Abstract：
Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2-3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 μg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 μg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 μl of GNP conjugate and 50 μl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.
摘要：
日本脑炎（JE）是一种蚊子传播的病毒性人畜共患疾病。猪的血清转化发生在人类感染前2-3周，因此，猪是预测人类JE爆发的合适前哨。进行本研究的目的是开发免疫层析试纸条（ICS）测定法，以检测猪群中日本脑炎病毒（JEV）的最新感染。ICS测定的两种形式被标准化。在第一种格式中，将金纳米颗粒（GNP）与山羊抗猪IgM（50μg/ml）缀合，然后在NCM上点样JEV的重组NS1蛋白（1mg/ml）作为测试线，蛋白G（1mg/ml）作为对照线。在格式II中，将GNP与rNS1蛋白(50μg/ml)缀合,随后点样山羊抗猪IgM(1mg/ml)作为测试线,并点样针对rNS1的IgG(1mg/ml)作为对照线。为了减少非特异性结合，血清和硝酸纤维素膜（NCM）的阻断是使用PBS-T和1％BSA中的5％SMP，分别。当10μl的GNP缀合物和50μl的1:10SMP阻断的血清在BSA阻断的NCM上反应,随后反应时间为15分钟时,观察到测定的最佳反应条件。显示测试线和对照线两者的样品被认为是阳性的,而仅显示对照线的样品被认为是阴性的。使用间接IgMELISA和开发的ICS测定筛选了总共318个野外猪血清样品。格式-I的相对诊断敏感性和特异性分别为81.25％和93.0％，而格式-II的相对诊断敏感性和特异性分别为87.50％和62.93％，分别。在测试的318个样本中，32例通过IgMELISA呈阳性，血清阳性为10.06％，而ICS形式I的血清阳性为8.1％。由于格式I的最佳灵敏度和更高的特异性，该方法在三个不同的实验室中得到验证,kappa一致性范围为0.80~1,这表明所开发的用于检测野猪血清样本以检测近期JEV感染的方法具有出色的可重复性.
