猪流行性腹泻病毒抗体化学发光免疫分析法的建立及应用

Establishment and application of a chemiluminescence immunoassay for antibodies against porcine epidemic diarrhea virus

摘要:

【背景】 猪流行性腹泻目前仍是中国乃至全球养猪业的主要疫病，了解和掌握该疫病发病和流行状况对于该病的防控具有重要意义。【目的】 以表达纯化的猪流行性腹泻病毒重组核衣壳蛋白为靶标抗原，建立一种高特异性、高灵敏度、高通量及高自动化程度的猪流行性腹泻病毒抗体化学发光免疫分析法，用于监测猪流行性腹泻病毒流行或感染状况。【方法】 通过无缝克隆技术构建重组质粒并诱导表达出核衣壳蛋白，与羧基磁珠进行偶联，形成免疫磁珠复合物。通过优化各项反应条件，建立猪流行性腹泻病毒抗体检测方法，并对该方法的特异性、灵敏度、重复性及符合率进行验证。【结果】 重组核衣壳蛋白为可溶性蛋白且具有良好的反应原性。建立的检测方法最优偶联pH值为8.0、最优偶联蛋白量为80 μg、最优封闭剂为20 μL的10%牛血清白蛋白、最优磁珠浓度为0.25 mg/mL、最优样品稀释度为10倍、最优酶稀释度为1:15 000、最优一抗孵育时间为10 min、最优二抗孵育时间为15 min、最优底物反应时间为3 min。该方法灵敏度高于酶联免疫吸附试验，并且与11种病原抗体阳性标准血清均无交叉反应。重复性试验中，批内变异系数为3.09%-8.80%，批间变异系数为4.87%-9.17%，均 < 10%。符合率试验中，阳性符合率为93.4%、阴性符合率为99.2%、总符合率为98.2%。【结论】 本研究建立的猪流行性腹泻病毒抗体化学发光免疫分析法可用于临床样品中猪流行性腹泻病毒抗体的检测，以及猪流行性腹泻病毒流行状况的追踪了解，也为后续试剂盒开发提供了参考。

Abstract:

[Background] Porcine epidemic diarrhea (PED) remains a major disease in the swine industry in China and the globe. Therefore, understanding and monitoring the incidence and epidemic situation of this disease is of great significance for its prevention and control. [Objective] To establish a highly specific, sensitive, high-throughput, and highly automated chemiluminescent immunoassay for antibodies against porcine epidemic diarrhea virus (PEDV) using purified recombinant N protein of PEDV as the target antigen for monitoring PEDV epidemics or infections. [Methods] We constructed a recombinant plasmid by seamless cloning and induced the expression of N protein. The obtained protein was coupled with carboxy magnetic beads to form an immunomagnetic bead complex. After optimization of the reaction conditions, the PEDV antibody detection method was established. The specificity, sensitivity, reproducibility, and compliance rate of the established method were evaluated. [Results] The recombinant N protein was soluble and had good immunoreactivity. The optimal conditions for the established method were coupling pH 8.0, coupling protein amount of 80 μg, 20 μL 10% BSA as the blocking reagent, magnetic bead concentration of 0.25 mg/mL, sample dilution of 1:10, enzyme dilution of 1:15 000, incubation time of 10 min for the primary antibody, incubation time of 15 min for the secondary antibody, and substrate reaction time of 3 min. The established method showed higher sensitivity than ELISA and no cross-reactivity with 11 pathogen antibody-positive standard sera. The reproducibility test showed that the intra-batch and inter-batch coefficients of variation were 3.09%-8.80% and 4.87%-9.17%, respectively, which were both < 10%. The compliance test showed a positive compliance rate of 93.4%, a negative compliance rate of 99.2%, and an overall compliance rate of 98.2%. [Conclusion] The chemiluminescent immunoassay established in this study can be used to detect PEDV antibodies in clinical samples, track and understand the epidemic situation of PEDV, and provide a reference for subsequent reagent kit development.