SURFACE PLASMON RESONANCE BASED SYSTEMS

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1 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 () 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 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). 1

2 TABLE OF CONTENTS 1 Theory Biacore Surface preparation Assay types Experimental design Practical work Reagents preparation Protocol 1:Immobilization of the ligand on a sensor chip by amine coupling Protocol 2:Immobilization of the ligand on a sensor chip using ligand capture method Data analysis Immobilization Sensorgram Analysis Kinetic Analysis References

3 1 THEORY The optical phenomenon of Surface Plasmon Resonance () 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 () 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 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 Biacore s 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 System features Biacore s optical biosensors are designed around three core technologies: An optical detector system that monitors the changes in signal brought about by binding events in real time. An exchangeable sensor chip upon which one of the interacting biomolecules is immobilized or captured. The resulting biospecific surface is the site where biomolecular interactions occur. A microfluidic and liquid handling system that precisely controls the flow of buffer and sample over the sensor surface. These systems are contained within the processing unit that communicates with a computer equipped with control and data evaluation software. Figure 1 Surface Plasmon Resonance principle. 3

4 1.1.2 The detection system Surface Plasmon Resonance () is a phenomenon that occurs in thin conducting films placed at the interface between two media of different refractive indices. In Biacore systems, 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. 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.1). Under these conditions, the light leaks an electromagnetic component called an evanescent wave across the gold interface into the sample/buffer solution. At a certain angle of incident light, 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 ( angle). When a change in mass occurs near the sensor chip surface, e.g., as a result of a binding event, the angle of light at which occurs shifts due to a change in refractive index near the sensor chip surface. A sensorgram depicts changes in angle in real time, with responses measured in resonance units (RU). One RU corresponds to shift in angle, or to 1pg/mm 2 of molecule on the surface. Since only the evanescent wave penetrates the sample, measurements can be carried out with colored, turbid, or even opaque solutions without interference from conventional light absorption or light scattering. A sensorgram is a plot of the binding response in resonance units versus time in seconds, which is displayed and recorded as a change in mass occurs on the sensor chip surface (Fig. 2). The sensorgram provides essentially two kinds of information that are relevant to different types of applications: (1) the rate of interaction (association, dissociation, or both), which provides information on kinetic rate constants and analyte concentration; and (2) the binding level, which provides information on affinity constants and can be used for qualitative or semi-quantitative applications. 4

5 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 The microfluidic system Biacore systems make use of an integrated microfluidic cartridge (IFC) to deliver samples and buffer to the sensor surface. The IFC consists of a series of micro-channels encased in a plastic housing. In automated systems, samples are transferred through a needle into the IFC. Flow cells are created through pressure contact between molded channels in the IFC and the sensor chip surface. A series of computer-controlled pneumatic valves meters precise access to the channels and the flow cells. The design of the IFC, as well as the number and configuration of flow cells, is specific to each instrument platform. Pulse-free syringe pumps ensure a continuous flow of buffer over the sensor surface whenever samples are not being injected. There are two flow cells in the Biacore X instrument. 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. Dual-channel detection of the signal and serial flow allow simultaneous monitoring of both 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. Sample bound to the sensor chip surface can be recovered for complementary analysis and/or identification, e.g., using mass spectrometry. Biacore X is well suited for laboratory environments with low-sample throughput where many users handle different types of samples. 1.2 Surface preparation Sensor Chips Although can be generated in thin films made from conducting metals, all Biacore sensor chips are coated with a thin, uniform gold layer. 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 signal, gold is amenable to covalent attachment of surface matrix layers and, in physiological buffer conditions, gold is mostly inert. A variety of sensor surfaces are available to give a range of alternative possibilities for different experimental situations: 5

6 Figure3. 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. 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 Immobilization Chemistries A critical step in the development of reliable assays is the selection of the most suitable immobilization technique such that ligand activity is maintained and binding sites are available to interacting partners. Commonly utilized immobilization strategies are outlined here. Guidelines and a detailed review of immobilization chemistries have been published recently (Karlsson and Larsson, 2004). A. 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. All types of immobilization can be performed directly in the Biacore system, and a typical immobilization reaction usually takes <30 min to complete. 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. To ensure specificity, it is recommended that the ligand purity exceed 95%. a) Amine coupling Amine coupling is the most generally applicable covalent coupling chemistry used to immobilize protein ligands. 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. The dextran matrix on the sensor chip surface is first activated with a 1:1 mixture of 0.4 M 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS) to create reactive succinimide esters. The standard activation time is 7 min; this can be varied from 1 to 10 min to create fewer or more, respectively, reactive groups on the sensor chip surface depending on the immobilization level required. The ligand is then injected in low salt buffer lacking primary amines at a ph below the isoelectric point (pi) of the protein. Under these conditions, the ligand acquires a positive charge and is effectively preconcentrated into the negatively charged carboxymethyl dextran matrix. High local concentrations of ligand obtained through electrostatic preconcentration maximize the efficiency of amine coupling. Finally, unreacted esters are blocked with ethanolamine. The volume or 6

7 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, p38α and GSK3β, 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. Figure 4 Ammine coupling and Thiol coupling 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. 7

8 B. 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. 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.5). 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. 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). 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. Monoclonal antibodies are frequently used as capture molecules, e.g., anti- GST antibodies can be immobilized and used to capture GST-tagged molecules. In general, 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. Figure 5 Bioconjugation. 1.3 Assay types Binding Specificity Biacore is well suited to carry out qualitative studies to confirm the specificity of interactions as well as quantitative measurements for affinity, kinetics, and concentration determination. A small volume of analyte can be tested easily for selective binding to 2 to 400 targets simultaneously, depending on the instrument platform chosen. Furthermore, analyte activity is not compromised since interactants do not need to be labeled. 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. Examples of specificity assays include identification of binding sites (Jokiranta et al., 2000), monitoring steps involved in complex formation (Schuster et al., 1993; Clark et al., 2001; Thai and Ogata, 2005), and assessing cofactor requirements for an interaction to occur (e.g., Ca2+; Schlattner et al., 2001). technology is increasingly being used to monitor immune responses either to an immunotherapeutic protein, vaccine, or even whole virus in research and preclinical environments (Alaedini and Latov, 2001; Abad et al., 2002; Swanson, 2003; Rini et al., 2005; Thorpe and Swanson, 2005). One simple assay requiring 8

9 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) Kinetics and Affinity Analysis A hallmark of Biacore s biosensors is the ability to derive kinetic association and dissociation rate constants from real-time measurement of binding interactions, thereby providing valuable information regarding complex formation and complex stability that is not revealed through affinity measurements. Rate constants can provide a link between protein function and structure, e.g., through the evaluation of the impact of amino acid substitutions on an interaction. 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. The typical working range for affinity measurements with Biacore is picomolar to high micromolar for KD (M). Association rate constants can be measured ranging from 10^3 to 10^8 M 1 sec 1 and dissociation rate constants from 10 5 to 1 sec 1 (Karlsson, 1999). To measure rate constants accurately, proper experimental design is critical. As with all surface-based analysis methods, the phenomenon of mass transport should be considered. For analyte molecules to bind to a ligand on the sensor surface, they must be transported from the bulk analyte solution to the surface. Under laminar flow conditions used in Biacore, the rate of transport of the analyte to the surface is proportional to the cube root of the flow rate, and is also influenced by the dimensions of the flow cell and the diffusion properties of the analyte. If the rate of transport of analyte to the surface is much faster than the rate of analyte association with the ligand, the observed binding will be determined by the kinetic rate constants. However, if mass transport is much slower than association, 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). Optimal assay conditions to minimize mass transport limitations to measure rate constants are a combination of high flow rates and low surface binding capacity. High flow rates minimize the diffusion distance from the bulk flow to the surface, while low ligand densities reduce analyte consumption in the surface layer. In practice, 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. 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., 1998; Karlsson, 1999). Reliable detailed kinetic analysis requires data from four to six analyte concentrations, spanning the range of 0.1 to 10 times KD. Analyte concentrations must be accurately known to determine correct association rate constants. 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, so that can lead to low signal-to-noise ratios. This is often most easily achieved through dilution of a concentrated analyte stock into running buffer. Samples containing high refractive index solutions, such as high salt, glycerol, or DMSO, 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. 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, 1999) during data analysis. Although it is not necessary to reach equilibrium, it is recommended that the association times used be sufficient for at least one analyte concentration to reach steady state. To accurately determine dissociation rate constants, a measurable decrease in signal should occur during the dissociation period. If possible, kinetic experiments should be designed such that the data are described by the simplest interaction model. For example, in the case of an antibody-antigen interaction, 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. Avidity refers to the ability of an antibody to bind to two antigen molecules simultaneously, thus, the antibody may not dissociate from the antigen immobilized on the chip surface before binding 9

10 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 F alt, 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 (k obs ). If the concentration of the analyte is known, then the association rate constant of the interaction (k ass ) 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 receptor analyte complex is allowed to dissociate. Analysis of these data gives the dissociation rate constant (k diss ) 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 (K D = k diss /k ass ) 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 Concentration Analysis Most chemical and spectroscopic methods used to quantify proteins measure total protein content, do not distinguish active from inactive molecules, and cannot be used with unpurified samples. Since is a noninvasive technology (i.e., no light penetrates the sample), it is possible to measure sub-femtomole 10

11 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 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). 1.4 Experimental design Some experimental design questions should be considered when using Biacore to measure the binding kinetics of protein interactions Which interactant should be immobilized as the ligand? Often protein-protein interactions can be studied using X100 with either interactant immobilized as the ligand. General properties such as purity, quantity, mass, stability, valency, isoelectric point, and available tag should be considered when deciding which interactant to immobilize. The purity of the ligand is very important to ensure binding specificity as well as binding capacity. Impurities in the ligand preparation can be immobilized as well as the ligand, which will complicate the determination of ligand density. Impurities in the analyte material can complicate results by introducing nonspecific binding or by affecting the accurate determination of analyte concentration. If an interactant is of limited quantity, it could be used as the ligand, because immobilization, either direct or via capture, usually requires very small amounts of material (2 to 10 μg). Biacore systems can detect molecules as small as 200 Da; therefore, the size of the interactants is not usually a limiting factor. However, the Rmax equation (eq. 4.1 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. If one of the interactants has a protein purification tag (e.g., 6His), then that protein could be captured as the ligand using an anti-tag antibody surface. When using Biacore to measure the binding kinetics of an antibody-antigen interaction, the antibody should be immobilized as the ligand to avoid binding avidity effects that result from the bivalency of the antibody. Immobilization of the bivalent protein 11

12 enables determination of the kinetic rate constants by fitting the responses to a simple 1:1 (Langmuir) binding model 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; 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 1.2. 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 What is a good control surface? 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 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. 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-β2μ-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-β2μ-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 Is the immobilized ligand active? 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. 12

13 1.4.6 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. 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) How should the surface be regenerated? 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 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. 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 Bulk refractive index The 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 to changes in responses that are known as bulk refractive index changes (square-wave shaped signals superimpose to the binding curves). 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 high salt 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 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

14 procedure, which enables preconcentration. Note that prepared surfaces can have a negative charge during experiments, depending on sensor chip type and blocking procedure. Figure 7. 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 Amine coupling Buffer components that contain primary amines, such as Tris or sodium azide, must be avoided in amine coupling to prevent competition with the ligand for coupling. 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 Immobilization levels The binding capacity of the surface will depend on the level and activity of immobilized ligand. The response correlates with change in mass concentration on the sensor chip surface, and therefore, depends on the molecular weight (mass) of the analyte in relation to the number of ligand sites on the surface. The term Rmax describes the maximum binding capacity of the surface ligand for analyte in RU. The theoretical Rmax is calculated from Equation 4.1: Rmax = (analyte MW/ligand MW) RL Sm (4.1) 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

15 Kinetic constants measurable by direct Antibody affinity is defined as depicted in Figure 8. Normally, IgGs have one effective binding site if an affinity interaction is taking place. Thus, for a monovalent antigen, 1:1 affinity binding is to be expected. When both Fab fragments of the same IgG molecule interact with a multivalent antigen, then an avidity interaction is taking place and higher stabilization of the Ab-Ag antibody is observed. 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, such as IgM (Abbas et al., 1997). In the case of antibody natural antigen interactions, equilibrium constants usually range from 107 to 1010 M 1, and immunoglobulins with K A 104 M 1 for a particular antigen are ineffective. The range of rate constants amenable to study by varies with the molecular weight of the analyte and with the sensitivity of the particular biosensor employed. Figure 8 Schematic definition of the affinity constant in an antibody-antigen interaction. 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 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. 15

16 Baseline levels Baseline increase over repeated cycles can be observed. Inefficient regeneration steps are often to blame, with bound analyte not being fully washed off after each binding cycle; alternative regeneration agents must be tested and a cocktail approach (Andersson et al., 1999 a, b) may be required Fitting data Antigen-antibody (Fab) interactions are expected to display a Langmuirian behavior on the biosensor. Deviations from pseudo-first-order kinetics, one of the most difficult problems to solve in biosensor analysis (Morton et al., 1995; O Shannessy and Windzor, 1996; Hall et al., 1996), can result from several factors. Whatever they may be, one must keep in mind that, for kinetic studies, mass transport effects must be minimized. This can be achieved by decreasing ligand immobilization levels (to the minimum giving a satisfactory signal-to-noise ratio), increasing buffer flow rate (higher than 30 µl/min), and increasing analyte concentration (as long as surface capacity is not saturated). 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). Another precaution aimed at eliminating mass transport effects in complex dissociation (i.e., rebinding) consists of replacing buffer with ligand solution during the dissociation phase. Other common sources of deviation are ligand or analyte heterogeneity. 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. Analyte heterogeneity can be reduced through additional sample purification steps. The sources of deviation most difficult to deal with are those intrinsic to the binding partners or phenomena, such as analyte multivalency, avidity, or complex binding mechanisms (e.g., involving conformational changes). When these effects are present, the only way to take them into account is to use the more complex fitting models, although it may be difficult to judge whether a better fitting model corresponds to the real interaction mechanism (Schuck, 1997) 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. In terms of the time required to complete the assays as described here, one 8-hr day should be sufficient to accomplish the protocols for surface preparation and assay development, as well as set-up of the kinetic analysis, which can be run overnight. Data analysis requires 1 hr. Derivatized sensor chips can be stored at 4 C in a humid environment, however, the activity of the immobilized ligand after storage should be evaluated. Detailed time considerations (i.e., step by step) are as follows: Pre-concentration assays: 60 min; Covalent immobilization: 30 min; Evaluation of regeneration conditions: 30 min; Two blank runs: 20 min; One analyte run (sample plus regeneration agent): 20 min; Data analysis: 60 min (variable, depending on the quality of the fit); System priming: 10 min; Weekly desorb operation: 30 min; Monthly sanitize operation: 40 min; Signal calibration ( normalizing ): 40 min. 16

17 2. PRACTICAL WORK 2.1 Reagents preparation Materials HBS-P+ 1 mg/ml anti-β2μ-globulin antibody 10 mm sodium acetate, ph mm NaOH 100 μg/ml (8.5 μm) β2μ-globulin 25 µg/ml anti-mouse IgG antibody 10 mm sodium acetate, ph 5.0 Deionized water 10 mm sodium Acetate ph mm NaOH 1) Dilute 100 μl of 3M sodium acetate ph 5.5 into 29.9 ml milliq water. 2) Dilute 500 μl of 1M NaOH into 9500 μl milliq water 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-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. 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 μg/ml anti-β2μ-globulin antibody in 10 mm sodium acetate, ph ) Dilute 2 μl of 1 mg/ml anti-β2μ-globulin into 198 μl sodium acetate, 10mM ph 5.5. β2μ-globulin diluted series preparation (for Protocols 1 and 2) 18) Dilute 5.6 μl of 8.5 μm stock β2μ-globulin (100 μg/ml) into μl HBS-P+ to prepare the 32 nm sample. 19) Prepare two-fold serial dilutions of 32 nm β2μ-globulin down to 4 nm. To prepare the 16nM solution, take 600 μl of buffer and 600 μl of 32nM β2μ-globulin diluted solution. Proceed this way down to 4 nm concentration. 17

18 Also include a buffer-only sample (0 nm) that will be used for double-reference subtraction during data analysis.11 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 Stock solution Concentration [nm] Volumes needed [µl] Sample Buffer Protocol 2 Buffer preparation 1) Same buffer 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+. 25 µg/ml anti-mouse IgG antibody in 10 mm sodium acetate, ph 5.0 3) Dilute 5 μl of 1 mg/ml anti-mouse IgG antibody into 195 μl sodium acetate, ph PROTOCOL 1: Immobilization of the ligand on a sensor chip by amine coupling As explained in paragraph 1.2, several coupling chemistries are available for covalent immobilization of the ligand to the sensor chip surface. Amine coupling is the most widely applicable approach for immobilization of ligands. There are three steps in amine coupling: 1) Activation of the surface, 2) Contact of the ligand with the activated surface, 3) Blocking of unreacted sites. After identification of the optimal ph for preconcentration of the ligand, the active and reference surfaces are prepared on Sensor Chip CM5 using amine-coupling chemistry. The immobilization surface preparation application wizard is used to create active and reference surfaces automatically. The active surface will contain the anti-β2μ-globulin antibody ligand, and the reference surface will contain no ligand, but is activated and blocked with the same chemistry as the active surface. Generally, 75% surface activity can be expected for ligands immobilized directly by amine coupling. Note that when using the Aim for Immobilized Level option in the application wizard, the control software performs an injection of ligand to test for preconcentration prior to activating the surface to ensure that the 18

19 desired immobilization level can be achieved during amine coupling. If the preconcentration response is too low to achieve the target immobilization level, the run will be stopped. Materials 200 ml HBS-P+ 10 μg/ml anti-β2μ-globulin antibody in 10 mm sodium acetate, ph mm sodium acetate, ph 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 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 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-β2μ-globulin antibody - Set the target level at 1200 RU 19

20 - Enter 50mM NaOH for the Wash Solution. 7) Check Prime before run to flush the flow system with fresh buffer at the start of the run. 8) Click on Next. 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 values. Make sure you don t make bubbles while putting the reagents into the vials. 10) 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. 11) Click Load Samples to release the sample rack so that you can load samples. 12) Wait until the Rack Locked indicator on the instrument is turned off before attempting to remove the sample rack. 13) The sample rack can be removed from the sample compartment when the Rack locked indicator on the instrument is not lit. 14) Replace the rack and click OK to lock the rack. 15) Click on Next on the Immobilization- Rack Positions window. 16) Prepare Run Protocol : in this window are shown the preparations you need to complete before starting the run. 17) Once you checked it is correct, click on Start. 20

21 21

22 2.2.2 Binding kinetics Materials Diluted series of 100 μg/ml (8.5 μm) β2μ-globulin in HBS-P+ 10 mm glycine, ph ) 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. 19) Define the Injection Sequence. Chose CM5 sensor chip and Multi-cycle kinetics type. Set 1 regeneration and then click on Next. 20) 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. 21) 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. 22) Define Samples. Set B2micro as Sample id. Set 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. 23) Click on Next. 24) 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. 22

23 25) 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. 26) 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. 27) Click on Next on the Immobilization- Rack Positions window. 28) 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-β2μ-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-β2μ-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 µg/ml anti-mouse IgG antibody in 10 mm sodium acetate, ph M ethanolamine 5 µg/ml solution of anti-β2μ-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. 23

24 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 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. 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. Note: The position marked H2O requires an uncapped vial with MQ water. 24

25 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 : in this window are shown the preparations you need to complete before starting the run. 19) Once you checked it is correct, click on Start Binding kinetics Materials Diluted series of 8.5 μm stock β2μ-globulin in HBS-P+ 10 mm glycine, ph μg/ml solution of anti-β2μ-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. 25

26 21) Define the Injection Sequence. Chose CM5 sensor chip and Multi-cycle kinetics type. Check Ligand capture. Set 1 regeneration and then click on Next. 22) 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. 23) 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. 24) Define Samples. Set B2micro as Sample id. Set 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. 25) Click on Next. 26) Prepare the rack. 27) 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. 26