SCINTILLATION PROXIMITY ASSAY MANUAL
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SCINTILLATION PROXIMITY ASSAY
MANUALNOTICE This Manual contains materials and information protected by copyright. No part of this document may be disclosed, photocopied, reproduced or translated to another language without the prior written consent of Amersham Biosciences. Sepharose, Cytostar-T and Quan-T-RT are trademarks of Amersham Biosciences. Amersham Biosciences is a trademark of Amersham plc MicroBeta and RackBeta are trademarks of Perkin Elmer Lifesciences. TopCount is a trademark of Packard BioScience Triton is a trademark of Union Carbide Chemicals. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them. A copy of these terms and conditions is available on request. Amersham Biosciences Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE-751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D- 79111 Freiburg, Germany Amersham Biosciences KK Sanken Building 3-25-1 Hyakunincho Shinjuku-ku Tokyo zip 169-0073 Japan. Scintillation Proximity Assay (SPA) Technology is covered by US Patent No. 4568649, European Patent No. 0154734 and by Japanese Patent No. 1941524.
SCINTILLATION PROXIMITY ASSAY MANUAL
SECTION 1 SAFETY
SECTION 2 SCINTILLATION PROXIMITY ASSAY: The-
ory and practical application
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 A brief history of scintillation proximity assay . . . . . . . . . . . 8
2.3 Scintillation Proximity Assay (SPA) Beads . . . . . . . . . . . . 11
2.3.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.3.2 Yttrium silicate (YSi) based SPA beads . . . . . . . . . . . 13
2.3.3 Poly(vinyl toluene) (PVT) based SPA beads . . . . . . . 15
2.3.4 Bead Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.3.4.1 The yttrium silicate polylysine bead . . . . . . . . . 16
2.3.4.2 The protein A beads . . . . . . . . . . . . . . . . . . . . . 16
2.3.4.3 The Secondary antibody beads . . . . . . . . . . . . 17
2.3.4.4 The wheat germ agglutinin (WGA) beads . . . . . 18
2.3.4.5 PVT WGA PEI (polyethyleneimine) beads . . . . 20
2.3.4.6 Streptavidin beads . . . . . . . . . . . . . . . . . . . . . . 20
2.3.4.7 Glutathione beads . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.4.8 Copper his-tag beads . . . . . . . . . . . . . . . . . . . . 23
2.3.4.9 RNA binding beads . . . . . . . . . . . . . . . . . . . . . . 24
SECTION 3 THE APPLICATION OF SPA TECHNOLO-
GY TO ENZYME ASSAYS
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2 Assay design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2.1 Signal decrease assay . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.2 Signal increase assay . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3 Solid phase versus solution phase SPA enzyme assays . 30
3.3.1 Solid phase SPA enzyme assays. . . . . . . . . . . . . . . . 31
3.3.2 Solution phase SPA enzyme assays . . . . . . . . . . . . . 33
3.3.3 Non-specific binding (NSB) and non proximity effect (NPE). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3.4 Stopping the reaction . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.4 Coupling strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.4.1 Incorporation of biotin into proteins and peptides. . . . 35
3.4.2 Incorporation of biotin into oligonucleotides and nucleic
acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.5 Summary of enzyme assay design procedure . . . . . . . . . 37
3.5.1 Source of enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.5.2 Design of substrate . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.5.3 Signal increase or decrease assay . . . . . . . . . . . . . . 38
3.5.4 On or off bead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.5.5 Optimize incubation conditions. . . . . . . . . . . . . . . . . . 38
3.5.6 Bead type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.5.7 Optimize bead amount . . . . . . . . . . . . . . . . . . . . . . . . 38
3.5.8 Optimize assay structure . . . . . . . . . . . . . . . . . . . . . . 39
3.5.9 Performance criteria . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5.10 Validate assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5.11 Color quench curve . . . . . . . . . . . . . . . . . . . . . . . . . 39
SECTION 4 THEORY OF SCINTILLATION COUNTING
AND COLOUR QUENCHING
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.1.1 The conversion of radioactivity into light . . . . . . . . . . 41
4.1.2 The detection of the photons . . . . . . . . . . . . . . . . . . . 41
4.1.3 Dual PMT coincidence counting. . . . . . . . . . . . . . . . . 42
4.1.4 Time resolved pulse discrimination . . . . . . . . . . . . . . 43
4.1.5 The pulse height spectrum . . . . . . . . . . . . . . . . . . . . . 45
4.1.6 The effect of quenching on the pulse height spectrum 48
4.2 Theory of colour quenching . . . . . . . . . . . . . . . . . . . . . . . 49
4.2.1 Chemical quenching. . . . . . . . . . . . . . . . . . . . . . . . . . 494.3 Color quenching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.4 Quench correction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.5 General Guidelines for performing Color Quench Correction
using Labelled Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
SECTION 5 THE APPLICATION OF SPA TECHNOLO-
GY TO RECEPTOR BINDING ASSAYS
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.2 Coupling strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.2.1 Membranes derived from tissues . . . . . . . . . . . . . . . . 59
5.2.2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.2.3 Cell membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.2.4 Solubilized receptors . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.2.5 Soluble receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.3 Assay development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.1 Use of second antibody beads in non RIA applications . . 69
SECTION 6 THE APPLICATION OF SPA TO RADIOIM-
MUNOASSAY
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.2 Assay design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.2.1 Choice of SPA bead . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.2.2 Optimization of SPA bead and antibody. . . . . . . . . . . 76
6.2.3 Choice of tracer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.2.4 Incubation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.2.5 Incubation temperature . . . . . . . . . . . . . . . . . . . . . . . 77
6.2.6 Sample preparation and color quench . . . . . . . . . . . . 77
SECTION 7 ReferencesSAFETY 1
SECTION 1 SAFETY
Scintillation proximity assay (SPA) is a radioisotopic technique and,
therefore, requires the use of radioactive material. Product safety
information for all Amersham Biosciences products is contained
within a "Safety Warnings and Precautions" section of the pack
leaflet or specification sheet that accompanies each product. Please
follow the instructions relating to the safe handling and use of these
and other materials in the product. In addition, most countries have
legislation governing the handling, use, storage, disposal, and
transportation of radioactive materials. The safety information
provided is intended to complement local regulations or codes of
practice. Such legislation may require that a person be nominated to
oversee radiological protection. Users of radioactive products must
make themselves aware of and observe the local regulations or
codes of practice that relate to such matters.
All Amersham Biosciences products contain the following warning.
Warning: For research use only. Not recommended or intended for
diagnosis of disease in humans or animals. Do not use internally or
externally in humans or animals.
For those products that contain radioactive material or are for use
with radioactive material, then the following handling instructions are
recommended.
"Instructions relating to the handling, use, storage, and disposal of
radioactive materials".
1. Upon receipt, vials or ampoules containing radioactive material
should be checked for contamination. All radioactive materials
should be stored in specially designated areas and suitable
shielding should be used where appropriate. Access to these
areas should be restricted to authorized personnel only.
2. Only responsible persons in authorized areas should use
radioactive material. Care should be taken to prevent ingestion
or contact with skin or clothing. Protective clothing such as
laboratory overalls, safety glasses, and gloves should be worn
whenever radioactive materials are handled. Where this is
appropriate, the operator should wear personal dosimeters to
measure radiation dose to the body and fingers.
3. No smoking, drinking, or eating should be allowed in areas where
radioactive materials are used. Avoid actions that could lead to
the ingestion of radioactive materials, such as the pipetting of
page 1SAFETY 1
radioactive solutions by mouth.
4. Vials containing radioactive materials should not be touched by
hand; wear thin surgical gloves as normal practice. Use forceps
when handling vials containing "hard" beta emitters such as
phosphorus-32 or gamma emitting labelled compounds.
Ampoules likely to contain volatile radioactive compounds should
be opened only in a well-ventilated fume cabinet.
5. Work should be carried out on a surface covered with absorbent
material or in enamel trays of sufficient capacity to contain any
spillage. Working areas should be monitored regularly.
6. Any spills of radioactive material should be cleaned immediately
and all contaminated materials should be decontaminated or
disposed of as radioactive waste via an authorized route.
Contaminated surfaces should be washed with a suitable
detergent to remove traces of radioactivity.
7. After use, all unused radioactive materials should be stored in
specifically designated areas. Any radioactive product not
required or any materials that have come into contact with
radioactivity should be disposed of as radioactive waste via an
authorized route.
8. Hands should be washed after using radioactive materials.
Hands and clothing should be monitored using appropriate
instruments to ensure that no contamination has occurred before
leaving the designated area. If radioactive contamination is
detected, hands should be washed again and rechecked. Any
contamination persisting on hands and clothing should be
reported to the responsible person so that suitable remedial
actions can be taken.
9. Certain national/international organizations and agencies
consider it appropriate to have additional controls during
pregnancy. Users should check local regulations.
Amersham Biosciences uses scintillant beads that are based either
on yttrium silicate or on poly (vinyl toluene). Yttrium silicate is
classified as harmful when in particulate forms such as dust or beads.
All yttrium silicate based SPA products carry the following warnings.
Warning: Contains yttrium compounds. Harmful by inhalation,
contact with skin and if swallowed.
These scintillation proximity reagents contain yttrium compounds.
Care should be taken to prevent ingestion, contact with skin, or
page 2SAFETY 1
inhalation of the dried powder. Use in a well-ventilated enclosure.
Wear suitable protective clothing such as laboratory overalls, safety
glasses, and gloves. In the event of contact with skin or eyes wash
the affected area thoroughly. If swallowed, take large amounts of
water and seek medical attention.
The total yttrium compounds present in each pack is given in the
appropriate pack leaflet.
Poly (vinyl toluene) beads are not known to be harmful, but they
should be considered as a potential irritant in dried form as a dust or
powder. In this case the warning statement will be:
"This product contains one or more chemical substances supplied in
small quantities. In the form supplied, these substances are not
classified as dangerous within the meaning of the definitions of the
Council of European Communities Directive 67/548/EEC and
subsequent amendments.
All chemicals should be considered as potentially hazardous. We
recommend that only those persons who have been trained in
laboratory techniques handle these products and that they are used
in accordance with the principles of good laboratory practice. Wear
suitable protective clothing such as laboratory overalls, safety
glasses, and gloves. Care should be taken to avoid contact with skin
or eyes. In case of contact with skin or eyes wash immediately with
water."
page 3SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
SECTION 2 SCINTILLATION
PROXIMITY ASSAY: Theory and
practical application
2.1 Introduction
When a radioactive atom decays it releases sub-atomic particles
such as electrons, and depending upon the isotope, other particles
and various forms of energy such as γ-rays. The distance these
particles will travel through water is limited and is dependent upon the
energy of the particle, which is normally expressed in MeV.
Scintillation proximity assay (SPA) relies upon this limitation.
For example, when a tritium atom decays it releases a β-particle. If
the [3H] atom is within 1.5 µm of a suitable scintillant molecule, the
energy of the β-particle will be sufficient to reach the scintillant and
excite it to emit light. If the distance between the scintillant and the
[3H] atom is greater than 1.5 µm, the β-particles will not have sufficient
energy to travel the required distance. In an aqueous solution,
collisions with water molecules dissipate the β-particle energy and it
therefore cannot stimulate the scintillant. Normally the addition of
scintillation cocktail to samples containing radioactivity ensures that
the majority of [3H] emissions are captured and converted to light. In
SPA, the scintillant is incorporated into small fluomicrospheres.
These microspheres or "beads" are constructed in such a way as to
bind specific molecules. If a radioactive molecule is bound to the
bead it is brought in close enough proximity that it can stimulate the
scintillant to emit light as depicted in Fig 2.1. The unbound
radioactivity is too distant from the scintillant and the energy released
is dissipated before reaching the bead and therefore these
page 4SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
disintegrations are not detected
Radioligand is in close proximity, stimu- Unbound radioligand does not stimulate
lating the bead to emit light the bead
Fig 2.1: Diagrammatic representation of SPA (not to scale).
Although many isotopes have emissions with appropriate energies,
few are practical for application in SPA. Tritium is ideally suited for
SPA because its β-particle has an extremely short pathlength through
water of only 1.5 µm. This means that the background obtained from
unbound tritium molecules is normally low, even when relatively large
amounts of activity are used. This low energy does have some
drawbacks. If a [3H] ligand binds to a receptor on a membrane, which
is in turn attached to the bead, the ligand may be held so far away
from the bead that a substantial portion of the energy of the emitted
radiation is dissipated before it reaches the bead and the efficiency
of detection may be lowered. As the distance the β-particle can travel
is dependent upon the properties of the material it must travel
through, the membrane tends to reduce the average pathlength of
the particles. This effectively means that the efficiency of detection
of the radioactive emissions is lowered for [3H] in these
circumstances.
Iodine-125 is another isotope that displays excellent properties for
use in SPA. The [125I] atom decays by a process termed "electron
capture". This type of decay gives rise to particles named Auger
electrons and these electrons may be detected by SPA. The
[125I]Auger electrons have pathlengths of approximately 1 µm and
17.5 µm. SPA assays with [125I] do not appear to display the
reduction in efficiency that is sometimes evident with [3H]. This is
page 5SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
presumably due to the energy of the Auger electrons being sufficient
to travel through the membrane and still be capable of stimulating the
bead to emit light. Indeed there is some evidence that a proportion
of the Auger electrons are too energetic and pass straight through the
bead. This effect is apparent when beads are allowed to settle and
become packed together. The observed number of counts increases
(see Fig 2.2 and Fig 2.3) because some Auger electrons travel away
from their bead of origin and instead are detected by adjacent beads
(see Fig 2.4).
CPM SQ P(I)
50% 1mg 20%
2mg
0.5mg 1mg
2mg 0.5mg
40% 16%
Iodine 0.125mg
Iodine 0.125mg
% Increase
30% 12%
% Increase 0.031mg
2mg
0.031mg
20% 8%
0.5mg
1mg 2mg
10% 0.125mg 4% 1mg
0.031mg Tritium 0.5mg
Tritium 0.125mg
0% 0% 0.031mg
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Time (h)
Time (h)
Fig 2.2: The effect of bead settling on count rate and quench parameter for Wallac MicroBeta™.
CPM tSIS
120%
50%
1mg
100% 0.5mg 1mg
Iodine 40%
Iodine 2mg
80% 2mg 0.5mg
30%
% Increase
0.125mg
% Increase
60% 0.125mg
20%
40% Tritium Tritium 0.031mg
2mg 10% 0.125mg
20% 1mg 0.5mg
0.5mg 1mg
0.125mg 0% 0.031mg
0% 0.031mg 2mg
0 2 4 6 8 10 12
0 2 4 6 8 10 12 0.031mg
-10%
-20%
Time (h) Time (h)
Fig 2.3: The effect of bead settling on count rate and quench parameter for Packard TopCount™.
page 6SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
a. Electron travels away from the bead b. More energetic electrons travel
of origin but interacts with adjacent through the bead of origin and interact
bead. Result is an increase in counts. with adjacent beads. The result is an
increase in the spectral quench
parameter of the isotope (SQP[I])or the
transformed spectral index of the
sample (tSIS) but not in counts because
the electron has already been
"detected" by the bead of origin during
the timeframe of counting.
Fig 2.4: a. Effect of bead on the count rate b. Effect of bead packing on the quench index parameter.
The SQP(I) or tSIS value for [125I] also increases SQP(I) and tSIS are
measures of the number of photons produced per disintegration,
which itself relates to the amount of the Auger electron's energy
absorbed by the bead. The more energetic Auger electrons pass
through their bead of origin, losing some energy and producing some
photons as they do so, but now can interact with adjacent beads
leading to more photons and a resulting increase in SQP(I) or tSIS
(see Fig 2.4a).
In the case of tritium, an increase in cpm is observed on packing
because of the effect in Fig 2.4a, but the β-particle is not energetic
enough to interact with more than one bead as in Figure 2.4b and an
increase in SQP(I) or tSIS is not seen.
These higher energy electrons will also tend to stimulate beads when
emitted from unbound isotopes resulting in a low-level background
termed non-proximity effect (NPE). Figure 2.5 illustrates where an
unbound molecule is close enough to the bead so that while the
majority of Auger electrons dissipate their energy to the medium, the
high-energy population are capable of exciting the scintillant giving a
background or NPE. This is often a component of a blank or non-
specific binding parameter in developed assays, but is a stable and
page 7SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
reproducible effect.
Fig 2.5: Diagrammatic representation of non-proximity effect (not to
scale).
2.2 A brief history of scintillation proximity assay
In 1978 Hart and Greenwald (1) presented data demonstrating an
agglutination assay in which [3H]-labelled polystyrene particles,
coated with human albumin, were incubated with scintillant-
impregnated polystyrene particles coated with anti-human albumin
antibodies. As the antibody-antigen interaction was allowed to
proceed, the scintillant particles became crosslinked with the
[3H]-labelled particles. The β-rays from the [3H] particles could
therefore strike the scintillant particles and the light emitted could be
detected by standard scintillation counting. They termed this method
scintillation proximity assay (SPA).
Further publications on the albumin/anti-albumin system (2, 3)
characterized this agglutination assay and added further explanation
to the SPA principle involving the limited pathlength of [3H]β-particles
through water. It was postulated that such agglutination assays
would be particularly useful as virus detection assays. This
aggregation method was subsequently patented (4).
Gruner and co-workers (5, 6) applied the SPA principle to monitor the
uptake of [3H]tetraphenylphosphonium ions (TPP) by Escherichia coli
membrane vesicles. In this application the scintillant beads were
page 8SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
encapsulated in a permeable gel that would allow penetration of the
labelled TPP but not the membrane vesicles. Uptake of TPP by the
vesicles could therefore be monitored as a decrease in the light
signal from the encapsulated beads.
The pioneering work of Hart was later taken up by Udenfriend and co-
workers who refined the method to involve a single particle or bead
containing scintillant. This was advantageous as the kinetics of
interactions of three or more macromolecules is a slow process and
ultimately the agglutination method of Hart was not amenable to
monitoring interactions between two individual molecules such as an
antibody-antigen interaction. Udenfriend et al (7) applied the SPA
technology to RIAs for enkephalins, thyroxin, and urinary morphine.
They termed their assay scintillation proximity radioimmunoassay
(SPRIA) and demonstrated that [125I] as well as [3H] was suitable for
SPA. Other isotopes were postulated as being amenable to SPA
such as 57Co, 75Se, and several other metals although the application
of these isotopes to SPA was unclear (8).
The beads used by Udenfriend were described as being "in no way
beads". They were in effect irregular particles and amorphous
aggregates of PVT . The surface methyl groups were oxidized to
carboxyl groups and various proteins were then linked to the beads
via carbodiimide coupling. The beads themselves were extremely
hydrophobic and were required to be washed in 3% TritonTM X-100*
before the derivatization was performed to enable the beads to be
dispersed in suspension (7). It was also noted that supposedly
similar beads from another supplier did not perform in the SPA
application.
The hydrophobic nature of the beads was exploited by Nelson (9) to
bind membranes or isolated acetylcholine receptor to the bead
surface. Using this approach, a SPA receptor-binding assay for
[125I]α-bungarotoxin to Torpedo membranes was developed. The
system potentially lacked robustness due to the non-specific
adsorption of the receptor to the bead but demonstrated the
application of a SPA receptor-binding assay.
During the period spanning the work of Udenfriend and Nelson (7, 8,
9), Bertoglio-Matte was granted a patent (10) incorporating the
principle of SPA and its application to RIA, receptor-binding assays,
and enzyme assays. The work presented in this patent described
SPA assays using a scintillant impregnated SepharoseTM bead. As
with the work of Udenfriend, the applications described elegantly
demonstrate the SPA principle. However, the bead design was crude
and did not offer a suitably generic coupling mechanism to be of
widespread application.
page 9SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
In 1988 Amersham Biosciences began work on evaluating the SPA
technology. The problems associated with the hydrophobic PVT
beads described by Udenfriend (7) became apparent immediately.
The beads aggregated in the absence of detergent and many
non-specific binding artifacts were encountered. In addition, the
hydrophobic adsorption method applied by Nelson (9) was found to
be limited in its application and the coupled material was often
displaced by non-specific protein or surfactants. In order to advance
the generic application of scintillation proximity assays, new bead
designs were required with defined and reproducible coupling
methods that would be useful for the attachment of a wide variety of
analytes.
The first application area addressed was radioimmunoassay (RIA).
Generic RIA beads were designed utilizing the base glass scintillator
yttrium silicate (11, 12). Proprietary methods were developed for
linking a variety of proteins to yttrium silicate particles resulting in a
range of "generic beads" for widespread use in RIA. These SPA
beads have protein A, sheep anti-mouse, donkey anti-rabbit, and
donkey anti-sheep antibodies coated on to their surfaces and are
therefore ideal for use in RIA. In 1989 Amersham Biosciences took
an exclusive worldwide license of the Bertoglio-Matte patent (10).
The RIA beads were launched in May of the same year.
Work on receptor-binding assays began in 1989. Early
experimentation with carboxylate derivatized PVT (7, 8) was found to
be irreproducible with poor coupling and high non-specific effects due
to the hydrophobic nature of the particles. Surface derivatization of
yttrium silicate with polycationic coatings produced particles suitable
for trapping negatively charged cell membranes thereby immobilizing
receptors tightly to the SPA beads. These polylysine yttrium silicate
beads were found to perform in a robust and reproducible fashion for
many applications in both tissues and cultured cell membranes.
Amersham Biosciences first launched products containing these
beads in 1990.
Yttrium silicate is an extremely dense material and SPA beads
manufactured with this material tend to settle very rapidly. For many
applications such as enzyme assays or soluble receptor assays, it is
preferable for the beads to remain in suspension for greater periods
of time. In 1990, work on a new PVT based bead was initiated. The
bead was designed to be a hydrophilic particle of approximately 5 µm
in diameter. Products incorporating this new PVT bead were first
launched in the same year. This was the first application of a SPA
assay for enzymes to be reported (13).
The scintillant in these beads was modified in 1991 to ensure that all
SPA assays designed with Amersham Biosciences SPA reagents
page 10SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
were fully compatible with current counting instrumentation.
Since 1991 an increasing number of application areas have been
addressed using SPA. Most recently, Amersham Biosciences has
extended the use of SPA to its use in scintillating microplates known
as Cytostar-TTM. These devices, which contain a scintillating base
plate, allow the study of a wide range of biochemical events in
cultured whole cells in contact with the base plate.
SPA has become the multidisciplinary technology alluded to by
Udenfriend (9) and Bertoglio-Matte (10). The current SPA beads
offer a variety of generic coupling mechanisms to link molecules of
interest easily and selectively to the bead surface, with low
non-specific binding properties. These beads have been
successfully applied to the areas of RIA, receptor-binding, enzyme
inhibition, protein-protein, protein-peptide, and protein-DNA
interaction assays providing simple, quick, and reliable technology
for the homogeneous assay of biochemical events.
2.3 Scintillation proximity assay beads
2.3.1 Introduction
Two types of material are used to make scintillation proximity assay
(SPA) beads. The first is yttrium silicate (YSi), which derives its
scintillation behavior from the luminescent properties of cerium ions
trapped within its crystal lattice. The second is poly(vinyl toluene)
(PVT) which acts as a solid "solvent" for the same types of organic
scintillators found in conventional liquid scintillation cocktails. Both of
these scintillators are compatible with all current scintillation counters
and both types of bead can be suitably derivatized for use in several
types of SPA technology. The various beads available and their
applications are summarized in Table 2.1.
Table 2.1: Types of SPA bead available.
Major application
Bead Type Code
area
Anti-rabbit YSi RIA RPN140 (500 mg)
PVT RIA RPNQ0016 (500 mg)
Anti-mouse YSi RIA RPN141 (500 mg)
PVT RIA RPNQ0017 (500 mg)
Anti-sheep YSi RIA RPN142 (500 mg)
PVT RIA RPNQ0018 (500 mg)
page 11SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
Table 2.1: Types of SPA bead available.
Anti-guinea pig PVT RIA RPNQ0178 (500 mg)
Protein A YSi RIA RPN143 (500 mg)
PVT RIA RPNQ0019 (500 mg)
Polylysine YSi Receptor-binding RPNQ0010 (1 g)
assays
WGA PVT Receptor-binding RPNQ0001 (500 mg)
assays
YSi Receptor-binding RPNQ0011 (250 mg)
assays
Streptavidin PVT Enzyme assays RPNQ0006 (50 mg)
RPNQ0007 (500 mg)
YSi Enzyme assays RPNQ0012 (250 mg)
Glutathione PVT Protein-binding RPNQ0030 (750 mg)
YSi Protein-binding RPNQ0033 (50 mg)
RPNQ0034 (500 mg)
WGA-PEI Type PVT Receptor-binding RPNQ0003 (500 mg)
A assays
WGA-PEI Type PVT Receptor-binding RPNQ0004 (500 mg)
B assays
Copper his-tag PVT Protein-binding RPNQ0095 (250 mg)
YSi Protein-binding RPNQ0096 (125 mg)
RNA-binding YSi RNA-binding RPNQ0014 (50 mg)
RPNQ0013 (500 mg)
Select-a-Bead WGA-PVT and Receptor-binding RPNQ0250 (100 mg
Kit YSi, WGA-PEI assays pots)
Type A and
Type B, and
YSi-Polylysine
beads
2.3.2 Yttrium silicate based SPA beads
Yttrium silicate (YSi) is supplied as irregular-shaped crystals with an
average particle size of approximately 2 µm in diameter. Figure 2.6
page 12SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
shows a photographic comparison of YSi and PVT beads. The
density of this material is 4.1 g/cm3 which means that the particles
settle quickly in aqueous buffers, and care must be taken when
pipetting YSi suspensions to ensure an even distribution of the solid.
For the same reason, assays using YSi beads usually have to be
shaken in order to facilitate equilibration.
Fig 2.6: Appearance of YSi and PVT SPA beads. Beads were
photographed in thin film suspension at a magnification of x400. a. YSi
beads b. PVT beads.
The beads have been derivatized in two ways, either by direct
coupling of proteins to the chemically activated surface; or by
pre-coating with polylysine or poly(ethyleneimine) (PEI),
cross-linking with glutaraldehyde, and coupling to the resulting free
aldehyde groups. The former direct coupling method is used for
protein A beads, whereas the latter indirect method is used for
coupling anti-rabbit, anti-sheep, and anti-mouse antibodies. The
protein A beads and second antibody beads have been employed in
page 13SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
several radioimmunoassays (14–19). Polylysine-coated beads have
themselves been used in receptor-binding assays. Yttrium silicate is
one of the most efficient solid scintillators known (11,12), and as
such, results in the highest signal output when used in SPA.
2.3.3 Poly(vinyl toluene) based SPA beads
Poly(vinyl toluene) (PVT) beads are made by a suspension
polymerization method that produces spherical particles having a
typical size distribution as depicted in Figure 2.7. In fact, 85% by
volume of particles are between 2 and 8 µm in diameter.
Fig 2.7: Typical particle size distribution (by volume) for PVT SPA
beads.
PVT is a hydrophobic polymer and to reduce non-specific binding the
bead surface has been coated with a polyhydroxy film. The
polyhydroxy coating confers a hydrophilic character to the bead. The
coating is activated by proprietary methods and various proteins may
then be covalently bound to the bead.
Although their scintillation efficiency is not quite as high as YSi; PVT
beads offer several advantages in terms of their density and surface
properties. The density of PVT is approximately 1.05 g/cm3, which
means that the beads stay in suspension much longer than YSi and
are more easily pipetted. Glycerol can be incorporated into the assay
buffers to match the densities of the beads and the buffer, which will
prevent the beads from settling out during the course of the assay.
page 14SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
2.3.4 Bead types
2.3.4.1 Yttrium silicate-polylysine beads
The polymeric form of lysine is known to interact with cellular
membranes (20) and other negatively charged species through ionic
binding. Polylysine is coated onto YSi SPA beads and cross-linked
in situ to form tightly bound polylysine molecules on the bead surface.
Yttrium silicate-polylysine SPA beads are routinely tested in a
receptor-binding assay to assesses the ability of the bead to bind a
known quantity of receptor bearing membranes.
The beads are supplied as aliquots of 1 g lyophilized from a solution
of 1% w/v sucrose. In this form, the beads may be stored at 2–8 °C
for up to 6 months before use. Once reconstituted, the beads should
be stored at 2–8 °C and used within 1–2 weeks. It is normally
advisable to include suitable antimicrobial agents in the
reconstitution buffer, particularly if proteins or other molecules are
precoupled to the beads.
Yttrium silicate beads are stable when frozen. However, in general,
proteins, membranes, and other biological molecules that have been
immobilized are less stable to repeated freeze-thaw cycles than if
they had been frozen from free solution. It is therefore not advisable
to repeatedly freeze-thaw polylysine beads.
2.3.4.2 Protein A beads
Protein A is isolated from the cell wall of a number of strains of the
bacteria Staphylococcus aureus. The protein consists of a single, 42
kDa polypeptide chain and contains little or no carbohydrate. Protein
A is characterized by its ability to bind to the IgG of most mammalian
species. However, it does not bind avian IgG and gives only a weak
interaction with ruminant IgG. Binding is through the Fc portion of the
immunoglobulin leaving the Fab region free for binding antigen.
Table 2.2 summarizes the binding specificity of protein A. The
protein A SPA bead is designed primarily for use in RIA applications;
particularly when the primary antibody is of rabbit, mouse, or guinea
pig origin.
Yttrium silicate- and PVT-protein A beads are supplied as 500 mg
aliquots. Yttrium silicate beads are lyophilized from 5% w/v lactose
solution whilst PVT beads are lyophilized from 10% w/v sucrose
solution. In this form the beads may be stored at 2–8 °C for up to 12
months before use. Once reconstituted, the beads should be stored
at 2–8 °C and used within 7 days. It is normally advizable to include
suitable antimicrobial agents in the reconstitution buffer under these
conditions.
page 15SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
Yttrium silicate beads are stable when frozen. For longer term
storage, reconstituted beads may be frozen at -15 to -30 °C. Avoid
repeated freeze- thaw cycles of reconstituted SPA beads.
PVT beads should not be frozen. Storage of PVT beads in frozen
form may alter the binding capacity and the non-specific binding
properties of the material.
Table 2.2: Binding specificity of protein A.
Type of
Species Subclass
immunogluobulin
Human IgG 1, 2, 4
IgA 2
IgM (some) -
Rabbit IgG (soluble com- -
plex)
Mouse IgG 1 (weakly),2a, 2b, 3
Rat IgG 1,2c
Guinea pig IgG 1,2
Bovine IgG 2 (weakly)
Sheep IgG 2 (weakly)
Goat IgG 2 (weakly)
Horse IgG a, b, c (all weakly)
Dog IgG a, b, c, d
IgA (some) -
IgM (some) -
2.3.4.3 Secondary antibody beads
There are four types of secondary antibody bead available for use in
RIA applications. These are sheep-anti-mouse, donkey-anti-rabbit,
donkey-anti-sheep, and sheep-anti-guinea pig. The latter is only
available coupled to PVT beads whereas the others are available
coupled to both PVT and YSi beads. All four bead types are coated
with an appropriate affinity purified IgG. The beads are routinely
tested in a RIA application prior to dispatch.
Yttrium silicate and PVT secondary antibody beads are supplied as
500mg aliquots. Yttrium silicate beads are lyophilized from 5% w/v
page 16SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
lactose solution whilst PVT beads are lyophilized from 10% w/v
sucrose solution. In this form the beads may be stored at 2–8 °C for
up to 12 months before use. Once reconstituted, the beads should
be stored at 2–8 °C and used within 7 days. It is normally advisable
to include suitable antimicrobial agents in the reconstitution buffer
under these conditions.
Yttrium silicate beads are stable when frozen. For longer term
storage reconstituted beads may be frozen at -15 to -30 °C. Avoid
repeated freeze-thaw cycles of reconstituted SPA beads.
PVT beads should not be frozen. Storage of PVT beads in frozen
form may alter the binding capacity and the non-specific binding
properties of the material.
2.3.4.4 Wheat germ agglutinin (WGA) beads
Lectins are proteins that are capable of agglutinating erythrocytes
and other types of cells. The agglutination produced by many lectins
is specifically inhibited by simple sugars and lectins and has been
shown to act by binding to sugar residues on the surface of cells
(21,22).
Wheat-germ agglutinin (WGA) is a lectin isolated from Triticum
vulgaris (wheat germ). The commercially available product is affinity
purified and contains no intrinsic protein-bound carbohydrate. In
solution at neutral pH the protein is a homodimer with a molecular
weight of about 35 000. WGA has an affinity for N-acetyl-
β-D-glucosaminyl residues and N-acetyl-β-D-glucosamine oligomers,
or glycoproteins (23).
For SPA, WGA is covalently bound to the PVT and YSi beads by a
simple process. Batches of WGA-SPA beads are tested routinely for
their ability to bind [3H]N,N',N''-triacetyl chitotriose, the trisaccharide
of N-acetyl-β-D-glucosamine.
It has been reported that N,N'-diacetyl chitobiose is 600-fold and
N,N',N''-triacetyl chitotriose 3 000-fold more potent in binding to WGA
than N-acetyl-β-D-glucosamine itself (24). The relative binding of
N-acetyl-β-D-glucosamine and its di, tri, and penta saccharides has
been investigated using WGA SPA beads and it has been shown that
the relative order of displacement of [3H]N,N',N''-triacetyl
glucosamine by these ligands is similar in magnitude and order to
that cited in the literature (24). This is an indication that the
immobilized WGA has retained its characteristic binding properties.
WGA SPA beads can therefore be used to investigate
receptor-ligand interactions with either partially purified cell
membrane preparations or with fractionated, solubilized receptor
preparations by immobilizing receptors and receptor bearing
page 17SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
membranes through the glycosylation of these components. The
performance of the bead is more critical to the effectiveness of the
solubilized receptors than to cellular membranes. This is because the
binding of membranes is a co-operative process with one membrane
fragment having many points of attachment. Soluble or solubilized
glycoproteins may have few binding sites, therefore the affinity for the
WGA bead is lower. The current specification set for WGA SPA
beads is based on a level of N,N',N''-triacetyl chitotriose binding per
unit bead.
YSi-WGA SPA beads are supplied as 250 mg aliquots whereas PVT-
WGA SPA beads are supplied as 500 mg aliquots. Both bead types
are lyophilized from 1% w/v sucrose solution. In this form and stored
at 2–8 °C protected from light, WGA SPA beads are stable for at least
6 months. Once reconstituted, the beads should be stored at 2–8 °C
and used within 1–2 weeks. It is normally advizable to include
suitable antimicrobial agents in the reconstitution buffer under these
conditions.
PVT beads should not be frozen. Storage of PVT-WGA beads in
frozen form may alter the binding capacity and the non-specific
binding properties of the material.
Fig 2.8: Displacement of [3H]-N,N',N"-triacetyl chitotriose with N-acetyl-
β-D-glucosamine and its di, tri and penta oligomers from PVT-WGA
SPA beads.
Assays were performed using 12.5 µg of PVT-WGA bead and 10 µCi
page 18SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
of [3H]N,N',N"-triacetyl chitotriose in 200 µl of buffer consisting of 25
mM MOPS, 150 mM NaCl, pH 7.1. The microplate was shaken for
30 min until equilibrium was achieved and counted for 10 min per
sample well in a PackardTM Microplate scintillation counter.
2.3.4.5 PVT-WGA polyethyleneimine beads
The treatment of PVT-WGA SPA beads with positively charged
polyethyleneimine (PEI) blocks potential non-specific binding sites
on the SPA bead surface.
There are two SPA bead types available with PEI treatment. The
PVT-WGA-PEI type A SPA beads (RPNQ0003) are treated with PEI
prior to the coupling of WGA to the PVT SPA bead. The PVT-WGA-
PEI type B SPA beads (RPNQ0004) are treated with PEI after the
WGA coupling stage
The PVT-WGA-PEI type A and type B SPA beads exhibit different
characteristics with regard to the non-specific binding of radiolabelled
ligand directly to the SPA bead. Therefore, both bead types should
be evaluated when deciding which SPA bead to use and both are
included in the Select-a-Bead Kit (see table 2.2). The binding
capacity of both bead types for cell membrane protein remains 10–
30 mg membrane protein per milligram of SPA bead.
PVT-WGA-PEI Type A and type B SPA beads are supplied as either
100 mg (in the Select-a-Bead Kit) or 500 mg aliquots, lyophilized from
1% w/v sucrose solution. In this form and stored at 2–8 °C protected
from light, WGA-PEI SPA beads are stable for at least 6 months.
Once reconstituted, the beads should be stored at 2–8 °C and used
within 1–2 weeks. It is normally advizable to include suitable
antimicrobial agents in the reconstitution buffer for storage under
these conditions.
PVT beads should not be frozen. Storage of PVT-WGA beads in
frozen form may alter the binding capacity and the non-specific
binding properties of the material.
2.3.4.6 Streptavidin beads
Streptavidin is a 60kDa biotin-binding protein derived from the
fungus Streptomyces avidinii (25). Streptavidin is carbohydrate-free,
unlike the related egg white-derived avidin, which is a glycoprotein
(26). The high affinity of streptavidin for biotin or biotinylated species
makes streptavidin an invaluable tool for a wide variety of assay
applications.
Streptavidin is widely available in a processed form that has been
affinity purified to give a tetrameric protein containing four biotin-
binding subunits (25). For SPA, streptavidin is covalently bonded to
page 19SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
beads by a simple process. This process appears to destroy or
obscure one of the four biotin binding sites. This generates a stable
SPA bead with streptavidin bound to it that has three of its four
originally available biotin-binding sites free for ligand or substrate
interaction. The dissociation rate constant for the streptavidin-biotin
complex on SPA beads has been measured in our laboratories and
is of the same order as that quoted in the literature (27).
The biotin-binding capacity of each batch of streptavidin SPA beads
is estimated by a [3H]biotin-binding assay. Figure 2.9 demonstrates
the saturation binding of PVT-streptavidin SPA beads with [3H]biotin.
Although three [3H]biotin-binding sites are theoretically free on
streptavidin bound to SPA beads, the apparent capacity of the beads
is likely to be affected by the properties of the biotinylated molecules
such that fewer equivalents of substrate may be bound. The binding
capacity of the biotinylated molecules for the streptavidin SPA bead
should be determined empirically in each case.
YSi-streptavidin SPA beads are supplied as 250 mg aliquots, PVT-
streptavidin SPA beads are supplied as either 50 mg or 500 mg
aliquots, both types are lyophilized from 1% w/v sucrose solution. In
this form and stored at 2–8 °C protected from light, streptavidin SPA
beads are stable for at least 6 months with constant biotin-binding
capacity. Once reconstituted, the beads should be stored at 2–8 °C
and used within 1–2 weeks. It is normally advizable to include
suitable antimicrobial agents in the reconstitution buffer for storage
under these conditions.
PVT beads should not be frozen. Storage of PVT-streptavidin beads
in frozen form may alter the binding capacity and the non-specific
page 20SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
binding properties of the material.
Fig 2.9: Saturation binding of PVT-streptavidin SPA beads by
[3H]biotin.
Assays were performed using 0.1 mg of PVT-streptavidin SPA beads
and the specified quantity of [3H]biotin in 125 µ l of phosphate
buffered saline. The reactions were carried out in Sarstedt microfuge
tubes and allowed to settle for 16 h prior to counting in a Wallac
RackBetaTM scintillation counter.
2.3.4.7 Glutathione beads
Glutathione (or g-glutamylcysteinylglycine) is a small tripeptide that is
capable of conjugating with glutathione-s-transferase (GST)
enzymes. This property has been developed for immobilization of
GST by affinity chromatography and has been extended to purify
GST-fusion proteins.
Using this concept for immobilizing GST-fusion proteins, the
glutathione SPA bead has been developed. The outer surface of the
bead has been modified by a coating of glutathione. This bead will
enable the trapping and quantification of GST-fusion proteins either
directly if they are radiolabelled, or indirectly via radiobelled binding
page 21SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
partners.
The ability to trap and quantify cloned tagged proteins is a desirable
target in the drug screening market, potentially replacing yeast
hybridization systems, immunoprecipitation, electrophoresis, and
blotting methods.
YSi-glutathione SPA beads are supplied as 50 mg or 500 mg aliquots
lyophilized from borate buffer. In this form and stored at 2–8 °C
protected from light, YSi-glutathione SPA beads are stable for at
least 3 months. Once reconstituted, the beads should be stored at
2–8 °C and used within 1–2 weeks. It is normally advizable to include
suitable antimicrobial agents in the reconstitution buffer under these
conditions.
PVT-glutathione SPA beads are supplied as 750 mg aliquots
lyophilized from borate buffer. In this form and stored at 2–8 °C
protected from light, PVT-glutathione SPA beads are stable for at
least 3 months. Once reconstituted, the beads should be stored at
2–8 °C and used within 1–2 weeks. It is normally advizable to include
suitable antimicrobial agents in the reconstitution buffer under these
conditions.
This bead has been used with [3H] and [125I], but can be adapted for
use with other isotopes, such as [33P].
2.3.4.8 Copper his-tag beads
Immobilized Metal Affinity Chromatography (IMAC) for the
separation of histidine-tagged proteins and oligopeptides has been
known for some years, originally using iminodiacetic acid (IDA) and
tris(carboxymethyl)ethylene diamine (TED) as well as those based
on ethylenediamine tetraacetic acid (EDTA). These bind a range of
first-row transition metals such as Zn2+, Ni2+ and Cu2+.
Using this concept, the his-tag beads developed by Amersham
Biosciences is a novel bead formulation where the outer surface of
the bead has been modified by a coating of a chemical chelate
(containing bound copper). This bead will enable the trapping and
quantitation of histidine-tagged-fusion proteins and their binding
partners.
In a direct assay format, the SPA beads could be used to trap and
quantitate the binding of a directly radiolabelled histidine (his)-tagged
fusion protein, peptide, or oligopeptide such as a kinase substrate,
using [33P]ATP as the donor molecule. In an indirect assay format,
the SPA beads could be used to trap and quantitate the association
of a radiolabelled binding partner to a histidine (his)-tagged fusion
protein, peptide, or oligopeptide.
page 22SCINTILLATION PROXIMITY ASSAY: Theory and practical application 2
Yttrium silicate-his-tag SPA beads are supplied as 125mg aliquots at
20mg/ml in water. In this form and stored at 2–8 °C protected from
light, YSi-his-tag SPA beads are stable for at least 6 months. Once
opened, the beads should be stored at 2–8 °C and used within 1–2
weeks.
PVT-his-tag SPA beads are supplied as 250 mg aliquots at 20 mg/ml
in water. In this form and stored at 2–8 °C protected from light, PVT-
his-tag SPA beads are stable for at least 6 months. Once opened the
beads should be stored at 2–8 °C and used within 1–2 weeks.
2.3.4.9 RNA-binding beads
Uncoated YSi beads have been shown to interact with primary
phosphate groups in nucleotides (e.g. ATP) and oligonucleotides,
DNA, and RNA. Membrane preparations can also be coupled to the
beads.
Yttrium silicate-RNA-binding SPA beads are supplied as 500 mg
aliquots at 100 mg/ml in water. In this form and stored at 2–8 °C
protected from light, YSi-RNA-binding SPA beads are stable for at
least 6 months. Once opened, the beads should be stored at 2–8 °C
and used within 1–2 weeks.
page 23THE APPLICATION OF SPA TECHNOLOGY TO ENZYME ASSAYS 3
SECTION 3 THE APPLICATION
OF SPA TECHNOLOGY TO
ENZYME ASSAYS
3.1 Introduction
The catalytic action of enzymes can be determined by scintillation
proximity assay (SPA). The basis of the majority of SPA enzyme
assays is the use of biotinylated substrates, which may either be
immobilized on, or subsequently captured by, streptavidin-coated
SPA beads. The biotin-streptavidin system is renowned for the
strength of binding involved and therefore gives a reliable,
reproducible, and high-affinity capture system for use in SPA enzyme
assays.
SPA enzyme assays have been developed for a number of enzyme
classes including hydrolases, transferases, polymerases, and
kinases. The technique is applicable to [3H], [125I], and [33P]-labelled
substrates. and as with other assays, optimization is required.
The conversion of the substrate to product is monitored by designing
the assay to either remove or add radioisotope with respect to the
component, which is captured on the SPA bead. Either the process
can involve the removal of radioactivity by the enzyme resulting in a
decrease in the SPA signal or, conversely, the reaction may involve
the addition of radioisotope causing an increase in the SPA signal. In
all cases, the discrimination of product from substrate does not
require the components to be separated because SPA is a
homogeneous technology. This has the advantage in some
instances that the incomplete recovery or detection of the product is
not an issue. As the entire reaction takes place in one tube there are
no errors incurred by transfer and separation steps, which are
traditionally employed for enzyme assays. SPA enzyme assays
thereforeshow high precision and reproducibility when compared to
methods such as precipitation, filtration, and HPLC.
SPA is a powerful technology when large numbers of samples are
required to be assayed in a limited time frame. In this instance, SPA
may be considered an enabling technology for many enzyme assays.
The removal of a laborious or cumbersome separation step means
that SPA enzyme assays are fast, simple, precise, and easy to
automate. The enzyme reaction may be terminated by methods such
as a pH shift or the addition of a chelator of essential cofactors. In
most instances, the "stop" reagent may be formulated with the SPA
beads present. Therefore, the reaction can be stopped and the
page 25THE APPLICATION OF SPA TECHNOLOGY TO ENZYME ASSAYS 3
signal generated by a single pipetting step.
SPA is a solid phase technology and the binding capacity of the bead
surface is finite. Therefore, the quantity of substrate that can be used
is also finite. It is important to balance the quantity of bead required
with the quantity of substrate in order to obtain an adequate signal
with a concentration of substrate that gives a kinetically competent
assay. As in other assay technologies, this will invariably involve
"trade offs" in assay volume, substrate concentration, signal
obtained, blank, and sensitivity, which will be particular to each
individual assay.
When designing a SPA enzyme assay, two options are available;
namely a solid phase ("on"-the-bead) format or a solution phase
("off"-the-bead) assay format. The format selected depends largely
on the assay being developed and the intended application of that
assay. For example, a solution phase assay lends itself more to
kinetic analysis compared with the solid phase format.
One problem that arises owing to the homogeneity of the assays is
the presence of color in the samples, which will not be separated
before the assay is counted. The issue of color quench will be
covered in section 4 of the course manual whereas this section will
cover the fundamental aspects of the design and development of
SPA enzyme assays.
3.2 Assay design
The fundamental aspects of designing a SPA enzyme assay are
similar to those involved in traditional methods. It is therefore useful
to consult the literature available for the enzyme of interest to
ascertain the requirements for pH, ionic strength, cofactors, and
substrate specificity. In addition, to design the appropriate SPA
assay, the substrate or product must be able to effectively bind to the
SPA bead, so the inclusion of a biotinylation site may be necessary.
This must not interfere with the activity of the enzyme. Another
aspect to consider is a route to terminate the enzyme reaction.
Enzymes that display critical pH dependence can be terminated by
an appropriate pH shift. Another approach may be to sequester an
important cofactor, such as divalent cations, by chelators like EDTA
or EGTA.
In general, SPA enzyme assays are designed using the streptavidin
SPA bead. The biotin-streptavidin reaction is stable and rapid over a
wide range of conditions and therefore provides the ideal capture
system for application in SPA enzyme assays. Another strategy is to
use SPA antibody-specific or protein A beads to capture a reaction
product using a specific antibody.
page 26THE APPLICATION OF SPA TECHNOLOGY TO ENZYME ASSAYS 3
The key component in the design of a SPA enzyme assay is the
substrate. If structure-activity studies have been performed on the
enzyme of interest, then this information can be extremely valuable
in designing the substrate. It is important to ascertain whether the
biotinylation or the radiolabelling interferes with the kinetics of the
enzyme action. This is normally determined by direct comparison of
rates of activity in a SPA format and a traditional method such as
HPLC. Biotinylation of substrates may be affected by a number of
reagents depending upon the moiety to be coupled (see section 3.4).
The molecule can be radiolabelled with [3H], [125I], or [33P] for SPA
enzyme assays.
3.2.1 Signal decrease assay
The signal decrease assay format is suitable for hydrolytic enzymes
that act at a single cleavage site on the chosen substrate. The
substrate is designed with a site for bead binding and a radiolabelling
site. These sites are separated by a cleavage sequence for the
enzyme. Figure 3.1a demonstrates how the labelled residue is
removed from the substrate by the enzyme, causing a decrease in
SPA signal proportional to the enzyme activity as shown in Figure
3.1b, which is a timecourse for the enzyme. Most signal decrease
assays can either be designed as solution phase or solid phase
assays, but not all are amenable to the solid phase format.
3.2.2 Signal increase assay
The signal increase assay format is suitable for polymerase and
transferase enzymes, and any assay where the labelled product of a
reaction can be coupled to a SPA bead. The signal increase in the
assay is proportional to the enzyme activity as shown in Figures 3.2b
and 3.3b.
Polymerase reactions: (e.g. reverse transcriptase [see Fig 3.2a]).
Labelled residues are added to an acceptor molecule, which can be
attached to a SPA bead (in this case via a biotin-streptavidin link).
Transferase reactions: (e.g. CETP). The acceptor molecule is
biotinylated for attachment to the streptavidin-SPA bead and a
radiolabelled molecule is transferred from donor to acceptor
molecule.
Product capture assays: (e.g. endothelin converting enzyme [see
Fig 3.3a]). The radiolabelled product of an enzyme reaction is
captured on a SPA bead, in this case by a second antibody
interaction. If an antibody is used for product capture it must be able
to discriminate adequately between a small amount of generated
product and the excess substrate present.
page 27THE APPLICATION OF SPA TECHNOLOGY TO ENZYME ASSAYS 3
The advantage of product-capture assays is that they can be used for
signal increase assay formats to measure hydrolytic enzyme
activities. However, the fact that there are multiple interactions
involved in the assay (e.g. product-antibody, antibody-second
antibody) may be an issue in screening applications.
For most assays, there is the option to design the assay in either
solution phase or solid phase..
Fig 3.1: Diagrammatic representation of a signal decrease SPA enzyme
assay
Fig 3.2: Timecourse analysis of HIV-1 proteinase SPA enzyme assay
The assay was performed as described in the protocol booklet for the
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