抑制性ELISA法检测药械组合产品中rhBMP-2的含量

目的：建立抑制性酶联免疫吸附分析法检测药械组合产品（骨植入物）中rhBMP-2的含量。方法：将含rhBMP-2的骨植入物匀浆后，先与rhBMP-2抗体溶液反应，再将剩余抗体与包被的rhBMP-2进行显色反应，骨修复材料对显色反应的抑制程度与其所含的rhBMP-2含量成正比，从而能准确测定组合产品中rhBMP-2的含量。通过对rhBMP-2包被浓度、rhBMP-2抗体浓度的优化，建立该方法的基本实验条件；并对检测方法的标准曲线范围、检测回收率、精密度、可重复性进行验证；用该方法对复合不同rhBMP-2含量的骨修复材料进行检测。结果：建立的抑制性酶联免疫吸附分析方法标准曲线线性良好（R²=0.998），质量浓度范围为0.078~10 μg·mL^-1，检测回收率均在理论值±20%范围内，实验内精密度和实验间精密度验证数据RSD均小于15%，不同操作人员对检测结果无显著影响。用该方法实现了对rhBMP-2含量分别5、10、50 μg·g^-1的骨修复材料的准确检测。结论：本研究建立了抑制性酶联免疫吸附分析方法，弥补了仅通过工艺参数计算间接确定药械组合产品中rhBMP-2含量的不足，实现了药械组合产品中rhBMP-2含量的直接准确检测。

Objective:To develop an inhibition ELISA for quantifying the rhBMP-2 in drug-device combination products (bone graft). Methods:After homogenization of the bone graft containing the rhBMP-2,the suspension of homogenate was first reacted with rhBMP-2 specific antibody solution. The residual antibody was collected and further reacted with coated rhBMP-2. The inhibition potency of the chromogenic reaction was proportional to the content of the rhBMP-2 contained in bone graft,thus the content of rhBMP-2 in the combination product could be determined. The basic experiment conditions for this method were established with the optimization of the concentration of coated solid rhBMP-2 and the rhBMP-2 antibody. The range of the standard curve,the detection recovery rate,precision,and reproducibility of the method were verified. Results:The standard curve of the established inhibition ELISA was linear (R²=0.998) and the detection concentration range was 0.078-10 μg·mL^-1. The results showed the recovery rate was between ±20% of theoretical value,and the intra-experimental precision and inter-experimental precision validation data indicated that the RSD were all less than 15%. Different operators had no significant impact on the detection results. The different content of rhBMP-2 contained in bone graft with 5,10 and 50 μg·g^-1 was accurately determined by this method,respectively. Conclusion:An inhibition ELISA was successfully established to detect the contents of BMP-2 in drug-device combination of bone graft products,which made up for the deficits of the indirect determination of the rhBMP-2 content in the combination products with the calculation of process parameters.