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首页> 外文期刊>BMC Biotechnology >Estimation of affinities of ligands in mixtures via magnetic recovery of target-ligand complexes and chromatographic analyses: chemometrics and an experimental model
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Estimation of affinities of ligands in mixtures via magnetic recovery of target-ligand complexes and chromatographic analyses: chemometrics and an experimental model

机译：通过目标配体配合物的磁回收和色谱分析估计混合物中配体的亲和力：化学计量学和实验模型

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Background The combinatorial library strategy of using multiple candidate ligands in mixtures as library members is ideal in terms of cost and efficiency, but needs special screening methods to estimate the affinities of candidate ligands in such mixtures. Herein, a new method to screen candidate ligands present in unknown molar quantities in mixtures was investigated. Results The proposed method involves preparing a processed-mixture-for-screening (PMFS) with each mixture sample and an exogenous reference ligand, initiating competitive binding among ligands from the PMFS to a target immobilized on magnetic particles, recovering target-ligand complexes in equilibrium by magnetic force, extracting and concentrating bound ligands, and analyzing ligands in the PMFS and the concentrated extract by chromatography. The relative affinity of each candidate ligand to its reference ligand is estimated via an approximation equation assuming (a) the candidate ligand and its reference ligand bind to the same site(s) on the target, (b) their chromatographic peak areas are over five times their intercepts of linear response but within their linear ranges, (c) their binding ratios are below 10%. These prerequisites are met by optimizing primarily the quantity of the target used and the PMFS composition ratio. The new method was tested using the competitive binding of biotin derivatives from mixtures to streptavidin immobilized on magnetic particles as a model. Each mixture sample containing a limited number of candidate biotin derivatives with moderate differences in their molar quantities were prepared via parallel-combinatorial-synthesis (PCS) without purification, or via the pooling of individual compounds. Some purified biotin derivatives were used as reference ligands. This method showed resistance to variations in chromatographic quantification sensitivity and concentration ratios; optimized conditions to validate the approximation equation could be applied to different mixture samples. Relative affinities of candidate biotin derivatives with unknown molar quantities in each mixture sample were consistent with those estimated by a homogenous method using their purified counterparts as samples. Conclusions This new method is robust and effective for each mixture possessing a limited number of candidate ligands whose molar quantities have moderate differences, and its integration with PCS has promise to routinely practice the mixture-based library strategy.
机译：背景技术就成本和效率而言，在混合物中使用多个候选配体作为库成员的组合文库策略是理想的，但需要特殊的筛选方法来估计此类混合物中候选配体的亲和力。在本文中，研究了一种筛选混合物中摩尔浓度未知的候选配体的新方法。结果拟议的方法涉及用每种混合物样品和外源参考配体制备筛选处理混合物（PMFS），启动配体之间的竞争性结合，使PMFS与固定在磁性颗粒上的靶标结合，并在平衡状态下回收靶标配体复合物通过磁力提取和浓缩结合的配体，并通过色谱法分析PMFS和浓缩提取物中的配体。每个候选配体与其参考配体的相对亲和力通过一个近似方程式估算，假设（a）候选配体及其参考配体与靶标上的相同位点结合，（b）色谱峰面积超过5乘以它们的线性响应截距，但在其线性范围内;（c）它们的结合率低于10％。通过主要优化目标使用量和PMFS组成比，可以满足这些先决条件。以混合物中生物素衍生物与固定在磁性颗粒上的链霉亲和素的竞争结合为模型，测试了该新方法。通过平行组合合成（PCS）无需纯化，或通过合并单个化合物，即可制备出每种混合物样品，其中每种样品均含有限数量的摩尔浓度差异适中的候选生物素衍生物。一些纯化的生物素衍生物用作参考配体。该方法显示出对色谱定量灵敏度和浓度比变化的抵抗力。验证近似方程的最佳条件可以应用于不同的混合物样品。在每个混合物样品中具有未知摩尔量的候选生物素衍生物的相对亲和力与通过均质方法使用其纯化的对应物作为样品估算的亲和力一致。结论该新方法对于每种混合物都具有有限的候选配体，而这些配体的摩尔量具有适度的差异，该方法是可靠且有效的，并且其与PCS的整合已有望常规实施基于混合物的文库策略。

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