测抗体（鼠抗人 IgG/FITC 缀合物或兔抗犬 IgG/FITC 缀合物）对目标病毒抗体进行定量分析。结果表明，当使用更高浓度的目标分析物时，荧光强度增加，但对照几乎没有荧光，与常规 ELISA 非常相似。对于人狂犬病免疫球蛋白、CDV McAbs 和 CPV McAbs，最低检测浓度分别为 0.2 IU/mL、0.3 ng/mL 和 0.5 ng/mL。所有这些结果表明，该测定法可用于确定犬病毒特异性抗体的存在。此外，这里设计的方法可以作为其他细菌和病毒标志物分析的一般方案使用。
