Note: Descriptions are shown in the official language in which they were submitted.
CA 02198470 1997-02-25
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The resent invention relates to a method of labelling chemical materials,
particularly
biological materials, for the purpose of chemical, and particularly biological
assay, and more
particularly specific binding assay, such as immunoassay and hybridisation
probing
techniques and for incorporation of labels into specific amplification
products e.g. using
PCR in assay formats.
The firefly luciferase mediated cleavage of luciferin (Eqn 1 ) has a high
quantum yield and
stable light output allowing the enzyme itself to be detected at very' low
concentrations using
relatively simple instruments (see McCapra. Potential applications of
bioluminescence and
chemiluminescence in Turner et al (Edit.) Biasensors: fundamentals and
applications:
Oxford University Press, (19$8): 617-37). Many methods have been developed
using
luciferase as an indirect label (see Wannlund and DeLuca, 'Bioluminescence
immunoassays:
Use of luciferase antigen conjugates far determination of methoxylate and DNP'
in Deluca
and McElroy (Edits). Bioluminescence and Chemiluminescence: Basic chemistry
and
analytical applications. London: Academic Press, (1981 ): 693-696; Geiger and
Miska,
Bioluminescence enhanced enzyme immunoassay: New ultrasensitive detection
system for
enzyme immunoassay, Clin. Chem. Clin. Biochem. J. ( 19$7) 25. 3:l-38 and
Murakami et al,
Development of a bioluminescent detection system using adenylate; kinase and
firefly
luciferase in Szalay et al (Edits.) Bioluminescence and cherniluminescence:
Status Report,
Chichester: John Wiley and Sons, (1993) 296-300.
The present inventors have noted that assay design could be much simplified
whilst
maintaining sensitivity by using luciferase as a direct label, but that
methods of coupling
it to assay binding agents are required that do not result in inactivation of
what is well
known to be a very labile enzymatic activity. The standard covalent coupling
reagents
such as glutaraldehyde, S14ICC and S1VIPB rapidly and irreversibly inhibit
luciferase
activity.
AMENDED SHEET
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2
Luciferase, eg that from Photinus pyralis, contains four
cysteine residues (see de Wet et al (1987) Molecular and
Cellular Biology 7, 725-737) two of which are near to or
part of the active site (see Deluca and McElroy (1978)
Methods in Enzymology, 57, 3-15). The present inventors
have determined that binding of covalent coupling reagents
to these residues may cause the observed inactivation and
have provided a method for coupling luciferase to assay
agents that protects the enzyme from irreversible
inhibition.
Thus in a first aspect of the present invention
there is provided a method for conjugating firefly
luciferase to a chemical entity, particularly to a specific
binding agent such as an antibody, antigen or a nucleic
acid, and more particularly an antibody, comprising (a)
mixing the luciferase with one or more of D-luciferin,
magnesium ions and adenosine triphosphate, such that the
concentrations of magnesium ions and adenosine triphosphate
are greater than 0.2 mmol/1 and 0.05 mmol/1 respectively and
(b) performing a covalent coupling reaction between the
luciferase and the chemical entity using a covalent coupling
reagent.
More specifically, in this first aspect, the
invention provides a method for conjugating firefly
luciferase to an antibody or nucleic acid, comprising: (a)
mixing the luciferase with a reagent selected from the group
consisting of (i) D-luciferin, (ii) magnesium ions and
adenosine triphosphate, and (iii) D-luciferin, magnesium
ions and adenosine triphosphate, such that the
concentrations of magnesium ions and adenosine triphosphate
where present are greater than 0.2 mmolll and 0.05 mmol/1,
respectively, and (b) performing a covalent coupling
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2a
reaction between the luciferase and the antibody or nucleic
acid using a covalent coupling reagent.
In a further method aspect, the invention provides
a method for conjugating firefly luciferase to an antibody,
antigen or nucleic acid, comprising the steps of: (a) mixing
firefly luciferase with D-luciferin, magnesium ions and
adenosine triphosphate, such that the concentrations of
magnesium ions and adenosine triphosphate are greater than
0.2 mmol/1 and 0.05 mmol/l, respectively; and (b) covalently
coupling the luciferase and the antibody, antigen or nucleic
acid using a covalent coupling reagent.
In a still further method aspect, the invention
provides a method for conjugating firefly luciferase to an
antibody, antigen or nucleic acid, comprising the steps of:
(a) mixing firefly luciferase with D-luciferin; and (b)
covalently coupling the luciferase and the antibody, antigen
or nucleic acid using a covalent coupling reagent.
In a yet further method aspect, the invention
provides a method for conjugating firefly luciferase to an
antibody or nucleic acid, comprising the steps of: (a)
mixing firefly luciferase with D-luciferin or a combination
of magnesium ions and adenosine triphosphate, such that the
concentrations of magnesium ions and adenosine triphosphate
where present are greater than 0.2 mmol/1 and 0.05 mmol/1,
respectively; and (b) covalently coupling the luciferase and
the antibody, antigen or nucleic acid using a covalent
coupling reagent.
In another further method aspect, the invention
provides a method for conjugating firefly luciferase to an
antibody or nucleic acid, comprising the steps of: (a)
mixing firefly luciferase with D-luciferin, magnesium ions
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2b
and adenosine triphosphate, such that the concentrations of
magnesium ions and adenosine triphosphate are greater than
0.2 mmol/1 and 0.05 mmol/1, respectively; and (b) covalently
coupling the luciferase and the antibody, antigen or nucleic
acid. using a covalent coupling reagent; wherein oxygen is
excluded from the reaction.
In a kit aspect, the invention provides a test kit
for use in a specific binding assay comprising an antibody
or a nucleic acid conjugated to active luciferase by the
method of the invention and a light guide or light detector
array onto which antibodies, antigens or nucleic acids have
been immobilised.
In a further kit aspect, the invention provides a
test kit for use in a specific binding assay comprising an
antibody conjugated to active luciferase and a light guide
or light detector array onto which antibodies, antigens or
nucleic acids have been immobilised.
Preferably the step (a) is carried out by mixing
the luciferase with its substrates in solution and
preferably both magnesium and adenosine triphosphate are
present as magnesium adenosine triphosphate (Mg2+ ATP),
optionally together with D-luciferin. Preferably only one
of Mg2+ ATP or D-luciferin is present. If all three of Mg2+,
ATP .and luciferin are present then it is preferable to
exclude oxygen from the reaction mixture.
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In a second aspect of the present invention there is provided a labelled
chemical entity
comprising a chemical entity conjugated to active firetly~ luciferase as
provided by the
method of the present invention. Preferably the chemical entity is a specific
binding agent
suitable for use in a specific binding assay, preferably being an antibody.
antigen or nucleic
acid. When the binding agent is a nucleic acid it is preferably an
oligonucleotide, but may be
;~ polynucleotide or a nucleoside, and may be used as a hybridisation probe or
a chain
extension primer, eg a PCR primer. Most advantageously the entity is an
antibody as
previous attempts to couple antibodies to luciferase have resulted in almost
complete
inactivity.
.~ particular advantage of provision of luciferase labelled chemical entities,
and particularly
luciferase labelled antibodies, is the enablement of performance of light
guide associated
capture assays. In one preferred such assay a capture antibody for a target
antigen is
immobilised upon a lightguide such as an optic fibre that is arranged to input
light falling
upon it to a light measuring device eg a photomultiplier; an antigen to be
measured is
applied to the lightguide in a liquid sample and a second antibody,
characterised in that it is
labelled with luciferase, is contacted in solution with the lightguide
whereupon it becomes
bound to the already captured antigen that has been bound to the capture
antibody.
In order to determine the presence and/or amount of the captured antigen it is
only necessary
to contact D-luciferin and MgZ+ AT'P in solution with the surface of the
lightguide, which
has the antigen-antibody complex bound to it, and to measure the amount of
light emitted
and transferred to the light measuring device eg photomultiplier. In this
manner it is possible
for luminometric assays of greater Sensitivity to be carried out, with
multiple specificity
provided by use of several lightguides each capable of capturing a different
antigen and
placed in a single sample chamber such that several different immunoassays can
be carried
out simultaneously by adding several different luciferase labelled antibodies
in the same
step.
AMENDED SHEET
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Alternatively a charge couple device (CCD) or equivalent such as diode arrays
or
photomultipliers might be used to monitor a number of discrete areas on a 1 or
2
dimensional detector array surface simultaneously, each with a different
immobilised
antibody specific for a different antigen, such that presence of a particular
antigen might be
detected by the position of light emission. Similarly, use of such lightguides
or charge
couple devices onto which antigens or anti-immunoglobulin antibodies specific
for a target
antibody have been immobilised allows competition binding assays for specific
antibodies
wherein the amount of luciferase labelled antibody bound to the antigen on the
lightguide
will be reduced when competing antibody is added at the same time in the form
of a sample.
On removal of the sample and luciferase labelled antibody and exposure of the
lightguide to
D-luciferin and Mg'-+ ATP substrate solution the amount of light detected at
the
photomultiplier may be related to the amount of antibody in the sample that is
specific for
the immobilised antigen or anti-immunoglobulin antibody.
It will be realised that if lightguides having oligonucleotide probes bound to
their surfaces
(see method in applicant's WO 930621) and oligonucleotides labelled with
luciferase are
employed that assay of oligonucleotides and polynucleotides will be possible
in analogous
manner to the antibody-antigen assays described above. I~urthermore, using
different
lightguides or array areas, simultaneous assay of antigens and nucleic acids
will be possible
from a single sample.
AMENDED SHEET
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WO 96/07100 PGT/GB95102038
A particular advantage of performing luciferase labelled binding assays on the
surface
of a light guide such as a planar waveguide or an o~>tic fibre (these may be
multiplexed) is that the need to separate reagents from the species of
interest, ie the
bound species, is reduced as light generated within a few hundred nanometers
of the
lightguide surface, is preferentially detected by the detector while light
generated in
bulk solution is not. Thus assays using such format (usually termed
evanescence)
would require no wash steps or sequential additions of reagents, and thus
could be
carried out very quickly and optionally in a flow of liquid using a flow cell.
The labelled agents of the present invention, the method for producing them
and a
method for their use will now be exemplified by way of illu:~tration only by
reference
to the following non-limiting Examples and Figures.
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6
FIGURES
Figure 1 shows a plot of luminescence v time obtained after incubation of
luciferase
with covalent coupling reagent with protection br varying quantities of
MgZ+ATP as
described in Example 1.
Figure 2 shows a plot of luminescence v amount of ricin for an assay as
carried out as
described in Example 1. The upper plot is that obtained using a 1/100 dilution
of the
IgG-luciferase conjugate while the lower plot uses a 1/1000 dilution.
LHZ + ATP + E M~ LI-I;-AMP.E + PP,
LH~-AMP.E + O, -~- L + CO~ + AMP + Light
where LHZ is luciferin, E is luciferase, AMP is adenosine monophosphate, PP,
is
inorganic pyrophosphate. and L is oxyluciferin.
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EXAMPLE 1
A Multilite~ Luminometer and 3.5m1 polystyrene tubes (Biotrace Bridgend UK)
were used
for all light measurements. Firefly luciferase (L-5?56), I7TT, BSA (Fraction
V), ATP and
ricin were obtained from Sigma (Poole, UK); D-luciferin was obtained from
Fluka
(Gillingham, UK) and sulphosuccinamidyl-4-(N-maleirnidomethyl)cyclohexane-1-
carboxylate (sulfo-SMCC) was obtained from Pierce and Warriner (Chester, UK).
Sheep-
anti-ricin antibodies were produced by conventional techniques and other
reagents were of
analytical grade.
Luciferase substrate was l Ummol/litre HEPES buffer pH7.75 containing
0.4mmol/litre D-
luciferin, 40mmo1/litre magnesium sulphate, 2mmo1/litre. AT'P, 2mmo1/litre
EDTA,
2mmo1/litre DTT, 0.2% BSA and 2gmol/litre sodium pyrophosphate. Substrate was
prepared fresh daily from stock solutions. The Luciferase activity assay was
carried out by
adding 2 x 10-61 of the sample to be assessed to 200 x 10-f'1 of substrate
(described above) in
a 3.5m1 polystyrene tube and luminescence was measured after a 5 second delay,
integrated
over a 10 second period.
Substrate protection method: Luciferase at 4.2gmo1/litre was mixed with sulfo-
SMCC at
630gmo1/litre in lOmmol/litre HEPES buffer at pH 7.75 , and the rriixture
incubated at room
temperature for 30 minutes. At timed intervals a sample was removed from the
reaction
mixture and diluted 100-fold in buffer and the luciferase activity of the
sample measured.
Various concentrations of Mg'+ ATP or D-luciferin were added to the mixture
and their
Lffect on activity monitored to determine the most effective protection
against inhibition.
AI4tEyDED S~?EET
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8
Covalent coupling with sulfo-SMCC: sheep IgG raised against ricin was reduced
to
release thiol groups by reaction with 2-mercaptoethylamine-HC1 at 37°C
for
90 minutes (see Pierce Warriner kit instructions). 4mg luciferase was prepared
in
lml phosphate buffer pH 7.0 containing O.Smmo!/litre ATP and 2.SmmoUlitre
magnesium sulphate. SOpI of 20mmoUlitre sulfo-SMCC in phosphate buffer pH 6.0
was added and the mixture incubated at room temperature for 30 minutes. The
activated luciferase was purified using a Pierce GF-5 desalting column and
activated
luciferase and reduced IgG were combined at a molar ratio of 1:1 in phosphate
buffer
pH 7.0 containing 0.25mmol/litre EDTA, O.Smmolllitre ATP and 2.Smmo1/litre
magnesium sulphate. The reaction was allowed to proceed at room temperature
for 30
minutes and then stopped by adding 1 Opl 1 molllitre cysteine for 20 minutes
before
purifying the luciferase -antibody conjugate on a Pierce GF-5 desalting
column. The
product was stored at 4°C in sodium phosphate buffer pH 7.0 containing
0.25mmoUlitre EDTA until required.
The activity remaining in the conjugated luciferase was assessed by
performance of an
ELISA assay as set out below:
Tube-based bioluminescent ELISA: Polystyrene tubes were coated with 100p1
ricin
in carbonate-bicarbonate buffer pH 9.6 containing 0.02% thiomersal overnight
at 4°C.
The luciferase-antibody conjugate was diluted in phosphate buffered saline
(PBS) and
after blocking of tubes with 200p1 1% BSA, 100p1 labelled antibody was added
and
incubation carried out for 1 hour at 37°C. The tubes were washed five
time in PBS
containing 5~ TweenT"" 20. 200,1 substrato was added to each
tube and luminescence measured immediately.
CA 02198470 1997-02-25
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9
Results: The results obtained from substrate protection by Nlg'+ A'TP are
shown in Figure 1
where % remaining activity as indicated by luminometer readings is plotted
against time as
derived by removal of samples (3 repeats each point] trom the reaction mixture
which
contained 0.63 x 10-6 mol/litre sulfc> -SMCC, 4.2 x 106 cnolilitre luciferase
and Mg2+ ATP.
Plots are shown for reaction with no Mg2+ or AT'P; 0.03mmo1/litre ATP and
0.2mmo1/litre
Mgz+, 0.25mmo1/litre ATP and 2.Ommo1/litre Mg7~, 0.25mmo1/litre ATP and
2.Smmol/litre
Mgz+. D -luciferin was also assessed for protective effect (not shovvn) and
was found to give
some retention of activity but significantly less than that of Nlg'+ ATP. The
maximum D-
luciferin concentration tested was 1 mmol~'litre and allowed retention of 11 %
of initial
luciferase activity after 30 minutes exposure to sulfo-SMCC as compared to
over 40% using
the Mg Z+ A'TP.
Tube based bioluminescent ELISA results are shown in Figure 2. 7.'he
polystyrene tubes
were used to allow light measurements to be made directly in the Multilite~
luminometer
but microtitre plates and a plate luminometer could equally be used. The
results show that an
active luciferase-antibody conjugate is produced and that the antibody retains
its binding
properties.
AMENDED SHEET
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EXAMPLE 2
.A sheep polyclonal antibody raised to ricin was covalently Coupled to an
optic fibre
connected to a photomultiplier tube; the section of the fibre with antibody
coupled to it
residing within a chamber capable of being filled with a variety of solutions
and emptied as
required. The assay followed the following cycle:
I;i) D-luciferin containing substrate solution added to the chamber such that
any
luciferase present would cause light to be emitted;
(ii) substrate solution replaced by test sample including ricin;
(iii) test sample solution replaced with solution containing sheep anti-ricin
IgG
labelled with luciferase;
(iv) sheep anti-ricin solution replaced with D-luciferin containing substrate
solution;
(v) substrate solution replaced by regeneration buffer.
Using this format it is possible to determine the amount of ricin in the test
sample by
relating the increase in signal from the photomultiplier tube during t:he step
(iv) over the
I>rior signal and relate that to the amount of ricin, eg using a standard
curve obtained using
signal increase for known amounts of ricin.
The method for conjugating luciferase and the labelled chemical entities
described in this
application may also be suitable for the luciferases described in WG 9525798.
Similarly the
method may also be suitable for other luciferases.
AMENDED SHEET
