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首页> 外文期刊>Frontiers in Molecular Neuroscience >Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders
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Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

机译：人单克隆抗体的设计和表征，可调节与耳聋和皮肤疾病有关的突变型连接蛋白26半通道

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Figure 1. Selection of antibodies in phage. (A) Optical density readout from a plate reader vs. panning round for a negative control antigen (BSA, red) and the pepEC1.1 bait peptide (blue); only pepEC1.1 shows a signal increase after two rounds of enrichment. (B) Phage ELISA results showing optical density readout from the plate reader vs. colony number, ranked by increasing signal intensity. Positive clones (pepEC1.1/BSA ratio > 2) were sequenced and a phylogenetic tree based on the heavy-chain CDR3 sequences was constructed (inset; clones are labeled by the protocol used to obtain them and the clone number of each protocol). (C,D) After purification, the binding specificity of the candidate antibody, abEC1.1, was tested by ELISA, against pepEC1.1 (C), and by western blot, against Cx26 proteins (D), fused with a 10*His and flag tag (29.4 kDa) and purified from a Sf9 expression system (left), or from proteins obtained from HeLa DH transfectants expressing the 52 kDa chimeric protein Cx26-Venus (right). The negative control peptide in (C) corresponds to residues 172 to 184 of Cx26 (sequence: AWPCPNTVDCFVSR), which form part of EC2.
机译：图1.噬菌体中抗体的选择。 （A）从板读取器中的光密度读数对阴性对照抗原（BSA，红色）和pepEC1.1诱饵肽（蓝色）的淘选回合;经过两轮富集后，只有pepEC1.1显示信号增加。 （B）噬菌体ELISA结果显示从读板器读取的光密度与菌落数的关系，按信号强度增加排序。对阳性克隆（pepEC1.1 / BSA比> 2）进行测序，并构建基于重链CDR3序列的系统进化树（插图;通过用于获得克隆的方案标记克隆，以及每个方案的克隆编号）。 （C，D）纯化后，通过ELISA检测候选抗体abEC1.1对pepEC1.1的结合特异性（C），并通过Western blot检测与10x融合的Cx26蛋白（D）的结合特异性His和flag标签（29.4 kDa）并从Sf9表达系统（左），或从表达52 kDa嵌合蛋白Cx26-Venus的HeLa DH转染子中获得的蛋白中纯化（右）。 （C）中的阴性对照肽对应于Cx26的残基172至184（序列：AWPCPNTVDCFVSR），其形成EC2的一部分。

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