BIAcore analysis

The surface matrix of a carboxymethylated sensor chip CMS (Pharmacia Biosensor, Uppsala, Sweden) was activated by injection of 35 pL of 0.05 M N-hydroxy-succinimide (NHS)/0.2 M N-ethyl-N (dimethylaminopropyl)carbodiimide (EDC). The BIAcore IFC forms four parallel flow cells on the sensor chip. Three of them were derivatized with 40 mM cystamin-dihydrochloride/ethanolamine in the ratios 

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Screening in early work sought to identify high affinity of the antibody for the TSA, using a process known as ELISA. This search can now be performed more quantitatively by BIAcore analysis, based on surface plasmon resonance methodology (Lof s and Johnsson, 1990). A subsequent development is the catELISA assay (Tawfik et al., 1993), which searches for product formation and hence the identification of abzymes that can generate product. 

As discussed above, not all of the proteins identified by mode-of-action studies employing the SMART approach are suitable drug targets. However, the identification of enzymes of the glycolytic complex by affinity chromatography and their validation by BIAcore analysis finally led to the working hypothesis that Leflimo-... 

Nieba L., Nieba-Axmann S. E., Rersson A., Hamalainen M., Edebratt F., Hansson A., Lidholm J., Magnusson K., Karlsson A. F., and Rliickthun A., BIAcore analysis of histidine-tagged proteins using a chelating NTA sensor chip, Anal. Biochem., 252, 217-228, 1997. 

Qualitative BIAcore analysis is very easy, fast, and requires only small amounts of protein. It is an ideal analytical tool to answer the question does protein X bind to DNA y or DNA z or both, not more not less Therefore, BIAcore analysis can be used for fast screening of protein mutants [21]. A variation of this theme is studying the influence of other factors on DNA-protein interaction. For example, BIAcore analysis revealed that two protein complexes, checkpoint clamp and its loader, interact with DNA only when mixed together and that ATP/Mg is required for this reaction. Neither checkpoint clamp nor its loader alone can bind DNA, regardless of the presence of ATP/Mg [22],... 

Although fully devoted to the Health Sector, Biacore offers a set of services, which might be of great interest to developmental activities in other industrial areas. Then-focus is on systems for protein interaction analysis, which yield data on the interactions between proteins and other molecules. Protein functionality and the elucidation of reaction mechanisms play an important key role in the development and production of industrial processes. Currently, their products are used in antibody characterization,... 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

Molecular weight of heavy and light chains Peptide mapping Amino acid analysis Intrinsic fluorescence spectroscopy Thermal denaturation monitored by fluorescence Fourier transfrome infrared spectroscopy Binding (e.g., ELISA, BiaCore, etc) Potency (e.g., cell based, ELISA)... 

Very few immunosensors are commercially available. The commercial immunosensors are either the detector or bioanalyzer types. The PZ 106 immunosensor from Universal Sensors Inc. (New Orleans, LA) has been used as a detector to measure antibody-antigen reaction. Ohmicron (Newtown, PA) developed a series of pesticide immuno-bioanalyzers that have been used in field tests. Pharmacia Biosensor USA (Piscataway, NJ) recently introduced BIAcore immunodetection system. A combination of a unique flow injection device and surface plasmon resonance (SPR) detection technique provides a real time analysis. A carboxylmethyldextran layer added to plasmon generating gold film is a hydrophobic, activatable, and flexible polymer that provides high antibody and low non-specific bindings. System demonstration at the Institute of Food Technologists (IFT) 1994 meeting in Atlanta drew attention of food scientists. It should easily be adapted for food protein characterization. 

SPR method is currently well established and used in analysis of affinity interactions. The commercial instruments produced by Biacore AB or Texas Instruments are available in the market. Substantial progress in SPR instrumentation is connected also with the possibility to detect simultaneously the affinity interaction from several surfaces [72], This approach has been proved equally effective for detection of thrombin-DNA aptamer interactions [36]. 

Samsonova, J.V., N.A. Uskova, A.N. Andresyuk, et al. 2004. Biacore biosensor immunoassay for 4-nonylphenols Assay optimization and applicability for shellfish analysis. Chemosphere 57 975-985. 

Myska, D.G. (2000) Kinetic, Equilibrium and Thermodynamic Analysis of Macromolecular Interactions with BIACORE, Methods Enzymol. 323, 325-340. 

Another technique used for the analysis of receptor-ligand interaction is surface plasmon resonance (SPR), with its first commercially available application in the BIAcore instruments [44] (Fig. 5.8). Like PCS, it allows the determination of kinetics by monitoring the association and dissociation of a receptor-ligand complex in real time. The interaction partners do not necessarily have to be labeled, which is an advantage of the technique. The principle of SPR measurements is based on an optical phenomenon. The core unit in this technique is a sensor chip consisting of a thin gold film with a modified surface attached on one side. One reactant is attached to the modified sensor surface, whereas the other reaction partner flows past this surface in solution. When the two interaction partners form a com-... 

Papalia GA, Giannetti AM, Arora N, Myszka DG (2008) Thermodynamic characterization of pyrazole and azaindole derivatives binding to p38 mitogen-activated protein kinase using Biacore T100 technology and van t Hoff analysis. Anal Biochem 383 255-264... 

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