目的 建立定量检测重组猪胰蛋白酶(recombinant porcine trypsin, RPT)的双抗体夹心ELISA，并进行方法学验证及初步应用。 方法 通过抗体筛选获得可配对的抗RPT鼠源单克隆抗体对，并确定包被抗体及酶标抗体的最佳工作浓度、封闭液种类和浓度，建立可定量检测RPT含量的双抗体夹心ELISA，确定该方法的线性范围、灵敏度、准确度、精密度和特异性。用建立的方法检测疫苗制品生产过程中病毒背景液中RPT含量，并验证该方法对不同品牌的天然提取猪胰蛋白酶的检出效率。 结果 确定了包被抗体（RPT-5C2D12）最佳工作浓度为6.000 μg/mL、酶标抗体（RPT-1E10A5）最佳稀释比为1∶5 000；封闭液为质量分数1%牛血清白蛋白；四参数逻辑拟合的标准曲线决定系数≥0.990，检测范围为0.156~40.000 ng/mL；定量限为0.156 ng/mL；准确度加标回收率为92.3%~106.7%；精密度验证批间变异系数≤6.7%，批内变异系数≤5.9%；除RPT外，与其他对照品蛋白均无交叉反应；3批次疫苗病毒背景液样品的加标回收率在87.4%~98.2%。2种品牌天然提取猪胰蛋白酶相对RPT的平均检出效率分别为59%和68%。结论 成功建立了RPT双抗体夹心ELISA，该方法具有良好的线性关系、灵敏度、准确度、精密度及特异性，可用于疫苗类生物制品的猪胰蛋白酶残留量评估。

Objective To establish a double antibody sandwich ELISA for quantitative detection of recombinant porcine trypsin (RPT), and to perform methodological validation and preliminary application. Methods A double antibody sandwich ELISA for quantitative detection of RPT content was established.Paired mouse monoclonal antibodies against RPT were selected through antibody screening, optimal working concentrations of coating and enzyme-labeled antibodies as well as type and concentration of blocking agent were determined. The linear range, sensitivity, accuracy, precision and specificity of the method were determined. This established method was used to detect the content of RPT in viral background liquid during the vaccine production, and to verify the detection efficiency of this method for different brands of natural extracted porcine trypsin. Results The coating antibody was determined to be RPT-5C2D12 with optimal working concentration of 6 000 μg/mL， the enzyme-labled antibody was RPT-1E10A5 with optimal dilution of 1∶5 000, and the blocking solution was 1% bovine serum albumin. A four-parameter logistic fitting standard curve achieved correlation coefficient ≥0.990 and detection range of 0.156-40.000 ng/mL. Limits of quantification was 0.156 ng/mL. Spike recovery rate for accuracy ranged from 92.3% to 106.7%. Precision validation showed inter-assay coefficient of variation (CV) ≤6.7% and intra-assay CV ≤5.9%. Except for RPT, there was no cross-reactivity with other control proteins. The spike recovery rate for three batches of viral background fluid samples ranged from 87.4% to 98.2%. The relative detection efficiency for two brands of natural extracted porcine trypsin was 59% and 68% compared to RPT. Conclusion The established double antibody sandwich ELISA for quantitative detection of RPT has good linearity, sensitivity, accuracy, precision, and specificity, and can be used for assessing trypsin residues in vaccine type biological products.