U.S. patent application number 10/353721 was filed with the patent office on 2004-02-05 for electrochemical assays.
Invention is credited to Badley, Robert Andrew, Porter, Robert Andrew.
Application Number | 20040020791 10/353721 |
Document ID | / |
Family ID | 8229630 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040020791 |
Kind Code |
A1 |
Porter, Robert Andrew ; et
al. |
February 5, 2004 |
Electrochemical assays
Abstract
Disclosed is a component for a device for detecting the presence
of an analyte of interest in a sample, the component comprising an
electrically conducting solid support having immobilised thereon a
chemical moiety, said chemical moiety comprising an electroactive
portion with an electrochemical property capable of being directly
modulated in a detectable manner by the binding thereto of a
binding partner having a specific binding activity for the
electroactive portion, together with apparatus comprising the
component, and a method of detecting the presence of an analyte of
interest.
Inventors: |
Porter, Robert Andrew;
(Northamptonshire, GB) ; Badley, Robert Andrew;
(Bedford, GB) |
Correspondence
Address: |
OPPEDAHL AND LARSON LLP
P O BOX 5068
DILLON
CO
80435-5068
US
|
Family ID: |
8229630 |
Appl. No.: |
10/353721 |
Filed: |
January 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10353721 |
Jan 28, 2003 |
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09554765 |
May 19, 2000 |
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6551495 |
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09554765 |
May 19, 2000 |
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PCT/GB98/03495 |
Nov 23, 1998 |
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Current U.S.
Class: |
205/777.5 ;
204/403.01; 204/403.1; 205/792 |
Current CPC
Class: |
C07D 209/86 20130101;
C07K 2317/622 20130101; C07K 16/44 20130101; C07K 2317/22 20130101;
G01N 33/5438 20130101; C07K 2317/31 20130101; Y10S 436/806
20130101; C07K 16/26 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
205/777.5 ;
205/792; 204/403.01; 204/403.1 |
International
Class: |
G01N 027/26 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 21, 1997 |
EP |
97309425.3 |
Claims
1. A component for a device for detecting the presence of an
analyte of interest in a sample, the component comprising an
electrically conducting solid support having immobilised thereon a
chemical moiety, said chemical moiety comprising an electroactive
portion with an electrochemical property capable of being directly
modulated in a detectable manner by the binding thereto of a
binding partner having a specific binding activity for the
electroactive portion.
2. A component according to claim 1, wherein the electrically
conducting solid support comprises gold, platinum, metal oxide,
carbon/graphite, silicon or silicate.
3. A component according to claim 1 or 2, wherein the modulation of
the electrochemical property involves oxidation or reduction of the
chemical moiety.
4. A component according to any one of claims 1, 2 or 3, wherein
the electroactive portion comprises an immunogen or a hapten, and
the binding partner comprises an immunoglobulin or an effective
antigen-binding portion thereof with binding specificity for the
electroactive portion.
5. A component according to any one of the preceding claims,
wherein the chemical moiety comprises an organic, moderately
hydrophobic entity.
6. A component according to any one of the preceding claims,
wherein the chemical moiety comprises a conjugated system of
delocalized electrons.
7. A component according to any one of the preceding claims,
wherein the chemical moiety comprises an organometallic compound or
a heteroaromatic compound.
8. A component according to any one of the preceding claims,
wherein the chemical moiety comprises a monomer, dimer or polymer
comprising one or more of the following: carbazoles, ferrocenes,
pyrroles, furans, and thiophenes.
9. A component according to any one of the preceding claims,
wherein the electroactive portion of the chemical moiety is
separated from the electrically conducting solid support by a
pendant chain portion.
10. A component according to claim 9, wherein the pendant chain
portion comprises a conjugated system of delocalised electrons, to
facilitate transport of electrons between the electroactive portion
and the electrically conducting solid support.
11. A component according to claim 9, wherein the pendant chain
portion is alkyl, alkenyl, or substituted alkyl or alkenyl.
12. A component according to claim 11, wherein the chain portion
conforms to the general formula (--CH.sub.2--).sub.n, where n is an
integer from 3 to 14, preferably from 5 to 12, inclusive.
13. A component according to any one of the preceding claims,
wherein the chemical moiety is a molecule which can form a
self-assembling monolayer on the electrically conducting solid
support.
14. An assay device for detecting the presence of an analyte of
interest, comprising a component in accordance with any one of the
preceding claims.
15. An assay device according to claim 14, further comprising one
or more of the following: sample receiving means for accepting a
sample under test; a binding partner having specific binding
activity for the electroactive portion of the chemical moiety;
detection means for detecting a modulation in an electrochemical
property of the electroactive portion of the chemical moiety; data
processing means for processing data output from the detection
means; and data display means for displaying the assay result.
16. An assay device according to claim 14 or 15, comprising a
binding partner releasably immobilised on a solid support, said
binding partner having specific binding activity for the
electroactive portion of the chemical moiety, and being released
from the solid support in the presence of the analyte of
interest.
17. A component for use in a device according to claim 14, said
component comprising a solid support having releasably immobilised
thereon a binding partner having specific binding activity for an
electroactive portion of chemical moiety, the binding partner being
released from the solid support in the presence of the analyte of
interest, and wherein binding of the binding partner to the
electroactive portion directly modulates an electrochemical
property of the electroactive portion in a detectable manner.
18. A method of detecting the presence of an analyte of interest in
a sample, the method comprising the steps of: providing an
electrically conducting solid support having immobilised thereon a
chemical moiety having an electroactive portion with an
electrochemical property capable of being modulated in a detectable
manner directly by the binding thereto of a binding partner having
specific binding activity for the electroactive portion; and
causing the binding partner to contact the electroactive portion of
the chemical moiety as a result of the presence in the sample of
the analyte of interest.
19. A method according to claim 18, comprising use of a component
according to any one of claims 1-13, or an assay device according
to any one of claims 14, 15 or 16.
20. A method according to claim 18 or 19, wherein the binding
partner is present as a binding entity and comprises a first
specific binding activity for the electroactive portion of the
chemical moiety, and a different, second specific binding
activity.
21. A method according to any one of claims 18, 19 or 20, wherein
the binding partner comprises a bispecific antibody or bispecific
antigen-binding immunoglobulin portion.
22. A chemical moiety, comprising an electroactive portion, having
the structure shown in FIG. 13 wherein R.sub.1 and R.sub.2 are,
indepenently, H; OH; C.sub.1-C.sub.14 alkyl, aryl, alkenyl or
alkoxy (all optionally substituted); halide; amide; or amine; and
further wherein the heteroaromatic ring structure may be optionally
substituted at one or more positions with alkyl, aryl, alkenyl, or
alkoxy groups (all themselves optionally substituted), acid groups
(organic or inorganic), halide, amide or amine.
23. A chemical moiety according to claim 22, wherein R.sub.1 is
alkyl, preferably ethyl, propyl or butyl.
24. A chemical moiety according to claim 22 or 23, wherein R.sub.2
is C.sub.1-C.sub.12 alkyl, preferably C.sub.4-C.sub.8 alkyl.
25. A chemical moiety according to any one of claims 22, 23 or 24,
wherein R.sub.1 or R.sub.2 is substituted so as to comprise a
terminally positioned reactive substituent thiol, carboxyl, amide,
amine, halide, aldehyde, ketone, epoxide, or succinimide group, or
other protein coupling agent which facilitates coupling of the
moiety to other entities.
26. A molecule having specific binding activity for a chemical
moiety in accordance with any one of claims 22-25.
27. A molecule according to claim 26, comprising an immunoglobulin
molecule or an effective portion thereof.
28. A molecule according to claim 26 or 27, comprising at least one
further specific binding activity, which further specific binding
activity is for an analyte of interest of for an antibody directed
against an analyte of interest.
Description
FIELD OF THE INVENTION
[0001] This invention relates, inter alia, to an assay method and
to an assay device.
BACKGROUND OF THE INVENTION
[0002] This invention relates in general to electrochemical assays.
In such assays, the presence of an analyte of interest in a sample
causes a measurable change in an electrochemical property of a
sensor device. Typically, electrochemical assays are classified as
"potentiometric" or "amperometric", which measure changes in either
potential or current respectively.
[0003] A number of electrochemical assays have been described. For
example, enzyme electrodes have been used for the direct
measurement of biomolecules such as glucose, urea, amino acids, and
others in physiological samples. These enzyme electrodes include a
selective enzyme layer immobilized at the surface of a
potentiometric or amperometric device that senses the steady state
concentration of a product formed in the immobilized layer as the
substrate for the enzyme diffuses into this reactive film.
[0004] Other assays involve the use of Nafion.TM. (E.I. Du Pont de
Nemours and Co. Inc.) film. Nafion.TM. is a polyanionic
perfluorosulphonate ionomer with permselective properties: large
hydrophobic cations rather than small, hydrophilic cations tend to
accumulate in the polymer via cationic exchange.
[0005] Limoges & Degrand (1993 Analytical Chemistry 65,
1054-1060) described a model system is which a Nafion.TM.-coated
electrode was used to detect the presence of amphetamine. The assay
took the form of a competitive immunoassay, in which amphetamine in
a sample competed with known amounts of labelled amphetamine for
binding to amphetamine--specific antibodies. The competitor
amphetamine was labelled with cobalticenium, which is a redox
label.
[0006] The basis for the assay is that once bound by antibody, the
labelled amphetamine is excluded from the Nafion.TM. film because
of the large size of the resulting amphetamine/antibody complex.
Thus, in the presence of high concentrations of amphetamine in the
sample, there is more free labelled amphetamine available for ion
exchange and incorporation into the Nafion.TM. film. Accordingly,
the current corresponding to the oxidation or reduction of the
cobalticenium-label in the film is proportional to the
concentration of amphetamine in the introduced sample.
[0007] This sort of assay has several disadvantages and accordingly
has not been widely adopted. In particular it requires the
performance of a number of reaction steps before detection can
effected by the Nafion.TM.-coated electrode. The assay apparatus
disclosed by Limoges & Degrand comprised a sensor which was not
disposable or re-usable.
[0008] A different sort of electrochemical assay involves the use
of an "Antibody responsive membrane electrode", as described by
Solsky & Rechnitz (1979 Science 204, 1308; 1981 Anal. Chim.
Acta 123, 135). The antibody responsive membrane comprised
ionophores (crown ethers) within a polyvinyl chloride matrix, the
ionophores being conjugated to a hapten in such a way that the
haptens projected from the surface of the membrane. The membrane
was mounted in the tip of a conventional potentiometric membrane
electrode. The addition of a sample containing antibodies specific
for the hapten would allow antibodies to bind to the hapten which,
in some unknown way, altered the electrochemical properties of the
ionophores, changing the potential across the membrane.
[0009] The manner in which the device works is not understood,
making it impossible rationally to design improvements thereon.
Also, the associated detection system is large and cumbersome and
not readily re-usable.
[0010] WO 89/11649 discloses a device for use in an electrochemical
assay, the device comprising an electroactive polymer layer, within
which layer are entrapped antibody molecules having binding
specificity for an analyte of interest. Binding of the analyte of
interest to the antibody inhibits the flow of counter ions from the
environment surrounding the device into the space around the
electroactive polymer, hence inhibiting electron flow to or from
the polymer during a redox reaction. There is no disclosure of the
electrochemical properties of the polymer being affected by the
binding of a binding partner directly to the polymer.
[0011] WO 95/29199 discloses an electrode having a similar
arrangement, wherein binding of a binding partner to a chemical
moiety attached to an electroactive polymer can indirectly affect
the electrochemical properties of the polymer. There is no
disclosure of a binding partner having binding specificity for the
electroactive polymer itself. A similar arrangement is disclosed in
EP 0239969.
[0012] WO 93/25907 discloses a competition assay system involving
an antigen of interest, and a derivatised antigen carrying a redox
label, competing for binding to limiting amounts of antibody.
Excess redox-labelled antigen is bound electrostatically to a
polymer-coated film, so as to alter the redox potential across the
film, which is measured in a conventional manner. The polymeric
layer is not electroactive and essentially non-conducting.
[0013] EP 0402917 discloses a biosensor operating on a very similar
principle to that disclosed in WO 89/11649: a conducting surface
with an electroactive surfactant coating is modified by inclusion
of one member of a specific binding pair. The analyte of interest
is the other member of the specific binding pair. Binding of the
reciprocal members of the specific binding pair blocks the movement
of counter ions. There is no binding event involving binding
directly to the electroactive surfactant.
[0014] WO 97/27474 discloses a method of determining the presence
of an analyte of interest, whereby an electrode is coated with a
member of a specific binding pair. In the absence of analyte of
interest (which is the reciprocal member of the specific binding
pair), an electroactive redox molecule can come into proximity with
the electrode and donate electrons to, or accept electrons from,
the electrode. This process is inhibited by the analyte of
interest, which blocks the redox molecule from coming into
proximity with the electrode. Thus there is no disclosure of the
direct binding of a binding partner to an electroactive molecule so
as to modify the electrochemical properties thereof. (All documents
cited in the present specification are incorporated herein by
reference). The present invention aims to provide an improved type
of electrochemical assay, particularly one which will be suitable
for forming the basis of disposable, easy-to-use assay devices.
SUMMARY OF THE INVENTION
[0015] In a first aspect the invention provides a method of
detecting the presence of an analyte of interest in a sample, the
method comprising the steps of: providing an electrically
conducting solid support having immobilised thereon a chemical
moiety having an electroactive portion with an electrochemical
property capable of being modulated in a detectable manner directly
by the binding thereto of a binding partner having specific binding
activity for the electroactive portion; and causing the binding
partner to contact the electroactive portion of the chemical moiety
as a result of the presence in the sample of the analyte of
interest.
[0016] In a second aspect, the invention provides a component for a
device for detecting the presence of an analyte of interest in a
sample, the component comprising an electrically conducting solid
support having immobilised thereon a chemical moiety, said chemical
moiety comprising an electroactive portion with an electrochemical
property capable of being directly modulated in a detectable manner
by the binding thereto of a binding partner having specific binding
activity for the electroactive portion.
[0017] The method of the invention may be used qualitatively, so as
to indicate the presence or absence of the analyte of interest.
Alternatively, and preferably, the method may be used
quantitatively so as to indicate the amount (in relative or
absolute units) of the analyte present. It will be apparent that
all sorts of substances may be analytes of interest, although
biological molecules (i.e. molecules present in or produced by
living organisms) will typically be of most interest. These include
nucleic acids (DNA and RNA or chimeras thereof, carbohydrates,
enzymes, antigens, allergens), hormones (both protein and steroid
varieties), especially sex and fertility hormones such as human
chorionic gonadotrophin (hCG), estrone-3-glucuronide (E3G),
progesterone-3-glucuron- ide (P3G), luteinising hormone (LH) and
follicle stimulating hormone (FSH); and disease markers and
diagnostic indicators (e.g. antibodies). Alternatively, the analyte
of interest may be particulate e.g. bacterium, virus, yeast,
fungus.
[0018] A particular advantage of the present invention is that it
allows for assays to be conducted using samples which may be
turbid, which samples can not be assayed using conventional
colourimetric techniques. Examples of samples which are, or may be,
turbid include: serum; whole blood; food and drink samples (e.g.
milk); samples containing turbid growths of micro-organisms, and
the like.
[0019] For the purposes of the present specification, the
"electroactive portion" of the chemical moiety is defined as that
portion which can donate and/or accept an electrical charge when
the chemical moiety undergoes a redox reaction. Typically, the
electrochemical property which is modulated in the assay will
typically be one or more redox potentials. The electroactive
portion may comprise one or more groups capable of undergoing
oxidation or reduction under the assay conditions, the redox
potential of any one or more of such groups being modulated by
binding of the binding partner to the electroactive portion.
Conveniently the one or more altered redox potentials may be
detected by potentiometric or amperometric methods, known to those
skilled in the art. Preferably amperometric detection methods (such
as chronoamperometry) are employed, as these generate results which
are generally easier to measure than potentiometric methods.
[0020] Preferably, the modulation of the electrochemical property
of the electroactive portion is detected and/or measured by
determining the amount of charge transferred to or from the
electrically conducting solid support at a particular potential
difference. The charge may be transferred between the electroactive
portion and the electrically conducting solid support by movement
of electrons, and/or ions, and/or other charged particles.
[0021] Conveniently, the electroactive portion of the chemical
moiety, and the binding partner, are members of a specific binding
pair. Advantageously the binding partner comprises an
immunoglobulin or an effective portion thereof retaining specific
binding activity for the chemical moiety. Effective portions of
immunoglobulins therefore include, for example Fv, scFv, Fab,
Fab.sub.2, and heavy chain variable regions (Hcv, such as those
available from llamas and camels) or a chimeric molecule comprising
any one or more of the aforementioned portions. Conveniently, the
binding partner may be prepared by means of monoclonal antibody
techniques or by selection and isolation from an appropriate
library of nucleic acid sequences encoding binding partners (e.g.
phage display libraries), which are both well known to those
skilled in the art.
[0022] The electroactive portion of the chemical moiety is
conveniently an intrinsic immunogen, or is at least capable of
acting as an immunogen (i.e. is a hapten) when conjugated to
appropriate carrier molecules (such as bovine serum albumin, or
plant peptide derivative etc.). This facilitates the production of
immunoglobulins (or effective portions thereof) having specific
binding activity for the chemical moiety, which immunoglobulins may
act as binding partners in the method of the invention.
[0023] In general, the chemical moiety will typically comprise as
an electroactive portion an organic, moderately hydrophobic
molecule. Examples include organometallic compounds (such as
cobalticenium, and ferrocene), and heteroaromatic compounds (such
as carbazoles, pyrroles, furans and thiophenes). Desirably the
electroactive portion will typically comprise one or more groups
readily capable of undergoing oxidation and/or reduction (oxidation
being thought of as the removal of an electron, and reduction as
the addition of an electron).
[0024] Oxidation or reduction will normally result in the creation
of electrically charged entities, the effects of which may be
stabilised by a conjugated system of electron orbitals in the
moiety ("delocalised electrons"). Accordingly, it is preferred
that, in the conditions of the assay, the chemical moiety comprises
a conjugated system of delocalised electrons. Particular examples
of such chemical moieties include pyrroles, furans, thiophenes, and
analogues and/or multimers thereof (as described, for example, by
Diaz et al., 1979 J. Chem. Soc. Chem. Commun. p635; Niziurski-Mann
et al., 1993 J. Am. Chem. Soc. 115, 887; and Waltman et al., 1984
J. Phys. Chem. 88, 4343). The non-bonding electrons of the
respective Nitrogen, Oxygen and Sulphur atoms contribute to the
conjugated system of electron orbitals. A preferred moiety is
N-alkyl carbazole or analogues thereof (in monomeric, dimeric or
polymeric forms). FIGS. 1A-D show the general structure of pyrrole,
furan, thiophene and carbazole monomers, respectively.
[0025] In general it will be desired for the chemical moiety to be
immobilised upon a solid support (e.g. an assay dipstick, or a
capillary fill chamber). Preferably the chemical moiety will be
immobilised upon an electrically conducting portion of the solid
support, which electrically conducting portion may comprise, for
example, a thin strip of gold, platinum or other conducting metal,
a metal oxide, carbon/graphite, silicon, or silicate.
[0026] The immobilisation of the chemical moiety upon the solid
support may be accomplished in several ways. The present inventors
have found that one method is to place a solid support in a
solution of a suitable chemical moiety precursor, and then to cause
in situ formation of the chemical moiety upon the solid support.
Typically, such a process may take the form of causing
polymerisation of precursor monomers upon the solid support,
resulting in the formation of a mesh-like coating of chemical
moiety upon the solid support.
[0027] Alternatively, and more preferably the chemical moiety may
be immobilised upon the solid support by means of an intervening
"pendant" chain portion, preferably one which allows for
self-assembly as a monolayer on a supporting surface (as described,
for example, by Sabatini & Rubinstein, 1987 J. Phys. Chem. 91,
6663-6669; von Velzen et al., 1994 J. Am. Chem. Soc. 116,
3597-3598; Chidsey et al., 1990 J. Am. Chem. Soc. 112, 4301-4306;
and Rubinstein et al., 1988 Nature 332, 426-429). The chain portion
may be thought of as forming an integral part of the chemical
moiety.
[0028] The use of a pendant portion or molecule is preferred as it
separates the electroactive portion from the electrically
conducting surface of the electrode (whilst preferably, but not
necessarily, retaining a degree of electrical conductivity through,
for example a system of delocalized electrons). The pendant portion
is typically substantially linear. A preferred pendant portion is
an alkyl or alkenyl group (typically comprising 3 to 14, preferably
5 to 12, carbon atoms), which may be substituted or
unsubstituted.
[0029] In a particular embodiment, an alkyl or alkenyl pendant
group is attached to the electrode via a sulphydryl or thiol
group--other chemical attachments may be equally suitable. Another
advantage of the use of pendant groups or portions is that they
tend to fill in "pin holes" (minor irregularities in the surface of
the conducting layer of the electrode), which can have a
detrimental effect on the reproducibility of results obtained. It
may also be preferred to include excess pendant molecules attached
to the electrode (i.e. not every pendant group will necessarily be
joined to an electroactive portion). The presence of excess pendant
groups is throught to improve the stability and rigidity of the
monolayer, which optimises the method/device of the invention. In
particular embodiments, the inventors have found that a ratio of
four pendant portions to three electroactive portions, may provide
optimum results. The excess pendant molecule need not be identical
to the pendant portion attached to the electroactive portion: thus,
excess pendant "spacer" molecules may be deliberately introduced.
Such spacer molecules will conveniently be of a similar nature to
the pendant portion of the chemical moiety (e.g. alkyl or alkenyl),
but will be no longer in chain length, and possibly shorter, than
the pendant portion of the chemical moiety, so as to avoid the
possibility that the spacer molecules cause steric hindrance when
the binding partner attempts to bind to the electroactive
portion.
[0030] It is highly preferred that the assay method is such that
the analyte of interest need not be identical to the electroactive
portion of the chemical moiety to which the binding partner binds.
In this way, the assay method may be adapted to detect the presence
of any analyte of interest, with the binding partner (typically
immunoglobulin) specific for the electroactive portion being
required simply as the last step of the assay to generate a
detectable signal by binding to the electroactive portion. This
preferred feature may be obtained, for example, by utilising
competition or displacement type methodologies, as will be
explained below.
[0031] Accordingly, in preferred embodiments, the binding partner
is present as a binding entity, which binding entity comprises
first and second specific binding activities. The first specific
binding activity is for the electroactive portion of the chemical
moiety as aforesaid. The second specific binding activity is
typically for the analyte of interest, or for a molecule (such as
an immunoglobulin) which itself has specific binding activity for
the analyte of interest, such that the presence of the analyte of
interest tends to displace the binding entity from a solid support.
The binding entity may also possess further specific binding
activities, but these are not essential to performance of the
invention.
[0032] Conveniently the binding entity will be a bispecific
antibody or "Diabody" or other bispecific immunoglobulin fragment
(e.g. double headed scFv, double headed HCV or a chimeric molecule
comprising an scFv and/or an HCV fragment). Alternatively, the
binding entity may comprise a non-binding component, to which are
attached first and second binding partners having respective first
and second specific binding activities. These binding partners may
comprise conventional immunoglobulin molecules, such as
monospecific antibodies, or effective binding portions thereof
(e.g. scFv etc.). The non-binding component may be any substance
large enough, and with appropriate chemical properties, for the
first and second binding partners to be attached thereto. The
non-binding component may be, for example, a peptide, a
polypeptide, a liposome or, more conveniently, a particle such as a
latex bead. Methods of attachment of immunoglobulins to latex beads
are well-known to those skilled in the art.
[0033] Conveniently, the method step of causing the binding partner
to contact the electroactive portion as a result of the presence of
the analyte of interest in the sample is effected by the analyte
causing displacement or release of the binding partner from a solid
support to which the bindng partner is releasably immobilised prior
to introduction of the sample. The analyte will typically displace
the binding partner from, or compete with the binding partner for
binding to, binding sites on the solid support by means of which
the binding partner is releasably immobilised.
[0034] Thus, in particular embodiments, the assay method involves
use of a first solid support upon which is immobilised the chemical
moiety, and a second solid support (which may be a separate portion
of the first solid support, or a discrete component) upon which is
immobilised an analogue of the analyte of interest. The analogue is
such that the second specific binding activity of the binding
entity will bind to the analogue, albeit with lower affinity than
for the analyte of interest. Accordingly, prior to performance of
the assay, the binding entity is reversibly immobilised upon the
second solid support via its second specific binding activity.
[0035] In the presence of free analyte of interest, the analyte
will compete with the immobilised analogue for binding to the
binding entity via the second specific binding activity. Typically,
the affinity of the binding entity for the analyte is greater than
that for the analogue, such that the binding entity will be
displaced from the second solid support if the analyte of interest
is present in the sample. The displaced binding entity is then free
to react, via its first specific binding activity, with the
electroactive portion of the chemical moiety immobilised on the
first solid support, thereby modulating an electrochemical property
of the electroactive portion in a detectable manner as
aforesaid.
[0036] Accordingly, in preferred embodiments, there is an affinity
difference between the binding affinity of the analogue and the
analyte of interest respectively, such that the presence of the
analyte, even at low concentration, will tend to displace the
binding entity from the analogue. Alternatively there may be no
difference in binding affinity, and the binding entity is displaced
from the analogue by competition, e.g. because the analyte of
interest is present in the sample at a greater effective
concentration than the analogue.
[0037] The displaced binding entity may be allowed to diffuse from
the second solid support to the first solid support, especially
where the first and second supports are in close proximity (e.g. 50
.mu.m-5 mm, preferably 50 .mu.m-1 mm). Alternatively, the binding
entity may be transported by capillary action along or through a
porous medium, or may be transported by a flow of a fluid sample
(e.g. a body fluid such as blood or urine and the like), as
disclosed in, for example, WO 91/05262. A pump means (e.g. syringe
pump or peristaltic pump) may be provided, if appropriate, to pump
fluid comprising released binding entity from the second solid
support to the first solid support.
[0038] Use of analogues of analytes of interest, in a slightly
similar manner, is disclosed and taught, for example, in EP 0 324
540, and in PCT/EP95/04518. Those skilled in the art will
appreciate that the analyte of interest will typically be a
biological molecule, such as a peptide or polypeptide, or a steroid
hormone or the like. Conveniently, the analogue of the analyte of
interest will be an epitope mimic, i.e. a molecule (typically
smaller than the analyte of interest) generally of synthetic
origin, such as a short peptide, which behaves in a manner
comparable to the binding site of the analyte to which the binding
partner binds. Examples of analytes and suitable analogues are
disclosed in EP 0 324 540 and PCT/EP95/04518. The embodiment
described above is a variant of known displacement/competition type
assays, disclosed inter alia in WO 91/05262 and EP 0 383 313.
[0039] In a further embodiment the binding entity is such that the
second specific binding activity is for an antibody directed
against the analyte of interest. The binding entity may comprise,
for example, an analogue of the analyte of interest, which analogue
is relatively loosely bound by antibody specific for the analyte of
interest, which antibody is immobilised on the second solid
support. Alternatively the binding entity may comprise an
anti-idiotypic antibody specific for the binding site of the
immobilised antibody. In any event, the immobilised antibody
convniently (but not essentially) has a greater affinity for the
analyte than for the binding entity, such that the presence of the
analyte of interest in the test sample will tend to cause the
displacement of the binding entity from the second solid support.
The displaced binding entity is then free to bind to the first
solid support, via its first specific binding activity, as outlined
above.
[0040] Methods of immobilising antibodies on solid supports are
known to those skilled in the art. Useful discussion is provided by
G Hermanson, in "Bioconjugate Techniques" (Academic Press, 1996).
Typically the immobilised antibody is covalently coupled by an
added functional group. Conveniently the antibody for the analyte
may be immobilised by attachment to a further antibody-specific
antibody immobilised on the solid support. The first solid support
will typically comprise an electrode. The second solid support may
be any of those routinely used in assays and include, for example,
synthetic plastics materials, microtitre assay plates, latex beads,
filters, glass or plastics slides, dipsticks etc. Advantageously,
the second solid support comprises a wettable surface.
[0041] Conveniently the component of the second aspect of the
invention is used to perform an assay of the type disclosed in our
co-pending European patent application No. 97309409.7, filed on
Nov. 21st, 1997. The component might take the form of a relatively
cheap, disposable or replacable part for use and interaction with
other components (e.g. a separate signal detection means., acting
as a reader for the assay result). Alternatively, the component may
be provided as an integral part of a larger device.
[0042] In a third aspect, the invention provides an assay device
comprising the component of the second aspect. Conveniently the
device further comprises one or more of the following: sample
receiving means for accepting a sample under test; a binding
partner having specific binding activity for the electroactive
portion of the chemical moiety; detection means for detecting a
modulation in an electrochemical property of the electroactive
portion of the chemical moiety; data processing means for
processing data output from the detection means; and data display
means for displaying the assay result, preferably in a numerical
form.
[0043] The assay device will conveniently comprise a capillary-fill
reaction chamber as part of the sample receiving means. In a
preferred embodiment the chamber is at least partly defined by the
first and second solid supports of the preferred method aspect.
Capillary-fill devices which may be adapted for use in the present
invention are taught, for example, in U.S. Pat. No. 5,141,868.
[0044] In a fourth aspect the invention provides a component for
use in the device defined above, said component comprising a solid
support having releasably immobilised thereon a binding partner
having specific binding activity for an electroactive portion of a
chemical moiety, the binding partner being displaced from the solid
support in the presence of the analyte of interest, and wherein
binding of the binding partner to the electroactive portion
directly modulates an electrochemical property of the electroactive
portion in a detectable manner. As with the component of the second
aspect of the invention, the component may be provided as a
relatively cheap, disposable or replacable part for use and
interaction with other components (e.g. a separate signal detection
means, acting as a reader for the assay result), or may be provided
as an integral part of a larger device.
[0045] In a fifth aspect, the invention provides a chemical moiety,
comprising an electroactive portion, having the structure shown in
FIG. 13 wherein: R.sub.1 and R.sub.2 are independently H; OH;
C.sub.1-C.sub.14 alkyl, aryl, alkenyl or alkoxy (all optionally
substituted); halide; amide; or amine; and further wherein the
heteroaromatic ring structure may be optionally substituted at one
or more positions with alkyl, aryl, alkenyl, or alkoxy groups (all
themselves optionally substituted), acid groups (organic or
inorganic), halide, amide or amine.
[0046] Advantageously R.sub.1 is alkyl, preferably ethyl, propyl or
butyl and preferably R.sub.2 is C.sub.1-C.sub.12 alkyl, more
preferably C.sub.4-C.sub.8 alkyl, and most preferably C.sub.6
alkyl. It is generally preferred that R.sub.1 and R.sub.2 are not
identical. In a preferred embodiment R.sub.2 comprises a
(preferably terminally positioned) reactive substituent such as a
thiol, carboxyl, amide, amine, halide, aldehyde, ketone, epoxide,
or succinimide group, or other protein coupling agent (e.g. as
mentioned in Hermanson, "Bioconjugate Techniques", Academic Press,
1996) which facilitates coupling of the moiety to other entities
(e.g. solid surfaces). In a preferred embodiment, the chemical
moiety is 3,3(N-[6-thiol hexyl]carbazole)N-ethyl carbazole.
[0047] The chemical moiety of the fifth aspect of the invention may
conveniently be immobilised on an electrically conducting solid
support, so as to form a component in acordance with the second
aspect of the invention.
[0048] In a sixth aspect, the invention provides a molecule having
binding specificity for the chemical moiety of the fifth aspect
defined above. More particularly the molecule preferably has
binding specificity for the electroactive heteroaromatic ring
portion of the chemical moiety. The molecule will conveniently
comprise an immunoglobulin molecule or an effective portion thereof
which retains binding specificity for the chemical moiety--such
portions include, for example, Fv scFv, Fab, Fab.sub.2, HCV, or a
chimeric molecule comprising any one or more of the aforementioned
portions. In a preferred embodiment the molecule will comprise at
least two binding specificities: a first binding specificity for
the chemical moiety, as aforementioned; and a second binding
specificity for an analyte of interest or for an antibody (or
effective antigen-binding portion thereof) directed against an
analyte of interest. It will be apparent to the reader that
molecules in accordance with the sixth aspect of the invention will
conveniently be suitable for use as a binding partner in performing
the method of the first aspect and/or may conveniently be
releasably immobilised on a solid support so as to provide a
component in accordance with the fourth aspect of the
invention.
[0049] The invention will now be further described by way of
illustrative examples and with reference to the accompanying
drawings, in which:
[0050] FIGS. 1A-1D show the general structure of pyrroles, furans,
thiophenes and carbazoles, respectively;
[0051] FIGS. 2 and 3 are schematic representations of assays using
the method, component and assay device of the invention;
[0052] FIGS. 4A and 4B show the results of cyclic
voltammograms;
[0053] FIG. 5 is a bar chart of .DELTA.Q for different
concentrations of IgG;
[0054] FIG. 6 is a schematic representation of the reaction scheme
used to prepare a compound suitable for use in performing the
method of, and making a component in accordance with, the present
invention;
[0055] FIGS. 7 to 9 are graphs of mC against time;
[0056] FIGS. 10-12 are schematic representations of plasmid
constructs useful for obtaining reagents suitable for use in the
method of the present invention;
[0057] FIG. 13 is a schematic representation of a chemical moiety
of use in performing various aspects of the invention;
[0058] FIG. 14 is a schematic representation of an electrochemical
cell used to investigate electrochemical properties of various
electrode coatings;
[0059] FIG. 15 is a graph showing a dose response curve for an
assay device in accordance with the invention used in accordance
with the method of the invention to detect the presence of an E3G
analyte of interest; and
[0060] FIG. 16 is a graph of .mu.C against time.
EXAMPLES
Example 1
[0061] This example relates to an illustration of how an assay in
accordance with the invention may be performed.
[0062] Referring to FIG. 2, there is provided an electrically
conducting first solid support 10, upon which is immobilised a
chemical moiety 12. The chemical moiety 12 is attached to the first
solid support by a "pendant" portion 14, such that a
self-assembling monolayer of the chemical moiety 12 is formed on
the first solid support 10.
[0063] There is also provided a second solid support 16, upon which
is immobilised an analogue 18 of the analyte of interest 20. A
binding partner 22 is releasably immobilised, via analogue 18, to
the second solid support 16. The binding partner 22 is a bispecific
antibody, having a first and a second specific binding activity
(denoted as 24 and 24' respectively). The first specific binding
activity 24 is for the chemical moiety 12. The second specific
binding activity 24' is a relatively high binding affinity for the
analyte of interest 20, and also confers a relatively low binding
affinity for the analogue 18.
[0064] Accordingly, upon introduction of a sample comprising the
analyte of interest 20, the binding partner 22 is specifically
displaced from the second support 16 and is free to bind to the
chemical moiety 12 via the second binding activity 24. Binding of
the binding partner 22 to the chemical moiety 12 directly modulates
a detectable electrochemical property of the chemical moiety 12
(e.g. a redox potential), such that a difference (e.g. in charge,
current or potential difference) is detected at the first support
10.
[0065] Conveniently first and second solid supports 10, 16 form
part of a capillary-fill chamber, into which a test sample is
introduced when performing an assay. The physical separation
between the binding partner 22 displaced from the second solid
support 16 will readily be transported, in a passive manner by
diffusion, to the first support 10.
Example 2
[0066] A different embodiment of the method of the invention is
illustrated in FIG. 3. Functionally comparable integers are denoted
in FIG. 3 by the same reference numerals used in FIG. 2. As with
Example 1, the arrangement is conveniently configured for use in a
capillary-fill assay device.
[0067] In the embodiment illustrated in FIG. 3, the chemical moiety
12 is immobilised on a first solid support 10 as described above.
The second solid support 16 is coated with a plurality of molecules
of a monoclonal antibody 18 specific for the analyte of interest
20.
[0068] The binding partner 22 is a fusion protein comprising an
antibody molecule portion 24, and a mimotope portion 24'. The
mimotope portion 24' is a peptide "mimic" of the epitope recognised
by monoclonal antibody 18 (hence "mimotope"). However, the binding
affinity of the antibody 18 is considerably higher (e.g. 10 to 100
times) for the analyte 20 than for the mimotope 24'. Accordingly,
the presence of the analyte of interest displaces the binding
partner 22 from the solid support 16. The assay then proceeds as
described for the embodiment illustrated in FIG. 2.
[0069] In some respects, the embodiment shown in FIG. 3 may be
preferred, as the binding partner 22 is not bound to the analyte 20
when it binds to the chemical moiety 12--this may be preferable as
the presence of the analyte 20 may effect the manner in which the
binding partner 22 modulates the electrochemical property of the
chemical moiety 12.
[0070] Those skilled in the art will appreciate that, using a
single chemical moiety 12, the assay method of the invention may be
modified so as to be used in the detection of any analyte of
interest: one can vary the mimotope portion 24' (in FIG. 3) or the
second binding activity 24' (in FIG. 2), such that displaement
occurs in response to the presence of the relevant analyte, whilst
the first binding activity 24 of the binding partner 22 can remain
the same.
[0071] Those skilled in the art will also appreciate that Examples
1 and 2 could also be performed as "competition" type assays, in
which the concentration of free analyte of interest is sufficient
to cause displacement of the binding partner from the second solid
support, even though the binding partner may not have a greater
binding affinity for the analyte of interest than for the analogue
18 (in Example 1) or antibody 18 (in Example 2).
Example 3
[0072] Production of Anti-Carbazole Monomer Antibodies and
Electrochemical Characterization thereof on a Polymeric Carbazole
Electrode Surface in Organic Solvent
[0073] This example relates to the preparation of an electrode,
coated with a carbazole electroactive chemical moiety by means of
electroplating, which electrode is stable in organic solvents (e.g.
dichloromethane). Unless otherwise stated, all reagents are
commercially obtainable, and were purchased from Aldrich, USA.
[0074] 3.1 Synthesis of N-(6-Hexanoic Acid) Carbazole.
[0075] Carbazole (3.34 g, 20 mmol) was added with sodium hydroxide
(4.8 g) to a mixture of water (20 ml) and toluene (20 ml).
Cetyltrimethylammonium bromide (1.46 g, 14 mmol) was added as a
surfactant to combine the two phases. To this 6-bromo-hexanoic acid
was added (5.9 g, 30 mmol) and the solution was left to reflux for
48 hours. The resulting solution was washed with diethyl-ether
(3.times.50 ml) to remove the organic phase. The solvent was then
removed via rotary evaporation.
[0076] The product was subjected to chromatography over silica gel
(acidified) with diethyl-ether:petrol (6:4) and the eluate
collected in 2 ml aliquots which were checked for presence of
product by TLC and detected by UV fluorescence. IR: 3500-2500
(broad) OH (acid), 3000, 2800 (sharp) tertiary amine, 1750 C.dbd.O
(acid), 1500 tertiary amine. .sup.1H NMR: (CDCL.sub.3) 10.10 (H, m,
OH), 8.06 (2H, m, ArH), 7.62 (6H, t, J 6.35, ArH), 4.05 (2H, t, J
8.82, CH.sub.2) 2.15 (2H, m, CH.sub.2), 1.75 (2H, m, CH.sub.2),
1.56 (4H, m, CH.sub.2) m/z 281.2 (m.sup.+) 180.1 N-methyl carbazole
cation.
[0077] The acid group in the compound facilitates coupling to
carrier molecules, such as bovine serum albumen (BSA) and plant
protein derivative (PPD) for various purposes.
[0078] 3.2 Preparation of Immunogen by Attachment of N-(6-Hexanoic
Acid) Carbazole to PPD
[0079] In order to prepare anti-carbazole antibodies (useful as
binding partners to modulate the electrochemical properties of the
carbazole), it was necessary to couple the N-(6-hexanoic acid)
carbazole to a larger carrier molecule, as the carbazole compound
is too small by itself to elicit an antibody response. PPD was
selected as the carrier molecule.
[0080] The reaction mixture for coupling PPD (plant peptide
derivative) was prepared by dissolving
1-ethyl-3-(3-dimethylaminopropyl)carbodimide (40 mg) in MES buffer
[2-(N-morpholine)ethane sulfonic acid (aqueous saline pack)](1 ml).
Half of this solution (500 .mu.l) was taken and was dissolved in
DMF (500 .mu.l). To this N-(6-hexanoic acid)carbazole in DMF (100
mg/ml, 20 mg) was added. This mixture was stirred and watched
carefully for any signs of precipitation occurring. If
precipitation did appear a small amount of DMF was added to the
solution to redissolve the precipitate. To this reaction solution
PPD (500 .mu.l) was added slowly and stirred for 24 hours in the
dark. The reaction mixture after this time was dialysed for 24
hours against phosphate buffered saline. A fluorimeter scan was
taken for the this N-(6-hexanoic acid)carbazole PPD and also for
PPD alone on a Perkin Elmer model 5050 fluorimeter (200-400 nm).
Peaks were noted for PPD: 200 nm and 390 nm, and for PPD carbazole:
200, 270, 292, 329, 335, 345, 350 and 390 nm. This demonstrated
that carbazole was effectively coupled to the carrier molecule.
[0081] 3.3 Production of Antibodies to Carbazole
[0082] Polyclonal antibodies against carbazole were made in a
rabbit. A preimmunisation bleed sample was taken to establish
background immunity. The rabbit was injected with carbazole/PPD
sample (1 mg/ml), with Freund's complete adjuvant at four sites
(250 ml each site) and after one week a post immunisation bleed was
taken. After one month a second immunisation was made with Freund's
incomplete adjuvant with the carbazole/PPD sample (again at four
sites). A second post immunisation bleed was one week later. A
third bleed was taken one month after the second bleed (with no
additional immunisation). Immunisation sites were either side of
the spine, two at the front, two at the back. Bleeds were taken
from the marginal ear vein. Blood was allowed to clot and serum
tested for antibody activity by ELISA, using plates coated with
BSA-conjugated carbazole, as described below. Use of the BSA
conjugate facilitated coating of the plates with the hapten, whilst
the use of a different carrier molecule to that used in the
production of antibodies (PPD) ensured that any antibody detected
in the ELISA was due to an anti-hapten response rather than an
anti-carrier response.
[0083] 3.4 Synthesis of BSA N-(6-Hexanoic) Carbazole
[0084] The N-(6-hexanoic acid) carbazole (50 .mu.l of 100 .mu.g/ml
in DMF) was added to N-methyl-morpholine (10 .mu.l). To this
isobutyl chloroformate (10 .mu.l) was added and cooled over ice for
5 minutes. The mixture was then transferred (47 .mu.l) to a
solution of BSA (2 .mu.l, taken from a stock solution of 6.7 mg of
BSA in 500 .mu.l of water and 100 .mu.l of DMF) and stirred for
four hours. The sample was transferred to a dialysis tube and
dialysed for 48 hours against PBS, changing the PBS solution once.
This gave a ratio of 150:1 N-(6-hexanoic acid) carbazole to BSA.
Fluorimeter scan: BSA 285 nm, BSA carbazole 285, 329, 335, 345,
352, 380, demonstrating effective coupling had taken place.
[0085] 3.5 ELISA Using BSA N(6-Hexanoic) Carbazole Coated
Plates
[0086] Microtitre plates were sensitised with the BSA
N(6-hexanoic)carbazole (10 mg/ml) made up in sodium carbonate
buffer (0.1M, pH 9.6) in PBST (phosphate saline buffer 0.15% tween
20 (Sigma), 200 ml to each well) and incubated at 37.degree. C. for
one hour. The plates were then emptied and washed three times with
PBST. The plates were then blocked with a 1% solution (in PBST) of
skimmed milk powder [Marvel.TM.] 200 ml to each well, and incubated
(37.degree. C. for one hour). After this time the plates were
emptied and washed in PBST three times as before. The serum was
then added at a range of dilutions to the plates (150 ml). This was
again left to incubate (37.degree. C. for one hour). The plates
were then washed in PBST as before and a {fraction (1/1000)}
dilution of goat anti-rabbit alkaline phosphatase as conjugate
(Zymed Laboratories Inc.) in PBST was added to each well (150 ml).
This was left at 37.degree. C. for one hour and the plates washed
in PBST as before.
[0087] The enzyme substrate was a solution of paranitrophenol
phosphate (Sigma Diagnostics) (one tablet) was made up in a buffer
solution (5 ml) (diethylamine, pH 9.8, magnesium chloride 50:1).
This substrate solution (150 ml) was added to each assay well and
the colour was allowed to develop over one hour at room
temperature. The results were read on a Dynatech model MR7000 plate
reader W/L MODE:dual, Test filter: 405 nm, Ref. filter 570 nm. The
results indicated (data omitted for brevity) that a significant
anti-hapten response followed boosting, and that anti-hapten
antibody levels reached background (control) levels only at
dilutions of {fraction (1/3,200)} or higher.
[0088] 3.6 Synthesis of Hexakis[6-(2)-(3)-(Carbazol-9-yl)Hexyl]
.beta.-Cyclodextrin.
[0089] In order to cast electrodes, by electroplating of gold or
platinum surfaces in organic solvents, the above
carbazole-containing compound was prepared. .beta.-cyclodextrin was
readily available in the laboratory, and it was believed that its
inclusion would facilitate the formation of a mesh-like coating of
the carbazole-containing compound on the electrode.
[0090] Sodium hydride (0.15 g, 80% 5 mmol) was weighed out and
added to N,N-dimethyl formamide (DMF) (5 ml). .beta.-cyclodextrin
(0.567 g, 0.5 mmol) was added slowly to this DMF solution whilst
stirring, and left to dissolve completely for 10 minutes.
9-(6-bromohexyl)carbazole (1.25 g, 4 mmol) was added to the mixture
and stirred at room temperature for 48 hours.
[0091] To the crude product mixture diethyl-ether (200 ml) was
added and shaken, leaving an immiscible layer. The organic layer
was then removed and water added. This was then left to stir for 90
minutes and allowed to settle for a further 30 minutes. The product
was then filtered off, near to dryness, and left in a dessicator at
reduced pressure until completely dry (0.78 g, 59.2%). .sup.1H NMR
400 mhz (DMSO): 8.10 (2H, m, position 4 and 5, ArH), 7.36 ( 4H, m,
position 2,3,6,7, ArH), 7.10 (2H, m, position 1 and 8, ArH),
5.8-5.7 (secondary alcohol in the unsubstituted
.beta.-cyclodextrin), 4.81 (anomeric proton 1H, OCHO, position 1
.beta.-cyclodextrin), 4.5 (primary alcohols in the unsubstituted
.beta.-cyclodextrin), 4.36 (2H, m, N--CH.sub.2), 4.16 (2H, m,
R--O--CH.sub.2--CH.sub.2), 3.61-3.28 (5H, m, aliphatic protons from
the .beta.-cyclodextrin), 1.61 (2H, m, CH.sub.2(pendant)) 1.32 (6H,
m, 3CH.sub.2(pendant)) m/z (FAB.sup.+) (The ions corresponding to
the hexa-substituted derivative were the most intense in the mass
spectrum) 1407 (carb-hexyl).beta.CD+Na, 1656
(carb-hexyl).sub.2.beta.CD+Na, 1906 (carb-hexyl).sub.3.beta.CD+Na,
2158 (carb-hexyl).sub.4.beta.CD+Na, No peak for
(carb-hexyl).sub.5.beta.CD+Na, 2628 (carb-hexyl).sub.6.beta.CD,
2676 (carb-hexyl).sub.6.beta.CD+2Na (M.sup.+) 2664
2x(carb-hexyl).sub.6.beta.CD+3Na.sup.2+.
[0092] 3.7 Synthesis of Hexakis[6-(2)-(3)-(Carbazol-9-yl)Butyl]
.beta.-Cyclodextrin.
[0093] For comparison, a similar compound to that described in 3.6
above was prepared, but using a butyl pendant portion rather than a
hexyl pendant portion. The method of preparation was essentially
identical to that described above.
[0094] 3.8 General Cyclic Voltammetry for Electroplating in Organic
Solvents--Experimental Details
[0095] Cyclic voltammetry was performed on a Princeton Applied
Research Corporation Scanning Potentiostat (model 362) together
with a Bryans flat bed X--Y recorder (model A25000) and on a
EG&G model 273A Princeton Applied Research
Potentiostat/Galvanostat. (Using Echem and Lotus 1 2 3 to process
the data).
[0096] The electrochemical cell consisted of a 25 ml round bottomed
flask using a silver wire as a pseudo or quasi-reference electrode,
and an aluminium rod as the counter electrode. The working
electrode consisted of a clean gold or platinum wire. The
electrolyte solution comprised a 0.1 molar solution of
tetrabutylammonium hexafluorophosphate in dry dichloromethane
(dried over calcium chloride). The carbazole .beta.-cyclodextrin
monomer (0.01 g) was dissolved in the electrolyte solution (10 ml).
The three electrodes were then placed into the polymer solution and
one or two cyclic voltammetric scans (from 0 volts to 1.5 volts,
and then back to 0 volts, at 150 mV per second) were recorded in
order to create a polymer film on the working electrode. The
electrodes were then transferred to a clean flask with clean
electrolyte solution (0.1 molar solution of tetrabutylammonium
hexafluorophosphate in dry dichloromethane) (10 ml) and about seven
scans made or until polymer film showed a stable scan. The polymer
was then stably formed on the gold or platinum wire and the
electrode could be used for analysis.
[0097] 3.9 EIA Electrode Assays
[0098] The ELISAs performed previously involved binding of antibody
to carbazole presented on microtitre plates as a monomer, whereas
on the electroplated electrodes the carbazole is present as a
dimer. Therefore, in order to determine if the same antibody would
still bind to the dimeric carbazole present on the electrodes, EIA
electrode assays were performed.
[0099] Electrodes were cast in dichloromethane by performance of
cyclic voltammetry, as described above. These electrodes were
allowed to dry for ten minutes and dipped into anti-carbazole serum
at varying dilutions of serum in PBST, (250 .mu.l) and incubated
for one hour at 37.degree. C. After this time the electrodes were
washed in PBST three times and dried on a tissue. The electrodes
were then dipped into goat anti-rabbit alkaline phosphatase
conjugate ({fraction (1/1000)}, 250 .mu.l) and incubated for one
hour (37.degree. C.). The electrodes after this time were washed in
PBST as before and then dipped into the para nitrophenolphosphate
substrate solution (250 .mu.l) as described for the ELISA and the
colour read after one hour.
[0100] The results (data omitted for brevity) clearly indicated
that a significant amount of the anti-carbazole antibody bound to
the electrode, compared to control serum from the pre-immunisation
bleed.
[0101] 3.10 Electrochemical Studies of the Effect of Anticarbazole
Antibody by Cyclic Voltammetry and Chronoamperometry
[0102] Electrodes were cast in dichloromethane as described above
(3.8). A final cyclic voltammogram was made and recorded. These
electrodes were air dried for ten minutes. The rabbit
anti-carbazole serum was diluted to various concentrations, and an
electrode was added to each solution (250 .mu.l) for ten minutes.
The electrodes were removed and washed in PBST and dried with a
tissue. A second cyclic voltammogram was undertaken which was
compared to the original and differences could clearly be noted.
Sample results are shown in FIGS. 4A (pre-bleed) and 4B (immune
serum, containing anti-carbazole antibody).
[0103] FIG. 4A shows a graph of mA against mV. The thick plot is
the trace obtained in the absence of serum, the thin plot is the
trace obtained when pre-immunisation serum was added. It can be
clearly seen that the pesence of serum markedly dereases the peak
heights. FIG. 4B is a similar graph showing the results obtained
for polymer alone (in the absence of serum, thick plot) and in the
presence of immune serum containing anti-carbazole antibody (thin
plot). The effect of the anti-carbazole antibody was to increase
the heights of both oxidative peaks (one to a much greater extent
than the other), indicated by a greater minus value (i.e. loss of
electrons from the carbazole). (Similar experiments were performed
using the shorter (butyl) pendant portion carbazole-containing
moiety: results were less clear cut, suggesting that the butyl
pendant portion is sub-optimal, probably because it is too short to
facilitate binding of antibody to the carbazole moiety).
[0104] So as to minimise any non-specific effects of serum proteins
other than antibody, the antibody fraction was separated from serum
by using a protein A affinity chromatography column (PROSEP A,
protein A immobilised on glass beads, obtained from Porton
products), using conventional techniques. The IgG fraction was
eluted off by treating the column with 0.1 M citrate (pH 3.4) and
collected in 0.5 ml aliquots. These were then neutralised with
dilute sodium hydroxide. Elution samples were evaluated by on line
UV scanner (360 nM). Total IgG concentration was measured using a
Perkin-Elmer Lambda 16 UVDM scanner from 240 to 350 nM.
Measurements were taken at 280 nM and the peak absorption placed
into Beer's law (A=.epsilon.cl) where the absorption coefficient of
the constant region of IgG (.epsilon.) is 0.51.
[0105] The cyclic voltammetry experiments were then repeated using
the protein A treated, IgG-enriched material. Subtantially similar
results were obtained.
[0106] Cyclic voltammetry demonstrated that binding of
carbazole-specific IgG to the electrode increased the oxidative
peak height, but that such binding also lowered the redox
potentials of the carbazole moiety. It was decided that an
amperometric measurement method would be more appropriate, and
easier to use, than a potentiometric method such as cyclic
voltammetry. Accordingly, further analysis was performed by
chronoamperometry (i.e. application of a fixed voltage for a period
of time). This method can be used to generate a graph of current
(I) against time (seconds or milliseconds), and the area under the
curve gives a total coulombic charge. This is particularly useful,
as an electron has a known colombic charge
(1.602.times.10.sup.-19), thus one can determine the total number
of electrons transferred between the carbazole and the electrode
during the experiment.
[0107] From plots of coulombic charge against time (e.g. as shown
in FIG. 8), the difference .DELTA.Q between the baseline scan and
an experimental scan could be calculated. FIG. 5 is a bar chart
showing increase in total oxidative electron flow (as measured by
.DELTA.Q, in units of .mu.C) for different concentrations of IgG
(.mu.g/ml) prepared from immune serum. No change in electron flow
was observed using control pre-immuisation serum.
Example 4
[0108] Electrochemical Characterization of Rabbit Polyclonal
Anti-Carbazole Monomer Antibodies on a Carbazole Dimer Monolayer
Electrode Surface in Aqueous Conditions
[0109] Desirably an electrode for use in detecting the presence of
an analyte of interest should be stable in water or an aqueous
environment, as the majority of samples are, in practice, likely to
be aqueous. The inventors found that the electrodes described in
the preceding example were not particularly stable in water. It was
therefore decided to prepare an alternative electrode in which the
elctroactive moiety was covalently coupled to the conducting
surface, rather than being formed by electroplating and "cast" in
situ. In addition, so as to present a more uniform and ordered
surface (facilitating antibody binding), it was decided to use a
compound which was capable of forming a self-assembling
monolayer.
[0110] 4.1 Formation of N-(6-Bromo Hexyl) Carbazole
[0111] A mixture of carbazole (1.67 g, 10 mmol), sodium hydroxide
(5 g), water (5 mls), toluene (10 mls) and cetyltrimethyl ammonium
bromide (0.7 g, 2 mmol) was stirred at room temperature. To this
1-6 dibromohexane (2.3 ml, 30 mmol) was added dropwise (FIG. 6,
step 1). This was stirred and refluxed at 170.degree. C. for four
hours.
[0112] The mixture was allowed to cool and was cleaned, firstly by
adding dichloromethane (50 mls) and then by washing with water
(3.times.100 mls). The organic layer was then removed and dried
over anhydrous sodium sulphate. The organic layer was then filtered
and dichloromethane removed by rotary evaporation. The crude sample
was separated over silica gel (diethyl ether/hexane 2:8) and the
second spot taken and reduced under vacuum (yield 1.09 gm, 33%).
The identity of the compound was confirmed by mass spectrometry
(329 M+ion; 249 loss of Br; 180 N-methyl carbazole ion; 167
carbazole; 152 loss of nitrogen from carbazole; 69,55
C.sub.5H.sub.9 and C.sub.4H.sub.7 ions respectively, from alkyl
chain).
[0113] 4.2 Formation of 3,3 (N-[6-Bromo Hexyl] Carbazole) N-ethyl
Carbazole)
[0114] Electroplating of electrodes was believed to create a
surface which contained both monomeric and dimeric carbazole
entities. In order to remove any uncertainty, it was desired that a
dimeric carbazole entity was prepared at the outset in this
example.
[0115] Accordingly, N-[bromo hexyl] carbazole (1 g, 3 mmol) from
step 4.1 was taken and dissolved in glacial acetic acid (Acros) (75
mls) and perchloric acid (Acros) (70% ww 8 mls). Whilst stirring,
ground N-ethyl carbazole (0.8 g, 4 mmol) was added which was
followed by 2,3-dichloro-5,6 dicyano-p-benzoquinone (3 g, 13 mmol)
(FIG. 6, step 2). The mixture was stirred for one hour after which
time a green precipitate of the dimer carbazole was obtained and
filtered. The mother liquor was returned to the flask and left
overnight. The sample was filtered once again and the combined
filtrates were dissolved in acetone (200 ml). A saturated solution
of aqueous sodium dithionite (Aldrich) (500 ml) was added to the
mixture, which was stirred overnight. The solution changed from a
dark green colour to a brown cream colour.
[0116] The reaction mixture was then washed with dichloromethane
(4.times.100 mls) to remove the product. The organic layer was then
washed with a small amount of water (4.times.25 mls). The organic
layer was then dried over anhydrous sodium sulphate and the solvent
removed by rotary evaporation (crude mixture 1.5 gms, yield 83.3%).
The identity of the compound was confirmed by mass spectrometry
(522 M+ion; 422 loss of HBr; 388 N-ethyl dimer; 329
N(6-bromohexyl)carbazole ion; 179 N-methyl carbazole ion; 129 loss
of benzene ring from carbazole; 97 C.sub.6H.sub.11N, 83
C.sub.6H.sub.11, and 69 C.sub.5H.sub.9, ions respectively).
[0117] 4.3 Formation of 3,3 (N-[6-Thiol Hexyl] Carbazole) N-Ethyl
Carbazole
[0118] This step involves the addition of a thiol group to
facilitate covalent coupling to the gold surface of the
electrode.
[0119] The crude sample (1.5 g) from 4.2 above was disolved in
ethanol 95% (25 ml) and DMF (25 ml) to which thiourea (Aldrich)
(0.25 g, 3.3 mmol) was added (FIG. 6, step 3). This was refluxed (6
hours) and then cooled and stirred overnight. The DMF and ethanol
was removed by rotary evaporation.
[0120] Thin layer chromatography (TLC) of the crude product on
silica gel suggested purification could be achieved by first
removing the impurities with dichloromethane and eluting the
product off the column with DMF (FIG. 6, step 4). Once the product
was obtained the DMF was removed by rotary evaporation.
[0121] The sample was dissolved in DMF (25 ml) and refluxed. Whilst
refluxing sodium hydroxide (0.3 g in 15 ml) was added dropwise
(FIG. 6, step 5). This was then left to reflux (4 hours) and
allowed to cool. The reaction solution was filtered to remove any
precipitation. The sample was then cooled over ice and distilled
water (400 ml) added to the solution [some of this material was
separated and concentrated by rotary evaporation, to yield the 3,3
(N-[6-sodium mercaptan hexyl] carbazole) N-ethyl carbazole salt,
which was used to prepare conjugates, as described in 4.5
below].
[0122] To precipitate the 3,3 (N-[6-thiol hexyl] carbazole) N-ethyl
carbazole product concentrated sulphuric acid (10 ml) was added
dropwise (FIG. 6, step 6). The product was then filtered through a
5 mm filter and placed in a desiccator (dried indicator silica gel
as desiccant) and left under vacuum for three days. (0.22 gms, 94%,
pure 4%).
[0123] FIG. 6 is a schematic representation of the reaction scheme
used to prepare 3,3 (N-[6-thiol hexyl] carbazole) N-ethyl
carbazole. Step 1 shows the reaction with dibromo hexane, step 2
shows the reaction of N-[bromo hexyl] carbazole with glacial acetic
acid, perchloric acid and 2,3-dichloro-5,6 dicyano-p-benzoquinone.
Step 3 shows the reaction with thiourea. Step 4 represents the TLC
purification of the desired salt: 3,3 (N-[isothiouronium hexyl]
carbazole) N-ethyl carbazole; and steps 5 and 6 represent the
reactions with sodium hydroxide and sulphuric acid
respectively.
[0124] 4.4 Formation of 3,3 (N-[6-Thiol Undecyl] Carbazole) N-Ethyl
Carbazole
[0125] An essentially identical compound to that above was
prepared, except that the compound comprised a longer (unedcyl,
C.sub.11) pendant portion. The synthetic technique was essentially
identical as described in 4.2 and 4.3, except that 1-11
dibromo-undecane (0.7 g, 15 mmol) was used in place of dibromo
hexane.
[0126] 4.5 Formation of Conjugates Comprising Hexyl Carbazole
Dimers
[0127] Conjugates of the dimeric carbazole compounds were prepared
for the purposes of production and ELISA testing of antibodies,
since the polyclonal antiserum prepared previously (3.3) was raised
against monomeric carbazole and would not necessarily recognise the
compounds in dimeric form. Accordingly, the carbazole dimers were
used to prepare conjugates with keyhole limpet haemocyanin (KLH)
for immunisation, and to prepare conjugates with bovine serum
albumen (BSA), as described below.
[0128] (i) Formation of KLH (Keyhole Limpet Haemocyanin) Carbazole
Conjugate
[0129] Maleimide-activated KLH (Pierce 2 mg) was dissolved in PBS
(phosphate buffered saline) (200 .mu.l). To this, 3,3(N-[6-sodium
mercaptan hexyl]carbazole)N-ethyl cabazole (2 mg in 200 .mu.l of
PBS) was added and the mixture stirred at room temperature in a
ReactiVial.TM. (Pierce). The sample was then dialysed in a
Slide-A-Lyzer (Pierce) dialysis cassette in PBS for 2 hours. The
PBS was then changed and the sample left to dialyse overnight at
room temperature. The sample was then removed and made up to 2.5 ml
with PBS and placed onto a PD10 column (Pierce) and removed off the
column in a 3.5 ml aliquot of PBS. The sample volume was reduced by
spin filtration. UV scans taken showed that 50 to 60 carbazole
dimers were bound to the carrier protein, indicating that half the
available sites were filled. The KLH conjugate was used for
immunisation, essentially as described in 3.3.
[0130] (ii) Formation of BSA Carbazole Conjugate
[0131] BSA (Pierce, 8 mg) was dissolved in PBS (1 ml) and to this
solution was added SPDP
(N-succinimidyl-3-[2-pyridyldithio]propionate) (2.1 mg) in DMSO (25
.mu.l) and incubated, stirred at room temperature (1 hour). The
mixture was taken and made up to 2.5 ml in PBS and loaded onto a
PD10 column. The sample was then removed from the column with 3.5
ml of PBS. The sample was placed into a ReactiVial.TM. to which
3,3(N-[6-sodium mercaptan hexyl]carbazole)N-ethyl cabazole (4 mg in
200 ml of PBS) was added and stirred at room temperature overnight.
The sample was then dialysed in a Slide-A-Lyzer (Pierce) dialysis
cassette in PBS for 2 hours. The PBS was then changed and the
sample left to dialyse overnight at room temperature. The sample
was then removed and made up to 2.5 ml and placed onto a PD10
column (Pierce) and removed from the column in a 3.5 ml aliquot. UV
scans taken showed that 5 to 8 carbazole dimers were bound to the
carrier protein, indicating that half the available sites were
filled. (Essentially similar techniques were used to prepare
ovalbumen [OVA] conjugates, starting from maleimide activated OVA
(Pierce, 2 mgs), and OVA conjugates were occasionally used in place
of BSA conjugates).
[0132] The BSA conjugates were used to coat ELISA plates,
essentially as described previously. ELISAs performed using BSA
conjugate-coated Greiner microtitre plates demonstrated that the
antibody raised against the KLH conjugate successfully bound to the
dimeric carbazole molecules (data omitted for brevity).
[0133] 4.6 Formation of 3,3(N-(6-thiolhexyl)carbazole)N-ethyl
carbazole monolayer on a Gold Surface
[0134] A gold electrode surface was cleaned by polishing.
Monolayers were formed by immersing a polished screen printed
planar electrode (3 mm by 7 mm) into a solution of the
3,3(N-(6-thiol hexyl)carbazole)N-ethyl carbazole (approximately
0.01 M in DMF). This was left for 24 hours at room temperature in
the dark. The electrodes were removed and washed in dichloromethane
and then placed into clean dichloromethane to soak overnight in the
dark at room temperature.
[0135] In some experiments, the monolayer also comprised spacer
pendant molecules (without electroactive portions), formed by
dipping the electrode in a mixture of the 3,3(N-(6-thiol
hexyl)carbazole)N-ethyl carbazole solution together with a thiol
alkane (typically a C.sub.4 molecule).
[0136] 4.7 Electrochemistry of 3,3(N-(6-thiol
hexyl)carbazole)N-ethyl carbazole monolayer
[0137] Electrochemistry was performed using an EG&G model 273A
Princeton Applied Research Potentiostat/Galvanostat and using Echem
and Lotus 123 to process the data so obtained.
[0138] (i) Electrochemistry in Organic Solvent
[0139] The dichloromethane electrochemistry was performed with a
cell comprising a silver wire as a pseudo or quasi-reference
electrode, an aluminium rod as the counter electrode and the
monolayer on the gold surface as the working electrode. The
electrolyte solution comprised of a 0.1 molar solution of
tetrabutylanmonium hexafluorophosphate in dry dichloromethane. The
electrode was removed from the stock solution and rinsed in
dichloromethane. The electrode was then placed in dried
dichloromethane and left to soak overnight in the dark at room
temperature.
[0140] The electrode was dried in a stream of nitrogen. To check
the electrochemistry of the monolayer, the electrode was
transferred to a cell containing dried dichloromethane with
tetrabutylammonium hexafluorophosphate (0.1 M) as the electrolyte.
The cell contained a silver pseudo reference electrode and an
aluminium rod as the counter electrode. Cyclic voltammograms (100
mV/sec 0 to 1.1 to 0 V) were repeated until a stable, reproducible
scan could be achieved. The cyclic voltammograms of the monolayer
were found to be different to the electroplated carbazole polymer
cyclic voltammograms. The pure dimeric monolayer produced a single
redox peak process, rather than the two peak voltammogram observed
with the cast electrodes (described in Example 3).
[0141] (ii) Electrochemistry in Aqueous Conditions
[0142] Working electrodes were transferred through stages of
acetonitrile: water mixes before being placed in the aqueous
solution (for conducting electrochemical analysis in aqueous
environments), as follows: the electrode was left to soak for 15
minutes in acetonitrile. After this time the electrode was removed
and 8 mls of the solution was taken and 2 mls of the solution was
discarded. The solution was made up to 10 mls with MilliQ water and
the electrode returned to the acetonitrile:water mix to soak for 15
minutes. The process of discarding 2 mls of the acetonitrile:water
mix and making up to 10 mls with MiliQ water was repeated four
times, each time being soaked for 15 minutes. The
acetonitrile:water mix was then discarded and the electrode rinsed
in a stream of water and left for final soak in clean MiliQ water
for 15 minutes. This totalled six soaking periods from pure
acetonitrile to pure water. This had the effect of improving the
cyclic voltammograms obtained by allowing for gradual
acclimatization of the electrode to an aqueous environment, and
hence formation of a more stable monolayer.
[0143] Electrochemistry in an aqueous environment was performed
with a cell, the arrangement of which is illustrated schematically
in FIG. 14. Referring to FIG. 14, the cell was formed in a 100 ml
glass beaker (50) and comprised a Sure Flow.TM. (Orion, UK) Ag/AgCl
(inner)--KCl (outer) reference electrode (52), a clean, coiled
platinum counter electrode (54) and a gold working electrode with
the bound monolayer (56). The electrolyte solution (58) was sodium
hexafluorophosphate in deionised water (unles stated otherwise).
(The electrical connections of the components are omitted from FIG.
14 for the sake of clarity). The working monolayer electrode was
placed into this cell and a cyclic voltammogram taken at 100 mV/sec
from 0 to 0.6 V and then to -0.1V and back to 0 V. Repeated scans
(about six) were made until a stable cyclic voltammogram was
obtained.
[0144] As previously, the method of analysis was then changed to
chronoamperometry so that any current effects could be observed and
measured. Assays were again repeated until a stable, reproducible
scan could be formed to provide a base line.
[0145] 4.8 Using the Monolayer as an Analyte Detector in Aqueous
Conditions
[0146] An electrode, stabilized in aqueous conditions as described
above, was dried with tissue, taking care not to damage the
monolayer surface. A test or a control sample (with or without
anti-carbazole antibody) was then pipetted onto the electrode
surface (25 .mu.l, at 0.14 mgs/ml total Ig) and the electrode left
on a flat surface at room temperature for 15 minutes. After this
time the electrode was shaken dry and washed in a stream of water.
The electrode was then returned to the cell and a
chronoamperometric scan taken under the same condition as the
baseline scan. To enable comparison two samples were used: one
sample (the control) contained no anti-carbazole IgG
(pre-immunisation serum sample); the other sample contained the
relevant anti-carbazole IgG (the antibody sample). Thus, the
pre-immunisation serum sample is a negative control sample, whilst
the antibody sample was expected to show an effect from binding of
the relevant antibody to the carbazole.
[0147] The results show that, as anticipated, the negative control
scan gave a reduced peak value, as indicated in FIG. 7. FIG. 7
shows a graph of mC against time. The baseline plot is labelled 1,
and the plot obtained in the presence of pre-immune serum is
labelled 2. Conversely, duplicate samples of electrodes treated
with immune anti-carbazole antibody exhibited scans with a peak
height increased by 7.73 mC (FIG. 8) and 7.18 mC (FIG. 9)
respectively. Again, in FIGS. 8 and 9, baseline plots are labelled
1, experimental plots in the presence of antibody are labelled
2.
Example 5
[0148] Preparation of an Alternative Electrode Surface
[0149] Production of (Ferrocenylcarbonyloxyl)Undecyl Thiol
[0150] The above-mentioned material was synthesised for use as an
alternative electroactive chemical moiety for attachment to a solid
support. Ferrocene carboxylic acid (3.45 g, 15 mmole) was dissolved
in heptane (50 ml) and oxyalyl chloride (9.5 g, 75 mmole) and
stirred for one hour. The reaction mixture was then heated to
dissolve the remaining carboxylic acid and stirred for a further 45
minutes. The solvent was reduced under vacuum to remove the excess
oxalyl chloride. Heptane (30 mls.times.2) was added and reduced
under vacuum to make sure all remaining traces of oxalyl chloride
are removed. This left the resulting ferrocene carboxylic acid
chloride as a dark red compound.
[0151] The ferrocene carboxylic acid chloride was dissolved in
dichloromethane (150 mls) with 11-bromoundecenanol (3.46 g, 13.8
mmole) and triethylamine (3 g, 30 mmole) and stirred at room
temperature for 2 days. The reaction mixture was separated over
silica gel using dichloromethane as the solvent. The resulting
product was 11-(ferroceneyl carbonyloxyl)undecane bromide.
[0152] Sodium hydrosulphate hydrate (Aldrich 1 g.times.20 mmol) was
ground in to a powder and dissolved into DMF (50 ml) and stirred at
room temperature. To this 11-(ferroceneyl carbonyloxy)undecane
bromide (0.5 g, 10 mmol) was added and refluxed (60.degree. C.) for
one hour to yield the (ferrocenylcarbonyloxyl)undecyl thiol.
[0153] Additionally, conjugates of the ferrocenylcarbonyl undecane
compound were prepared, using KLH and BSA, in order to facilitate
the raising and testing of antibodies against the electroactive
hapten. KLH conjugates were prepared essentially as described
previously for the carbazole compound (4.5(i)), starting from
maleimide-activated KLH, except that the KLH was allowed to react
with (ferrocenylcarbonyloxyl)und- ecyl thiol (2 mg in 50 .mu.l
DMSO/150 .mu.l PBS), instead of the carbazole compound. Similarly,
BSA conjugates were prepared exactly as described in (4.5(ii)), but
using the ferrocenyl compound instead of the carbazole
compound.
[0154] The KLH conjugate was used to raise
anti-(ferocenylcarbonyloxyl)und- ecyl antibodies in rabbits, as
described above for the production of anti-carbazole antibodies
(3.3), and tested by ELISA on BSA conjugate-coated microtitre
plates, as described previously (3.5). The results (omitted for
brevity) showed that a good hapten-specific response was obtained,
and that polyclonal sera from boosted rabbits gave above-background
anti-hapten ELISA responses at serum dilutions in excess of
{fraction (1/1,000)}.
Example 6
[0155] Having demonstrated that binding of anti-carbazole
antibodies to the monolayer of carbazole dimers could be
successfully detected directly by electrochemical assay, the
inventors set out to devise an assay for an analyte of interest,
Estrone-3-glucuronide (E3G), based on this principle. The first
step in the formulation of such an assay was the preparation of a
bispecific anti-E3G/anti-carbazole antibody construct.
[0156] 6.1 Isolation of the scFv4155 Anti-E3G Antibody Fragment
[0157] The DNA encoding the scFv with a specificity towards E3G was
isolated from the hybridoma cell line 4155 and was assembled in an
E. coli expression plasmid pHEN, essentially as described by Ward
et al. (Nature 1989 341, 544). The active corresponding antibody
fragment was isolated via phage display as a fusion protein with
the gene III protein of modified M13. The scFv was tagged at the
C-terminus of the VL with a peptide sequence containing
polyhistidine residues for purification purposes and a sequence
recognised by a second antibody (anti hydrophil II) for detection.
(The antibody anti hydrohil II is disclosed, and a method of
obtaining it taught, in EP 0 456 790, wherein the antibody is
referred to as "anti hydrophilic tail"). The DNA sequence of the
resulting construct is shown as Seq. ID. NO: 1 in the attached
sequence listing. The amino acid sequence of the encoded
polypeptide is shown as Seq. ID. NO: 2 in the attached sequence
listing.
[0158] 6.2 Isolation of the Anti-Carbazole HCV Fragments HCV3 and
HCV24
[0159] The genes encoding the anti carbazole HC--V domains were
isolated essentially as described below. The genes were cloned into
an M13 phage display plasmid as a gene III fusion using the
restriction endonucleases Pst I and Bst EII via standard molecular
biological procedures. Briefly:
[0160] (i) Isolation of Gene Fragments Encoding Llama HC--V
Domains
[0161] A llama was immunised eight times at 2-4 week intervals with
carbazole coupled to the carrier PPD (250-500 .mu.g conjugate per
immunisation). Five days after the last immunisation, a blood
sample of about 200 ml was taken and an enriched lymphocyte
population was obtained via Ficoll (Pharmacia) discontinuous
gradient centrifugation. From these cells, total RNA was isolated
by acid guanidium thiocyanate extraction (e.g. via the method
described by Chomczynnski and Sacchi, 1987). After first strand
cDNA synthesis (using the Amersham first strand cDNA kit), DNA
fragments encoding HC--V fragments and part of the long or short
hinge region were amplified by PCR using specific primers
V.sub.H-2B, Lam-07 and Lam-08:
1 PstI V.sub.H-2B 5'-AGGTSMARCTGCAGSAGTCWGG-3- ' (SEQ. ID. NO: 3) S
= C and G, M = A and C, R = A and G, W = A and T, HindIII Lam-07
5'-AACAGTTAAGCTTCCGCTTGCGG- CCGCGGAGCTGGGGTCTTCGCTGTGGTGCG-3' (SEQ.
ID. NO: 4) HindIII Lam-08
5'-AACAGTTAAGCTTCCGCTTGCGGCCGCTGGTTGTGGTTTTGGTGTCTT- GGGTT-3'.
(SEQ. ID. NO: 5)
[0162] Upon digestion of the PCR fragments with PstI (coinciding
with codon 4 and 5 of the HC--V domain, encoding the amino acids
L-Q) and BstEII (located at the 3'-end of the HC--V gene fragments,
coinciding with the amino acid sequence Q-V-T), the DNA fragments
with a length between 300 and 400 bp (encoding the HC--V domain,
but lacking the first three and the last three codons) were
purified via gel electrophoresis and isolation from the agarose
gel. These PstI/BstEII fragments were inserted into a modified pHEN
based phage display vector linking the HCV genes to gene III of M13
via a HIS6-myc sequence. This library was transformed into E. coli
XL-1 Blue by electroporation yielding 2.7.times.10.sup.6 individual
transformants.
[0163] (ii) Selection of Carbazole Binding HCV Fragments using
Affinity Panning
[0164] Phages expressing HCV fragments at the tip were prepared by
starting 15 mL 2TY/Amp/Glucose with 50 .mu.L of the anti-carbazole
library and was grown until the culture had reached log-phase
(A.sub.600=0.3-0.5). M13K07 helper phage was added and the culture
was incubated for 30 minutes at 37.degree. C. without shaking. The
infected cells were spun at 5000 rpm for 10 minutes and the cell
pellet was resuspended in 200 mL 2xTY/Amp/Kan. After overnight
incubation at 37.degree. C. the cells were removed by
centrifugation. The phages were isolated fom the supernatant by PEG
precipitation:
[0165] add 1/5 volume PEG/NaCL (20% Polyethylene glycol 8000, 2.5M
NaCL) mix well and leave in ice-water for 1 hour; pellet the phage
particles by centrifugation at 8000 rpm for 30 minutes; resuspend
the phage pellet in 20 mL water and add 4 mL PEG/NaCl solution; mix
and leave for 15 minutes in ice-water; pellet the phage particles
by centrifugation at 5000 rpm for 15 minutes; and resuspend the
phage pellet in 2 mL PBS with 2% Marvel.
[0166] Nunc-immunotubes (5 mL) coated overnight at 37.degree. C.
with 1 mL OVA-carbazole in carbonate buffer (100 mg/ml), were
washed 3 times with PBS and blocked with PBS containing 2% Marvel
at 37.degree. C. for 1 hour. 1 mL phage suspension was added to the
tube (and to a control tube) and incubated for 2 hours at room
temperature with occasional shaking. Unbound phages were removed by
washing the tube 20 times with PBS-T followed by 20 washes with
PBS. Bound phages were eluted using 1 mL elution buffer (0.1M.
HCL/glycine pH2.2/1 mg/mL BSA). After 15 minutes at room
temperature the mixture was neutralised by adding 60 mL 2M Tris.
The eluted phage were recovered by adding 9 mL log-phase E. coli
XL-1 Blue (in addition 10 .mu.l of the eluted phages was used to
infect log phase E. coli D29AI). Also 4 mL log-phase E. coli XL-1
Blue was added to the immunotube. Both cultures were incubated for
30 minutes at 37.degree. C. without shaking to allow infection. The
fraction were pooled and serial dilutions of 10.sup.-1 to 10.sup.-6
were plated out on 2TY/Amp/Glucose selective plates.
2 Results of selection experiment: phage input per tube: 10.sup.13
phages recovered from control tube: 5 .times. 10.sup.4 phages
recovered from OVA-carbazole coated plate: 3 .times. 10.sup.6
[0167] (iii) Isolation of Specific Carbazole Binding HC--V
Domains
[0168] For the production of soluble HC--V fragments with a
HIS6myc-tail, individual colonies (in E. coli strain D29AI)
obtained after one round of panning, were grown in a 96 well
microtiter plate containing 200 .mu.l 2TY/Amp/Glucose medium per
well. Once the cultures reached OD.sub.600=0.5, 150 .mu.l of these
cultures was tranferred into a V-bottom 96 well plate and the cells
were pelleted by centrifugation. The E. coli cell pellets were
resuspended in 200 .mu.l 2TY/Amp/IPTG and incubated (with shaking)
at 25.degree. C. overnight. The presence of specific carbazole
binding HC--V fragments in the supernantants was determined as
follows:
[0169] Microtiter plates sensitized with OVA, BSA, OVA-carbazole or
BSA-carbazole in carbonate buffer (100 .mu.g/ml) were washed once
with PBS-T and incubated with 200 .mu.L blocking buffer (1% BSA in
PBS-T) per well for 1 hour at 37.degree. C., then: the E. coli
supernatants were mixed with equal volumes of blocking buffer;
50.mu.L of these samples was added to each well of the sensitized
microtiter plate; the antibody fragments were allowed to bind to
the antigen at 37.degree. C. for 1 hour; unbound HCVs were removed
by 4 washes with PBS-T; 100 .mu.L of an 1 .mu.g/mL solution of the
monoclonal-anti-myc antibody Nr 4111 (in blocking buffer) was added
to each well and incubated at 37.degree. C. for 1 hour; all unbound
antibody removed by 4 washes with PBS-T; 100 .mu.L of an
appropriate dilution of an alkaline-phosphatase conjugated
anti-mouse antibody (in blocking buffer) was added to each well
(incubate at 37.degree. C. for 1 hour); all unbound conjugated
antibody removed by 4 washes with PBS-T; and alkaline phosphatase
activity detected by adding 100 mL substrate solution to each well
(1 mg/ml pNPP in 1M diethanolamine/1 mM MgCl.sub.2).
[0170] In this way a number of specific anti-carbazole HC--V
fragments were isolated, among which were two termed HCV3 and HCV24
respectively. The DNA and amino acid sequences of HCV3 are shown as
SEQ. ID. NOS: 6 and 7 respectively in the attached sequence
listing. The DNA and amino acid sequences of HCV24 are shown as
SEQ. ID. NOS: 8 and 9 respectively in the attached sequence
listing. The antigen binding specificity of these fragments was
determined via ELISA, which showed that the fragments possessed the
desired anti-carbazole binding activity.
[0171] Ammonium thiocyanate (ATC) sensitivity (a relative measure
for binding strength) of the HCV-carbazole interaction was
determined to be 0.5M both for HCV3 and HCV24 (value=ATC
concentration at which binding signal was 50% of maximal) using the
protocol essentially as described above with inclusion of various
concentrations of ATC together with the HCV containing
supernatants.
[0172] 6.3 Expression and Purification of Anti-Carbazole HCV
Domains
[0173] The PstI/BstEII HCV3 and HCV24 gene fragments were subcloned
into the P. pastoris transformation/expresssion vector pPIC9. This
involved two cloning steps: in the first step the PstI/BstEII HCV3
and HCV24 fragments from the phage display vector pUR4536 were
subcloned into the pUC.Y{circumflex over ( )}HIS2t shuttle vector
thus yielding pUC.Y{circumflex over ( )}HCV3-HIS2t and
pUC.Y{circumflex over ( )}HCV24-HIS2t respectively. FIG. 10 shows a
map of plasmid Y{circumflex over ( )}HCV-HIS2t constructs. In a
subsequent step the XhoI/EcoRI HCV-HIS2t fragments were excised
from these intermediates and inserted into XhoI/EcoRI opened pPIC9
yielding pPIC.HCV3-HIS2t and pPIC. HCV24-HIS2t. FIG. 11 shows a map
of plasmid pPIC.HCV constructs. The amino acid sequence of the
expression products of pPIC.HCV3-HIS2t and pPIC.HCV24-HIS2t are
shown as SEQ. ID. NOS. 10 and 11 in the attached sequence
listing.
[0174] P. pastoris cells were transformed essentially as
recommended by the supplier of the P. pastoris expression system
(Invitrogen). Briefly, P. pastoris GS115 cells were grown overnight
at 30.degree. C. in 500 ml YPD medium (1% Yeast Extract, 2%
Peptone, 1% Glucose) to OD.sub.600=1.4. The cells were spun and the
pellet was washed with sterile distilled water before resuspending
in 100 ml KDT7 buffer (50 mM Potassium Phosphate pH7.5, 25 mM DTT).
After 15 minutes incubation at 37.degree. C. the cells were
pelleted (3 minutes, 3000 rpm) and resuspended in 100 ml ice-cold
STM buffer (92.4 g Glucose/l, 10 mM Tris.HCL pH7.5, 1 mM MgCl).
[0175] After 5 washes with this buffer the cell pellet was
resuspended in a final volume of 0.5 ml STM buffer. Approximately
2-5 .mu.g DNA in 2 .mu.l H.sub.2O (BglII digested pPIC constructs:
DNA purified via phenol/chloroform extractions and precipitation)
was mixed with 70 .mu.l of fresh competent P. pastoris cells (on
ice). The cells were electroporated in a 0.2 cm cuvette at 1.5 kV,
400 .OMEGA., 25 .mu.F in a BioRad Gene-Pulser. Immediately after
electroporation, 1 ml of YPD medium was added to the cells. After
recovery for 1 h at 30.degree. C., the cells were pelleted and
resuspended in 200 .mu.L 1M Sorbitol and plated out onto MD plates
(1.34% YNB, 4.times.10.sup.-5% Biotin, 1% Glucose, 0.15% Agar).
Colonies formed by transformed cells (His.sup.+) were visible
within 48 hours incubation at 30.degree. C. Transformed P. pastoris
cells GS115 were selected essentially as recommended by the
Invitrogen Pichia pastoris expression manual. The plates containing
the His.sup.+ transformants were used to screen for the Mut.sup.+
and Mut.sup.s phenotype as follows: Using sterile toothpicks,
colonies were patched on both an MM plate (1.34% YNB,
4.times.10.sup.-5% Biotin, 0.5% MeOH, 0.15% Agar) and an MD plate,
in a regular pattern, making sure to patch the MM plate first.
Approximately 100 transformants were picked for each construct.
After incubating the plates at 30.degree. C. for 2-3 days the
plates were scored. Colonies that grow normally on the MD plates
but show little or no growth on the MM plates were classified as
Mut.sup.s clones.
[0176] Transformed and selected P. pastoris clones were induced to
express HCV domains using the protocol outlined below: i) a single
colony from the MD plate was used to inoculate 10 ml of BMGY (1%
Yeast Extract, 2% Peptone, 100 mM potassium phosphate pH6.0, 1.34%
YNB, 4.times.10.sup.-5% Biotin, 1% Glycerol) in a 50 ml Falcon
tube; ii) the culture was grown at 30.degree. C. in a shaking
incubator (250 rpm) until the culture reached an OD.sub.600=2-8;
iii) cultures were spun at 2000 g for 5 min. and resuspended cells
in 2 ml of BMMY medium (1% Yeast Extract, 2% Peptone, 100 mM
potassium phosphate pH6.0, 1.34% YNB, 4.times.10.sup.-5% Biotin,
0.5% Glycerol); iv) cultures were returned to the incubator; v) 20
.mu.L of MeOH was added to the cultures after 24 h to maintain
induction; and vi) after 48 h the supernatant was harvested by
removing the cells by centrifugation.
[0177] Individual supernatants were assayed by SDS-PAGE and ELISA
and single HCV domain producing clones were used to scaled up this
process to yield larger amounts of antibody fragment.
[0178] Culture supernatants (200 mL, pH 6-8) were clarified through
a 0.45.mu. low protein binding cellulose acetate filter (Nalge Nunc
Intl.), applied to a Ni-NTA Superflow column (5 mL, Qiagen Ltd, UK)
at 2 mL/min, and washed with PBSA until the absorbance at 280 nm
reached baseline. Elution with a linear gradient of 0-500 mM
imidazole over 5 column volumes was followed by immediate buffer
exchange by passage down a column of G-25 Sepadex (150 mL bed
volume, Pharmacia) pre-equilibrated with PBSA, collecting 4 mL
fractions. Peak fractions were assayed by SDS-PAGE and ELISA then
combined and freeze dried in aliquots.
[0179] 6.4 Construction of the Bispecific scFv4155-HCV3 and HCV24
Constructs
[0180] The construction of the scFv4155-HCV3-HIS2t and
scFv4155-HCV24-HIS2t bispecific antibody fragments involved two
cloning steps. In the first step a synthetic XhoI/PstI fragment
(from pUR4124: a pUC vector containing VL-Lys-synthetic
linker-VH-Lys EcoRI/HindIII insert: the DNA and encoded amino acid
sequences are shown as SEQ. ID. NOS: 12 and 13 respectively in the
attached sequence listing), encoding a flexible polypeptide linker
which allows the fusion of the C-terminus of the scFv4155 and the
N-terminus of the HCV fragments, was inserted into XhoI/PstI opened
Y{circumflex over ( )}HCV3-HIS2t and Y{circumflex over (
)}HCV24-HIS2t thus yielding Y.link-HCV3-HIS2t and
Y.link-HCV24-HIS2t respectively. In the second step, the XhoI/EcoRI
fragments from the Y.link-HCV-HIS2t constructs were inserted into
BstEII/EcoRI opened pIC.scFv4155-HIS2t together with a VL4155
encoding BstEII/XhoI fragment (isolated from the same vector)
yielding pPIC.scFv4155-link-HCV3.HIS2t and
pPIC.scFv4155-link-HCV24.HIS2t respectively. FIG. 12 shows a map of
constructs based on plasmid pPIC.scFv4155-HCV. The amino acid
sequence of the expression product of pPIC.scFv4155-link-HCV3.HIS2t
and of pPIC.scFv4155-link-HCV24.HIS2t are shown as SEQ. ID. NOS: 14
and 15 respectively in the attached sequence listing.
[0181] P. pastoris transformants were isolated essentially as
described under 6.3 except that the pPIC DNA was digested with DraI
instead of BglII before transformation. The crude P. pastoris
supernatants were tested for the production of scFv-HCV fusion
protein via analysis on 12% acrylamide gels using the Bio-Rad
mini-Protean II system. E3G, carbazole and bispecific binding
activity was shown via ELISA as follows: (a) 96 well ELISA plates
(Greiner HC plates) were activated overnight at 37.degree. C. with
200 .mu.l/well of the OVA-E3G or OVA-carbazole conjugate; (b)
following one wash with PBST the wells were incubated for 1 hour at
37.degree. C. with 200 .mu.L blocking buffer per well (Blocking
buffer: 1% BSA in PBS-T); (c) serial dilutions of test samples (100
.mu.L) were mixed with equal volumes of blocking buffer and added
to the sensitised ELISA wells. Plates were incubated at 37.degree.
C. for 1-2 hours; (d) 100 .mu.L anti-hydrophil-II monoclonal (Clone
Nr 4890) in blocking buffer was added to each well and incubated at
37.degree. C. for 1 hours; (e) unbound antibody was removed by 4
washes with PBS-T; (f) 100 .mu.L of an appropriate dilution of an
alkaline-phosphatase conjugated anti-mouse antibody (in blocking
buffer) was added to each well (incubate at 37.degree. C. for 1
hour); and (g) unbound conjugated antibody was removed by 4 washes
with PBS-T. Alkaline phosphatase activity was detected by adding
100 mL substrate solution to each well (1 mg/ml pNPP in 1M
diethanolamine/1 mM MgCl.sub.2).
[0182] Alternatively, the presence of bispecific carbazole/E3G
binding HC--V fragments was detected by allowing the fragments to
bind to an OVA-carbazole coated plate and detecting E3G binding
activity by a subsequent incubation with E3G-AP conjugate.
Following one wash with PBST, captured E3G-AP was detected by
adding 100 .mu.l/well pNPP substrate (1 mg/mL pNPP in 1M
diethanolamine/1 mM MgCl.sub.2). In summary, the results showed
that the scFv4155-HCV3 and scFv4155-HCV24 bispecific molecules were
able to bind to both E3G and to carbazole.
Example 7
[0183] Assay for Estrone-3-Glucuronide
[0184] Having obtained a bispecific immunoglobulin molecule with
the desired two binding specificities, the inventors were able to
perform an assay to determine the presence of an E3G analyte of
interest in accordance with the invention.
[0185] 7.1 Construction of Electrochemical Sensing Layer
[0186] An electrode was prepared according to the description in
Example 4.6.
[0187] 7.2 Construction of Cell for Electrochemical
Measurements
[0188] (reference numerals refer to the integers shown in FIG.
2)
[0189] Strips of thin plastic sheet, approximately 0.5 mm thick,
were used as a spacer and stuck to the electrode support surface
surrounding the electrode (10 in FIG. 2) using double sided
adhesive tape. Thus a chamber 0.5 mm deep was formed surrounding
the gold electrode, creating a capillary-fill device. This allowed
50 .mu.l of sample to be placed over the electrode for incubations.
In a second version, a plastic lid was placed over the chamber but
leaving the two ends open. Checks showed that 10 .mu.l of liquid
was sufficient to fill the chamber and electrochemical measurements
could be performed to provide almost identical results to those
obtained with the lid absent.
[0190] 7.3 Formation of E3G and ED3G Conjugates
[0191] An estrone 3-glucuronide (E3G) or estradiol 3-glucuronide
(ED3G) ovalbumin conjugate was prepared by resuspending 2.6 mg of
E3G or ED3G in 2 ml of freshly prepared solution of EDC (1-ethyl
(dimethylaminopropyl) carbodimide, 0.1M) and NHS
(N-hydroxysuccinimide, 0.02M) and incubating for 15 mins at room
temperature.
[0192] To the E3G or ED3G solution, 2 ml of ovalbumin (10 mg/ml)
was added and this was incubated for 2.5 hr at room temperature
with constant mixing.
[0193] The conjugated E3G or ED3G ovalbumin solution was then
dialysed for 16 hr against 1L of phosphate buffered saline
containing 0.1% sodium azide.
[0194] 7.4 Construction of Immunochemical Surface
[0195] The surface of the lid (16) facing the electrode, which was
typically polycarbonate or polystyrene, was modified to form an
assay surface (18). Estrone-3-glucuronide (for competition assay)
or estradiol-3-glucuronide (for displacement assay) conjugated to
ovalbumin, as in example 7.3 above, was allowed to adsorb to the
plastic surface by incubating it with a 0.5 mg/ml solution in
phosphate buffered saline pH 7.2 (PBS) for 2 hours at room
temperature. The surface was rinsed with PBS to remove excess
conjugate and dried using a hot hair drier for thirty seconds. The
surface was then loaded with the double headed antibody
scFv4155-HCV3 (22), described in Example 6, by incubating the
surface with a solution (100 .mu.g/ml in PBS) of the double head
for thirty minutes at room temperature.
[0196] 7.5 Preparation of Cell for Electrochemical Measurements
[0197] A stable base line scan was obtained for a cell, without the
lid on, as described in Example 4.7 (ii).
[0198] 7.6 Assay for Estrone-3-Glucuronide (E3G)
[0199] The electrode was dried using a stream of nitrogen. Excess
antibody was rinsed off the surface with PBS and the lid (16)
shaken free of liquid, or in one experiment air dried without
noticable effect on the assay result, before attaching it over the
electrode chamber. Immediately, the chamber was filled with the
sample to be measured (20). In this example the samples were
solutions of E3G made up to known concentrations (0-50 .mu.g/ml) in
PBS. For samples containing E3G, double headed antibody was
displaced or competed off from the immunochemical surface thereby
allowing it to bind to the carbazole dimer groups (12) on the
electrode surface. The cell was established as described in 4.7
(ii) and left to react for 20 minutes.
[0200] 7.7 Measurement of the Electrochemical Response
[0201] The capillary was shaken empty and washed with distilled
water. The electrolyte solution was then returned to the
capillary-fill device and an electrical reading, here
chronoamperometric, measured (as in 4.7 (ii)), compared to the
background scan and the .DELTA.Q value calculated as described in
example 3.10.
[0202] 7.8 Assay Curve
[0203] FIG. 15 shows an assay curve (dose response, .DELTA..mu.Q
against concentration of E3G in .mu.g/ml) for a set of 8 cells
challenged with increasing concentrations of the analyte E3G. A
typical immunoassay curve shape is seen. A peak response was
obtained at a concentration of E3G between 10 and 20 .mu.g/ml. Two
important controls are also illustrated in the Figure: PBS (Con2)
or E3G (50 .mu.g/ml) in PBS (Con1), in the absence of double headed
antibody, caused no electrochemical changes.
[0204] 7.9 Assay with Reduced Chamber Depth
[0205] In a further experiment the chamber depth was reduced by
using double-sided adhesive tape alone as a spacer (giving about a
five fold reduction in chamber depth, to around 0.1 mm), and the
assay repeated substantially as described above. A beaker was
filled with E3G sample solution (25 .mu.g/ml), which solution
entered the capillary device by capillary action. A scan was
performed as soon as possible (after about 20 seconds), and further
scans made thereafter at about 1 minute intervals. The results,
shown in FIG. 16 (.mu.C against time in seconds) demonstrated very
rapid signal development (with a maximum value of .DELTA.Q obtained
after just one or two minutes), showing that the device allows for
real time monitoring and analysis of samples. Referring to FIG. 16,
(A) is the background scan, (B) the scan obtained after 20 seconds,
and (C), (D), (E) and (F) the scans obtained after 1, 2, 3 and 4
minutes respectively. It can be seen that there was essentially no
further change in the scan profile after 1-2 minutes.
Sequence CWU 1
1
15 1 804 DNA artificial anti-E3G antibody fragment 1 caggtgcagc
tgcaggagtc tgggggtggc ttggtgaacc ttggagggtc tatgactctc 60
tcctgtgtag cctctggatt cactttcaat acctattaca tgtcttgggt tcgccagact
120 ccagagaaga cgctggagtt ggtcgcagcc attaatagtg atggtgaacc
tatctattat 180 ccagacactt tgaagggccg agtcaccatc tctcgagaca
atgccaagaa gaccctatac 240 ctgcaaatga gcagtctaaa ctttgaggac
acagccttat attactgtgc aagacttact 300 tacgccgtgt atggtatgga
ctattggggc caagggacca cggtcaccgt ctcctcaggt 360 ggaggcggtt
caggcggagg tggctctggc ggtggcggat cggacatcga gctcacccaa 420
actccaccct ccctgcctgt cagtcttgga gatcaggttt ccatctcttg cagatctagt
480 cagagccttg tgtccaataa tagaaggaac tatttacatt ggtacctgca
gaagccaggc 540 cagtctccaa agctcgtgat ctacaaagtt tccaaccgat
tttctggggt cccagacagg 600 ttcagtggca gtggatcagg gacagatttc
acactcaaga tcagcagagt ggcggctgag 660 gatctgggac tttatttctg
ctctcaaagt tcacatgttc cgctcacgtt cggttctggg 720 accaagctcg
agatcaaacg gggatctcat caccatcacc atcacggatc cggtagcggg 780
aactccggta aggggtacct gaag 804 2 268 PRT artificial anti-E3G
antibody fragment 2 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val
Asn Leu Gly Gly 1 5 10 15 Ser Met Thr Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Asn Thr Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Thr
Pro Glu Lys Thr Leu Glu Leu Val 35 40 45 Ala Ala Ile Asn Ser Asp
Gly Glu Pro Ile Tyr Tyr Pro Asp Thr Leu 50 55 60 Lys Gly Arg Val
Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Leu Tyr 65 70 75 80 Leu Gln
Met Ser Ser Leu Asn Phe Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95
Ala Arg Leu Thr Tyr Ala Val Tyr Gly Met Asp Tyr Trp Gly Gln Gly 100
105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Thr
Pro Pro Ser 130 135 140 Leu Pro Val Ser Leu Gly Asp Gln Val Ser Ile
Ser Cys Arg Ser Ser 145 150 155 160 Gln Ser Leu Val Ser Asn Asn Arg
Arg Asn Tyr Leu His Trp Tyr Leu 165 170 175 Gln Lys Pro Gly Gln Ser
Pro Lys Leu Val Ile Tyr Lys Val Ser Asn 180 185 190 Arg Phe Ser Gly
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe
Thr Leu Lys Ile Ser Arg Val Ala Ala Glu Asp Leu Gly Leu 210 215 220
Tyr Phe Cys Ser Gln Ser Ser His Val Pro Leu Thr Phe Gly Ser Gly 225
230 235 240 Thr Lys Leu Glu Ile Lys Arg Gly Ser His His His His His
His Gly 245 250 255 Ser Gly Ser Gly Asn Ser Gly Lys Gly Tyr Leu Lys
260 265 3 22 DNA artificial primer 3 aggtsmarct gcagsagtcw gg 22 4
53 DNA artificial primer 4 aacagttaag cttccgcttg cggccgcgga
gctggggtct tcgctgtggt gcg 53 5 53 DNA artificial primer 5
aacagttaag cttccgcttg cggccgctgg ttgtggtttt ggtgtcttgg gtt 53 6 378
DNA artificial anti-carbazole HC-V fragments 6 caggtgcagc
tgcaggagtc agggggagga ttggtgcagc ctgggggctc tctgagactc 60
tcctgtgcag cttctggact cacattgact acctattcaa cgggctggtt ccgccaggct
120 ccagggaagg agcgtgaatt tgtaggaatg cttggatgga gtggtggtgg
caacacgtac 180 tacgcagact ccgtgaaggg ccgatttacc atctccagag
acaacgccaa gaatatggtg 240 tttctgcaaa tgagcagcct gaaacctgag
gacacggccg tttattactg tgcagcacga 300 caaccctacc gaggtagtta
cagtgatccg aataattatc attactgggg ccaggggacc 360 caggtcaccg tctcctca
378 7 126 PRT artificial anti-carbazole HC-V fragments 7 Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Leu Thr Thr Tyr 20
25 30 Ser Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45 Gly Met Leu Gly Trp Ser Gly Gly Gly Asn Thr Tyr Tyr
Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Met Val 65 70 75 80 Phe Leu Gln Met Ser Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Arg Gln Pro Tyr
Arg Gly Ser Tyr Ser Asp Pro Asn Asn 100 105 110 Tyr His Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 8 375 DNA
artificial anti-carbazole HC-V fragments 8 caggtgcagc tgcaggagtc
agggggagga ttggtgcagg ctgggggctc tctgagactc 60 tcctgtgcag
cctctggacg caccttcagt gtttatgccg tgggttggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtaggatac tttggcacgc gtggtggaag aacatactat
180 gcagactccg tgaagggccg attcaccatc gccatagaca acgctaagaa
cacggtgtat 240 ctgcaaatga atagcctgaa actagacgat acggccgttt
attactgcgc agtccgtatg 300 ccgtatagtg gtgattaccg atctagtggg
acatatgact actggggcca ggggacccag 360 gtcaccgtct cctca 375 9 125 PRT
artificial anti-carbazole HC-V fragments 9 Gln Val Gln Leu Gln Glu
Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Val Tyr 20 25 30 Ala Val
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45
Gly Tyr Phe Gly Thr Arg Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ala Ile Asp Asn Ala Lys Asn Thr Val
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Leu Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Val Arg Met Pro Tyr Ser Gly Asp Tyr Arg
Ser Ser Gly Thr Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 125 10 147 PRT artificial amino acid
sequence of the expression products of pPIC.HCV3-HIS2t 10 Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Leu Thr Thr Tyr 20
25 30 Ser Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45 Gly Met Leu Gly Trp Ser Gly Gly Gly Asn Thr Tyr Tyr
Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Met Val 65 70 75 80 Phe Leu Gln Met Ser Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Arg Gln Pro Tyr
Arg Gly Ser Tyr Ser Asp Pro Asn Asn 100 105 110 Tyr His Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser Gly Ser 115 120 125 His His His
His His His Gly Ser Gly Ser Gly Asn Ser Gly Lys Gly 130 135 140 Tyr
Leu Lys 145 11 146 PRT artificial amino acid sequence of the
expression products of pPIC.HCV4-HIS2t 11 Gln Val Gln Leu Gln Glu
Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Val Tyr 20 25 30 Ala Val
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45
Gly Tyr Phe Gly Thr Arg Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ala Ile Asp Asn Ala Lys Asn Thr Val
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Leu Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Val Arg Met Pro Tyr Ser Gly Asp Tyr Arg
Ser Ser Gly Thr Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser Gly Ser His 115 120 125 His His His His His Gly Ser
Gly Ser Gly Ala Ser Gly Lys Gly Tyr 130 135 140 Leu Lys 145 12 739
DNA artificial bispecific scFv4155-HCV3 and HCV24 construct 12
gaattcggcc gacatcgagc tcacccagtc tccagcctcc ctttctgcgt ctgtgggaga
60 aactgtcacc atcacatgtc gagcaagtgg gaatattcac aattatttag
catggtatca 120 gcagaaacag ggaaaatctc ctcagctcct ggtctattat
acaacaacct tagcagatgg 180 tgtgccatca aggttcagtg gcagtggatc
aggaacacaa tattctctca agatcaacag 240 cctgcaacct gaagattttg
ggagttatta ctgtcaacat ttttggagta ctcctcggac 300 gttcggtgga
accaagctcg agatcaaacg gggtggaggc ggttcaggcg gaggtggctc 360
tggcggtggc ggatcgcagg tgcagctgca ggagtcagga cctggcctgg tggcgccctc
420 acagagcctg tccatcacat gcaccgtctc agggttctca ttaaccggct
atggtgtaaa 480 ctgggttcgc cagcctccag gaaagggtct ggagtggctg
ggaatgattt ggggtgatgg 540 aaacacagac tataattcag ctctcaaatc
cagactgagc atcagcaagg acaactccaa 600 gagccaagtt ttcttaaaaa
tgaacagtct gcacactgat gacacagcca ggtactactg 660 tgccagagag
agagattata ggcttgacta ctggggcgaa ggcaccacgg tcaccgtctc 720
ctcatgataa gcttgtcac 739 13 241 PRT artificial bispecific
scFv4155-HCV3 and HCV24 construct 13 Asn Ser Ala Asp Ile Glu Leu
Thr Gln Ser Pro Ala Ser Leu Ser Ala 1 5 10 15 Ser Val Gly Glu Thr
Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile 20 25 30 His Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln 35 40 45 Leu
Leu Val Tyr Tyr Thr Thr Thr Leu Ala Asp Gly Val Pro Ser Arg 50 55
60 Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser
65 70 75 80 Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe
Trp Ser 85 90 95 Thr Pro Arg Thr Phe Gly Gly Thr Lys Leu Glu Ile
Lys Arg Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Val Gln 115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu
Val Ala Pro Ser Gln Ser Leu Ser 130 135 140 Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Gly Thr Gly Val Asn 145 150 155 160 Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile 165 170 175 Trp
Gly Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185
190 Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205 Ser Leu His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg
Glu Arg 210 215 220 Asp Tyr Arg Leu Asp Tyr Trp Gly Glu Gly Thr Thr
Val Thr Val Ser 225 230 235 240 Ser 14 409 PRT artificial
expression product of pPIC.scFv4 155-link-HCV3.HIS2t 14 Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Asn Leu Gly Gly 1 5 10 15 Ser
Met Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25
30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Thr Leu Glu Leu Val
35 40 45 Ala Ala Ile Asn Ser Asp Gly Glu Pro Ile Tyr Tyr Pro Asp
Thr Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys
Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Asn Phe Glu Asp
Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Leu Thr Tyr Ala Val Tyr
Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly
Gly Ser Asp Ile Glu Leu Thr Gln Thr Pro Pro Ser 130 135 140 Leu Pro
Val Ser Leu Gly Asp Gln Val Ser Ile Ser Cys Arg Ser Ser 145 150 155
160 Gln Ser Leu Val Ser Asn Asn Arg Arg Asn Tyr Leu His Trp Tyr Leu
165 170 175 Gln Lys Pro Gly Gln Ser Pro Lys Leu Val Ile Tyr Lys Val
Ser Asn 180 185 190 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Lys Ile Ser Arg Val Ala
Ala Glu Asp Leu Gly Leu 210 215 220 Tyr Phe Cys Ser Gln Ser Ser His
Val Pro Leu Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Gly Gly
Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 260 265 270 Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 275 280
285 Leu Thr Leu Thr Thr Tyr Ser Thr Gly Trp Phe Arg Gln Ala Pro Gly
290 295 300 Lys Glu Arg Glu Phe Val Gly Met Leu Gly Trp Ser Gly Gly
Gly Asn 305 310 315 320 Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp 325 330 335 Asn Ala Lys Asn Met Val Phe Leu Gln
Met Ser Ser Leu Lys Pro Glu 340 345 350 Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Arg Gln Pro Tyr Arg Gly Ser 355 360 365 Tyr Ser Asp Pro Asn
Asn Tyr His Tyr Trp Gly Gln Gly Thr Gln Val 370 375 380 Thr Val Ser
Ser Gly Ser His His His His His His Gly Ser Gly Ser 385 390 395 400
Gly Asn Ser Gly Lys Gly Tyr Leu Lys 405 15 408 PRT artificial
expression product of pPIC.scFv4 155-link-HCV4.HIS2t 15 Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Asn Leu Gly Gly 1 5 10 15 Ser
Met Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25
30 Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Thr Leu Glu Leu Val
35 40 45 Ala Ala Ile Asn Ser Asp Gly Glu Pro Ile Tyr Tyr Pro Asp
Thr Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys
Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Asn Phe Glu Asp
Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Leu Thr Tyr Ala Val Tyr
Gly Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly
Gly Ser Asp Ile Glu Leu Thr Gln Thr Pro Pro Ser 130 135 140 Leu Pro
Val Ser Leu Gly Asp Gln Val Ser Ile Ser Cys Arg Ser Ser 145 150 155
160 Gln Ser Leu Val Ser Asn Asn Arg Arg Asn Tyr Leu His Trp Tyr Leu
165 170 175 Gln Lys Pro Gly Gln Ser Pro Lys Leu Val Ile Tyr Lys Val
Ser Asn 180 185 190 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Lys Ile Ser Arg Val Ala
Ala Glu Asp Leu Gly Leu 210 215 220 Tyr Phe Cys Ser Gln Ser Ser His
Val Pro Leu Thr Phe Gly Ser Gly 225 230 235 240 Thr Lys Leu Glu Ile
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Gly Gly
Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly 260 265 270 Leu
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 275 280
285 Arg Thr Phe Ser Val Tyr Ala Val Gly Trp Phe Arg Gln Ala Pro Gly
290 295 300 Lys Gln Arg Glu Phe Val Gly Tyr Phe Gly Thr Arg Gly Gly
Arg Thr 305 310 315 320 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ala Ile Asp Asn 325 330 335 Ala Lys Asn Thr Val Tyr Leu Gln Met
Asn Ser Leu Lys Leu Asp Asp 340 345 350 Thr Ala Val Tyr Tyr Cys Ala
Val Arg Met Pro Tyr Ser Gly Asp Tyr 355 360 365 Arg Ser Ser Gly Thr
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr 370 375 380 Val Ser Ser
Gly Ser His His His His His His Gly Ser Gly Ser Gly 385 390 395 400
Asn Ser Gly Lys Gly Tyr Leu Lys 405
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