Rapid and sensitive detection of immunoglobulin M (IgM) and IgC antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay
Rapid and sensitive detection of immunoglobulin M (IgM) and IgC antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay
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DOI：
10.1128/jcm.37.4.1049-1056.1999
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发表时间：
1999-04-01
影响因子：
9.4
通讯作者：
Haas, L
中科院分区：
文献类型：
--
作者：
von Messling, V;Harder, TC;Haas, L
犬瘟热病毒（CDV）感染引起一种经常致命的全身性疾病在广泛的食肉动物物种，包括家狗。在CDV感染中，经典血清学提供诊断和预后价值的数据（血清转化动力学），也用于预测幼犬的最佳接种年龄，常规CDV血清学仍然基于时间和成本密集的病毒中和试验（V-NA）。在这里，我们描述了一种新的捕获夹心酶联免疫吸附试验（ELISA），该试验使用重组杆状病毒表达的CDV野生型分离物（2544/Han95）的核衣壳(N)蛋白检测犬血清中的CDV特异性抗体。重组抗原在绿芽海螺（Heliothis virescens）幼虫中高效产生。夹心捕获ELISA能够对196份和35份犬血清的cdv特异性免疫球蛋白G （IgG）和IgM血清状态进行明确的定性评价。间一致性分析（kappa = 0.988）表明，ELISA可替代V-NA，不受限制地定性测定cdv特异性IgG血清状态。为了半定量n特异性抗体，采用一步稀释（α法）igg特异性ELISA法。对来自德国北部犬血清的中欧CDV野生型分离物2544/Han95测定的V-NA滴度大于或等于50%中和剂量（ND50）的1/100时，Alpha值大于或等于50%，显示出非常好的一致性（kappa = 0.968）。ND50滴度为1/100被认为是一个阈值，而滴度大于或等于1/100表明具有弹性的保护性免疫。采用新开发的ELISA方法，在15份急性犬瘟热症状犬血清中的9份中检测到IgM类CDV n特异性抗体。在15只狗的5只白细胞（均为IgM阳性）中，用RT-PCR检测CDV RNA，重组夹心捕获ELISA检测Ige类n特异性抗体，具有较高的灵敏度和特异性，是传统CDV - na的快速、经济的替代方法，通过检测特异性IgM抗体，可作为RT-PCR和V-NA诊断急性犬瘟热感染的补充。
Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups, Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (kappa = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of greater than or equal to 50% showed very good inter-rater agreement (kappa = 0.968) with V-NA titers of greater than or equal to 1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of greater than or equal to 1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR, The recombinant capture-sandwich ELISA detecting N-specific antibodies of the Ige class provided superior sensitivity and specificity and thus represents a rapid and cost-effective alternative to classical CDV V-NA, By detection of specific IgM antibodies, the ELISA will be complementary to RT-PCR and V-NA in the diagnosis of acute distemper infections.