表面等离子共振技术分析抗A型肉毒毒素及抗破伤风毒素的单克隆抗体表位

Analysis of epitope of monoclonal antibodies against botulinum toxin and tetanus toxin by surface plasmon resonance technique

目的通过表面等离子共振(surface plasmon resonance,SPR)技术对抗A型肉毒毒素(botulinum toxin A,BONT/A)及抗破伤风毒素(tetanus toxin,TT)的单克隆抗体进行抗原表位分析。方法采用CM5芯片,通过氨基偶联抗BONT/A诊断血清,经Biacore T200捕获BONT/A,再进样抗BONT/A单克隆抗体A-13、A-14、C-5,通过响应值(response unit,RU)的变化来判断是否针对同一表位,并通过动物实验对抗BONT/A单克隆抗体进行生物学活性验证;采用CM5芯片,氨基偶联TT,经Biacore T200分别进样人源化抗TT单克隆抗体G2及G6,通过RU的变化来判断是否针对同一表位。结果抗BONT/A的3株单克隆抗体中,单克隆抗体A-13连续3次进样后,A-14继续进样的RU略降低,C-5进样的RU明显升高,A-13+A-14、A-13+C-5、A-14+C-5组合进样的相加指数(AI)分别为35. 8%、98. 2%、96. 0%。BONT/A剂量为2 000 LD_(50)时,A13+C5及A14+C5组小鼠在96 h内均死亡1只,A13+A14组小鼠24 h内全部死亡。G2连续3针进样后,G6进样的RU升高至825. 6;G6连续3针进样后,G2进样的RU升高至3 689. 2。结论 A-13及A-14针对同一抗原表位,与不同表位C-5之间抗体存在协同效应;抗TT的2株单克隆抗体针对不同抗原表位。

Objective To analyze the antigenic epitopes of monoclonal antibodies(McAbs)against botulinum toxin type A(BONT/A)and tetanus toxin(TT)by surface plasmon resonance(SPR)technique. Methods Biacore T200 and CM5 chip were employed for the experiment. Anti-BONT/A polyclonal antibody was linked covalently to dextran matrix by amine coupling. Then,the BONT/A was captured by Biacore T200. McAbs A-13,A-14 and C-5 against BONT/A were bound to BONT/A epitopes,of which the response units(RUs)were measured to determine whether the McAbs targeted to the same epitope,and the biological activities were verified in mice. Tetanus toxoid was linked covalently to dextran matrix by amine coupling,and anti-TT McAbs G2 and G6 were captured by Biacore T200 in turn,of which the RUs were measured to determine whether the McAbs targeted to the same epitope. Results After McAb A-13 was loaded for 3 times,the RU of A-14 decreased slightly,while that of C-5 increased significantly,and the additive indexes(AIs)of A-13 + A-14,A-13 + C-5 and A-14 + C-5 were 35. 8%,98. 2% and 96. 0% respectively. When the dosage of BONT/A was 2 000 LD50,one mouse in A13 + C5 group and one in A14 + C5 group died within 96 h,while all the mice in A13 + A14 group died within 24 h,after injection. After McAb G2 was loaded for 3 times,the RU of G6 increased to825. 6. However,after G6 was loaded for 3 times,the RU of G2 increased to 3 689. 2. Conclusion McAbs A-13 and A-14 targeted to the same epitope,which showed synergistic effect with C-5 targeting different epitope. However,the two McAbs against TT targeted to different antigenic epitopes.