Development of Indirect ELISA for Detecting Novel Duck Reovirus Using σB and σC Proteins as Coating Antigens
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摘要: 以纯化的新型鸭呼肠孤病毒（NDRV）NP03株重组σB和σC蛋白作为包被抗原，对反应条件进行优化，建立检测NDRV抗体的间接ELISA方法。优化后的最佳条件为：σB蛋白包被浓度为12.5 μg·mL-1；σC蛋白包被浓度为6.25 μg·mL-1；封闭液为15% FBS；血清稀释度为1：80；酶标二抗稀释度为1：400；底物37℃显色5 min。该方法对MDRV、DHV、MPV和MD-GPV阳性血清均无交叉反应，具有良好的特异性。该方法与NDRV全病毒间接ELISA相比较，符合率高达92.5%。本研究进一步丰富了NDRV抗体的检测方法。Abstract: An indirect ELISA was developed using the purified recombinant novel duck reovirus(NDRV)σB and σC proteins as coating antigens. The optimized conditions for the methodology were determined to include:concentrations of σB at 12.5 μg·mL-1 and σC at 6.25 μg·mL-1,a blocking buffer of 15% FBS, serum samples being diluted 80 times, goat anti-duck HRP-IgG being diluted 400 times,and substrate incubation at 37℃ for 5 min. The assay was found to be specific without any cross reaction with antibodies of MDRV, DHV, MPV, or MD-GPV. The coincidence rate between the indirect ELISA coated with the recombinant σB and σC proteins and that coated with NDRVwas 92.5%. It appeared that the newly developed ELISA exhibited satisfactory sensitivity and specificity on measurements,and could be an alternative means for detecting anti-NDRV antibodies.
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Key words:
- novel duck reovirus /
- prokaryotic expression /
- σB protein /
- σC protein /
- indirect ELISA
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图 1 纯化的σB和σC蛋白的SDS-PAGE及western blot分析
M为蛋白预染Marker；1~2为σB纯化蛋白的SDS-PAGE分析；3为σB纯化蛋白的western blot分析；4~5为σC纯化蛋白的SDS-PAGE分析；6为σC纯化蛋白的western blot分析。
Figure 1. SDS-PAGE and western blot analyses on purified σB and σC proteins
表 1 最佳抗原包被浓度及血清最佳稀释度的方阵滴定结果
Table 1. Optimal concentrations for coating antigens and serum dilution as determined by checkerboard titration method
血清稀释倍数 血清 蛋白 抗原质量浓度/(μg·mL-1) 50 25 12.5 6.25 3.125 1∶20 阳性 σB 1.742 1.738 1.730 1.721 1.693 阴性 0.267 0.267 0.260 0.255 0.212 阳性 σC 1.813 1.816 1.810 1.748 1.743 阴性 0.285 0.258 0.250 0.224 0.198 1∶40 阳性 σB 1.711 1.703 1.718 1.682 1.653 阴性 0.233 0.227 0.200 0.193 0.187 阳性 σC 1.769 1.773 1.737 1.692 1.737 阴性 0.212 0.194 0.188 0.166 0.158 1∶80 阳性 σB 1.698 1.691 1.664 1.594 1.487 阴性 0.188 0.176 0.162 0.157 0.148 阳性 σC 1.720 1.714 1.697 1.688 1.635 阴性 0.167 0.162 0.153 0.145 0.142 1∶160 阳性 σB 1.603 1.589 1.496 1.327 1.385 阴性 0.158 0.162 0.151 0.139 0.140 阳性 σC 1.607 1.524 1.470 1.415 1.430 阴性 0.142 0.137 0.130 0.126 0.126 1∶320 阳性 σB 1.305 1.312 1.217 1.153 1.008 阴性 0.148 0.138 0.121 0.121 0.110 阳性 σC 1.420 1.393 1.405 1.225 1.347 阴性 0.131 0.122 0.123 0.120 0.121 
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表 2 间接ELISA检测方法的反应条件的优化
Table 2. Optimized conditions for indirect ELISA
项目 σB蛋白 σC蛋白 封闭液 待检血清 羊抗鸭IgG-HRP 最佳质量浓度或稀释度 12.5 μg·mL-1 6.25 μg·mL-1 15% FBS 1∶80 1∶400 最佳反应条件 4℃/12 h 4℃/12 h 37℃/3 h 37℃/60 min 37℃/45 min
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表 3 间接ELISA特异性试验
Table 3. Specificity test on indirect ELISA
血清 OD490nm 值 S/P 值 结果 MDRV 0.242 0.039 阴性 DHV 0.230 0.031 阴性 MPV 0.191 0.006 阴性 MD-GPV 0.208 0.017 阴性 NDRV(+) 1.730 NDRV(-) 0.182
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表 4 间接ELISA重复性试验
Table 4. Repeatability test on indirect ELISA
血清 批内重复性试验 批间重复性试验 ${\bar{X}}$±SD CV/% ${\bar{X}}$±SD CV/% 1 1.429±0.012 5.21 1.442±0.008 4.78 2 0.641±0.006 3.17 0.625±0.008 6.43 3 1.337±0.010 6.06 1.387±0.010 5.10 4 0.801±0.018 4.14 0.803±0.016 6.25 5 1.511±0.011 2.93 1.523±0.010 4.97 6 1.007±0.007 3.76 1.127±0.007 3.21 7 1.454±0.008 5.21 1.429±0.018 6.78 8 0.403±0.010 6.20 0.415±0.014 6.78
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表 5 σB-σC-ELISA和NDRV-ELISA方法的比较试验
Table 5. Comparison between σB-σC-ELISA and NDRV-ELISA
项目 阳性 阴性 阳性率/% 符合率/% σB-σC-ELISA 31/80 49/80 38.75 92.5 NDRV-ELISA 33/80 47/80 41.25
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