新城疫病毒核蛋白单克隆抗体的制备及双抗体夹心ELISA的初步建立

目的制备新城疫病毒(NDV)核蛋白(NP)的单克隆抗体(单抗),并用于建立一种可定量检测NDV病毒含量的双抗体夹心酶联免疫吸附试验检测方法(NDV NP ELISA)。方法基于NDV病毒株F48E9获得NP基因,经原核表达方式制备出重组抗原rNP;免疫小鼠制备NDV NP特异性单抗;单抗经HRP标记和配对筛选,建立NDV NP ELISA,分析其特异性、灵敏度、精密度、准确度和检测线性,并分析本方法定量检测的NP含量与PFU病毒滴度的定量相关性。结果建立基于单抗3C10和4E7的NDV NP ELISA,其定量检测NDV rNP的最佳线性范围为0.015~0.250μg/ml(R2=0.9974),回收率在88.4%~106.01%,变异系数小于3.4%;该方法具有良好特异性;该方法定量检测NDV抗原含量与PFU病毒感染滴度有较好的相关性(R2=0.9209)。结论建立NDV NP ELISA,可准确定量检测NDV病毒中的NP抗原含量,为NDV病毒含量的测定提供一种可靠简便的分析方法。

Development of monoclonal antibodies against nucleoprotein of Newcastle disease virus and establishment of a quantitative double-antibody sandwich ELISA for NDV antigen

We developed the monoclonal antibodies against nucleoprotein (NP) of Newcastle disease virus (NDV),and established a double antibody sandwich ELISA method for quantitative determination of NP antigen of NDV (NDV NP ELISA).The recombination NP protein derived from strain F48E9 of NDV were prepared and used to immunize BLAB/c mice.The mouse splenic cells from immunized mice were fused with SP2/0 cells to generate monoclonal antibodies (mAb).The NDV NP specific mAbs were paired to establish a double antibody sandwich ELISA method.The performance of the NDV NP ELISA was evaluated,including specificity,sensitivity,precision,accuracy and linearity.The correlation between the ELISA and PFU virus titer was analyzed by regression analysis method.Two monoclonal antibodies 3C10 and 4E7 were selected to establish double antibody sandwich ELISA for NP antigen of NDV.The linearity and performance of the NDV NP ELISA was characterized.The detection linearity fell in the range of 0.015-0.250 μg/mL (R2 =0.997 4).The detection limit of the assay was 0.015 μg/mL.The recovery was between 88.4％ and 106.01％;the variation coefficient was below 3.4％.In testing of 50 NDV virus samples,this assay performed well and correlated comparably with PFU virus titer (R2 =0.920 9).The NDV NP ELISA for quantitative detection of NDV is a reliable quantifiable assay for detection of NDV NP protein;it provides a new approach for rapid and quantitative detection of Newcastle disease virus.