HIGH-RESOLUTION FUNCTIONAL-ANALYSIS OF ANTIBODY-ANTIGEN INTERACTIONS
HIGH-RESOLUTION FUNCTIONAL-ANALYSIS OF ANTIBODY-ANTIGEN INTERACTIONS
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DOI：
10.1016/0022-2836(92)90636-x
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发表时间：
1992-08-05
影响因子：
5.6
通讯作者：
WELLS, JA
中科院分区：
文献类型：
--
作者：
JIN, L;FENDLY, BM;WELLS, JA
采用综合突变分析法分析了对结合21种不同抗hGH小鼠单克隆抗体（MAb）至关重要的人生长激素（hGH）侧链，这些抗体的50%结合当量浓度（EC50）范围为~107至3 × 1010 m-1。使用同源物和丙氨酸扫描诱变与机器人辅助的酶联免疫吸附测定的组合来为每个单克隆抗体创建高分辨率的"功能表位"。每个功能表位定位于hGH的至少两个多肽片段，这些多肽片段在折叠的蛋白质中靠近在一起以形成补丁。尽管这些斑块有时重叠，但每个斑块都不同，表明没有两种MAb与hGH的结合相同。单克隆抗体结合到环和螺旋中的决定簇上，这些决定簇通常最容易被9 μ m半径的探针所接近。只有少数侧链支配每个功能表位，并且这些侧链倾向于Arg> Pro> Glu~Asp~Phe~Ile（未测试Ala、Cys和Trp）。我们的研究表明，大多数的hGH的可及表面在小鼠中是潜在的抗原性，并表明功能性表位是由较少的侧链比可能在接触表位占主导地位。
A comprehensive mutational analysis was used to analyze the side-chains on human growth hormone (hGH) important for binding 21 different anti-hGH mouse monoclonal antibodies (MAbs) whose equivalent concentrations for 50% binding (EC50) ranged from ~107to 3 × 1010m-1. A combination of homolog- and alanine-scanning mutagenesis coupled with a robot-aided enzyme-linked immunosorbant assay were used to create high resolution “functional epitopes” for each MAb. Every functional epitope mapped to at least two polypeptide segments of hGH that were close together in the folded protein to form a patch. Although these patches sometimes overlapped, each was different indicating no two MAbs bound identically to hGH. The MAbs bound to determinants in loops and helices that were generally most accessible to a 9 Å radius probe. Only a few side-chains dominated each functional epitope and these tended to be Arg > Pro > Glu~Asp~Phe~Ile (Ala, Cys and Trp were not tested). Our studies indicate that most of the accessible surface of hGH is potentially antigenic in the mouse and suggest that functional epitopes are dominated by fewer side-chains than may be in the contact epitope.