Beruflich Dokumente
Kultur Dokumente
Carlotta Guiducci
This text is based on references from Current Protocols in Immunology and Current Protocols in Protein Science, by John Wiley & Sons, Inc.
ABASTRACT
Biosensors are widely used in different applications nowadays and different properties of materials and interfaces are used to design them. Among the biosensors currently used, some are based on optical properties in particular on the refractive index and thus the surface Plasmon resonance (SPR) phenomenon of the device interface. The surface Plasmon resonance based systems are very efficient because they enable the detection and quantification of biological interactions in real time, without the use of labels. They can be used to analyze samples going from low-molecular-mass drugs to multiprotein complexes and bacteriophages and finally, can also be used to detect interactions with very low affinity (from millimolar to picomolar in strength). In this laboratory, you will be introduced in some aspects of Plasmon resonance based systems. Then in practical session, 2-globulin will be detected using a SPR based system via two different strategies of ligands (probes) immobilization on the sensor chip: by amine coupling (direct immobilization) and using ligand capture method (indirect immobilization).
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TABLE OF CONTENTS
1 Theory....................................................................................................................................................... 3 1.1 1.2 1.3 1.4 2 Biacore............................................................................................................................................... 3 Surface preparation ............................................................................................................................ 5 Assay types ......................................................................................................................................... 8 Experimental design ........................................................................................................................ 11
Practical work ........................................................................................................................................ 17 2.1 2.2 2.3 Reagents preparation ....................................................................................................................... 17 Protocol 1:Immobilization of the ligand on a sensor chip by amine coupling ................................ 18 Protocol 2:Immobilization of the ligand on a sensor chip using ligand capture method ................. 22
Data analysis .......................................................................................................................................... 26 3.1 3.2 Immobilization Sensorgram Analysis ............................................................................................. 26 Kinetic Analysis .............................................................................................................................. 26
References .............................................................................................................................................. 27
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THEORY
The optical phenomenon of Surface Plasmon Resonance (SPR) used by Biacore systems enables the detection and quantification of protein-protein interactions in real time, without the use of labels. Biacore systems are widely used for characterizing the interactions of proteins with other proteins, peptides, nucleic acids, lipids, and small molecules. When Biacore is used to measure protein interactions, one of the interactants is immobilized onto a sensor chip surface and the other interactant is passed over that surface in solution via an integrated microfluidic flow system. The immobilized interactant is referred to as the ligand, and the injected interactant in solution is referred to as the analyte. Binding responses are measured in resonance units (RU) and are proportional to the molecular mass on the sensor chip surface and, consequently, to the number of molecules on the surface. Surface Plasmon Resonance (SPR) was first shown to be amenable for the label-free study of interactions between biomolecules over 20 years ago (Lieberg et al., 1983). Biacore pioneered commercial SPR biosensors offering a unique technology for collecting high quality, information-rich data from biomolecular binding events. Since the release of the first instrument in 1990, researchers around the world have used Biacores optical biosensors to characterize binding events with samples ranging from proteins, nucleic acids, small molecules to complex mixtures, lipid vesicles, viruses, bacteria, and eukaryotic cells. Typical questions answered with Biacore instruments include: How specific is an interaction? How strong is an interaction? What is the affinity? How fast is an interaction? What are the association and dissociation rate constants? Why is the interaction that strong or that fast? What are the thermodynamic parameters for an interaction? What is the biologically active concentration of a specific molecule in a sample?
1.1
Biacore
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Figure 2 The sensorgram is a plot of response in resonance units (RU) versus time in seconds, which is presented continuously in real time. Upon injection of an analyte, if a binding interaction occurs, then an increase in mass occurs on the sensor chip surface and association is measured. At the end of the analyte injection, as the complex decays, a decrease in mass occurs and dissociation is measured. Association and dissociation are measured as changes in response.
1.2
Surface preparation
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Figure3.
The most popular type of sensor chip has a layer of carboxymethylated (CM) dextran on top of the gold layer carboxyl groups allow covalent attachment of ligands or capturing molecules and also provide a hydrophilic environment for the interaction. A range of techniques can be used for ligand immobilization to a CM surface. Covalent coupling of the ligand can be performed by a variety of methods via free amino groups, thiol-disulfide exchange, or aldehyde groups. An alternative to covalent immobilization is ligand capture, in which a molecule with high affinity for the ligand is chemically coupled to the sensor chip. The ligand is then adsorbed to the capturing molecule from a solution in a separate step. Sensor chips are reusable since noncovalently bound material can be removed from the Sensor Chip surface with an injection of a suitable regeneration solution.
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concentration of ligand injected may be varied to adjust the immobilization level. The ligand may be prevented from attaching to the surface via sites important to the interaction under study by having analyte present during immobilization. Stabilization of ligand activity during immobilization was recently reported by Casper et al. (2004), where conformationally sensitive protein kinases, p38 and GSK3, were immobilized on Sensor Chip CM5 by amine coupling in the presence of a specific reversible inhibitor. This treatment resulted in a more stable surface with much higher specific binding activity. In cases where amine coupling interferes with the binding site on the ligand or in the case of very acidic proteins, the ligand can be attached using alternative coupling chemistries or a high-affinity capture approach.
b) Thiol coupling Thiol coupling is based on exchange reactions between thiol and active disulfide groups. In the case where the ligand has a free thiol group (typically cysteine residues), a surface thiol approach can be used where a disulfide group is introduced on the sensor chip dextran by first activating the surface with NHS and EDC to amine couple 2-(2-pyridinyldithio) ethaneamine (PDEA). Injection of the ligand results in thiol-disulfide exchange and excess PDEA groups are inactivated with cysteine- HCl. If the ligand lacks a free thiol group, a reactive disulfide (PDEA) can be linked to carboxyl groups on the ligand. The pyridyldisulfide groups linked to the ligand are then coupled to thiol groups on the sensor surface that has been derivatized through injection of NHS and EDC, cystamine and subsequent reduction with DTE or DTT. Modification of carboxyl groups results in an increase in the isoelectric point of the proteins, which is of additional benefit in the case of acidic proteins. c) Maleimide coupling Maleimide coupling is an alternative form of thiol coupling, which involves the formation of a stable thioether bond between reactive maleimido groups on the sensor surface and the thiol groups of the ligand molecule. Such surfaces have the capacity to withstand basic pH (>9.5) as well as reducing agents such as heterobifunctional reagents are available for introduction of reactive maleimido groups to the sensor surface, including sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester), sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate), and GMBS (N(g-maleimidobutyryloxy)sulfosuccinimide ester). d) Aldehyde coupling Aldehyde coupling involves the formation of a hydrazone bond via condensation of hydrazide groups on the sensor surface with aldehyde groups on the ligand molecule. These aldehyde moieties may be native to the protein or introduced through mild oxidation of cis-diols present in the ligand molecule. Aldehyde coupling is particularly useful for site directed immobilization of glyco-conjugates, glyco-proteins, and polysaccharides, and may also be useful for orientation-specific immobilization of proteins containing functional groups that may be converted to aldehyde moieties.
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Figure 5 Bioconjugation.
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small sample volumes can provide information regarding antibody isotype and active or relative concentration in serum. Another advantage of using Biacore for immunogenicity studies is the ability of the technique to detect both high- and low affinity antibodies, whereas traditional endpoint assays (e.g., ELISA) often fail to detect fast-dissociating antibodies (Swanson, 2003; Thorpe and Swanson, 2005).
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another antigen molecule. Avidity effects will slow down the dissociation rate yielding enhanced affinity values compared to those measured from a 1:1 interaction. It is also important that both the ligand and analyte be as homogeneous as possible. Impurities from partially purified material can complicate the results by affecting the accurate determination of analyte concentration or introducing nonspecific binding. Lastly, analyte should be injected over both a reference surface and an active ligand surface. Reference surfaces are necessary to subtract bulk refractive index responses from the specific binding signal as well as to ensure that there is no nonspecific interaction with the sensor chip surface. Several excellent reviews on the topic provide detailed guidelines on experimental setup and interpretation of results (Karlsson and Falt, 1997; Myszka, 1999; Myszka, 2000; Rich and Myszka, 2001; Van Regenmortel, 2003; Karlsson and Larsson, 2004).
Figure 6 Binding kinetics: 1. Receptor molecules are immobilized on the sensor surface. 2. At t = 0 s, buffer is contacted with the receptor through a microfluidic flow cell or through a cuvette. 3. At t = 100 s, a solution of analyte in the running buffer is passed over the receptor. As the analyte binds to the surface, the refractive index of the medium adjacent to the sensor surface increases, which leads to an increase in the resonance signal. Analysis of this part of the binding curve gives the observed association rate (kobs). If the concentration of the analyte is known, then the association rate constant of the interaction (kass) can be determined. At equilibrium, by definition, the amount of analyte that is associating and dissociating with the receptor is equal. The response level at equilibrium is related to the concentration of active analyte in the sample. 4. At t = 320 s, the analyte solution is replaced by buffer, and the receptoranalyte complex is allowed to dissociate. Analysis of these data gives the dissociation rate constant (kdiss) for the interaction. 5. Many complexes in biology have considerable half-lives, so a pulse of a regeneration solution (for example, high salt or low pH) is used at t = 420 s to disrupt binding and regenerate the free receptor. 6. The entire binding cycle is normally repeated several times at varying concentrations of analyte to generate a robust data set for global fitting to an appropriate binding algorithm. The affinity of the interaction can be calculated from the ratio of the rate constants (KD = kdiss/kass) or by a linear or nonlinear fitting of the response at equilibrium at varying concentrations of analyte. In addition to determining the interaction affinities and kinetics, a thermodynamic analysis of a biomolecular interaction is also possible.
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amounts of analyte bound to the sensor chip surface from complex matrices such as food products, serum, and cell extracts, to name a few (Nelson et al., 2000). Instrument automation decreases operator involvement thereby leading to highly reproducible measurements. Various assay formats are possible. For analytes >5000 Da, a direct binding assay format can be used with the optional response enhancement from a secondary detecting molecule. Enhancement not only increases the dynamic range of the assay but can also improve assay specificity. An enhancement step can also be used to determine the isotype of antibodies in serum that are generated in response to a protein therapeutic or vaccine. Unlike many other immunoassays, concentration analysis with Biacore requires no separation and washing steps and, since binding responses are monitored continuously, it is possible to quantify fast-dissociating, low-affinity interactants. The point at which analyte concentration is measured can be chosen, giving flexibility in the assay design, which is not available with standard end-point assays. Inhibition or competition assay formats are well suited for quantification of low-molecular-weight molecules (<5000 Da). Inhibition assays rely on mixing the sample with a known concentration of a molecule that binds with high affinity, typically an antibody, then injecting the mixture over the target molecule immobilized on the sensor surface to determine the concentration of the high-molecular-weight binder in the mixture. Biacore evaluation software provides a direct readout of analyte concentration from a calibration curve generated using standard analyte concentrations. In a competition assay, the low-molecular-weight analyte competes with a fixed concentration of a high-molecular-weight molecule that shares the same binding site. Generally, concentration analysis is carried out at high ligand densities and slow flow rates. Efficient regeneration of the surface while maintaining ligand activity is paramount for successful concentration analysis measurements.
1.3.4 Thermodynamics
By studying temperature dependence of rate and affinity constants it is possible to determine thermodynamic parameters for a binding interaction. Not only can the equilibrium values for changes in enthalpy (_H) and entropy (_S) associated with complex formation be determined, but transition state energetics can also be evaluated (Roos et al., 1998).
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enables determination of the kinetic rate constants by fitting the responses to a simple 1:1 (Langmuir) binding model.
1.4.2
The two general techniques for immobilization of the ligand are: (1) direct coupling to the sensor chip surface using one of several surface chemistries; and (2) capture onto a surface that has been derivatized with a binding molecule (e.g., via a purification tag). The various surface chemistries for direct coupling are described in paragraph 2. Ligands that are directly coupled to the surface will have a random orientation; therefore, some binding sites may not be accessible. However, captured ligands often exhibit higher binding activity, which can be critical for some proteins. The higher binding activity of the captured ligand can be attributed to a homogeneous presentation of the ligand, availability of the binding site, and use of fresh ligand for each binding cycle (no damage due to regeneration solution). The ligand capture approach can thus offer advantages over direct immobilization.
1.4.3
Usually, a surface that is treated with the same coupling chemistries used to immobilize the ligand serves as a suitable control for the reference surface. When using the ligand capture approach, the capture molecule serves as a suitable control for the reference surface. For some interactions, a nonspecific ligand may act as an appropriate control, such as a scrambled peptide sequence for a protein-peptide interaction. The use of bovine serum albumin as a control ligand is not recommended due to its tendency to nonspecifically bind to many proteins.
1.4.4
A low ligand density surface is optimal for accurate measurement of the binding kinetics of an interaction. The maximum binding capacity (Rmax) of the immobilized ligand should be in the range of 50 to 150 RU. Use the rearranged Rmax equation (eq. 4.2 in the Immobilization level paragraph) to determine an appropriate immobilization level that will generate an Rmax of 100 RU. For the direct immobilization of the anti-2-globulin described in this protocol, 1200 RU was targeted, which results in a calculated Rmax of 189 RU. However, the experimental Rmax for the anti-2-globulin surface is 40 RU due to a lower-than-expected surface activity. Immobilization levels depend on five main factors, i.e., (1) ligand concentration, (2) pH, (3) activation time (EDC/NHS mixture), (4) injection time (ligand), and (5) ionic strength. Lower ligand binding levels can be reached by decreasing the first four factors or by increasing the fifth. Conversely, higher concentrations and higher activation or contact times, as well as lower ionic strength, contribute to increased ligand immobilization levels.
1.4.5
Surface activity should be calculated using eq. 4.3 in the Immobilization level paragraph. The BIAevaluation software generates an experimental Rmax value after curve fitting as described in the Evaluation of the Kinetic Analysis section. Generally, 75% surface activity can be expected for ligands immobilized directly by amine coupling. Poor surface activity (<50%) can result from denaturation of the ligand due to the preconcentration solution, or inactivation of the binding site due to the immobilization chemistry. In this case, employing an alternative surface chemistry should be considered to improve the surface activity. Impurities in the ligand preparation can also be immobilized when using a direct coupling approach, which could affect the binding capacity of the surface, thereby lowering the apparent surface activity. In this case, employing the ligand capture approach can add an on-chip purification step that will usually result in improved surface activity.
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1.4.6
Inspect the analyte response on the reference flow cell to identify nonspecific binding due to electrostatic or hydrophobic interactions with the sensor chip surface. The bulk refractive index has a square-shaped response, while nonspecific binding will typically have an increasing response on the reference. Electrostatic nonspecific binding can be minimized by the addition of NaCl to the sample and running buffer or by using a sensor chip with less carboxymethylation, such as CM4. Nonspecific binding to the CM-dextran can be minimized by addition of soluble CMdextran to the sample at 1mg/ml. Hydrophobic nonspecific binding often can be minimized by the addition of a detergent, such as 0.05% polysorbate 20 or 10 mMCHAPS, to the sample and running buffers. Nonspecific binding to a capture surface can be resolved by switching to another capture molecule. Nonspecific binding should be checked by one of the following methods. (1) Perform the sample injection on both the specific cell and a reference cell. This reference cell must be prepared in a manner as similar as possible to that used for the specific one (e.g., using same coupling chemistry to immobilize a similar amount of inactivated ligand). (2) Inject a nonspecific analyte (e.g., a peptide with randomized sequence).
1.4.7
If the analyte does not dissociate from the ligand within a reasonable time (e.g., 20 min); then the surface should be regenerated. Identify a solution that removes the analyte from the immobilized ligand without affecting the ligand activity. An acidic or basic solution or chaotropic salt solution injected at a high flow rate (50 l/min) for a short time (1 min) will usually be effective in removal of the analyte from the ligand without damaging the binding activity of the ligand. Determination of ideal regeneration conditions may require optimization. Combining solutions together into regeneration cocktails can often produce efficient regeneration conditions (Andersson, 1999). For example, a combination of low pH and high ionic strength solutions such as 10 mM glycine, pH 2.0, and 1 M sodium chloride; a combination of basic and chaotropic solutions such as 10 mM sodium hydroxide and 500 mM sodium thiocyanate; a combination of a low pH solution and detergent such as 10 mm glycine, pH 2.0, and 0.05% polysorbate 20. An immobilization and regeneration database that provides examples and suggestions for a wide range of interactants is available at the Biacore Website.
1.4.8
The SPR detection technology used in Biacore systems measures changes in the refractive index that are related to changes in mass at the sensor chip surface. Differences in the refractive index between the running buffer and injected sample buffer will give rise tom changes in responses that are known as bulk refractive index changes. Bulk refractive index effects are common and do not affect the measurement of the binding interaction when a reference surface is used to subtract the bulk refractive index from the binding response. It is recommended that differences in the bulk refractive index between the analyte and running buffer are minimized by either dialysis or dilution of the analyte into the running buffer. Samples containing high refractive index solutions, such as highsalt solutions, glycerol, or DMSO, should either be buffer-exchanged into the running buffer or the solutions should be added to the running buffer to match the composition of the samples.
1.4.9
Immobilization pH scouting
Immobilization by covalent coupling cannot be repeated on the same sensor chip surface. Therefore, preconcentration is an important strategy to control the immobilization level for experimental optimization and efficiency. Typically, the optimal pH for preconcentration will be a 0.5- to 1-pH unit below the pI of the protein. Many proteins exhibit limited stability in the low-ionic-strength, low-pH solutions used for preconcentration, therefore, dilution of the ligand just prior to use is recommended. Always perform pH scouting on the flow cell that will be used for the immobilization, not on the reference flow cell. When using standard amine coupling, 40% of the carboxymethyl groups are converted to reactive esters after a 7-min activation. Thus, the activated sensor chip surface maintains a net negative charge during the immobilization 13
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procedure, which enables preconcentration. Note that prepared surfaces can have a negative charge during experiments, depending on sensor chip type and blocking procedure.
The PH scouting sensorgram allows the determination of the optimum immobilization pH according to the immobilization level desired by comparing the responses (in RU) obtained at each pH of the sensorgram with the desired response in RU.
Where, MW is the molecular weight (mass) of the ligand and analyte, RL (ligand response) is the amount of immobilized ligand in RU, and Sm is the stoichiometry as defined by the number of binding sites on the ligand. Rearrangement of the Rmax equation provides a means of calculating an appropriate ligand density to aim for when performing an immobilization for kinetic analysis as shown by the following Equation 4.2: RL = (ligand MW/analyte MW) Rmax (1/Sm) (4.2)
The maximum binding capacity (Rmax) of the immobilized ligand should be in the range of 50 to 150 RU for measurement of the binding kinetics of an interaction. Use of a low ligand density will minimize mass transfer limitations during the interaction. Mass transfer limitation refers to an experimental situation in which the supply of analyte to the sensor chip surface is a limiting factor for the interaction due to the demand for analyte by a high ligand density. Mass transfer limitations can result in calculated binding kinetics that are slower than the true binding kinetics. Partial mass transport limitations can be accounted for in data analysis using a binding model that includes a mass transfer rate constant. An experimental Rmax must be determined to assess the percent activity of the immobilized ligand. The quality of the binding data that can be obtained is reflected in the ligand activity as shown in the following Equation 4.3: %ligand activity = experimental Rmax/theoretical Rmax 100% (4.3) 14
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Direct quantitative evaluation of interaction kinetics outside these limits is often impossible. The best alternative for direct kinetic analysis of small analyte binding is the surface competition assay. However, even this method has an important limitation: macromolecules sharing the same binding specificity with the small target analyte (e.g., viral proteins) are not easily available. Other alternative SPR methodologies are briefly commented in Figure 9.
Figure9 Possible studies for binding kinetics assays. 1) Direct assay: Suitable for high molecular weight molecules 2) Sandwich assay: To be selected for relatively high molecular weight antigens and when high affinity antibodies are available. 3) Competition assay: Designed for low molecular weight antigens that do not generate sufficient signal when they accumulate on the surface (Direct assay) and are too small for a sandwich assay. 4) Inhibition assay: Designed for low molecular weight antigens that do not generate sufficient signal when they accumulate on the surface (Direct assay) and are too small for a sandwich assay.
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2. PRACTICAL WORK
2.1 Reagents preparation
Materials HBS-P+ 1 mg/ml anti-2-globulin antibody 10 mM sodium acetate, pH 5.5 50 mM NaOH 100 g/ml (8.5 M) 2-globulin 25 g/ml anti-mouse IgG antibody 10 mM sodium acetate, pH 5.0 10 mM sodium Acetate pH 5.5 1) Dilute 100 l of 3M sodium acetate pH 5.5 into 29.9 ml milliQ water. 50 mM NaOH 2) Dilute 500 l of 1M NaOH into 9500 l milliQ water.
2.1.1 Protocol 1
Buffer preparation 3) Dilute 10 HBS-P+ from stock to 1. 4) Take a 200 ml graduated cylinder. 5) Put 20 ml of 10 HBS-P+ 6) Add 180 ml MQ water. 7) Mix the solution. 8) Filter the solution through a 0.2-m filter: 9) Open the vent hood (it will automatically switch on) 10) Connect the stericup to the vacuum tube under vent hood 11) Switch on the vacuum pump from the hood front panel. 12) Poor the solution in the top part of the stericup. 13) Wait until the solution is filtered. 14) Close immediately the bottle with the filtered solution. 15) Switch off the pump and close the vent hood. 16) Equilibrate to room temperature before use. 10 g/ml anti-2-globulin antibody in 10 mM sodium acetate, pH 5.5 17) Dilute 2 l of 1 mg/ml anti-2-globulin into 198 l sodium acetate, pH 5.5.
2-globulin diluted series preparation (for Protocols 1 and 2) 18) Dilute 5.6 l of 8.5 M stock 2-globulin (100 g/ml) into 1494.4 l HBS-P+ to prepare the 32 nM sample.
19) Prepare two-fold serial dilutions of 32 nM 2-globulin down to 4 nM. To prepare the 16nM solution, take 600 l of buffer and 600 l of 32nM 2-globulin diluted solution. Proceed this way down to 4 nM concentration.
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Referring to the serial dilution section in the Master handout, compute the different volumes and concentrations needed to perform the two serial dilutions and fill the values obtained beforehand in the following table:
2-fold dilution
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2.1.2 Protocol 2
Buffer preparation 1) Same as Protocol 1
5 g/ml anti-2-globulin antibody in HBS-P+ 2) Dilute 2 l of 1 mg/ml anti-2-globulin into 398 l HBS-P+.
25 g/ml anti-mouse IgG antibody in 10 mM sodium acetate, pH 5.0 3) Dilute 5 l of 1 mg/ml anti-mouse IgG antibody into 195 l sodium acetate, pH 5.0.
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Materials 200 ml HBS-P+ 10 g/ml anti-2-globulin antibody in 10 mM sodium acetate, pH 5.5 10 mM sodium acetate, pH 5.5 50 mM NaOH Sensor Chip CM5 1.5-ml polypropylene tubes with rubber caps (Biacore) 100 mM N-hydroxysuccinimide (NHS) 400 mM N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) 1 M ethanolamine 1) Remove sensor chips from the fridge at least 30 min before starting the experiment. 2) Change the sensor chip. Biacore is always kept with a maintenance chip inside. On the Control Software Tool Bar click on Undock Chip and follow the displayed instructions. When undocked, the sensor chip should easily slide out of the instrument. Note that the sensor chip indicator light on the front of the instrument is lit steadily when a chip is docked, flashing when a chip is inserted but undocked, and unlit when no chip is inserted. 3) Insert and dock the new Sensor Chip CM5. Click the Command tab and then select the Dock command.
2.2.1
Ligand Immobilization
4) Create a New Wizard Template. Click on Wizards and select the Immobilization wizard type from the list in the left-hand panel. Then click on New. 5) Define the Immobilization Setup. This step will determine how the ligand is immobilized on the surface. Choose the chip type CM5.
6)
7) 8)
Choose the Flow Cell for Immobilization. - For cell 1 select Blank Immobilization with the Amine method. - For cell 2 select Aim for immobilized level with the Amine method. - Enter the ligand name anti-2-globulin antibody - Set the target level at 1200 RU - Enter 50mM NaOH for the Wash Solution. Check Prime before run to flush the flow system with fresh buffer at the start of the run. Click on Next. 19
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9)
Prepare the rack. A new dialog shows you where the samples and the reagents should be placed in the rack and how much of each sample is needed. The listed volumes are minimum valuesNote: The position marked H2O requires an uncapped vial with MQ water. Make sure you dont make bubbles while putting the reagents into the vials. Close all the vials with the orange rubber caps and place them in the right order.
10)
Click Load Samples to release the sample rack so that you can load samples. Wait until the Rack Locked indicator on the instrument is turned off before attempting to remove the sample rack. The sample rack can be removed from the sample compartment when the Rack locked indicator on the instrument is not lit. Replace the rack and click OK to lock the rack. Click on Next on the Immobilization- Rack Positions window. Prepare Run Protocol : The following text describes the preparations you need to complete before starting the run. Once you checked it is correct, click on Start.
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2.2.2
Materials
Binding kinetics
Diluted series of 100 g/ml (8.5 M) 2-globulin in HBS-P+ 10 mM glycine, pH 2.5 18) Create a New Wizard Template. Click on Wizards and select the Kinetics/Affinity wizard type from the list in the left-hand panel. Then click on New. Define the Injection Sequence. Chose CM5 sensor chip and Multi-cycle kinetics type. Set 1 regeneration and then click on Next. Define System Preparation. Check Prime before run to flush the flow system with fresh buffer at the start of the run. Check Run startup cycles. Write buffer as solution and set 3 cycles. Click on Next. Define Injection parameters. For the sample: Set Contact time: 120 s. Set Dissociation time: 300 s. For the First regeneration: Set Solution: 10mM Glycine pH 2.5. Set Contact time: 30 s. Set Stabilization period: 60 s. Click on Next. Define Samples. Set B2micro as Sample id. Set 11800 Da as Molecular Weight. Enter the sample concentrations in ascending order. Include in addition at least one blank sample and a replicate of a non-zero concentration. Do not run replicate samples in consecutive cycles since this will reduce the chance of detecting assay drift. Samples will be run in the order entered. Click on Next. Prepare the rack. Close all the vials with the orange rubber caps and place them in the right order. Note: The position marked H2O requires an uncapped vial with MQ water.
19)
20)
21)
22)
23) 24)
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25)
26)
27) 28)
Click Load Samples to release the sample rack so that you can load samples. Wait until the Rack Locked indicator on the instrument is turned off before attempting to remove the sample rack. The sample rack can be removed from the sample compartment when the Rack locked indicator on the instrument is not lit. Replace the rack and click OK to lock the rack. Click on Next on the Immobilization- Rack Positions window. Perpare Run Protocol. Once you checked it is all correct, click on Start.
2.3 PROTOCOL 2: Immobilization of the ligand on a sensor chip using ligand capture method
An alternative approach to direct immobilization of the ligand to sensor chip surface is capture of the ligand onto the sensor chip surface using a high-affinity binding molecule that has been coupled directly to the chip. In this protocol, regeneration of the surface usually removes both the bound analyte and the captured ligand for each cycle of analyte binding. The use of an anti-mouse IgG surface to capture the mouse monoclonal anti-2-globulin antibody ligand is described here. Amine coupling is used to prepare an anti-mouse IgG surface on the reference and active flow cells. The volume of anti-2-globulin required to reach the desired ligand level must be determined. Regeneration conditions for the anti-mouse IgG surface are provided. Lastly, experimental set-up for kinetic analysis using ligand capture is described. Materials 100 mM N-hydroxysuccinimide (NHS) 400 mM N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) 25 g/ml anti-mouse IgG antibody in 10 mM sodium acetate, pH 5.0 1 M ethanolamine 5 g/ml solution of anti-2-globulin antibody in HBS-P+ 10 mM glycine, pH 1.7 Sensor Chip CM5 1.5-ml polypropylene tubes with rubber caps (Biacore) 1) Remove sensor chips from the fridge at least 30 min before starting the experiment. 2) Change the sensor chip. Biacore is always kept with a maintenance chip inside.
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On the Control Software Tool Bar click on Undock Chip and follow the displayed instructions. When undocked, the sensor chip should easily slide out of the instrument. Note that the sensor chip indicator light on the front of the instrument is lit steadily when a chip is docked, flashing when a chip is inserted but undocked, and unlit when no chip is inserted. 3) Insert and dock the new Sensor Chip CM5. Click the Command tab and then select the Dock command.
2.3.1
Ligand Immobilization
4) Create a New Wizard Template. Click on Wizards and select the Immobilization wizard type from the list in the left-hand panel. Then click on New. 5) Define the Immobilization Setup. This step will determine how the ligand is immobilized on the surface. Choose the chip type CM5.
6) Choose for Cell 1 and 2 immobilization through Amine method. 7) Enter anti IgG as a capturing molecule. 8) Choose Specify contact time and set contact time 420 s. 9) Check Prime before run to flush the flow system with fresh buffer at the start of the run. 10) Click on Next. 11) Prepare the rack. A new dialog shows you where the samples and the reagents should be placed in the rack and how much of each sample is needed. The listed volumes are minimum values. Note: The position marked H2O requires an uncapped vial with MQ water. Make sure you dont make bubbles while putting the reagents into the vials. 12) Close all the vials with the orange rubber caps and place them in the right order.
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13) Click Load Samples to release the sample rack so that you can load samples. 14) Wait until the Rack Locked indicator on the instrument is turned off before attempting to remove the sample rack. 15) The sample rack can be removed from the sample compartment when the Rack locked indicator on the instrument is not lit. 16) Replace the rack and click OK to lock the rack. 17) Click on Next on the Immobilization- Rack Positions window. 18) Prepare Run Protocol : The following text describes the preparations you need to complete before starting the run. 19) Once you checked it is correct, click on Start.
2.3.2
Binding kinetics
Materials Diluted series of 8.5 M stock 2-globulin in HBS-P+ 10 mM glycine, pH 1.7 5 g/ml solution of anti-2-globulin in HBS-P+ 20) Create a New Wizard Template. Click on Wizards and select the Kinetics/Affinity wizard type from the list in the left-hand panel. Then click on New. 24
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21)
22)
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Define the Injection Sequence. Chose CM5 sensor chip and Multi-cycle kinetics type. Check Ligand capture. Set 1 regeneration and then click on Next. Define System Preparation. Check Prime before run to flush the flow system with fresh buffer at the start of the run. Check Run startup cycles. Write buffer as solution and set 3 cycles. Click on Next. Define Injection parameters. For the capture: Set Contact time: 180 s. Set Stabilization period: 60 s. For the sample: Set Contact time: 120 s. Set Dissociation time: 300 s. For the First regeneration: Set Solution: 10mM Glycine pH 1.7. Set Contact time: 120 s. Set Stabilization period: 60 s. Click on Next. Define Samples. Set B2micro as Sample id. Set 11800 Da as Molecular Weight. Enter the sample concentrations in ascending order. Include in addition at least one blank sample and a replicate of a non-zero concentration. Do not run replicate samples in consecutive cycles since this will reduce the chance of detecting assay drift. Samples will be run in the order entered. Click on Next. Prepare the rack. Close all the vials with the orange rubber caps and place them in the right order. Note: The position marked H2O requires an uncapped vial with MQ water.
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Click Load Samples to release the sample rack so that you can load samples. 25
ADVANCED SPR
Carlotta Guiducci
Wait until the Rack Locked indicator on the instrument is turned off before attempting to remove the sample rack. The sample rack can be removed from the sample compartment when the Rack locked indicator on the instrument is not lit. Replace the rack and click OK to lock the rack. Click on Next on the Immobilization- Rack Positions window. Perpare Run Protocol. Once you checked it is all correct, click on Start.
3 DATA ANALYSIS
3.1 Immobilization Sensorgram Analysis
. Q1. Discuss the immobilization sensorgram curve: explain in your lab notebook step by step the immobilization process using the immobilization sensorgrams Q2. Discuss the role of pH (of sodium acetate) during the immobilization process. Explain briefly what pH scouting is, and why it is important. Why do we use different pH of sodium acetate for the two protocols? Q3. Explain briefly the differences and advantages of subsequent/continuous injections of ligand (or capture molecule). Q4. Which is the theoretical binding capacity of the sensor chip surface? Does it correspond to the experimental data? If not, why this difference? Q5. From the Immobilization Sensorgrams, check if the RU bound to the surface corresponds to the data settings. To how many molecules per cm2 do they correspond? (Hint: 1RU = 1pg/mm2 , MWligand = 150000Da) Q6. Why do we add ethanolamine during immobilization? What might happen if this step is skipped?
ADVANCED SPR
Carlotta Guiducci
Q12. In some cases, the sensorgram is affected by bulk effect. Why does it occur? How do you identify and get rid of it? Q13. What kind of interaction are we analyzing? Why is the pH of glycine so important? Why do we use different pH glycine for the two protocols? Q14. Why do we use two different methods to study binding kinetics? What are the advantages of each method compared to the other? Can you extract this information from the sensorgrams? Q15. Why in kinetics analysis you need serial dilutions?
4 REFERENCES
Fundamentals of biochemical sensing systems, EPFL Master BE course, Prof.Guiducci, 2010. http://www.biacore.com/lifesciences/index.html Biacore catalog. https://www.biacore.com/lifesciences/service/online support/irdb/index.html Immobilization and regeneration database. http://www.biacore.com/lifesciences/service/training/courses/usa canada/biacorebasics/index
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