生物分子相互作用分析技术应用实例（一）——配体垂钓和抗体测定

BIA技术(biomolecular interaction analysis)——即生物分子相互作用分析技术的应用范围相当广泛.先介绍利用BIAcore分离得到了ECK-酪氨酸激酶受体的蛋白配体.另一应用实例是利用BIAcore直接从杂交瘤细胞上清液中测定单克隆抗体的活性、亲和力和动态常数.     

生物分子相互作用分析技术应用实例（二）——多聚物和转录蛋白的研究

BIA技术(biomolecular interaction analysis)即生物分子相互作用分析技术的应用范围相当广泛.这里介绍利用BIAcore研究信号传导中各蛋白质之间的相互作用及多聚物的形成及机理以及转录调节蛋白与启动子（DNA）的研究.     

SPR技术分析生物分子相互作用的研究方法

生物分子的活性功能是通过分子之间的相互作用来实现的,了解这种相互作用的关系时生命科学的研究及揭示生命发生发展的基本机制具有着重要的意义.基于表面等离子共振(SPR)的分析分子相互作用(BIA)的技术是新型的生物传感技术,其无需标、能实时跟踪检测生物分子间结合、解离的整个过程,通过分析传感图谱获取分子相互作用的模式和动力学常数等方面的信息.SPR是研究生物分子相互作用的强有力工具,SPR技术已被广泛应用于生命科学领域的研究,并且显示出广阔的应用前景.概述了SPR技术原理、分析方法及其评述了其存在的问题.     

分子动态模拟及其在生物大分子研究中的应用

生物分子动念模拟技术是运用计算机对生物大分子的结构、功能、质子运动轨迹以及生物分子间的相互作用进行预测,是研究生物分子结构和功能的重要手段.该文介绍分子动态技术的原理及其在生命科学研究中的应用和研究进展,分析目前存在的问题,并提出对未来工作的展望.     

太赫兹(THz)光谱在生物大分子研究中的应用

太赫兹(THz)辐射是一种新型的远红外相干辐射源,近年来,在生物大分子研究中得到了广泛的应用,特别是在生物分子的结构和动力学特性等方面有着巨大的应用潜力.结合THz光谱的特点,介绍了利用THz光谱对蛋白质、糖类及DNA等生物大分子的探索研究,以及THz技术在测定水环境与生物分子相互作用等方面的应用.探讨了该技术在生物学领域应用中有待解决的问题及发展前景.     

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。     

分子信标在DNA生物传感器中的应用

DNA传感器是基于DNA分子相互作用原理设计而成的一种新型的检测技术,具有快速,简单等优点,在基因分析及其他应用领域已显示出越来越重要的价值.分子信标是一种具有发卡式结构的寡核苷酸,由于其能够很好地识别单碱基错配序列,基于发卡式DNA的传感器较传统的单链DNA传感器有更好的检测特异性,目前得到广泛的研究.本文介绍了DNA生物传感器及分子信标的有关原理,并着重介绍了发卡式DNA的结构及其在DNA生物传感器中的应用.     

分子印迹技术用于生物大分子的识别

分子印迹技术是一种人工合成具有分子识别功能的介质的一种新技术,近年来在许多领域都得到很大的发展。本文介绍了分子印迹技术的发展现状,尤其对生物大分子的分子印迹技术进行了详细论述,对生物大分子印迹采用的功能单体,印迹分子的种类,印迹的方法,印迹的机理,存在的问题和应用的前景等分别进行了讨论。     

生物分子相互作分析技术应用实例（一）：配体垂钓和抗体测定

ＢＩＡ技术－即生物分子相经作用分析技术的应用范围相当广泛,先介绍利用ＢＩＡｃｏｒｅ分离得到ＥＣＫ－酪氨酸激酶受体的蛋白配体。另一应用实例是利用ＢＩＡｃｏｒｅ直接从杂交瘤细胞上清液中测定单克隆抗体的活性、亲和力和动态常数。     

生物分子相互作用分析法测定甜菜组织中的钙调素

以甜菜为实验材料,经甜菜坏死黄脉病毒（beet necrotic yellow vein virus, BNYVV）侵染后,用生物分子相互作用分析（biomolecular interaction analysis,BIA）技术测定植物组织中钙调素（calmodutin,CaM）含量,分析在BNYVV侵染下植物组织中CaM的变化。该方法可以实时检测生物分子之间的相互作用,受杂质影响小,可简化样品的前处理,能够快速高通量分析大量的蛋白质样品,灵敏度高,准确性好。     

Advances in surface plasmon resonance biomolecular interaction analysis mass spectrometry (BIA/MS)

Ongoing, worldwide efforts in genomic and protein sequencing, and the ability to readily access corresponding sequence databases, have emphatically driven the development of high‐performance bioanalytical instrumentation capable of characterizing proteins and protein–ligand interactions with great accuracy, speed and sensitivity. Two such analytical techniques have arisen over the past decade to play key roles in the characterization of proteins: surface plasmon resonance biomolecular interaction analysis (SPR‐BIA) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF). SPR‐BIA is used in the real‐time investigation of biomolecular recognition events, and is thereby capable of providing details on the association and dissociation kinetics involved in the interaction, information ultimately leading to the determination of dissociation constants involved in the event. MALDI‐TOF is used in the structural characterization, identification and sensitive detection of biomolecules. Although the two techniques have found many independent uses in bioanalytical chemistry, the combination of the two, to form biomolecular interaction analysis mass spectrometry (BIA/MS), enables a technique of analytical capabilities greater than those of the component parts. Reviewed here are issues of concern critical to maintaining high‐levels of performance throughout the multiplexed analysis, as well as examples illustrating the potential analytical capabilities of BIA/MS. Copyright © 1999 John Wiley & Sons, Ltd.     

Innovative analytical system for screening on lectins

Lectins are proteins or glycoproteins from plants, animals or microorganisms, which typically bind specifically to sugar residues, e.g., those located in cell walls or membranes. This reaction might change the physiology of the cell wall and influences the metabolism inside the cell. Some lectins of plants stimulate the immune system by unspecific activation of T-cells or influence cell division; others cause agglutination of cells (e.g., erythrocytes) and are therefore from therapeutic interest. In a new approach, biomolecular interaction analysis (BIA) was utilized for a screening program on lectins. The BIA has been done by a new interferometric biosensor based on spectral-phase interference (SPI). The system can be used either for characterisation of lectin binding domains or for a screening on lectins obtained from natural sources. Several lectin binding surfaces on the basis of SPI have been established.     

SPR studies of carbohydrate-lectin interactions as useful tool for screening on lectin sources

Lectins are proteins or glycoproteins from plants, animals or microorganisms, which typically bind specifically to sugar residues, e.g., located in cell walls or membranes. This reaction might change the physiology of the cell wall and influences the metabolism inside the cell. Some lectins of plants stimulate the immune system by unspecific activation of T-cells or influence cell division; others cause agglutination of cells (e.g., erythrocytes) and are therefore from therapeutic interest.

In a new approach, biomolecular interaction analysis (BIA) was utilised for a screening program on lectins. The BIA has been done by surface plasmon resonance (SPR). The system can be used either for characterisation of lectin-binding domains or for a screening on lectins from natural sources. Several lectin-binding surfaces on the basis of SPR have been established.     

Recent progress in biomolecular engineering

During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.     

The N-terminus of collagenase MMP-8 determines superactivity and inhibition: a relation of structure and function analyzed by biomolecular interaction analysis.

Tissue inhibitors of metalloproteinases (TIMPs) are the physiological, specific inhibitors of matrix metalloproteinases (MMPs) forming tight, noncovalent complexes. Therefore they control the proteolytic activity of MMPs toward the extracellular matrix. To analyze the inhibition of the "activated" and "superactivated" variants of human neutrophil collagenase (MMP-8) by TIMP-2, we determined complex dissociation constants using biomolecular interaction analysis (BIA). As it is known that the association rate constants can exceed the limits of the BIA instruments, the biomolecular interaction analysis was used to examine the equlibrium situation. The dissociation constants were determined by fitting the parameters of the mathematical term for the binding of collagenase onto the TIMP-coupled sensor chip surface to the saturation curve derived from individual sensorgrams. The resulting values are in the nanomolar range and correlate with the results of fluorescence kinetics. These data reveal that TIMP-2 (the recombinant inhibitory domain of human TIMP-2 and bovine TIMP-2 isolated from seminal plama) is a better inhibitor of the activated neutrophil collagenase than of the superactivated variant (the recombinant catalytic domain of human MMP-8). It has been demonstrated by X-ray analysis that the N-terminal heptapeptide only of superactivated MMP-8 is attached by a salt bridge and hydrophobic interaction to the C-terminal helix. Because these interactions have to be disrupted in the complex formation with TIMP we assume that the activated variant enables higher flexibility and a tighter induced fit in the complex formation. Therefore superactivation of MMP-8 correlates with weaker inhibition by TIMP-2.     

Delineating protein-protein interactions via biomolecular interaction analysis-mass spectrometry

The utility of biomolecular interaction analysis-mass spectrometry (BIA/MS) in screening for protein-protein interactions was explored in this work. Experiments were performed in which proteins served as ligands for screening of possible interactions with other proteins from human plasma and urine. The proteins utilized were beta-2-microglobulin, cystatin C (cysC), retinol binding protein (RBP), transthyretin (TTR), alpha-1-microglobulin, C-reactive protein, transferrin and papain. The immobilization of functionally active proteins was confirmed via interactions with antibodies to the corresponding proteins. Various dilutions of human urine and plasma were injected over the protein-derivatized surfaces. It was observed that the urine injections generally yielded smaller SPR responses than those observed after the plasma injections. The BIA/MS experiments did not reveal novel protein-protein interactions, although several established interactions (such as those between RBP and TTR, and cysC and papain) were validated. Few protein ligand deficiencies (such as truncations) leading to false negative and false positive BIA/MS results were also discovered.     

Detection of Staphylococcal enterotoxin B via biomolecular interaction analysis mass spectrometry

Detection of Staphylococcus enterotoxin B (SEB) by biomolecular interaction analysis mass spectrometry (BIA/MS) is presented in this work. The BIA/MS experiments were based on a surface plasmon resonance (SPR) MS immunoassay that detects affinity-captured SEB both via SPR and by means of exact and direct mass measurement by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Experiments were performed with standard samples and food samples to assess the BIA/MS limit of detection for SEB and to set the experimental parameters for proper quantitation. Single and double SPR referencing was performed to accurately estimate the amount of the bound toxin. Reproducible detection of 1 ng of SEB per ml, corresponding to affinity capture and MS analysis of approximately 500 amol of SEB, was readily achieved from both the standard and mushroom samples. A certain amount of SEB degradation was indicated by the signals in the mass spectra. The combination of MS with SPR-based methods of detection creates a unique approach capable of quantifying and qualitatively analyzing protein toxins from pathogenic organisms.     

Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays

The feasibility of multi-affinity ligand surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS) was explored in this work. Multi-protein affinity surfaces were constructed by utilizing antibodies to beta-2-microglobulin, cystatin C, retinol binding protein, transthyretin, serum amyloid P and C-reactive protein. In the initial experiments, all six antibodies were immobilized on a single site (flow cell) on the sensor chip surface, followed by verification of the surface activity via separate injections of purified proteins. After an injection of diluted human plasma aliquot over the antibodies-derivatized surfaces, and subsequent MALDI-TOF MS analysis, signals representing five out of the six targeted proteins were observed in the mass spectra. Further, to avoid the complexity of the spectra, the six proteins were divided into two groups (according to their molecular weight) and immobilized on two separate surfaces on a single sensor chip, followed by an injection of human plasma aliquot. The resulting mass spectra showed signals from all proteins. Also, the convolution resulting from the multiply charged ion species was eliminated. The ability to create such multi-affinity surfaces indicates that smaller-size ligand areas/spots can be employed in the BIA/MS protein interaction screening experiments, and opens up the possibilities for construction of novel multi-arrayed SPR-MS platforms and methods for high-throughput parallel protein interaction investigations.