Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries
Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries
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DOI：
10.1128/iai.65.9.3743-3752.1997
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发表时间：
1997-09-01
影响因子：
3.1
通讯作者：
Marks, JD
中科院分区：
文献类型：
--
作者：
Amersdorfer, P;Wong, C;Marks, JD
为了制备能够中和A型肉毒杆菌神经毒素（BoNT/A）的抗体，利用噬菌体抗体库在分子水平上表征了小鼠对BoNT/A结合结构域（H-C）的体液免疫应答。用BoNT/A H-C免疫小鼠，收获脾脏，构建了基于免疫球蛋白重链和轻链可变区基因的单链抗体（scFv）噬菌体抗体库，通过在固定的BoNT/A和BoNT/AH-C上选择来分离表达BoNT/A结合scFv的噬菌体。通过酶联免疫吸附试验和DNA测序鉴定了28个BoNT/AH-C结合单链抗体。在BIAcore中使用表面等离子体共振进行的表位作图显示，28个scFv仅结合4个非重叠表位，平衡常数（Kd）范围为7.3 x 10（-8）至1.1 x 10（-9）M。在小鼠半横膈测定中，与毒素对照相比，scFv结合表位1和2显著延长了神经麻痹的时间，分别为1.5倍和2.7倍，与表位3和4结合的scFv未显示针对神经麻痹的保护。结合表位1和表位2的组合对神经麻痹时间具有累加效应，与对照相比增加至3.9倍。结果表明，H上存在两个“生产性"受体结合位点，其导致毒素内化和毒性。用单克隆抗体阻断这两个表位可能提供针对BoNT/A中毒的有效免疫预防或治疗。
To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H-C) was characterized at the molecular level by using phage antibody libraries, Mice were immunized with BoNT/A H-C, the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes, Phage expressing BoNT/A binding scFv were isolated by selection on immobilized BoNT/A and BoNT/A H-C. Twenty-eight unique BoNT/A H-C binding scFv were identified by enzyme-linked immunosorbent assay and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (K-d) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5-and 2.7-fold, respectively, compared to toxin control, scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuroparalysis, which increased to 3.9-fold compared to the control, The results suggest that there are two ''productive'' receptor binding sites on H, which lead to toxin internalization and toxicity, Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.