基于ELISA与NMR方法的降钙素基因相关肽抗体的抗原表位鉴定

Epitope identification of calcitonin gene-related peptide antibodies based on ELISA and NMR methods

目的建立基于间接ELISA法的肽扫描与核磁共振光谱(nuclear magnetic resonance,NMR)技术相结合的方法,鉴定降钙素基因相关肽(calcitonin gene-related peptide,CGRP)抗体的抗原表位。方法以CGRP各截短片段为包被抗原,间接ELISA法测定2种抗体(新CGRP抗体和对照抗体)与各抗原的结合活性,根据四参数曲线的EC50值分析线性抗原表位。NMR技术采集CGRP的二维氢-氮相关(2D1H-15N HSQC)谱,利用抗体对核磁信号的扰动,分析抗体与CGRP上精氨酸的结合。液相色谱-质谱联用(liquid chromatography-mass spectrum,LC-MS)和NMR技术检测精氨酸专属性修饰,通过修饰速率的比对,获得CGRP上2个精氨酸的信号指认。结果2种抗体与CGRP(1-37)、CGRP(19-37)、CGRP(25-37)均存在量效关系,且符合四参数方程,但与CGRP(1-18)、CGRP(19-24)、无C-端酰胺的CGRP(25-37)均无量效关系;确定2种抗体的线性抗原表位均位于CGRP的C-末端。CGRP与对照抗体结合后,2D1H-15N HSQC谱中精氨酸ε-NH的信号消失;CGRP与新抗体结合后,精氨酸信号依然存在;确定抗原上的精氨酸R11和R18可与对照抗体结合,但不与新抗体结合。通过修饰速率的排序比对获得精氨酸ε-NH信号归属,即信号A对应R11,信号B对应R18。结论本研究采用ELISA法与NMR技术相结合鉴定了新CGRP抗体及对照抗体的线性表位和构象表位,为设计新的靶向CGRP抗体药物提供了理论依据。

Objective To develop an indirect ELISA-based peptide scanning method combined with nuclear magnetic resonance(NMR)technique for the epitope identification of calcitonin gene-related peptide(CGRP)antibodies.Methods The antigen binding activities of two antibodies(new CGRP antibody and control antibody)were determined by indirect ELISA using each truncated CGRP fragment as coating antigen,and the linear epitope was analyzed according to the EC_(50)value of four-parameter curve.Two-dimensional hydrogen-nitrogen correlation(2D1H-15N HSQC)spectrum of CGRP were acquired by NMR technique,and the binding of antibodies to the arginine of CGRP were analyzed through the disturbance of the antibodies to CGRP signals.Specific arginine modifications were detected by liquid chromatography-mass spectrum(LCMS)and NMR technique,and two arginine resonances were assigned on CGRP by correlating the rank order of the modification rate.ResultsThe antigen binding activities of two antibodies with CGRP(1-37),CGRP(19-37)and CGRP(25-37)showed dose-response relationships,and were fitted with four-parameter equation.However,there were no significant antigen binding with CGRP(1-18),CGRP(19-24)and CGRP(25-37)without C-terminal amide.The linear epitopes of both antibodies were located at the C-terminal of CGRP.The resonances of arginineε-NH in 2D1H-15N HSQC spectrum disappeared in the presence of the control antibody;and the resonances appeared in the presence of the new antibody.The arginine R11 and R18 of CGRP could bind to the control antibody,but not to the new antibody.The NMR assignment for the arginine resonances were made by correlating the relative ranking of the modification rate where signals A and B arose from R11ε-NH and R18ε-NH respectively.ConclusionIn this study,the linear and conformational epitopes of new CGRP antibody and control antibody were identified based on the methods of ELISA and NMR,which may provide a theoretical basis for the design of the candidate therapeutic CGRP antibodies.