A high-affinity monoclonal antibody with functional activity against the 5-hydroxytryptaminergic (5-HT4) receptor
A high-affinity monoclonal antibody with functional activity against the 5-hydroxytryptaminergic (5-HT4) receptor
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DOI：
10.1016/j.bcp.2005.07.005
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发表时间：
2005-10-01
影响因子：
5.8
通讯作者：
Hoebeke, J
中科院分区：
文献类型：
--
作者：
Kamel, R;Eftekhari, P;Hoebeke, J
用与5-HT4受体胞外第二环相对应的合成肽免疫BALB/c小鼠，取脾细胞与SP2/O骨髓瘤细胞融合，制备抗5-HT4受体的单抗。该单抗为IgG2b亚型。该抗体通过免疫印迹和表达5-HT4受体亚型的中国仓鼠卵巢(CHO)细胞的免疫荧光识别5-HT4(G)(h5-HT4(G))受体。抗体的表位图谱表明识别了构象表位，包括第二个细胞外环的N-端和C-端片段。表面等离子体共振动力学实验表明，该抗体与其同源肽具有皮摩尔亲和力。用同样的方法进行的抑制实验证实了这种相互作用的特异性。该抗体在500 PM时竞争性抑制反向激动剂GR 113808的结合，并对表达5-HT4(，)受体的CHO细胞的基础活性表现出反向激动剂作用。该抗体在500和50 PM时降低5-羟色胺的作用，但在下午5时升高5-羟色胺诱导的cAMP水平单抗的双重作用可归因于受体的单价或双价识别。这里描述的抗体是第一个5-HT4受体的高亲和力调节剂的例子。(C)2005 Elsevier Inc.保留所有权利。
Splenocytes from a BALB/c mouse immunised with a synthetic peptide corresponding to the second extracellular loop of the 5-HT4 receptor were fused with SP2/O myeloma cells to produce a monoclonal antibody. The monoclonal antibody was of the IgG2b isotype. The antibody recognised the human 5-HT4(g) (h5-HT4(g)) receptor by immunoblots and by immunofluorescence on chinese hamster ovary (CHO) cells expressing this 5-HT4 receptor isoform. Epitope mapping of the antibody suggested the recognition of a conformational epitope, encompassing the N- and C-terminal fragments of the second extracellular loop. Kinetic experiments using surface plasmon resonance showed that the antibody had a picomolar affinity for its cognate peptide. Inhibition experiments using the same methodology confirmed the specificity of the interaction. The antibody at a concentration of 500 pM competitively inhibited inverse agonist GR 113808 binding and showed an inverse agonist effect on the basal activity of CHO cells expressing the 5-HT4(,) receptor. The antibody decreased the effect of 5-HT at 500 and 50 pM concentrations but it increased 5-HT-induced cAMP levels at 5 pM. The dual effect of the monoclonal antibody could be ascribed to mono- or bivalent recognition of the receptor. The antibody described here is the first example of a high-affinity modulator of the 5-HT4 receptor. (c) 2005 Elsevier Inc. All rights reserved.