Preparation and Characterization Analysis of Monoclonal Antibody Against the Avian Tembusu Virus
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摘要: 为进一步建立基于禽坦布苏病毒E囊膜蛋白的快速检测方法，本研究通过纯化的禽坦布苏病毒为抗原免疫BALB/c小鼠，获得2株杂交瘤细胞株（2F6和4E11），其分泌的单抗效价可达1:64 000，并可有效识别禽坦布苏病毒感染的Vero细胞；原核表达的囊膜E经SDS-PAGE电泳后可与单抗进行Western-blot发生反应，初步表明制备的单克隆抗体为禽坦布苏病毒E蛋白单抗，为该病分子流行病学研究及新型诊断方法建立奠定基础。Abstract: This study aimed to prepare and characterize monoclonal antibody (MAb) against avian Tembusu virus. BALB/c mice were immunized with the antigen of purified avian Tembusu virus, and then two MAbs named as 2F6 and 4E11 were selected. The two MAbs, with the titers of up to 1:64, 000, were reacted with Vero cells infected with avian Tembusu virus. The Western blot analysis showed that both MAbs 2F6 and 4E11 were able to react with fusion envolope protein by prokaryotic expression, indicating that the two MAbs recognized the envelope protein. The prepared MAbs could be used to further explore the molecular epidemiology and development of new diagnostic methods for avian Tembusu virus infection.
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Key words:
- Avian Tembusu virus /
- monoclonal antibody /
- envelope protein
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图 1 单抗与Vero细胞作用的间接免疫荧光检测结果
注：A为鼠抗禽坦布苏病毒与病毒感染细胞反应；B为2F6株单抗与禽坦布苏病毒感染细胞反应；C为4E11株单抗与禽坦布苏病毒感染细胞反应；D为阴性对照。
Figure 1. Reaction of infected cells and monoclonal antibody determined by indirect immunofluorescence
图 2 禽坦布苏病毒E融合蛋白与4E11单抗反应
注：M为预染蛋白marker；1为禽坦布苏病毒pET-E蛋白。
Figure 2. Reaction of expressing E protein of avian Tembusu virus with MAb 4E11
表 1 方阵法确定抗原最佳包被浓度和抗体最佳稀释浓度(OD450)
Table 1. Optimal concentration antigen and antibody determined by matrix titrations (OD450)
包被浓度/(μg·mL-1) 阴、阳性血清稀释倍数 1:200
(+)1:200
(-)1:400
(+)1:400
(-)1:800
(+)1:800
(-)1:1600
(+)1:1600
(-)1:3200
(+)1:3200
(-)10 1.345 0.189 1.215 0.158 1.112 0.132 0.902 0.112 0.732 0.078 5 1.282 0.152 1.131 0.132 1.041 0.105 0.795 0.098 0.595 0.065 2.5 1.212 0.124 1.012 0.109 0.912 0.098 0.724 0.092 0.524 0.062 1.25 0.832 0.099 0.879 0.093 0.759 0.083 0.656 0.078 0.406 0.046 0.625 0.592 0.082 0.567 0.075 0.534 0.072 0.532 0.069 0.322 0.037 0.3125 0.435 0.075 0.415 0.069 0.405 0.063 0.396 0.058 0.272 0.032 注：“-”、“+”分别表示阴、阳性血清。表 3同。 
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表 2 方阵法确定病毒抗原最佳包被浓度和抗体最佳稀释浓度(P/N)
Table 2. The P/N value of optimal concentration antigen and antibody determined by matrix titrations
包被浓度/(μg·mL-1) 阴、阳性血清稀释倍数 1:200 1:400 1:800 1:1600 1:3200 10 7.12 7.69 8.42 8.05 9.38 5 8.43 8.57 9.91 8.11 9.15 2.5 9.77 9.28 9.31 7.87 8.45 1.25 8.40 9.45 9.14 8.41 8.83 0.625 7.22 7.56 7.42 7.71 8.70 0.3125 5.80 6.01 6.43 6.83 8.50
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表 3 间接ELISA检测细胞融合后杂交瘤细胞上清效价
Table 3. Titers of hybridoma cell culture supernatant determined by indirect ELISA
项目 1C2 1F7 2F6 2G5 3D11 4E11 4H8 5G3 5B3 6C4 + - OD450 0.46 0.62 0.75 0.53 0.65 0.94 0.75 0.53 0.56 0.61 1.23 0.18 P/N 2.56 3.44 4.17 2.94 3.61 5.22 4.17 2.94 3.11 3.39 6.83 1.00
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