Biacore

BIAcore AB is the main supplier of automated SPR detection systems (BIAcore 3000, TlOO, and more, see www.BIAcore.com) other suppliers are Texas Instruments (Spreeta and TISPR-I) and Nippon Laser and Electronics (SPR-670). In 2004, 88% of publications that reported the use of SPR indicated that the data were generated with BIAcore instruments [4]. Therefore, in the following the focus will be on BIAcore instruments and BIAcore nomenclature will be used. 

Since the first commercially available Biacore system (Biacore AB, Uppsala, Sweden) was introduced back in 1990, the technology has advanced to become a standard method in studying biomolecular interactions. This Chapter describes the function and practical utilization of this technology, discusses typical user questions and offers suggestions for problem solving. Studies of oligosaccharide interactions are presented, while the whole application range includes all kinds of biomolecules. 

Since applications are continually optimized and new reagents and consumables are being developed, detailed protocols will not be given. The reader is referred to updated protocols provided by the supplier, to the hterature cited in the text, and to the current reference list on the Internet under http //www.biacore.com. 

1 Real-time Analysis by Surface Plasmon Resonance 

Inside the instrument, the chip matrix forms a roof of small flow cells. In current instrument configurations, each sensor chip serves two or four of these flow channels, giving two or four separate measuring surfaces. The integrated flow system provides a continuous flow of buffer or samples over the chip surface. Sample injections and measurements run automatically only specimen loading to a sample loop can be 

Because detection directly reacts to substance accumulation, the signal depends both on the molecular weight and concentration of the injected partner, and on the number of binding sites present on the matrix. However, detectable limits are pico-molar concentrations or a minimal mass of about 180 Da [2]. 

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, 

Kinetics evaluation software generates the values of ka (rates of complex formation) and kd (rates of complex dissociation) by fitting the data to interaction models. In a sensorgram, if binding occurs as sample passes over a prepared sensor surface, the response increases and is registered upon equilibrium, a constant signal is reached. The signal decreases when the sample is replaced with buffer, since the bound molecules dissociate. 

The affinity (interaction strength), multiple interactions, and the changes in concentration can be also monitored from those studies. To deliver data in real time, the natural phenomenon of surface plasmon resonance (SPR) is employed. Since the refractive index (r ) at the interface changes as molecules are immobilized on the sensor surface, instant measure of r provides real-time assessment. The Tlcxchip platform exploits grating-coupled SPR (GC-SPR) for this purpose. 

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Figure 7.9. Schematic diagram of a surface plasmon resonance biosensor. One of the binding partners is immobilized on the sensor surface. With the BIACORE instrument, the soluble molecule is allowed to flow over the immobilized molecule. Binding of the soluble molecule results in a change in the refractive index of the solvent near the surface of the sensor chip. The magnitude of the shift in refractive index is related quantitatively to the amount of the soluble molecule that is bound.

New developments in immobilization surfaces have lead to the use of SPR biosensors to monitor protein interactions with lipid surfaces and membrane-associated proteins. Commercially available (BIACORE) hydrophobic and lipophilic sensor surfaces have been designed to create stable membrane surfaces. It has been shown that the hydrophobic sensor surface can be used to form a lipid monolayer (Evans and MacKenzie, 1999). This monolayer surface can be used to monitor protein-lipid interactions. For example, a biosensor was used to examine binding of Src homology 2 domain to phosphoinositides within phospholipid bilayers (Surdo et al., 1999). In addition, a lipophilic sensor surface can be used to capture liposomes and form a lipid bilayer resembling a biological membrane. 

Gershon P.D., Khilko S., Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector, J Immun Methods 1995 183 65-76. 

SPR chips coated with a carboxymethyl dextran matrix are supplied commercially by Biacore, a leading manufacturer of SPR instruments. Similar technique can be used for preparation of carboxymethyl dextran matrix on Me(Si)Ox surfaces on which -OH groups are generated by reaction with 3-glycidoxypropyltrimethoxysilane13. 

Fig. 10. A SPR Detection realized in a BIAcore system. A fan of polarized light passes a prism and is focused at the interface to an aqueous phase under conditions of total reflection. An evanescent wave enters the solvent phase. If the prism is coated with a thin gold layer at the interface the free electrons in the metal absorb energy from the evanescent wave for a distinct angle, depending on the refractive index of the solvent near the interface. B The gold layer can be modified with, e.g., a carboxydextrane matrix, where catcher molecules can be immobilized by standard chemistry. If a ligand is applied with the aqueous phase it may interact with the catcher and accumulate in the matrix, causing a shift in the resonance angle. If no specific binding occurs the refractive index in proximity of the sensor is less affected...

As described for stopped flow experiments above, all commercially available SPR systems work under (pseudo) first-order conditions as well. This is realized either by a large excess of free ligand (in the large volume of the cuvette) compared with a nanoliter volume of the sensor layer [156] or by continuous replacement of free ligand in a flow injection system (e.g.,BIAcore [157]). 

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. 

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. 

Figure 9. SPR sensor with four parallel sensing channels (provided hy S. Lolas, Biacore AB.)...

Incubahon with sAV led to the creation of the sAV monolayer. This surface was used exclusively with the BlAcore system (Biacore International... 

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