Protein BIAcore

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. 

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,... 

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. 

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. 

The avidin-biotin interaction has also been used to immobilize antibodies and proteins, especially in commercial systems based on surface plasmon resonance (SPR) measurements (e.g., the BIAcore). The extraordinary affinity (Kl 10-15 M) of avidin (or its bacterial relative, streptavidin) for the vitamin biotin is the basis of this immobilization procedure. A solid support (e.g., glass beads, sensor chip, optical fiber) covered with avidin can be used as an activated carrier for a very sturdy immobilization of previously biotinylated antibodies. In spite of the many methods for biotinylating proteins described in the literature, the use of biotinyl N-hydroxysuccinimide ester (BNHS) and similar derivatives, remains the most useful [65]. 

An important issue in the neutralization assay is the amount of therapeutic protein product to add. This dose should be based on the dose-response curve of the protein in the assay. The reduction should be in the linear part of the curve and be significant enough to be reliably testable in the assay. The titer of the neutralization is often expressed in units. The highest dilution of the serum with a significant inhibition is defined to contain one neutralizing unit. Sometimes the activity is expressed in arbitrary units using an animal antiserum as reference. Further characterization of the antibodies may include evaluation of Ig isotype and affinity. Although alternative methods may be employed, the BIAcore assay has become the standard for these types of analyses. 

Figure 10.4 Binding diagram of LDH, GAPDH, pyruvate kinase and DHODH to the derivatized BIAcore chip at different protein concentrations (x-axis protein concentration, y-axis resonance units).

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-... 

Bioconjugates play an important role in several fields of biomolecular and biomedicinal sciences. Protein/polypeptide-based conjugates with covalently attached epitope peptides are considered as potential synthetic vaccine candidates and/or target antigens in affinity-based bioassays (e.g., ELISA, BIACORE) (1,2). Drugs such as daunomycin, methotrexate, and a GnRH analog coupled either to monoclonal antibodies or to synthetic linear or branched polypeptides... 

Studies of the interaction of IL-6 and the sIL-6R were monitored by SPR detection using a BIAcore instrument [1] (Pharmacia, Uppsala). Immobilisation of the respective proteins to the carboxymethylated dextran matrix coating the gold sensor chip was performed using the EDC/NHS coupling chemistry as previously described for IL-6 and sIL-6R [3, 7, 8]. Regeneration of the sensor surface for IL-6 and sIL-6R was performed with lOmM HCl for 3 min [8] and 4M MgCl2 in lOmM Tris-HCl buffer, pH 7.4 for 1 min respectively. 

The K138C mutants (-1.5 mg in 0.5 mL of PBS) were treated with 50 mM DTT (removed by gel filtration) and then biotinylated using biotin-maleimide (Sigma) (2 1 molar ratio). The biotinylated product was purified by gel filtration on Superdex 75. Evidence of biotinylation was routinely obtained by western blots probed with an avidin-HRP conjugate (Pierce) and by binding of the proteins to streptavidin-coated BIAcore chips (Pharmacia SA5). 

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