Effect of serum heat-inactivation and dilution on detection of anti-WNV antibodies in mice by West Nile virus E-protein microsphere immunoassay.
Effect of serum heat-inactivation and dilution on detection of anti-WNV antibodies in mice by West Nile virus E-protein microsphere immunoassay.
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DOI：
10.1371/journal.pone.0045851
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Nerurkar VR
中科院分区：
文献类型：
--
作者：
Namekar M;Kumar M;O'Connell M;Nerurkar VR
利用西尼罗病毒（WNV）小鼠模型进行免疫发病机制研究对于开发西尼罗病毒抗病毒药物和疫苗具有重要意义。由于小鼠在西尼罗河病毒感染早期产生的抗体对于清除外周病毒至关重要，因此检测早期和持久的抗西尼罗河病毒抗体非常重要。ELISA和斑块减少中和试验传统上分别用于检测抗西尼罗河病毒抗体和西尼罗河病毒中和抗体。虽然这些检测是敏感和特异性的，但它们是昂贵和耗时的。微球免疫测定（MIA）灵敏、特异、允许高通量、具有成本效益、比其他方法需要更少的时间，并且需要低血清体积。一些测定参数，如血清热失活（HI）和稀释度可以改变西尼罗河病毒MIA的敏感性。我们检测了这些参数对WNV e -蛋白MIA （WNV E-MIA）的影响，以增强抗WNV IgM和IgG抗体的检测。采用连续稀释接种西尼罗河病毒小鼠不同时间点的HI和非HI （NHI）血清进行西尼罗河病毒E-MIA研究。与NHI血清相比，HI显著提高了IgM和IgG抗体的检测。感染后第3天较早检测到HI血清中WNV IgM和IgG抗体，IgM抗体持续到感染后第24天。1∶20稀释的HI血清与较高稀释度的血清相比，最适合检测IgM和IgG抗体。此外，在HI血清中加入外源性补体可降低西尼罗河病毒E-MIA的敏感性。这些结果表明，血清hi和最佳稀释通过消除补体干扰增强了西尼罗河病毒E-MIA的敏感性，从而在感染的早期和晚期检测到低滴度的抗西尼罗河病毒抗体。这种改进的MIA也可以很容易地用于检测其他感染因子和宿主蛋白的低滴度抗体。
Immunopathogenesis studies employing West Nile virus (WNV) mice model are important for the development of antivirals and vaccines against WNV. Since antibodies produced in mice early during WNV infection are essential for clearing virus from the periphery, it is important to detect early and persistent anti-WNV antibodies. ELISA and plaque reduction neutralization tests are traditionally used for detection of anti-WNV antibodies and WNV-neutralizing antibodies, respectively. Although these assays are sensitive and specific, they are expensive and time consuming. Microsphere immunoassays (MIA) are sensitive, specific, allow for high throughput, are cost effective, require less time to perform than other methods, and require low serum volumes. Several assay parameters such as serum heat-inactivation (HI) and dilution can alter WNV MIA sensitivity. We examined the effect of these parameters on WNV E-protein MIA (WNV E-MIA) for the enhanced detection of anti-WNV IgM and IgG antibodies. WNV E-MIA was conducted using serial dilutions of HI and non-HI (NHI) serum collected at various time points from mice inoculated with WNV. HI significantly enhanced detection of IgM and IgG antibodies as compared to NHI serum. WNV IgM and IgG antibodies in HI sera were detected earlier at day 3 and IgM antibodies persisted up to day 24 after infection. HI serum at 1∶20 dilution was found to be optimal for detection of both IgM and IgG antibodies as compared to higher-serum dilutions. Further, addition of exogenous complement to the HI serum decreased the WNV E-MIA sensitivity. These results suggest that serum-HI and optimal dilution enhance WNV E-MIA sensitivity by eliminating the complement interference, thereby detecting low-titer anti-WNV antibodies during early and late phases of infection. This improved MIA can also be readily employed for detection of low-titer antibodies for detection of other infectious agents and host proteins.
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DOI：
10.1128/cdli.12.8.959-969.2005
发表时间：
2005-08-01
期刊：
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY
影响因子：
--
作者：
Dias, D;Van Doren, J;Esser, MT
通讯作者：
Esser, MT
影响因子：
6.2
作者：
Schnaidt, Martina;Weinstock, Christof;Wernet, Dorothee
通讯作者：
Wernet, Dorothee
影响因子：
11.8
作者：
Roehrig, JT;Nash, D;Campbell, GL
通讯作者：
Campbell, GL
影响因子：
5.5
作者：
Ledizet, M;Kar, K;Koski, RA
通讯作者：
Koski, RA
影响因子：
8.8
作者：
Prince, Harry E.;Lape-Nixon, Mary;Busch, Michael P.
通讯作者：
Busch, Michael P.