SPR生物传感器及其应用进展

基于表面等离子体共振 (SPR)技术的光学生物传感器是进行生物分子相互作用分析的一种先进手段。与传统的超速离心、荧光法等相比 ,它具有实时检测、无需标记、耗样最少等特点 ,在药物筛选、临床诊断、食物及环境监控和膜生物学等领域中的新兴应用日益扩大 ,并且已成为生命科学和制药研究的一种标准的生物物理学工具。综述了近几年国际上生物传感器的应用进展情况 ,并简要展望了该技术的发展和应用前景     

Kinetic parameters of monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 on coagulation factor VIII and their influence on factor VIII activity

The murine monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 specifically bind and inhibit the procoagulant activity of human coagulation factor VIII (FVIII). They are frequently used as a model of inhibitors which are raised against injected FVIII in about 25% of hemophiliacs as a serious side effect of substitution therapy. However, binding kinetics of the interaction of these antibodies with FVIII and their influence on FVIII activity (inhibition) have not yet been examined systematically. For this, we examined association and dissociation of protein:antibody interaction using surface plasmon resonance (SPR) and determined their ability to inhibit the FVIII activity in a one‐stage and a two‐stage assay. SPR‐analysis revealed that the equilibrium dissociation constants (K_D) of ESH8 and ESH4 are low and in a similar range (ESH8: K_D(ESH8) = 0.542 nM; ESH4: K_D(ESH4) = 0.761 nM). A 5.7 times higher K_D than for ESH4 was observed for ESH2 (4.33 nM), whereas ESH5 showed the highest K_D of 28.8 nM. In accordance with the lowest K_D, ESH8, and ESH4 reduced FVIII activity of normal human plasma almost completely in a one‐stage clot inhibition assay (ESH8: 91.9%; ESH4: 90.1%). However, ESH8 inhibited FVIII activity more efficiently as only 1.0 µg/ml ESH8 was sufficient to obtain maximum inhibition compared to up to 600 µg/ml of ESH4. Despite its attenuated K_D, ESH2 inhibits FVIII:C still efficiently, reducing 61.3% of FVIII activity at a concentration of 9 µg/ml in the one‐stage clotting assay. However, a discrepancy of inhibitory efficiency was found depending on the method used to measure FVIII activity. These effects seem to be mainly caused by differences of activation time of FVIII during both FVIII activity assays. The systematic assessment of these results should support FVIII interaction studies, and can provide data to rationally test peptides/mimotopes to remove or neutralize inhibitors of FVIII activity. Copyright © 2009 John Wiley & Sons, Ltd.     

SPR-MS in functional proteomics

The mapping of protein networks and the establishment of thefunctional relationships between expressed proteins and theireffects on cellular processes represents a great challenge forfunctional or interaction proteomics. The combination of surfaceplasmon resonance (SPR)-based technology with mass spectrometry(MS) has created a unique analytical tool for functional proteomicsinvestigations. Proteins are affinity purified, quantified andcharacterised in terms of their interactions, while the massspectrometer identifies and structurally characterises the biomolecules.Recent developments have led to a closer integration of thesekey technologies, providing a combined approach which enablesidentification of proteins selected on the basis of their functionalbinding criteria. In addition to a historical overview of thisfield, some recent detailed examples of combined SPR-MS approacheswill be reviewed in a number of key application areas, includingligand fishing, peptide sequence and post-translational modificationanalysis by SPR-MS/MS and enzyme inhibitor screening.      

Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)

GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.     

基于SPR生物传感器的免疫学检测

利用一种全新的生物大分子相互作用检测仪表面等离子激元共振（SPR）生物传感器,对乙肝表面抗原,抗体,破伤风类毒素,抗体等生物制品进行生物特异性相互作用分析（BIA）,并对其在免疫学检测上的特征进行了探讨。     

蛋白质组学研究中的一种新方法——配体垂钓

基于表面等离子共振技术的配体垂钓技术能在蛋白质组水平上研究蛋白质的相互作用与功能,提供控制细胞功能的新靶标．其通过将受体固定在芯片表面,当被检测样品流过芯片表面时,配体与受体相结合, 实现俘获未知的相互作用的伙伴蛋白或复合体,并结合质谱技术鉴定出未知蛋白及其序列．     

苦绳（Dregea sinensis）中多氧化孕烷糖苷与免疫蛋白相互作用的研究

目的 基于化学结构特点研究植物苦绳（Dregea sinensis）中多氧孕烷糖苷的生物活性,以及化合物分子与靶蛋白分子间的作用机理。方法 本研究采用分子对接和表面等离子体共振方法,对191个多氧化孕烷糖苷类成分（>800 u）进行了免疫活性及其与免疫蛋白的动力学评价。结果 通过分子对接方法筛选出7个配体分子（6、18、23、30、78、79和80）和3个免疫相关蛋白（IL-2Rα、TLR4和TNF-α）。研究表明,化合物30和78在SPR实验中与靶蛋白IL-2Rα和TLR4具有显著的结合趋势,与IL-2Rα的结合常数K_D分别为2.41×10^-6和2.14×10^-6 mol/L,与TLR4则分别为1.96×10^-5和5.60×10^-6 mol/L。分子动力学研究进一步表征SPR阳性分子6、18、30、78和80与靶蛋白之间的相互作用基团。结论 研究揭示多氧化孕烷糖苷类成分可以通过形成氢键和Pi-Pi相互作用与靶蛋白结合。研究内容对快速评价多氧化孕烷糖苷类成分的活性具有重要意义,为揭示低丰度药效物质的潜在作用机制进行了有益探索。     

Kinetic Analysis of Analyte Binding by Optical Biosensors: Hydrodynamic Penetration of the Analyte Flow into the Polymer Matrix Reduces the Influence of Mass Transport

I examined the penetration of the hydrodynamic flow into a polymer matrix immobilized by grafting to a surface, such as used in optical biosensors designed to measure binding reactions in real time. I show that the flow penetrates with an appreciable velocity into a region located at the tip of such a polymer brush, corresponding to about 10 to 15% of the total mass of the grafted polymer. Furthermore, under the conditions recommended for kinetic measurements, the concentrations of both polymer and immobilized ligands are low in these regions of the matrix, where crowding effects are negligible. Under such conditions, the hydrodynamic flow penetrating into the dextran matrix flow will bring the analytes close to their targets, thus considerably reducing transport problems.     

The investigation of recognition interaction between phenylboronate monolayer and glycated hemoglobin using surface plasmon resonance

Glycated hemoglobin (HbA1c) is formed by a nonenzymatic reaction of glucose with the N-terminal valine of adult hemoglobin's beta-chain. The amount of HbA1c reflects the average concentration of glucose variation level over the preceding 2 to 3 months. Because the boronate has antibody mimicking for HbA1c, often it is used to detect HbA1c. However, factors such as the ratio of the phenylboronic acid derivatives and diol composition, the pH of the solution, and the stereostructure of phenylboronic acid derivatives could influence the interactions between phenylboronic acid derivatives and diol composition. In this study, the factors were evaluated using surface plasmon resonance (SPR). The results show that pH value is an important factor affecting HbA1c and phenylboronic acid to form the complex and Lewis bases. This could change the stereostructure of phenylboronic acid to form B(OH)(3) for binding with saccharine easily. In addition, linear response appeared in HbA1c in the range of 0.43 to 3.49 mug/ml, and the detection limit was 0.01 microg/ml. The results also demonstrated that an SPR biosensor can be used as a sensitive technique for improving the accuracy and correctness of HbA1c measurement.     

Study of the inhibitory effect of fatty acids on the interaction between DNA and polymerase β

The binding of human DNA polymerase β (pol β) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol β inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol β inhibitors. In the interaction between pol β and DNA, pol β could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol β inhibitors prevented the binding of pol β to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol β to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol β-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol β to DNA was mainly due to 7-fold increase in the dissociation rate. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 1000–1006. These authors contributed equally.