surface Plasmon resonance based systems

ADVANCED BIOENGINEERING METHODS LABORATORY

SPR

Carlotta Guiducci

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 (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).


SPR

Carlotta Guiducci

2

TABLE OF CONTENTS

1 Theory ....................................................................................................................................................... 3

1.1 Biacore ............................................................................................................................................... 3

1.2 Surface preparation ............................................................................................................................ 5

1.3 Assay types ......................................................................................................................................... 8

1.4 Experimental design ........................................................................................................................ 11

2 Practical work ........................................................................................................................................ 17

2.1 Reagents preparation ....................................................................................................................... 17

2.2 Protocol 1:Immobilization of the ligand on a sensor chip by amine coupling ................................ 18

2.3 Protocol 2:Immobilization of the ligand on a sensor chip using ligand capture method ................. 22

3 Data analysis .......................................................................................................................................... 26

3.1 Immobilization Sensorgram Analysis ............................................................................................. 26

3.2 Kinetic Analysis .............................................................................................................................. 26

4 References .............................................................................................................................................. 27


SPR

Carlotta Guiducci

3

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, without the use of labels. Biacore

systems are widely used for characterizing the interactions of proteins with other proteins, peptides, nucleic

acids, lipids, and small molecules.

When Biacore is used to measure protein interactions, one of the interactants is immobilized onto a sensor

chip surface and the other interactant is passed over that surface in solution via an integrated microfluidic

flow system. The immobilized interactant is referred to as the ligand, and the injected interactant in solution

is referred to as the analyte. Binding responses are measured in resonance units (RU) and are proportional to

the molecular mass on the sensor chip surface and, consequently, to the number of molecules on the surface.

Surface Plasmon Resonance (SPR) was first shown to be amenable for the label-free study of interactions

between biomolecules over 20 years ago (Lieberg et al., 1983). Biacore pioneered commercial SPR

biosensors offering a unique technology for collecting high quality, information-rich data from biomolecular

binding events. Since the release of the first instrument in 1990, researchers around the world have used

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

1.1.1 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.

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.


SPR

Carlotta Guiducci

4

1.1.2 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. 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 (SPR

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 SPR occurs shifts due to a change in refractive index near the sensor chip surface.

A sensorgram depicts changes in SPR angle in real time, with responses measured in resonance units (RU).

One RU corresponds to 0.0001◦ shift in SPR angle, or to 1pg/mm2 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.


SPR

Carlotta Guiducci

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.

1.1.3 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 SPR 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

1.2.1. Sensor Chips

Although SPR 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 SPR 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:


SPR

Carlotta Guiducci

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.

1.2.2 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. 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


SPR

Carlotta Guiducci

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.


SPR

Carlotta Guiducci

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

1.3.1 Binding Specificity

Biacore is well suited to carry out qualitative studies to confirm the specificity of interactions as well as

quantitative measurements for affinity, 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).

SPR 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


SPR

Carlotta Guiducci

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).

1.3.2 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, 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


SPR

Carlotta Guiducci

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 (kobs). If the concentration of the analyte is

known, then the association rate constant of the interaction (kass) can be determined. At equilibrium, by definition, the

amount of analyte that is associating and dissociating with the receptor is equal. The response level at equilibrium is related

to the concentration of active analyte in the sample.

4. At t = 320 s, the analyte solution is replaced by buffer, and the receptor–analyte complex is allowed to dissociate. Analysis

of these data gives the dissociation rate constant (kdiss) for the interaction.

5. Many complexes in biology have considerable half-lives, so a pulse of a regeneration solution (for example, high salt or

low pH) is used at t = 420 s to disrupt binding and regenerate the free receptor.

6. The entire binding cycle is normally repeated several times at varying concentrations of analyte to generate a robust data

set for global fitting to an appropriate binding algorithm. The affinity of the interaction can be calculated from the ratio of

the rate constants (KD = kdiss/kass) or by a linear or nonlinear fitting of the response at equilibrium at varying concentrations

of analyte. In addition to determining the interaction affinities and kinetics, a thermodynamic analysis of a biomolecular

interaction is also possible.

1.3.3 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 SPR is a

noninvasive technology (i.e., no light penetrates the sample), it is possible to measure sub-femtomole


SPR

Carlotta Guiducci

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.

1.3.4 Thermodynamics

By studying temperature dependence of rate and affinity constants it is possible to determine

thermodynamic parameters for a binding interaction. Not only can the equilibrium values for changes in

enthalpy (_H) and entropy (_S) associated with complex formation be determined, but transition state

energetics can also be evaluated (Roos et al., 1998).

1.4 Experimental design

Some experimental design questions should be considered when using Biacore to measure the binding

kinetics of protein interactions.

1.4.1 Which interactant should be immobilized as the ligand?

Often protein-protein interactions can be studied using X100 with either interactant immobilized as the

ligand. 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


SPR

Carlotta Guiducci

12

enables determination of the kinetic rate constants by fitting the responses to a simple 1:1 (Langmuir)

binding model.

1.4.2 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; 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.

1.4.3 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.

1.4.4 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.

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.

1.4.5 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.


SPR

Carlotta Guiducci

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).

1.4.7 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.

1.4.8 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. 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.

1.4.9 Immobilization pH scouting

Immobilization by covalent coupling cannot be repeated on the same sensor chip surface. Therefore,

preconcentration is an important strategy to control the immobilization level for experimental optimization

and efficiency. Typically, the optimal pH for preconcentration will be a 0.5- to 1-pH unit below the pI of the

protein. Many proteins exhibit limited stability in the low-ionic-strength, low-pH solutions used for

preconcentration, therefore, dilution of the ligand just prior to use is recommended. Always perform pH

scouting on the flow cell that will be used for the immobilization, not on the reference flow cell. When using

standard amine coupling, ∼40% of the carboxymethyl groups are converted to reactive esters after a 7-min

activation. Thus, the activated sensor chip surface maintains a net negative charge during the immobilization


SPR

Carlotta Guiducci

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.

1.4.10 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.

1.4.11 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, 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)


SPR

Carlotta Guiducci

15

1.4.12 Kinetic constants measurable by direct SPR

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 KA ≤104 M–1

for a particular antigen are ineffective. 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.

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 SPR methodologies are

briefly commented in Figure 9.

Figure9 Possible studies for binding kinetics assays.

1) Direct assay: Suitable for high molecular weight molecules

2) Sandwich assay: To be selected for relatively high molecular weight antigens and when high affinity antibodies are available.

3) Competition assay: Designed for low molecular weight antigens that do not generate sufficient signal when they accumulate on the

surface (Direct assay) and are too small for a sandwich assay.

4) Inhibition assay: Designed for low molecular weight antigens that do not generate sufficient signal when they accumulate on the

surface (Direct assay) and are too small for a sandwich assay.


SPR

Carlotta Guiducci

16

1.4.13 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.

1.4.14 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).

1.4.15 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.


SPR

Carlotta Guiducci

17

2. PRACTICAL WORK

2.1 Reagents preparation

Materials

HBS-P+

1 mg/ml anti-β2μ-globulin antibody

10 mM sodium acetate, pH 5.5

50 mM NaOH

100 μg/ml (8.5 μM) β2μ-globulin

25 µg/ml anti-mouse IgG antibody


Deionized water

10 mM sodium Acetate pH 5.5

1) Dilute 100 μl of 3M sodium acetate pH 5.5 into 29.9 ml milliQ water.

50 mM NaOH

2) Dilute 500 μl of 1M NaOH into 9500 μl milliQ water.

2.1.1 Protocol 1

Buffer preparation

3) Dilute 10× HBS-P+ from stock to 1×.

4) Take a 200 ml graduated cylinder.

5) Put 20 ml of 10× HBS-P+

6) Add 180 ml MQ water.

7) Mix the solution.

8) Filter the solution through a 0.2-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 5.5

17) 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 1494.4 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.


SPR

Carlotta Guiducci

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

1 2 3 4

Concentration [nM] 8500 32 4

Volumes

needed [µl]

Sample

Buffer

2.1.2 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 5.0.

2.2 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


SPR

Carlotta Guiducci

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 5.5


50 mM NaOH

Sensor Chip CM5

1.5-ml polypropylene tubes with rubber caps (Biacore)

100 mM N-hydroxysuccinimide (NHS)

400 mM N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)

1 M ethanolamine

1) Remove sensor chips from the fridge at least 30 min before starting the experiment.

2) Change the sensor chip.

Biacore is always kept with a maintenance chip inside.

On the Control Software Tool Bar click on “Undock Chip” and

follow the displayed instructions. When undocked, the sensor

chip should easily slide out of the instrument.

Note that the sensor chip indicator light on the front of the instrument is lit steadily when a chip is

docked, flashing when a chip is inserted but undocked, and unlit when no chip is inserted.

3) Insert and dock the new Sensor Chip CM5. Click the Command tab and then select the Dock

command.

2.2.1 Ligand Immobilization

4) Create a New Wizard Template.

Click on “Wizards” and select the “Immobilization” wizard type from the list in the left-hand panel.

Then click on “New”.

5) Define the Immobilization Setup. This step will determine how the ligand is immobilized on the

surface. Choose the chip type CM5.

6) 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


SPR

Carlotta Guiducci

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”.


SPR

Carlotta Guiducci

21


SPR

Carlotta Guiducci

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 2.5


Click on “Wizards” and select the “Kinetics/Affinity” wizard type from the list in the left-hand

panel.


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 “Stabilization period”: 60 s.


22) Define Samples.

Set “B2micro” as “Sample id”.

Set “11800” Da as Molecular Weight.

Enter the sample concentrations in ascending order. Include in addition at least one blank

sample and a replicate of a non-zero concentration.

Do not run replicate samples in consecutive cycles since this will reduce the chance of

detecting assay drift. Samples will be run in the order entered.


24) Prepare the rack.

Close all the vials with the orange rubber caps and place them in the right order.



SPR

Carlotta Guiducci

23


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.


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 5.0

1 M ethanolamine

5 µg/ml solution of anti-β2μ-globulin antibody in HBS-P+


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.


SPR

Carlotta Guiducci

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.

2.3.1 Ligand Immobilization


Click on “Wizards” and select the “Immobilization” wizard type from the list in the left-hand panel.


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.


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.




SPR

Carlotta Guiducci

25


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.


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”.

2.3.2 Binding kinetics

Materials

Diluted series of 8.5 μM stock β2μ-globulin in HBS-P+


5 μg/ml solution of anti-β2μ-globulin in HBS-P+


Click on “Wizards” and select the “Kinetics/Affinity” wizard type from the list in the left-hand

panel. Then click on “New”.


SPR

Carlotta Guiducci

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.


23) Define Injection parameters.

For the capture:



For the sample:


Set “Dissociation time”: 300 s.

For the First regeneration:

Set “Solution”: 10mM Glycine pH 1.7.




24) Define Samples.

Set “B2micro” as “Sample id”.

Set “11800” Da as Molecular Weight.

Enter the sample concentrations in ascending order. Include in addition at least one blank

sample and a replicate of a non-zero concentration.

Do not run replicate samples in consecutive cycles since this will reduce the chance of detecting

assay drift. Samples will be run in the order entered.


26) Prepare the rack.




SPR

Carlotta Guiducci

27


Wait until the “Rack Locked” indicator on the instrument is turned off before attempting to

remove the sample rack.

29) 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.


31) Perpare Run Protocol. Once you checked it is all correct, click on “Start”.

3 DATA ANALYSIS

3.1 Immobilization Sensorgram Analysis

.

Q1. Discuss the immobilization sensorgram curve: explain in your lab notebook step by step the

immobilization process using the immobilization sensorgrams

Q2. Discuss the role of pH (of sodium acetate) during the immobilization process. Explain briefly what pH

scouting is, and why it is important. Why do we use different pH of sodium acetate for the two protocols?

Q3. Explain briefly the differences and advantages of subsequent/continuous injections of ligand (or capture

molecule).

Q4. Which is the theoretical binding capacity of the sensor chip surface? Does it correspond to the

experimental data?

Q5. From the Immobilization Sensorgrams, check if the RU bound to the surface corresponds to the data

settings. To how many molecules per cm2 do they correspond? (Hint: 1RU = 1pg/mm

2 , MWligand =

150000Da)

Q6. Why do we add ethanolamine during immobilization? What might happen if this step is skipped?

3.2 Kinetic Analysis

Q7. Discuss the kinetics curve: explain in your lab notebook step by step the process

Q8. What kind of curve have you obtained? Which fitting is suitable for the actual case? Discuss briefly the

other types of fitting.

Q9. Identify the association and dissociation constant from the data. Compare the values between two

protocols and discuss.

Q10. Explain briefly what the mass transfer phenomena is, and how to detect it. How do mass transfer

phenomena can affect the binding kinetics analysis?

Q11. How can you identify and reduce nonspecific binding? Discuss the possibility to use another type of


SPR

Carlotta Guiducci

28

sensor chip.

Q12. In some cases, the sensorgram is affected by bulk effect. Why does it occur? How do you get rid of it?

Q13. What kind of interaction are we analyzing? Why is the pH of glycine so important? Why do we use

different pH glycine for the two protocols?

Q14. Why do we use two different methods to study binding kinetics? What are the advantages of each

method compared to the other? Can you extract this information from the sensorgrams?

Q15. Why in kinetics analysis you need serial dilutions?

4 REFERENCES

Fundamentals of biochemical sensing systems, EPFL Master BE course, Prof.Guiducci, 2010. http://www.biacore.com/lifesciences/index.html

Biacore catalog.

https://www.biacore.com/lifesciences/service/online support/irdb/index.html

Immobilization and regeneration database.

http://www.biacore.com/lifesciences/service/training/courses/usa canada/biacorebasics/index

http://www.biacore.com/lifesciences/index.html

Documents

surface Plasmon resonance based systems