电化学发光免疫分析法在评价治疗性单抗与FcγRⅠ结合活性中的应用

目的：建立电化学发光免疫分析（electrochemiluminescence immunoassay，ECLI）测定贝伐珠单抗与免疫球蛋白G Fc段受体Ⅰ（FcγRⅠ）结合活性的方法，并评价贝伐珠单抗生物类似药（similar biotherapeutic products，SBP）候选物和其原研药（reference biotherapeutic product，RBP）与FcγRⅠ结合活性的相似性。方法：先使用封闭液对链霉亲和素（streptavidin，SA）预包被的96孔MSD板进行封闭，再将生物素（biotin）标记的FcγRⅠ与之结合，之后加入不同稀释倍数的贝伐珠单抗，最后加入SULFO-TAG（Ru（bpy）₃²⁺）标记的羊抗人检测抗体上机读数。按照试验设计分别对FcγRⅠ浓度、单抗样品初始浓度、倍比稀释倍数、检测抗体浓度等试验参数进行优化，并对优化后方法的准确性和精密性进行初步验证，还将建立的方法用于评价贝伐珠单抗SBP候选药和RBP与FcγRⅠ结合活性的相似性。结果：采用优化后的方法检测，贝伐珠单抗与FcγRⅠ的结合呈现良好的剂量效应曲线，R²>0.99。该方法具有较好的准确性和精密性，靶值为50%~150%样品的回收率在95.2%~103.7%之间；RSD均小于10%。贝伐珠单抗SBP候选药与FcγRⅠ的相对结合活性均在为RBP相对结合活性均值±3SD之间。结论：电化学发光免疫分析法能够用于评价抗体与FcγRⅠ的结合活性，并可应用于对SBP候选药和RBP与FcγRⅠ结合活性的相似性分析。该方法的灵敏度和准确性较好，为评价抗体与FcγRⅠ的结合提供了新的技术手段。  

Objective:To establish an electrochemiluminescence immunoassay(ECLI) method for the detection of the binding activity of bevacizumab to immunoglobulin G Fc receptorⅠ (Fcγ RⅠ),and to evaluate the similarity of the binding activity to FcγRⅠ between the similar biotherapeutic products (SBP) candidates of bevacizumab monoclonal antibody and the original reference biotherapeutic product (RBP). Methods:96 well MSD plate precoated with streptavidin (SA) was blocked firstly,then combined with biotin labeled FcγRⅠ,and added with different bevacizumab dilutions. Finally,SULFO-TAG (Ru(bpy)₃²⁺) labeled sheep anti-human antibody was added for value reading. According to the experimental design,the experimental parameters such as FcγRⅠ concentration,the initial concentration and dilution ratio of mAb,and the concentration of detected Ab were optimized,and the accuracy and precision of the optimized method were preliminarily verified. The established method was also used to evaluate the binding activity similarity between the bevacizumab SBP candidates and bevacizumab RBP to FcγRⅠ. Results:According to the optimized detection method,binding of bevacizumab to FcγRⅠ showed a good dose-response curve with R²>0.99. The method possessed good accuracy and precision. The recovery of the sample with target value of 50%~150% was between 95.2% and 103.7%,and the coefficient of variation was less than 10%. The relative binding activities of bevacizumab SBP candidates to FcγRⅠ were in the range of mean±3SD of that of RBP. Conclusion:ECLI can be used to evaluate the binding activity of bevacizumab to FcγRⅠ,and to analyze the similarity of the binding activity of SBP candidates and RBP to FcγRⅠ. The sensitivity and accuracy of this method are good,which provides a new technique for evaluating the binding activity of antibody to FcγRⅠ. 

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