Immobilization techniques Introduction - MichalakMethods

Immobilization techniques

Immobilization

Introduction Introduction
How much ?
First step in the interaction analysis is the immobilization of one of the interactants on the
Which method ? sensor chip surface. This immobilization can be permanent in the form of a covalent bond
or transient by means of capturing.
Conditions The immobilization technique used depends on the ligand type (protein, sugar, DNA,
Ligand prep. LMW-substance), the analyte to be used (small or large interactants) and the purpose of
References the study (specificity, concentration, affinity, kinetics). In addition the ligand must retain
his biological activity (2).

Information about the ligand size, pI, amino acid composition, pH stability and possible
sites for oriented coupling are beneficial in determine the immobilization method to be
used.
Size and amino acid composition are normally well known. Both are used to determine
how and how much ligand should be immobilized in order to obtain a proper signal. Not
knowing the pI is not critical because with relative simple and fast pre-concentration
experiments, the proper immobilization pH is easy established.
The amount of ligand to be immobilized depends on the application. Below a figure
depicting the relative immobilization levels and applications.

Immobilization levels for different applications
Low High

Specificity
Concentration
Affinity
Kinetics
LMW binding

BiaSymposium ‘98

For specificity measurements, almost any ligand density will do as long as it gives a good
signal.
Concentration measurements need the highest ligand density to enhance mass transfer
limitation. In a total mass transfer controlled experiment binding will depend on the
analyte concentration and not on the binding kinetics between the ligand and analyte.
Affinity ranking can be done with low to moderate density sensor chips. The important
factor is that the analyte should saturate the ligand within a proper timeframe.
Kinetics are done with the lowest ligand density still giving a good response without being
disturbed by secondary factors such as mass transfer or steric hindrance.
LMW-binding is done with high density sensor chips to bind as much as possible of the
component to gain a proper signal.

January 28, 2004

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Immobilization techniques

Immobilization

Introduction Covalent coupling chemistries 13
How much ? Several covalent coupling chemistries are available to
immobilize the ligand depending on the available reactive OO
Which method ? groups. The amine (-NH2), thiol (-SH2) and aldehyde (-COOH) C ON
coupling chemistries are well established procedures. Covalent
Conditions coupling is stable and needs in general no modification of the O
Ligand prep. ligand. The immobilization level is easily controlled and the
References ligand consumption is low.

Unidirectional immobilization O O COOH
The standard covalent immobilization techniques are prone to
random orientating the ligand to the dextran surface. Further C ON O COOH O O COOH
more, free reactive groups close to the binding site can be
obscured reducing or eliminating affinity for de analyte (9). O O C CON O
Even a homogenous ligand will artificially be converted to a C
heterogeneous ligand. Unidirectional immobilization is used to CO O
reduce or eliminate this induced heterogeneity. C
OO O
Unidirectional immobilization can somewhat be enforced by COOH C
biotinylation of the ligand. The biotin is coupled with the help of
NHS-SS-biotin to free amino groups like the N-terminus and COOH NOC
lysine residues. The protein of interest should be purified to one
form with one biotin attached to get the maximum unidirectional O
immobilization. It is also a good idea to check if the protein is
still functional (4). 1 free binding site 2

A different approach is to use capturing antibodies. In this 2 less easy to reach binding site
affinity capturing system, the antibody should have a sufficient
affinity to the ligand in order to have a stable complex. In 3 binding site is modified by immobilisation
addition, the antibody may not interfere with the function or
binding place of the ligand. VH

Antibodies can also be digested with papain to form F(ab')2 VL CH1
fragments containing the two Fab fragments connected by the CL
disulfide bond. Reducing this bond gives the single Fab'
fragments that can be unidirectional covalently bound to the hinge
sensor chip using the thiol chemistry (9).
ss
The introduction of other functional groups to the ligand of
interest gives more capturing possibilities. For instance, with the
introduction of a 6 x HIS group the protein can be captured with
the NTA group.

Modifying the sensor chip surface with hydrophobic compound
makes it possible to capture vesicles. The vesicles can be used
for research or as a platform to insert proteins with hydrophobic
part which are otherwise difficult to immobilize.

The affinity capturing systems are general applicable but the
ligand has to be modified in some cases. Some of the systems
are totally regenerable but then the ligand consumption is high
and the ligand capturing is not always totally stable.

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Immobilization techniques

Immobilization

Introduction How much ligand to immobilize?
How much ?
As stated above, the desired ligand density on the sensor chip surface depends on the
Which method ? application.

Conditions For kinetic measurements is 0.07 pmolligand/mm2 a good starting point. The response of
Ligand prep. 0.07 pmol/mm2 can be calculated with (5):
References
(Mwligand/15) = response in RU which equals 0.07 pmolligand/mm2

In general for kinetic measurements, a total response of 100 – 500 RU when the analyte is
injected (8) is good. With the Rmax at the desired response the ligand response to be
immobilized can be calculated with:

Rmax = (Mwanalyte /Mwligand) x Rligand x Vligand

Because of the linear relation between response and amount of protein coated to the sensor
surface, the theoretical amount of ligand sites can be calculated (6).

Ligand sites (pmol/mm2) ≡ (Rligand /Mwligand x Vligand) Mwanalyte molecular weight analyte
Activityligand = (Rmax/Rligand) * (Mwligand/Mwanalyte) MWligand molecular weight ligand
Analyte response ≡ Mwanalyte x analyte molecules Rligand response due to the ligand
1000 RU ≡ 1 ng/mm2 (proteins) Vligand valency of the ligand,
amount of binding sites
Rmax response due to injecting the
analyte

It is theoretical because this formula assumes that all immobilized ligand molecules are
fully accessible and functional, which is probably not the case with standard amine
coupling.
A low analyte response can be caused by a low affinity; an impure ligand or the binding
sites are affected by the immobilization procedure. To high response can be induced by
non-specific binding or analyte aggregates (1).

Because the matrix has a significant extension (of the order of 100 nm for sensor chip
CM5, "surface concentrations" on the sensor chip surface are strictly speaking volume
concentrations. Assuming an extension of 100 nm for the surface layer, a ligand surface
concentration of 1 ng/mm2 corresponds to a volume concentration in the matrix of 10
mg/ml (5,10)

conc (mol/liter) = conc (RU) Conc: concentration ligand
100 x Mw (Da) Mw: molecular weight ligand

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Immobilization techniques

Immobilization

Introduction Choosing an immobilization method
How much ?
Choosing an immobilization method depends mostly on the nature of the ligand. Below a
Which method ? table with the recommended chemistries from Biacore (2).

Conditions Biomolecules Amine Thiol Aldehyde streptavidin-
Ligand prep.
References biotin

acidic peptides/proteins - + - (#)

neutral peptides/protein + (+) (#) (#)

basic peptides/proteins + (+) (#) (#)

nucleic acids - - -#

polysaccharides - - -#

+ recommended, (+) acceptable, - unsuitable, # requires ligand modification

Functional groups Amine Thiol Aldehyde streptavidin-

biotin

peptides/proteins

-NH2 + # - (#)

-SH - + - (#)

-COOH - (#) - (#)

-CHO - - # (#)

polysaccharides

-CHO - - (#) #

-COOH - (#)

+ recommended, (+) acceptable, - unsuitable, # requires ligand modification

Amine coupling is the most general applicable coupling chemistry and is recommended as
the first choice. Most macromolecules contain amine groups, which can be used in amine
coupling. Situations where amine coupling is less suitable is with acidic ligands (pI < 3.5),
ligands where an amine is in the active site and with molecules possessing a lot of amine
groups.

The choice of thiol coupling depends mostly on the availability of thiol groups on the
ligand. However, it is relatively easy to introduce the thiol groups on the ligand. The thiol
chemistry is more robust than the amine so the coupling conditions are less critical. The
thiol coupling cannot be used with strong reducing conditions, since the disulfide bond is
unstable under such conditions.

The choice of aldehyde coupling comes is specific cases. For instance with
polysaccharides and glycoconjugates, which have cis-diols and sialic acids. These groups
are relatively easy oxidized to aldehydes.

The choice of streptavidin-biotin coupling follows when the amine or thiol coupling is
unsatisfactory or unsuitable. Nucleic acids, polysaccharides and glycoconjugates are
relatively easy biotinylated using a variety of reagents and functional groups. Acidic and
acid sensitive proteins can be coupled at neutral pH with high efficiency.

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Immobilization techniques

Immobilization

Introduction Immobilization parameters
How much ?
After choosing an immobilization chemistry and sensor chip type some factors remain that
Which method ? influence the amount of ligand that will be immobilized.

Conditions In case of a covalent binding chemistry longer activation of the sensor chip surface will
Ligand prep. activate more carboxyl groups making it possible to bind more ligand. The pH of the
References coupling buffer, which will change the surface and ligand charges, regulates the amount
and speed in which the ligand goes towards the sensor chip surface (pre-concentration).
The reactivity of the ligand at the chosen pH determines how fast the ligand will bind to
the activated surface. A high ionic strength of the buffer will mask the charges of the
surface and ligand diminishing the pre-concentration effect. Ligand concentration is
directly related to the speed of pre-concentration. To high ligand concentrations makes it
difficult to immobilize the proper amount. The contact time of the ligand with the
activated surface is not linear as the sensor chip reaches saturation. The deactivation
conditions will block all remaining activated sites with an excess of reagents and because
of the high ionic strength wash away most of the electrostatic bound ligand.

In case of an affinity binding procedure the buffer strength, pH, ligand concentration, flow
rate and contact time will determine how much ligand is immobilized. For systems in
which it is not possible to remove the ligand from the sensor chip (avidin-biotin binding)
low ligand concentration (0.5 - 5 nM) are recommended during capturing.

In case of a lipid adsorption procedure, the cleanness of the sensor chip surface and the
machine is very important. The immobilization of the lipids begins with a cleaning step to
remove adsorbed compounds after which directly the lipids are injected. Buffer strength,
pH, lipid concentration, flow rate and contact time are important parameters. With lipid
adsorption longer injection times are needed. Normally injections goes on until the
binding levels out (± 1000 - 1500 RU) depending on the lipids used.
For a more thorough discussion of the parameters involved with immobilization, see BIA-
Applications Handbook chapter 3. For a list and explanation of several immobilization
principles see BIA-Applications Handbook chapter 4 (3). A nice article about factors
involving immobilization is written by Johnsson et al. (7).

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Immobilization techniques

Immobilization

Introduction Preparing the ligand
How much ?
Before immobilization, the ligand has to be brought in the right form and formulation.
Which method ? Most ligands can be immobilized without modification. However sometimes a
modification is required to use a specific immobilization technique. Some commonly
Conditions used modifications are:
Ligand prep.
References ♦ adding a disulfide group
♦ adding a thiol group
♦ adding an aldehyde group
♦ adding a biotin group

The proper formulation has to do with the buffer. For most immobilization methods, a
low salt buffer with the right pH is required (see pre-concentration). In addition, the
buffer components should not interfere with the immobilization.

Adding a disulfide group
By modifying an amine group with NHS/EDC followed by PDEA, a reactive disulfide
group is introduced. This group can react with a thiol group, which is on the sensor chip
surface (surface thiol method).

Adding a thiol group
By modifying an amine group with NHS/EDC followed by DTE and cysteamine, a thiol
group is introduced. This group can react with a reactive disulfide group, which is on
the sensor chip surface (ligand thiol method).

Adding an aldehyde group
For polysaccharides and glycoconjugates aldehyde coupling is a good alternative for
amine or avidin-biotin coupling. Especially for molecules containing sialic acid because
these residues are easily oxidized to aldehydes.
Aldehydes can be introduces by oxidizing cis-diols with sodium metaperiodate resulting
in an aldehyde group (11). This group can react with a hydrazide group introduced on
the sensor chip surface and the formed hydrazone bond is stabilized by reduction with
cyanoborohydrid.

Adding a biotin group
For the biotinylation of macromolecules are several commercial kits available with
instructions for use. A more thorough discussion of biotinylation can be found in (12).
More about biotinylation of nucleic acids can be found in Methods in Molecular
Biology vol. 4. (1988).

There are two important points in context with biomolecular interaction analysis.
The degree of biotinylation should be kept low (0.5 - 1.5 moles biotin/ligand). At higher
levels the binding kinetics are faster but the amount of ligand bound is reduced. Higher
levels of biotinylation may help stabilize the immobilized ligand against regeneration
conditions (2).
Excess of free biotin must be removed efficiently. Gel filtration on a desalting column,
dialysis or centrifugal ultrafiltration is recommended for medium-sized to large ligands
(Mw > 5000 Da).

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Immobilization techniques

Immobilization

Introduction References
How much ?
Reference List
Which method ?
1. ; BIA Symposium '98; 1998
Conditions 2. BIACORE AB; BIACORE Application Handbook; 1998
Ligand prep. 3. BIACORE AB; BIACORE Instrument Handbook; 1998
References 4. De Crescenzo, G., Grothe, S., Lortie, R., Debanne, M. T., and O'Connor-

McCourt, M.; Real-Time Kinetic Studies on the Interaction of Transforming
Growth Factor alpha with the Epidermal Growth Factor Receptor Extracellular
Domain Reveal a Conformational Change Model; Biochemistry; (39): 9466-
9476; 2000
5. Elwing, H.; Protein absorption and ellipsometry in biomaterial research;
Biomaterials; (19): 397-406; 1998
6. He, X., Ye, J., Esmon, C. T., and Rezaie, A. R.; Influence of Arginines 93, 97,
and 101 of thrombin to its functional specificity; Biochemistry; (36): 8969-8976;
1997
7. Johnsson, B., Lofas, S., and Lindquist, G.; Immobilization of proteins to a
carboxymethyldextran-modified gold surface for biospecific interaction analysis
in surface plasmon resonance sensors; Anal.Biochem.; (198): 268-277; 1991
8. Karlsson, R., Michaelson, A, and Mattson, L; Kinetic analysis of monoclonal
antibody-antigen interactions with a new biosensor based analytical system;
J.Immunol.Methods; 229-240; 1991
9. Kortt, A. A., Oddie, G. W., Iliades, P., Gruen, L. C., and Hudson, P. J.;
Nonspecific amine immobilization of ligand can Be a potential source of error in
BIAcore binding experiments and may reduce binding affinities; Anal.Biochem.;
(253): 103-111; 1997
10. Muller, K. M., Arndt, K. M., and Pluckthun, A.; Model and simulation of
multivalent binding to fixed ligands; Anal.Biochem.; (261): 149-158; 1998
11. O'Shannessy, D. J. and Wilchek, M.; Immobilization of glycoconjugates by their
oligosaccharides: use of hydrazido-derivatized matrices; Anal.Biochem.; (191):
1-8; 1990
12. Wilchek, M. and Bayer, E. A.; Introduction to avidin-biotin technology;
Methods Enzymol.; (184:5-13.): 5-13; 1990

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