目的 制备针对呼吸道合胞病毒(RSV) N蛋白的兔多克隆抗体，以其作为检测抗体建立基于ELISA的快速中和抗体检测方法。 方法 构建pET30a-N质粒，表达纯化N蛋白，免疫新西兰兔制备针对RSV N蛋白的多克隆抗体作为检测抗体。阳性血清系列稀释后与100半数组织培养感染剂量(TCID50)/孔的RSV中和，接种Hep-2细胞培养，80%丙酮固定细胞，ELISA方法检测感染细胞中病毒的N蛋白，当每孔的吸光度值低于临界值时，视为中和试验阳性孔，阳性孔血清的最高稀释度为该血清的中和抗体滴度。优化抗体稀释度、检测时间、细胞密度及中和时间，建立基于N-ELISA的中和抗体检测方法，对建立的实验方法进行细胞代次、边缘孔效应、准确性、重复性以及精密度验证，初步应用于人RSV IgG阳性血清检测，分析与微量中和法的相关性。 结果 成功构建出pET30a-N质粒，表达纯化的N蛋白纯度较高，特异性良好；三免后兔血清效价为1∶51 200，可特异性结合RSV；制备的抗RSV N兔多抗的效价为1∶51 200，特异性良好，建立的快速中和抗体检测方法在4 d就可检测，中和时间可缩短至30 min，细胞代次、96孔板位置(边缘孔和非边缘孔)对该方法检测无影响，重复性、精密性、准确度良好，建立的方法和微量中和法检测64份RSV IgG阳性人血清，相关系数为0.929 6，证明两种方法具有良好的正相关性。 结论 成功建立基于ELISA的快速中和抗体检测方法，可用于评价RSV疫苗接种后的血清抗体水平。 Objective To prepare rabbit polyclonal antibodies against respiratory syncytial virus (RSV) N protein and use them as the detection antibodies to establish a N-ELISA-based method for rapid detection of neutralizing antibodies. Methods A plasmid of pET30a-N for the expression of RSV N protein was constructed. After purification, the protein was immunized into New Zealand rabbits to prepare polyclonal antibodies, which were used as the detection antibodies. Positive serum samples were diluted and used to neutralize RSV (100 TCID50/well). Hep-2 cells were inoculated and cultured, and then the cells were fixed with 80% acetone. ELISA was performed to detect RSV N protein in infected cells. When the absorbance value of a well was below the cut-off value, it was regarded as the positive well in the neutralization test. The highest dilution of a positive well serum was the neutralizing antibody titer. After optimizting the antibody dilution, detection time, cell density and the duration of neutralization, the method for neutralizing antibody detection was established based on N-ELISA. The established method was verified by analyzing the influences of different cell generations and edge effects, and calculating the accuracy, repeatability and precision. The correlation between the established method and microneutralization method was analyzed by detecting human RSV IgG-positive serum. Results The plasmid pET30a-N was successfully constructed, and the expressed N protein showed high purity and good specificity. After the third immunization, the antibody titer in rabbit serum was 1∶51 200, and the antibodies could specifically bind to RSV. The prepared rabbit anti-RSV N polyclonal antibodies had a titer of 1∶51 200, and showed good specificity. The neutralizing antibodies could be detected on day 4 with the established method, and the duration of neutralization was shortened to 30 min. Cell generations and the position of wells in the 96-well plate (edge well and non-edge well) had no significant effect on the method, and the repeatability, precision and accuracy of the method were good. In the detection of 64 RSV IgG-positive human serum samples by the established method and microneutralization method, the correlation coefficient was 0.929 6, indicating a good positive correlation between the two methods. Conclusions A N-ELISA-based method for rapid neutralizing antibody detection is successfully established, which can be used to evaluate the serum antibody level after RSV vaccination.
Establishing N-ELISA-based method for rapid detection of neutralizing antibodies against respiratory syncytial virus
Objective To prepare rabbit polyclonal antibodies against respiratory syncytial virus (RSV) N protein and use them as the detection antibodies to establish a N-ELISA-based method for rapid detection of neutralizing antibodies. Methods A plasmid of pET30a-N for the expression of RSV N protein was constructed. After purification, the protein was immunized into New Zealand rabbits to prepare polyclonal antibodies, which were used as the detection antibodies. Positive serum samples were diluted and used to neutralize RSV (100 TCID50/well). Hep-2 cells were inoculated and cultured, and then the cells were fixed with 80% acetone. ELISA was performed to detect RSV N protein in infected cells. When the absorbance value of a well was below the cut-off value, it was regarded as the positive well in the neutralization test. The highest dilution of a positive well serum was the neutralizing antibody titer. After optimizting the antibody dilution, detection time, cell density and the duration of neutralization, the method for neutralizing antibody detection was established based on N-ELISA. The established method was verified by analyzing the influences of different cell generations and edge effects, and calculating the accuracy, repeatability and precision. The correlation between the established method and microneutralization method was analyzed by detecting human RSV IgG-positive serum. Results The plasmid pET30a-N was successfully constructed, and the expressed N protein showed high purity and good specificity. After the third immunization, the antibody titer in rabbit serum was 1∶51 200, and the antibodies could specifically bind to RSV. The prepared rabbit anti-RSV N polyclonal antibodies had a titer of 1∶51 200, and showed good specificity. The neutralizing antibodies could be detected on day 4 with the established method, and the duration of neutralization was shortened to 30 min. Cell generations and the position of wells in the 96-well plate (edge well and non-edge well) had no significant effect on the method, and the repeatability, precision and accuracy of the method were good. In the detection of 64 RSV IgG-positive human serum samples by the established method and microneutralization method, the correlation coefficient was 0.929 6, indicating a good positive correlation between the two methods. Conclusions A N-ELISA-based method for rapid neutralizing antibody detection is successfully established, which can be used to evaluate the serum antibody level after RSV vaccination.
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