生物薄膜干涉技术和ELISA法检测抗体药物免疫原性的方法学比较

采用生物薄膜干涉技术(BLI)开发快速检测治疗性单抗(抗EGFR单抗,Cetuximab)的抗抗体(ADAs)的检测方法。本研究利用ForteBio公司开发的Octet系统,在光纤制成的生物传感器底端覆盖生物分子相容层,偶联配体,形成生物膜层。当分析物结合传感器底端偶联的配体时,生物膜层厚度增加,反射光干涉光谱曲线产生可测量的漂移,从而可以实现对分子间相互作用的实时测量,同时以传统的抗体桥ELISA法作为平行对照。SA(Streptavidin)传感器结合生物素化的单抗,用免疫原性试剂孵育血清样品后,利用传感器检测血清样品的ADA。分别应用BLI和ELISA两种方法确定cutpoint,并进行剂量反应曲线的分析。研究结果表明BLI快速分析法具有同ELISA法极其相似的免疫原性检测行为,均能准确检测出筛选分析中的20例正常人血清的基线反应值。同时,BLI法在竞争抑制实验中体现出了很高的特异性。与ELISA相比,BLI技术用于治疗性抗体的临床免疫原性分析,更加省时,快捷,准确,而且可避免低亲和力ADA易受洗板干扰的弊端。因此,BLI技术可能成为抗体药物免疫原性检测的一种新的有效方法。

Methodological comparison of ELISA and BLI for the quantitation of anti-drug antibodies in human serum

We assessed the utility of the FortéBio Octet system for detection of anti-drug antibodies(ADAs) against therapeutic monoclonal antibody Cetuximab(an anti-EGFR monoclonal antibody).The Fort Bio Octet@system is based on a proprietary technique called BioLayer Interferometry(BLI),which is an optical technique that analyzes the interference pattern of white light reflected from two surfaces: the layer of immobilized protein on the biosensor tip,and the internal reference layer.The binding between a ligand captured on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip,which results in a wavelength shift,and a change in thickness of the biological layer.It can be measured in real-time.And the traditional ELISA was taken as the control.The binding between biotinylated monoclonal Abs and SA(streptavidin) Biosensor was construct in the experiment,and detect the ADAs from blood serum after incubation.The paralleled experiment was performed by ELISA,which used the same agentia and samples.Screening the cut point and sigmoidal dose-response curve equation with Fort Bio Octet@system and ELISA were performed.The results indicated that Fort Bio Octet@system had the same ability to detect immunogenicity of mAbs as did the ELISA immunoassay.Both of the two screening systems can detect the cut point from twenty normal human serum exactly.And the sensitivity of Fort Bio Octet@system was satisfactory in the detection of drug interference.The BLI assay system showed more convenient and accuratissime than the other methods we used before.In addition,there is no need to incorporate wash steps during which low-affinity Abs may dissociate and therefore failed to be detected.Accordingly,Fort Bio Octet@system has potential for detectation of ADAs with different affinity by providing multiple protocols saving any plate washing steps.