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ADVANCED SPR

BIOENGINEERING METHODS LABORATORY

Carlotta Guiducci

This text is based on references from Current Protocols in Immunology© and Current Protocols in Protein Science©, by John Wiley & Sons, Inc.

SURFACE PLASMON RESONANCE BASED SYSTEMS

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, β2μ-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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ADVANCED SPR

BIOENGINEERING METHODS LABORATORY

Carlotta Guiducci

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

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Data analysis .......................................................................................................................................... 26 3.1 3.2 Immobilization Sensorgram Analysis ............................................................................................. 26 Kinetic Analysis .............................................................................................................................. 26

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References .............................................................................................................................................. 27

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Since the release of the first instrument in 1990.  A microfluidic and liquid handling system that precisely controls the flow of buffer and sample over the sensor surface. information-rich data from biomolecular binding events. peptides. 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. nucleic acids. and the injected interactant in solution is referred to as the analyte..ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 1 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. 3 . 1983). without the use of labels.1. researchers around the world have used Biacore’s optical biosensors to characterize binding events with samples ranging from proteins. to the number of molecules on the surface. nucleic acids. bacteria. Figure 1 Surface Plasmon Resonance principle.1 Biacore 1.1 System features Biacore’s optical biosensors are designed around three core technologies:  An optical detector system that monitors the changes in SPR signal brought about by binding events in real time. These systems are contained within the processing unit that communicates with a computer equipped with control and data evaluation software. lipid vesicles. small molecules to complex mixtures. The resulting biospecific surface is the site where biomolecular interactions occur. The immobilized interactant is referred to as the ligand. lipids. and small molecules.  An exchangeable sensor chip upon which one of the interacting biomolecules is immobilized or captured. viruses. Binding responses are measured in resonance units (RU) and are proportional to the molecular mass on the sensor chip surface and. When Biacore is used to measure protein interactions. and eukaryotic cells. consequently. 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. Biacore systems are widely used for characterizing the interactions of proteins with other proteins. 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. Biacore pioneered commercial SPR biosensors offering a unique technology for collecting high quality.

A sensorgram depicts changes in SPR angle in real time. which provides information on affinity constants and can be used for qualitative or semi-quantitative applications. which is displayed and recorded as a change in mass occurs on the sensor chip surface (Fig.2 The SPR detection system Surface Plasmon Resonance (SPR) is a phenomenon that occurs in thin conducting films placed at the interface between two media of different refractive indices. A sensorgram is a plot of the binding response in resonance units versus time in seconds. as a result of a binding event. and (2) the binding level. which provides information on kinetic rate constants and analyte concentration.1. a 50-nm layer of gold on the sensor chip is sandwiched between the glass layer of a sensor chip and the sample solution flowing through the microfluidic cartridge.g. 4 . Since only the evanescent wave penetrates the sample. When a change in mass occurs near the sensor chip surface. or both). Under these conditions. the light leaks an electromagnetic component called an evanescent wave across the gold interface into the sample/buffer solution. In Biacore systems. or to 1pg/mm2 of molecule on the surface. dissociation.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 1.0001◦ shift in SPR angle. turbid..1). measurements can be carried out with colored. e. The sensorgram provides essentially two kinds of information that are relevant to different types of applications: (1) the rate of interaction (association. the angle of light at which SPR occurs shifts due to a change in refractive index near the sensor chip surface. the evanescent wave field excites electrons in the gold film resulting in the formation of surface plasmons (electron charge density waves) within the gold film with a concomitant drop in the intensity of the reflected light at this angle (SPR angle). Plane polarized light from a near-infrared LED is focused on the back of the sensor chip under conditions of total internal reflection and a diode array detector monitors the intensity of the reflected light (Fig. At a certain angle of incident light. 2). One RU corresponds to 0. or even opaque solutions without interference from conventional light absorption or light scattering. with responses measured in resonance units (RU).

At the end of the analyte injection. gold is amenable to covalent attachment of surface matrix layers and. Sensor Chips Although SPR can be generated in thin films made from conducting metals. as the complex decays.2. all Biacore sensor chips are coated with a thin. 1.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci Figure 2 The sensorgram is a plot of response in resonance units (RU) versus time in seconds..2 Surface preparation 1. Sample bound to the sensor chip surface can be recovered for complementary analysis and/or identification. e. Dual-channel detection of the SPR signal and serial flow allow simultaneous monitoring of both flow cells. if a binding interaction occurs. is specific to each instrument platform. The design of the IFC. in physiological buffer conditions. A variety of sensor surfaces are available to give a range of alternative possibilities for different experimental situations: 5 . Association and dissociation are measured as changes in response.3 The microfluidic system Biacore systems make use of an integrated microfluidic cartridge (IFC) to deliver samples and buffer to the sensor surface. A series of computer-controlled pneumatic valves meters precise access to the channels and the flow cells. The instrument control software can be set to automatic in-line reference subtraction of the control surface signal from the same sample injection. uniform gold layer. Two different molecules can be immobilized on one sensor chip or one flow cell can be used as a reference and the other immobilized with a molecule of interest. gold is mostly inert. Pulse-free syringe pumps ensure a continuous flow of buffer over the sensor surface whenever samples are not being injected. as well as the number and configuration of flow cells. Upon injection of an analyte. using mass spectrometry. Biacore X is well suited for laboratory environments with low-sample throughput where many users handle different types of samples. Flow cells are created through pressure contact between molded channels in the IFC and the sensor chip surface. 1. samples are transferred through a needle into the IFC.1. then an increase in mass occurs on the sensor chip surface and association is measured. Gold has a number of advantages in that it results in a well-defined reflectance minimum when a visible light source is used to generate the SPR signal. which is presented continuously in real time. a decrease in mass occurs and dissociation is measured.1. The IFC consists of a series of micro-channels encased in a plastic housing. There are two flow cells in the Biacore X instrument.g. In automated systems.

dimethylaminopropyl) carbodiimide (EDC) and 0. Immobilization is via free primary amine groups such as lysine residues that are abundant in most proteins or the N-terminus of proteins and peptides. in which a molecule with high affinity for the ligand is chemically coupled to the sensor chip. Different types of sensor chips 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.2. and a typical immobilization reaction usually takes <30 min to complete. Finally. A. The ligand is then adsorbed to the capturing molecule from a solution in a separate step.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci Figure3. reactive groups on the sensor chip surface depending on the immobilization level required. the ligand acquires a positive charge and is effectively preconcentrated into the negatively charged carboxymethyl dextran matrix. A range of techniques can be used for ligand immobilization to a CM surface. The ligand is then injected in low salt buffer lacking primary amines at a pH below the isoelectric point (pI) of the protein. or aldehyde groups.2 Immobilization Chemistries A critical step in the development of reliable SPR assays is the selection of the most suitable immobilization technique such that ligand activity is maintained and binding sites are available to interacting partners. Immobilization is usually carried out at low flow rates (5 to 10 μl/min) and the concentration of ligand used for immobilization is typically in the range of 1 to 100 μg/ml. this can be varied from 1 to 10 min to create fewer or more. Direct immobilization chemistries A number of covalent immobilization techniques are available to covalently attach proteins or other biomolecules to the dextran on the sensor chip surface. unreacted esters are blocked with ethanolamine. An alternative to covalent immobilization is ligand capture. Commonly utilized immobilization strategies are outlined here. 1. High local concentrations of ligand obtained through electrostatic preconcentration maximize the efficiency of amine coupling.4 M 1-ethyl-3-(3. respectively. Guidelines and a detailed review of immobilization chemistries have been published recently (Karlsson and Larsson. Covalent coupling of the ligand can be performed by a variety of methods via free amino groups. All types of immobilization can be performed directly in the Biacore system. it is recommended that the ligand purity exceed 95%. The volume or 6 .1 M N-hydroxysuccinimide (NHS) to create reactive succinimide esters. The standard activation time is 7 min. a) Amine coupling Amine coupling is the most generally applicable covalent coupling chemistry used to immobilize protein ligands. 2004). Under these conditions. The dextran matrix on the sensor chip surface is first activated with a 1:1 mixture of 0. Sensor chips are reusable since noncovalently bound material can be removed from the Sensor Chip surface with an injection of a suitable regeneration solution. thiol-disulfide exchange. To ensure specificity.

HCl. the ligand can be attached using alternative coupling chemistries or a high-affinity capture approach. 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). cystamine and subsequent reduction with DTE or DTT. Figure 4 Ammine coupling and Thiol coupling b) Thiol coupling Thiol coupling is based on exchange reactions between thiol and active disulfide groups. If the ligand lacks a free thiol group. 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.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci concentration of ligand injected may be varied to adjust the immobilization level. In the case where the ligand has a free thiol group (typically cysteine residues). (2004). Stabilization of ligand activity during immobilization was recently reported by Casper et al. and may also be useful for orientation-specific immobilization of proteins containing functional groups that may be converted to aldehyde moieties. Such surfaces have the capacity to withstand basic pH (>9. and polysaccharides. were immobilized on Sensor Chip CM5 by amine coupling in the presence of a specific reversible inhibitor. The ligand may be prevented from attaching to the surface via sites important to the interaction under study by having analyte present during immobilization. a reactive disulfide (PDEA) can be linked to carboxyl groups on the ligand. c) Maleimide coupling Maleimide coupling is an alternative form of thiol coupling. glyco-proteins. Injection of the ligand results in thiol-disulfide exchange and excess PDEA groups are inactivated with cysteine. including sulfo-MBS (m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester). and GMBS (N(g-maleimidobutyryloxy)sulfosuccinimide ester). Modification of carboxyl groups results in an increase in the isoelectric point of the proteins. 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. 7 . p38 α and GSK3β. These aldehyde moieties may be native to the protein or introduced through mild oxidation of cis-diols present in the ligand molecule. which is of additional benefit in the case of acidic proteins. where conformationally sensitive protein kinases. 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.5) as well as reducing agents such as heterobifunctional reagents are available for introduction of reactive maleimido groups to the sensor surface. 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. Aldehyde coupling is particularly useful for site directed immobilization of glyco-conjugates. sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate).

2005. Figure 5 Bioconjugation..3 Assay types 1. 2001.. Indirect (capture) immobilization a) General capture methods Capture approaches provide an alternative to covalent immobilization and take advantage of tags commonly used for ligand purification. monitoring steps involved in complex formation (Schuster et al. SPR technology is increasingly being used to monitor immune responses either to an immunotherapeutic protein.g. Thorpe and Swanson.. kinetics. This technique involves high-affinity capture of the ligand onto a capturing molecule that has been covalently immobilized using one of the techniques described earlier (Fig. 2001). regeneration of the surface removes both the ligand and the analyte at the end of an assay cycle such that fresh ligand must be captured for a new cycle. Ca2+. A small volume of analyte can be tested easily for selective binding to 2 to 400 targets simultaneously. 2003. 2002.GST antibodies can be immobilized and used to capture GST-tagged molecules. Another benefit of capture approaches is the creation of a homogenous surface since all ligands are similarly oriented through a common site on the ligand (the tag). depending on the instrument platform chosen. 1993. 2001.g.1 Binding Specificity Biacore is well suited to carry out qualitative studies to confirm the specificity of interactions as well as quantitative measurements for affinity. Examples of specificity assays include identification of binding sites (Jokiranta et al. Thai and Ogata. analyte activity is not compromised since interactants do not need to be labeled.. anti. Monoclonal antibodies are frequently used as capture molecules.3. 2005). One simple assay requiring 8 . 2005)..ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci B. The requirement for ligand purity is less stringent for capture approaches than for covalent immobilization since the capture step can also provide a ligand purification step. e. 1. Clark et al. and concentration determination. It is possible to monitor a number of sequential binding events since each yields a concomitant increase in mass on the sensor chip surface and all stages in the binding process can thus be monitored. or even whole virus in research and preclinical environments (Alaedini and Latov.. and assessing cofactor requirements for an interaction to occur (e.. Swanson. Schlattner et al. The affinity of the ligand for the capture molecule should be sufficiently high to ensure that little or no ligand dissociates from the surface for the duration of an analysis cycle. Abad et al. Furthermore. vaccine.. 2000). In general.5). Rini et al.

Samples containing high refractive index solutions. the observed binding will be determined by the kinetic rate constants.1 to 10 times KD. If the rate of transport of analyte to the surface is much faster than the rate of analyte association with the ligand. The typical working range for affinity measurements with Biacore is picomolar to high micromolar for KD (M). It is important to note that mass transport is a well-understood physical property of the system and partial mass transport limitations can be accounted for during data analysis (Myszka et al. 2005). should either be exchanged into the running buffer or the concentration of the high refractive index component should be matched precisely in the continuous flow running buffer. 1. 1998. the binding interaction will be limited by the rate of analyte transport and there will be partial or no kinetic information in the binding data (mass transport limitation). thereby providing valuable information regarding complex formation and complex stability that is not revealed through affinity measurements. if mass transport is much slower than association. In practice. Although it is not necessary to reach equilibrium. the antibody should be immobilized or captured on the surface and the antigen used as analyte to avoid avidity effects resulting from the bivalent nature of the antibody. Rate constants can provide a link between protein function and structure. the phenomenon of mass transport should be considered.. Kinetic assays should include a series of start-up cycles using buffer as analyte to equilibrate the surface as well as cycles with zero concentration of analytes as part of the concentration series for the purposes of double-referencing (Myszka. For analyte molecules to bind to a ligand on the sensor surface.3. To measure rate constants accurately. Analyte concentrations must be accurately known to determine correct association rate constants. through the evaluation of the impact of amino acid substitutions on an interaction. whereas traditional endpoint assays (e. glycerol. thus. a measurable decrease in signal should occur during the dissociation period.. while low ligand densities reduce analyte consumption in the surface layer. in the case of an antibody-antigen interaction. To accurately determine dissociation rate constants. This is often most easily achieved through dilution of a concentrated analyte stock into running buffer. or DMSO. kinetic experiments should be designed such that the data are described by the simplest interaction model. they must be transported from the bulk analyte solution to the surface. spanning the range of 0.. Optimal assay conditions to minimize mass transport limitations to measure rate constants are a combination of high flow rates and low surface binding capacity. the rate of transport of the analyte to the surface is proportional to the cube root of the flow rate. Reliable detailed kinetic analysis requires data from four to six analyte concentrations. e. Under laminar flow conditions used in Biacore.and low affinity antibodies.2 Kinetics and Affinity Analysis A hallmark of Biacore’s SPR biosensors is the ability to derive kinetic association and dissociation rate constants from real-time measurement of binding interactions. For example.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci small sample volumes can provide information regarding antibody isotype and active or relative concentration in serum. Association rate constants can be measured ranging from 103 to 108 M−1 sec−1 and dissociation rate constants from 10−5 to 1 sec−1 (Karlsson. such as high salt. this translates to using ligand densities that result in a maximum analyte binding response no greater than 50 to 150 RU and flow rates >30 μl/min. the antibody may not dissociate from the antigen immobilized on the chip surface before binding 9 . Avidity refers to the ability of an antibody to bind to two antigen molecules simultaneously. Thorpe and Swanson. proper experimental design is critical. 1999). so that can lead to low signal-to-noise ratios. 1999). Affinity values can be derived either from interactions that have reached equilibrium or from the ratio of the dissociation and association rate constants in cases where the system does not reach steady state during the time frame of the experiment. If possible. However. ELISA) often fail to detect fast-dissociating antibodies (Swanson. Karlsson. Analytes should be in the same buffer as the continuous flow buffer to minimize bulk refractive index differences to avoid the so called bulk effect. As with all surface-based analysis methods. High flow rates minimize the diffusion distance from the bulk flow to the surface. 1999) during data analysis. it is recommended that the association times used be sufficient for at least one analyte concentration to reach steady state. and is also influenced by the dimensions of the flow cell and the diffusion properties of the analyte.g.g. Another advantage of using Biacore for immunogenicity studies is the ability of the technique to detect both high. 2003.

Since SPR is a noninvasive technology (i. 4. 1997. At t = 100 s. a thermodynamic analysis of a biomolecular interaction is also possible. Impurities from partially purified material can complicate the results by affecting the accurate determination of analyte concentration or introducing nonspecific binding. 1999. the refractive index of the medium adjacent to the sensor surface increases. 2001. by definition. 2003. In addition to determining the interaction affinities and kinetics. At t = 0 s. and the receptor–analyte complex is allowed to dissociate. Analysis of this part of the binding curve gives the observed association rate (kobs). 2000. 5. the analyte solution is replaced by buffer. 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. 1. a solution of analyte in the running buffer is passed over the receptor.. Analysis of these data gives the dissociation rate constant (kdiss) for the interaction. Many complexes in biology have considerable half-lives.e. which leads to an increase in the resonance signal.3. Myszka. If the concentration of the analyte is known. Several excellent reviews on the topic provide detailed guidelines on experimental setup and interpretation of results (Karlsson and F¨alt. Receptor molecules are immobilized on the sensor surface. no light penetrates the sample). it is possible to measure sub-femtomole 10 . 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. 2004). Myszka. buffer is contacted with the receptor through a microfluidic flow cell or through a cuvette.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci another antigen molecule. Lastly. 3. Karlsson and Larsson. As the analyte binds to the surface. At t = 320 s. high salt or low pH) is used at t = 420 s to disrupt binding and regenerate the free receptor. Van Regenmortel. then the association rate constant of the interaction (kass) can be determined. 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. Rich and Myszka. It is also important that both the ligand and analyte be as homogeneous as possible. Figure 6 Binding kinetics: 1. Avidity effects will slow down the dissociation rate yielding enhanced affinity values compared to those measured from a 1:1 interaction. and cannot be used with unpurified samples.3 Concentration Analysis Most chemical and spectroscopic methods used to quantify proteins measure total protein content. At equilibrium. 2. analyte should be injected over both a reference surface and an active ligand surface. do not distinguish active from inactive molecules. the amount of analyte that is associating and dissociating with the receptor is equal. 6. The response level at equilibrium is related to the concentration of active analyte in the sample. so a pulse of a regeneration solution (for example.

usually requires very small amounts of material (2 to 10 μg). therefore. and available tag should be considered when deciding which interactant to immobilize. For analytes >5000 Da. General properties such as purity. mass. the low-molecular-weight analyte competes with a fixed concentration of a high-molecular-weight molecule that shares the same binding site. The purity of the ligand is very important to ensure binding specificity as well as binding capacity. either direct or via capture. Enhancement not only increases the dynamic range of the assay but can also improve assay specificity. typically an antibody. which will complicate the determination of ligand density. the Rmax equation (eq. 6His). However. 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.. Inhibition or competition assay formats are well suited for quantification of low-molecular-weight molecules (<5000 Da). to name a few (Nelson et al.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci amounts of analyte bound to the sensor chip surface from complex matrices such as food products. which is not available with standard end-point assays. Generally. then that protein could be captured as the ligand using an anti-tag antibody surface.. 2000). concentration analysis with Biacore requires no separation and washing steps and..3. If one of the interactants has a protein purification tag (e. When using Biacore to measure the binding kinetics of an antibody-antigen interaction. 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. The point at which analyte concentration is measured can be chosen. since binding responses are monitored continuously. Unlike many other immunoassays. 1. Impurities in the analyte material can complicate results by introducing nonspecific binding or by affecting the accurate determination of analyte concentration. giving flexibility in the assay design. Biacore evaluation software provides a direct readout of analyte concentration from a calibration curve generated using standard analyte concentrations. 1. 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. stability. valency. 1. Various assay formats are possible. because immobilization. In a competition assay. quantity. low-affinity interactants. Biacore systems can detect molecules as small as 200 Da. it could be used as the ligand. and cell extracts. Immobilization of the bivalent protein 11 .1 in the Immobilization level paragraph) should be used to calculate whether the ratio of the molecular masses of the interactants would limit the response of the interaction. Instrument automation decreases operator involvement thereby leading to highly reproducible measurements. Inhibition assays rely on mixing the sample with a known concentration of a molecule that binds with high affinity. the size of the interactants is not usually a limiting factor. the antibody should be immobilized as the ligand to avoid binding avidity effects that result from the bivalency of the antibody. it is possible to quantify fast-dissociating.4 Thermodynamics By studying temperature dependence of rate and affinity constants it is possible to determine thermodynamic parameters for a binding interaction. Impurities in the ligand preparation can be immobilized as well as the ligand. If an interactant is of limited quantity. 1998). 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. a direct binding assay format can be used with the optional response enhancement from a secondary detecting molecule.1 Which interactant should be immobilized as the ligand? Often protein-protein interactions can be studied using X100 with either interactant immobilized as the ligand. serum.g.4 Experimental design Some experimental design questions should be considered when using Biacore to measure the binding kinetics of protein interactions.4. 4. isoelectric point.

Generally. contribute to increased ligand immobilization levels. the capture molecule serves as a suitable control for the reference surface. 75% surface activity can be expected for ligands immobilized directly by amine coupling. Ligands that are directly coupled to the surface will have a random orientation.g. However. Conversely. 1. 1. In this case. The various surface chemistries for direct coupling are described in paragraph 2. which results in a calculated Rmax of 189 RU. In this case. a surface that is treated with the same coupling chemistries used to immobilize the ligand serves as a suitable control for the reference surface.3 in the Immobilization level paragraph. 1.4 How much of the ligand should be immobilized? A low ligand density surface is optimal for accurate measurement of the binding kinetics of an interaction. such as a scrambled peptide sequence for a protein-peptide interaction. some binding sites may not be accessible. and (5) ionic strength. The maximum binding capacity (Rmax) of the immobilized ligand should be in the range of 50 to 150 RU. The ligand capture approach can thus offer advantages over direct immobilization. The BIAevaluation software generates an experimental Rmax value after curve fitting as described in the Evaluation of the Kinetic Analysis section.4. (3) activation time (EDC/NHS mixture).5 Is the immobilized ligand active? Surface activity should be calculated using eq. (1) ligand concentration. the experimental Rmax for the anti-β2μ-globulin surface is ∼ 40 RU due to a lower-than-expected surface activity.e. i. higher concentrations and higher activation or contact times. Lower ligand binding levels can be reached by decreasing the first four factors or by increasing the fifth. The higher binding activity of the captured ligand can be attributed to a homogeneous presentation of the ligand. Immobilization levels depend on five main factors. Use the rearranged Rmax equation (eq. employing the ligand capture approach can add an on-chip purification step that will usually result in improved surface activity. 1200 RU was targeted.2 in the Immobilization level paragraph) to determine an appropriate immobilization level that will generate an Rmax of ∼100 RU. employing an alternative surface chemistry should be considered to improve the surface activity. 4. For the direct immobilization of the anti-β2μ-globulin described in this protocol. For some interactions.3 What is a good control surface? Usually. When using the ligand capture approach.. 4. 12 .4. 1. The use of bovine serum albumin as a control ligand is not recommended due to its tendency to nonspecifically bind to many proteins. which can be critical for some proteins.4. and (2) capture onto a surface that has been derivatized with a binding molecule (e.. via a purification tag).ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci enables determination of the kinetic rate constants by fitting the responses to a simple 1:1 (Langmuir) binding model.2 How should the ligand be immobilized? The two general techniques for immobilization of the ligand are: (1) direct coupling to the sensor chip surface using one of several surface chemistries. Impurities in the ligand preparation can also be immobilized when using a direct coupling approach. which could affect the binding capacity of the surface. captured ligands often exhibit higher binding activity. (2) pH. therefore. thereby lowering the apparent surface activity. availability of the binding site. as well as lower ionic strength. a nonspecific ligand may act as an appropriate control. Poor surface activity (<50%) can result from denaturation of the ligand due to the preconcentration solution. However. or inactivation of the binding site due to the immobilization chemistry. (4) injection time (ligand).4. and use of fresh ligand for each binding cycle (no damage due to regeneration solution).

4.0. 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.to 1-pH unit below the pI of the protein.7 How should the surface be regenerated? If the analyte does not dissociate from the ligand within a reasonable time (e. the activated sensor chip surface maintains a net negative charge during the immobilization 13 .g. 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. or DMSO. Nonspecific binding to a capture surface can be resolved by switching to another capture molecule. 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.. the optimal pH for preconcentration will be a 0. An acidic or basic solution or chaotropic salt solution injected at a high flow rate (50 μl/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. not on the reference flow cell.8 Bulk refractive index 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. Determination of ideal regeneration conditions may require optimization.9 Immobilization pH scouting Immobilization by covalent coupling cannot be repeated on the same sensor chip surface. An immobilization and regeneration database that provides examples and suggestions for a wide range of interactants is available at the Biacore Website. 1. pH 2.4.4. Combining solutions together into “regeneration cocktails” can often produce efficient regeneration conditions (Andersson. 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) Perform the sample injection on both the specific cell and a reference cell. such as CM4.5. a combination of basic and chaotropic solutions such as 10 mM sodium hydroxide and 500 mM sodium thiocyanate. using same coupling chemistry to immobilize a similar amount of inactivated ligand). Identify a solution that removes the analyte from the immobilized ligand without affecting the ligand activity.05% polysorbate 20. pH 2. Many proteins exhibit limited stability in the low-ionic-strength. preconcentration is an important strategy to control the immobilization level for experimental optimization and efficiency. and 1 M sodium chloride. a combination of low pH and high ionic strength solutions such as 10 mM glycine.0. Nonspecific binding to the CM-dextran can be minimized by addition of soluble CMdextran to the sample at 1mg/ml. then the surface should be regenerated. For example. to the sample and running buffers. such as high–salt solutions.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 1.. 20 min). This reference cell must be prepared in a manner as similar as possible to that used for the specific one (e. 1999). Samples containing high refractive index solutions. When using standard amine coupling. Therefore. 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. ∼40% of the carboxymethyl groups are converted to reactive esters after a 7-min activation. 1.g. a peptide with randomized sequence). 1. low-pH solutions used for preconcentration. Typically. a combination of a low pH solution and detergent such as 10 mm glycine. glycerol. dilution of the ligand just prior to use is recommended. Nonspecific binding should be checked by one of the following methods. Thus. and 0.. (2) Inject a nonspecific analyte (e.4. therefore. The bulk refractive index has a square-shaped response. Hydrophobic nonspecific binding often can be minimized by the addition of a detergent. such as 0.6 Is binding of the analyte specific to the ligand? Inspect the analyte response on the reference flow cell to identify nonspecific binding due to electrostatic or hydrophobic interactions with the sensor chip surface. Always perform pH scouting on the flow cell that will be used for the immobilization.05% polysorbate 20 or 10 mMCHAPS.g. while nonspecific binding will typically have an increasing response on the reference.

11 Immobilization levels The binding capacity of the surface will depend on the level and activity of immobilized ligand. The SPR response correlates with change in mass concentration on the sensor chip surface.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.3: %ligand activity = experimental Rmax/theoretical Rmax × 100% (4. and therefore. Partial mass transport limitations can be accounted for in data analysis using a binding model that includes a mass transfer rate constant. Mass transfer limitations can result in calculated binding kinetics that are slower than the true binding kinetics. The quality of the binding data that can be obtained is reflected in the ligand activity as shown in the following Equation 4. such as Tris or sodium azide.1: Rmax = (analyte MW/ligand MW) × RL × Sm (4.10 Amine coupling Buffer components that contain primary amines. Figure 7. Use of a low ligand density will minimize mass transfer limitations during the interaction. MW is the molecular weight (mass) of the ligand and analyte. 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. The theoretical Rmax is calculated from Equation 4.3) 14 . 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. An experimental Rmax must be determined to assess the percent activity of the immobilized ligand. which enables preconcentration. depending on sensor chip type and blocking procedure. The term Rmax describes the maximum binding capacity of the surface ligand for analyte in RU.4. PH Scouting / concentration scouting sensorgram 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. Note that prepared surfaces can have a negative charge during experiments.4. Ligand contact should be completed within 15 min after activation of the surface with EDC/NHS to ensure coupling before the reactive esters on the surface hydrolyze. depends on the molecular weight (mass) of the analyte in relation to the number of ligand sites on the surface. and Sm is the stoichiometry as defined by the number of binding sites on the ligand. 1. RL (ligand response) is the amount of immobilized ligand in RU.2: RL = (ligand MW/analyte MW) × Rmax × (1/Sm) (4. must be avoided in amine coupling to prevent competition with the ligand for coupling. 1.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci procedure.1) Where.

Avidity phenomena are extremely important and must be considered for multivalent antigens (natural antigens binding to several Ig molecules and forming the so-called immune complexes) and for multivalent Ig molecules.4.. 15 .. 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. equilibrium constants usually range from 107 to 1010 –1 M . However. The best alternative for direct kinetic analysis of small analyte binding is the surface competition assay. even this method has an important limitation: macromolecules sharing the same binding specificity with the small target analyte (e. such as IgM (Abbas et al.g. The range of rate constants amenable to study by SPR varies with the molecular weight of the analyte and with the sensitivity of the particular biosensor employed. Direct quantitative evaluation of interaction kinetics outside these limits is often impossible. 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. 1:1 affinity binding is to be expected. Figure9 Possible studies for binding kinetics assays. Figure 8 Schematic definition of the affinity constant in an antibody-antigen interaction. and immunoglobulins with KA ≤104 M–1 for a particular antigen are ineffective. Thus. Normally. 1997). In the case of antibody–natural antigen interactions. IgGs have one effective binding site if an affinity interaction is taking place. for a monovalent antigen.12 Kinetic constants measurable by direct SPR Antibody affinity is defined as depicted in Figure 8. 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.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 1. viral proteins) are not easily available. When both Fab fragments of the same IgG molecule interact with a multivalent antigen. Other alternative SPR methodologies are briefly commented in Figure 9. then an avidity interaction is taking place and higher stabilization of the Ab-Ag antibody is observed.

. sample buffer should resemble the running buffer as closely as possible.  Signal calibration (“normalizing”): 40 min. as well as set-up of the kinetic analysis.. the activity of the immobilized ligand after storage should be evaluated. avidity. Deviations from pseudo-first-order kinetics.. Derivatized sensor chips can be stored at 4◦C in a humid environment. involving conformational changes). increasing buffer flow rate (higher than 30 µl/min). such as analyte multivalency.  Data analysis: 60 min (variable. 1.  Weekly “desorb” operation: 30 min. however. Thus. Hall et al.4..4. depending on the quality of the fit). Another precaution aimed at eliminating mass transport effects in complex dissociation (i. or complex binding mechanisms (e.4. Such bulk RI response may be eliminated by subtraction of a blank run. especially those including organic solvents. Data analysis requires ∼1 hr. In terms of the time required to complete the assays as described here.. Analyte heterogeneity can be reduced through additional sample purification steps. 16 . This can be achieved by decreasing ligand immobilization levels (to the minimum giving a satisfactory signal-to-noise ratio). 1995. one must keep in mind that. 1997)..  Monthly “sanitize” operation: 40 min. alternative regeneration agents must be tested and a cocktail approach (Andersson et al. O’Shannessy and Windzor. Mass transport limitations can be tested through analysis of effects of different buffer flow rates on initial binding rates (slopes at the beginning of association steps). Detailed time considerations (i. mass transport effects must be minimized. 1996. preventing binding phenomena from occurring. 1996). The first is mainly due to random immobilization procedures and can be minimized by lowering binding levels or using oriented methodologies such as streptavidin–biotin or anti-Fc–Fc indirect immobilization. When these effects are present.  System priming: 10 min.  One analyte run (sample plus regeneration agent): 20 min. Inefficient regeneration steps are often to blame.e. 1.13 Baseline levels Baseline increase over repeated cycles can be observed.4. Beware of “exotic” buffers. step by step) are as follows:  Pre-concentration assays: 60 min. which can be run overnight. 1.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 1. 1999 a.  Evaluation of regeneration conditions: 30 min. and increasing analyte concentration (as long as surface capacity is not saturated).14 Buffer refractive index response When sample and running buffers are different. can result from several factors.e. b) may be required. these can cause dramatic compression effects on the dextran hydrogel matrix. the only way to take them into account is to use the more complex fitting models. for kinetic studies. although it may be difficult to judge whether a better fitting model corresponds to the real interaction mechanism (Schuck. nonspecific bulk refractive index (RI) jumps take place (square-wave shaped signals superimpose to the binding curves).g. with bound analyte not being fully washed off after each binding cycle. one of the most difficult problems to solve in biosensor analysis (Morton et al. rebinding) consists of replacing buffer with ligand solution during the dissociation phase. Other common sources of deviation are ligand or analyte heterogeneity.  Two blank runs: 20 min. Whatever they may be. The sources of deviation most difficult to deal with are those intrinsic to the binding partners or phenomena. but useful information from stages immediately after the injection pulse may be lost. one 8-hr day should be sufficient to accomplish the protocols for surface preparation and assay development.16 Time Considerations The overall time that it takes to develop and conduct kinetic analysis of a protein interaction using Biacore will depend on the nature and behavior of the interactants.15 Fitting data Antigen-antibody (Fab) interactions are expected to display a Langmuirian behavior on the biosensor.  Covalent immobilization: 30 min.

4 μl HBS-P+ to prepare the 32 nM sample. pH 5. 12) Poor the solution in the top part of the stericup. 2.9 ml milliQ water.0 10 mM sodium Acetate pH 5.5 μM stock β2μ-globulin (100 μg/ml) into 1494. 8) Filter the solution through a 0. 13) Wait until the solution is filtered. 4) Take a 200 ml graduated cylinder. 15) Switch off the pump and close the vent hood.1. β2μ-globulin diluted series preparation (for Protocols 1 and 2) 18) Dilute 5. 16) Equilibrate to room temperature before use. pH 5.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 2. Proceed this way down to 4 nM concentration. 10 μg/ml anti-β2μ-globulin antibody in 10 mM sodium acetate.5 1) Dilute 100 μl of 3M sodium acetate pH 5. 17 .5. 7) Mix the solution.5 50 mM NaOH 100 μg/ml (8. pH 5.6 μl of 8. pH 5.2-μm 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. 19) Prepare two-fold serial dilutions of 32 nM β2μ-globulin down to 4 nM.5 μM) β2μ-globulin 25 µg/ml anti-mouse IgG antibody 10 mM sodium acetate.1 Reagents preparation Materials HBS-P+ 1 mg/ml anti-β2μ-globulin antibody 10 mM sodium acetate. 5) Put 20 ml of 10× HBS-P+ 6) Add 180 ml MQ water.5 17) Dilute 2 μl of 1 mg/ml anti-β2μ-globulin into 198 μl sodium acetate.5 into 29.1 Protocol 1 Buffer preparation 3) Dilute 10× HBS-P+ from stock to 1×. 50 mM NaOH 2) Dilute 500 μl of 1M NaOH into 9500 μl milliQ water. take 600 μl of buffer and 600 μl of 32nM β2μ-globulin diluted solution. PRACTICAL WORK 2. To prepare the 16nM solution. 14) Close immediately the bottle with the filtered solution.

but is activated and blocked with the same chemistry as the active surface. 75% surface activity can be expected for ligands immobilized directly by amine coupling. pH 5. Amine coupling is the most widely applicable approach for immobilization of ligands.0. 25 µg/ml anti-mouse IgG antibody in 10 mM sodium acetate. The immobilization surface preparation application wizard is used to create active and reference surfaces automatically. 2) Contact of the ligand with the activated surface. the active and reference surfaces are prepared on Sensor Chip CM5 using amine-coupling chemistry.2. 2. The active surface will contain the anti-β2μ-globulin antibody ligand.0 3) Dilute 5 μl of 1 mg/ml anti-mouse IgG antibody into 195 μl sodium acetate.1. 3) Blocking of unreacted sites. pH 5. the run will be stopped.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci Referring to the serial dilution section in the Master handout. Note that when using the Aim for Immobilized Level option in the application wizard. 18 . 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 Stock solution 8500 1 2 3 4 Concentration [nM] Volumes needed [µl] Sample Buffer 32 4 2. and the reference surface will contain no ligand. several coupling chemistries are available for covalent immobilization of the ligand to the sensor chip surface. Generally.2 Protocol 2 Buffer preparation 1) Same as Protocol 1 5 μg/ml anti-β2μ-globulin antibody in HBS-P+ 2) Dilute 2 μl of 1 mg/ml anti-β2μ-globulin into 398 μl HBS-P+. the control software performs an injection of ligand to test for preconcentration prior to activating the surface to ensure that the desired immobilization level can be achieved during amine coupling. If the preconcentration response is too low to achieve the target immobilization level. There are three steps in amine coupling: 1) Activation of the surface.2 PROTOCOL 1: Immobilization of the ligand on a sensor chip by amine coupling As explained in paragraph 1. After identification of the optimal pH for preconcentration of the ligand.

pH 5.Set the target level at 1200 RU .2. Biacore is always kept with a maintenance chip inside.For cell 1 select “Blank Immobilization” with the “Amine” method. 19 . and unlit when no chip is inserted.5 10 mM sodium acetate.Enter 50mM NaOH for the Wash Solution.For cell 2 select “Aim for immobilized level” with the “Amine” method. When undocked. . 6) 7) 8) Choose the Flow Cell for Immobilization. Check “Prime before run” to flush the flow system with fresh buffer at the start of the run. Click the Command tab and then select the Dock command.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci Materials 200 ml HBS-P+ 10 μg/ml anti-β2μ-globulin antibody in 10 mM sodium acetate. the sensor chip should easily slide out of the instrument.5 50 mM NaOH Sensor Chip CM5 1. On the Control Software Tool Bar click on “Undock Chip” and follow the displayed instructions. 2. Note that the sensor chip indicator light on the front of the instrument is lit steadily when a chip is docked. 5) Define the Immobilization Setup. . pH 5. This step will determine how the ligand is immobilized on the surface. . Choose the chip type CM5. Click on “Next”.Enter the ligand name “anti-β2μ-globulin antibody” . Click on “Wizards” and select the “Immobilization” wizard type from the list in the left-hand panel. flashing when a chip is inserted but undocked. 2) Change the sensor chip.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. 3) Insert and dock the new Sensor Chip CM5. Then click on “New”.1 Ligand Immobilization 4) Create a New Wizard Template.

Prepare Run Protocol : The following text describes the preparations you need to complete before starting the run. Replace the rack and click OK to lock the rack. The sample rack can be removed from the sample compartment when the “Rack locked” indicator on the instrument is not lit.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 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. 10) 11) 12) 13) 14) 15) 16) 17) Click “Load Samples” to release the sample rack so that you can load samples. 20 . Once you checked it is correct. The listed volumes are minimum valuesNote: The position marked H2O requires an uncapped vial with MQ water. Make sure you don’t make bubbles while putting the reagents into the vials. Click on “Next” on the “Immobilization. Wait until the “Rack Locked” indicator on the instrument is turned off before attempting to remove the sample rack. Close all the vials with the orange rubber caps and place them in the right order. click on “Start”.Rack Positions” window.

5. Click on “Next”. Note: The position marked H2O requires an uncapped vial with MQ water. For the First regeneration:  Set “Solution”: 10mM Glycine pH 2. Check “Run startup cycles”. 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.  Set “B2micro” as “Sample id”. Define Samples. pH 2. Define Injection parameters. Click on “Wizards” and select the “Kinetics/Affinity” wizard type from the list in the left-hand panel.  Set “Dissociation time”: 300 s. Chose “CM5 “ sensor chip and “Multi-cycle” kinetics type. Click on “Next”. Samples will be run in the order entered. Define System Preparation. Check “Prime before run” to flush the flow system with fresh buffer at the start of the run. Define the Injection Sequence.5 μM) β2μ-globulin in HBS-P+ 10 mM glycine. For the sample:  Set “Contact time”: 120 s.2. Close all the vials with the orange rubber caps and place them in the right order. Set 1 regeneration and then click on “Next”. 19) 20) 21) 22) 23) 24) 21 . Then click on “New”.  Set “Stabilization period”: 60 s.5 18) Create a New Wizard Template.  Enter the sample concentrations in ascending order. Prepare the rack.  Set “11800” Da as Molecular Weight. Click on “Next”. Write “buffer” as solution and set 3 cycles.  Set “Contact time”: 30 s.2 Materials Binding kinetics Diluted series of 100 μg/ml (8.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 2.

Materials 100 mM N-hydroxysuccinimide (NHS) 400 mM N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) 25 µg/ml anti-mouse IgG antibody in 10 mM sodium acetate. The use of an anti-mouse IgG surface to capture the mouse monoclonal anti-β2μ-globulin antibody ligand is described here. The volume of anti-β2μ-globulin required to reach the desired ligand level must be determined. Biacore is always kept with a maintenance chip inside. click on “Start”. experimental set-up for kinetic analysis using ligand capture is described. Wait until the “Rack Locked” indicator on the instrument is turned off before attempting to remove the sample rack. Amine coupling is used to prepare an anti-mouse IgG surface on the reference and active flow cells. Replace the rack and click OK to lock the rack. Lastly. Perpare Run Protocol. regeneration of the surface usually removes both the bound analyte and the captured ligand for each cycle of analyte binding.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.Rack Positions” window.5-ml polypropylene tubes with rubber caps (Biacore) 1) Remove sensor chips from the fridge at least 30 min before starting the experiment.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 25) 26) 27) 28) Click “Load Samples” to release the sample rack so that you can load samples. 2. pH 1. 2) Change the sensor chip. Once you checked it is all correct. 22 . pH 5. The sample rack can be removed from the sample compartment when the “Rack locked” indicator on the instrument is not lit. Regeneration conditions for the anti-mouse IgG surface are provided.7 Sensor Chip CM5 1.0 1 M ethanolamine 5 µg/ml solution of anti-β2μ-globulin antibody in HBS-P+ 10 mM glycine. Click on “Next” on the “Immobilization. In this protocol.

Then click on “New”. This step will determine how the ligand is immobilized on the surface.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci On the Control Software Tool Bar click on “Undock Chip” and follow the displayed instructions. 10) Click on “Next”. The listed volumes are minimum values. When undocked. Note that the sensor chip indicator light on the front of the instrument is lit steadily when a chip is docked. 11) Prepare the rack. Choose the chip type CM5.3. 9) Check “Prime before run” to flush the flow system with fresh buffer at the start of the run. 2. 5) Define the Immobilization Setup. Click on “Wizards” and select the “Immobilization” wizard type from the list in the left-hand panel. flashing when a chip is inserted but undocked. 23 . Note: The position marked H2O requires an uncapped vial with MQ water. 7) Enter “anti IgG” as a capturing molecule. 8) Choose “Specify contact time” and set contact time 420 s. and unlit when no chip is inserted. 6) Choose for Cell 1 and 2 immobilization through “Amine” method. 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.1 Ligand Immobilization 4) Create a New Wizard Template. Make sure you don’t 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. 3) Insert and dock the new Sensor Chip CM5. the sensor chip should easily slide out of the instrument. Click the Command tab and then select the Dock command.

16) Replace the rack and click OK to lock the rack.7 5 μg/ml solution of anti-β2μ-globulin in HBS-P+ 20) Create a New Wizard Template. 24 . 15) The sample rack can be removed from the sample compartment when the “Rack locked” indicator on the instrument is not lit. 14) Wait until the “Rack Locked” indicator on the instrument is turned off before attempting to remove the sample rack.2 Binding kinetics Materials Diluted series of 8.3.5 μM stock β2μ-globulin in HBS-P+ 10 mM glycine. 17) Click on “Next” on the “Immobilization. Then click on “New”. click on “Start”.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 13) Click “Load Samples” to release the sample rack so that you can load samples. 18) Prepare Run Protocol : The following text describes the preparations you need to complete before starting the run.Rack Positions” window. 2. 19) Once you checked it is correct. Click on “Wizards” and select the “Kinetics/Affinity” wizard type from the list in the left-hand panel. pH 1.

Click on “Next”. Note: The position marked H2O requires an uncapped vial with MQ water.  Enter the sample concentrations in ascending order. 25 . 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.  Set “11800” Da as Molecular Weight. Click on “Next”. Write “buffer” as solution and set 3 cycles. Check “Ligand capture”.  Set “Dissociation time”: 300 s. Close all the vials with the orange rubber caps and place them in the right order. Check “Run startup cycles”.  Set “Stabilization period”: 60 s. For the capture:  Set “Contact time”: 180 s.  Set “B2micro” as “Sample id”. For the First regeneration:  Set “Solution”: 10mM Glycine pH 1.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 21) 22) 23) 24) Define the Injection Sequence. Define Injection parameters. Define Samples. Chose “CM5 “ sensor chip and “Multi-cycle” kinetics type. Set 1 regeneration and then click on “Next”. Check “Prime before run” to flush the flow system with fresh buffer at the start of the run.  Set “Stabilization period”: 60 s. Include in addition at least one blank sample and a replicate of a non-zero concentration. Click on “Next”. For the sample:  Set “Contact time”: 120 s. Define System Preparation.  Set “Contact time”: 120 s.7. Prepare the rack. 25) 26) 27) 28) Click “Load Samples” to release the sample rack so that you can load samples.

3 DATA ANALYSIS 3.2 Kinetic Analysis Q7. Explain briefly the differences and advantages of subsequent/continuous injections of ligand (or capture molecule). Once you checked it is all correct. Replace the rack and click OK to lock the rack. Which is the theoretical binding capacity of the sensor chip surface? Does it correspond to the experimental data? If not.1 Immobilization Sensorgram Analysis . Q4. and how to detect it. why this difference? Q5. To how many molecules per cm2 do they correspond? (Hint: 1RU = 1pg/mm2 . check if the RU bound to the surface corresponds to the data settings. The sample rack can be removed from the sample compartment when the “Rack locked” indicator on the instrument is not lit. Perpare Run Protocol. Q9. 26 . Why do we use different pH of sodium acetate for the two protocols? Q3. Explain briefly what pH scouting is. Discuss the kinetics curve: explain in your lab notebook step by step the process Q8. How do mass transfer phenomena can affect the binding kinetics analysis? Q11. What kind of curve have you obtained? Which fitting is suitable for the actual case? Discuss briefly the other types of fitting. Explain briefly what the mass transfer phenomena is. Compare the values between two protocols and discuss. Q10.Rack Positions” window. Why do we add ethanolamine during immobilization? What might happen if this step is skipped? 3. Discuss the immobilization sensorgram curve: explain in your lab notebook step by step the immobilization process using the immobilization sensorgrams Q2. MWligand = 150000Da) Q6. Discuss the role of pH (of sodium acetate) during the immobilization process. From the Immobilization Sensorgrams. click on “Start”. and why it is important.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci 29) 30) 31) Wait until the “Rack Locked” indicator on the instrument is turned off before attempting to remove the sample rack. Q1. How can you identify and reduce nonspecific binding? Discuss the possibility to use another type of sensor chip. Identify the association and dissociation constant from the data. Click on “Next” on the “Immobilization.

biacore.com/lifesciences/index.Guiducci.biacore.com/lifesciences/service/training/courses/usa canada/biacorebasics/index 27 . http://www. Why does it occur? How do you identify and get rid of it? Q13. EPFL Master BE course. the sensorgram is affected by bulk effect. Why in kinetics analysis you need serial dilutions? 4 REFERENCES Fundamentals of biochemical sensing systems. In some cases. Prof. https://www. 2010. 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.html Biacore catalog. http://www.biacore. 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.ADVANCED SPR BIOENGINEERING METHODS LABORATORY Carlotta Guiducci Q12.html Immobilization and regeneration database.com/lifesciences/service/online support/irdb/index.