An enzyme-linked immunosorbent assay to compare the affinity of chemical compounds for 尾-amyloid peptide as a monomer
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- 作者：Chunyi Jiang ; Yu Feng ; Xiaotong Huang ; Yechun Xu ; Yaping Zhang ; Naiming Zhou ; Xu Shen ; Kaixian Chen ; Hualiang Jiang and Dongxiang Liu
- 关键词：Enzyme ; linked immunosorbent assay ; β ; Amyloid peptide ; Binding affinity ; Inhibitor ; Bcl ; xL
- 刊名：Analytical and Bioanalytical Chemistry
- 出版时间：March, 2010
- 出版年：2010
- 期刊代码：8_1618-2642
- 类别：en
- 卷：396
- 期：5
- 页码：1745-1754
- 数据来源：sp
摘要
Aβ1–42 is the proteolytic cleavage product of cleavage of the amyloid precursor protein by β- and γ-secretases. The aggregation of Aβ1–42 plays a causative role in the development of Alzheimer’s disease. To lock Aβ1–42 in a homogenous state, we embedded the Aβ1–42 sequence in an unstructured region of Bcl-xL. Both the N-terminus and the C-terminus of Aβ1–42 were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aβ1–42 but maintained the sequence as a monomer in solution. With Bcl-xL-Aβ42, we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aβ1–42. Bcl-xL-Aβ42 was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aβ1–42 inhibited the interaction between Bcl-xL-Aβ42 and antibody 4G8. The method can not only reproduce the activities of the reported Aβ1–42 inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aβ1–42. Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aβ1–42 and inhibits Aβ1–42 fibril formation. As the structure of Bcl-xL-Aβ42 monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aβ1–42. In addition, the monomeric form of the Aβ1–42 sequence in Bcl-xL-Aβ42 would also facilitate the identification of Aβ1–42 binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here.