Note: Descriptions are shown in the official language in which they were submitted.
WO 93/03367 PCT/US92/06249
2~~~~~~~~
DIFFERENTIAL BINDING AFFINITIES
AND DISSOCIATI0,~1 ASSAYS BASED THEREON
1. FIELD OF THE INVENTION
' 5 The present invention relates to methods
for determining the presence of an analyte in a
sample. More particularly, homogeneous liquid-phase
and heterogeneous liquid-phase/solid-phase release
assays that are highly specific and sensitive are
i0 provided.
2. BACKGROUND OF THE INVENTION
Immunoassays utilize the specific binding,
capabilities of antibodies to detect the presence of
15 target molecules in solution. Although the general
principle is applicable to a broad range of problems,
major commercial interest has centered on medical
diagnostic applications for a wide variety of analytes
in biological fluids such as blood, saliva, and urine.
Several types of immunoassays, useful for
distinct applications, already exist. Each such assay
type requires away of distinguishing whether binding
sites on an antibody are occupied or free. Typically
this is accomplished by means of label such as an
35 atom, molecule, enzyme or particle attached
permanently to either the antibody or to an analog of
the analyte.
Sensitivity and specificity are key
parameters of an immunoassay. Specificity relates
30 primarily to the antigen binding site of the antibody,
which is inherent to selection of variable region gene
segments and is independent of the assay
configuration. Sensitivity relates primarily to the
affinity of the antibody for its ligand(s) and to the
35 inherent detectability of the label. For example,
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radioisotopes, used for radioimmunoassay, can be
detected at significantly lower concentrations than
fluorescent molecules. Enzyme labels ark detectable
at concentrations similar to fluorescent labels. When
substrates that produce fluorescent or
chemiluminescent products are used with enzyme labels,
the sensitivity of resulting immunoassays is
comparable or greater than with radioisotope labels.
Many conventional assay techniques are
l0 considered competitive in that the analyte and labeled
component have comparable affinities for the antibody
binding site. One example of such a competitive
method is found in U.S. Patent 3,617,837 by Rubenstein
and Ullman which describes a technique in which ligand
and enzyme-bound-ligand compete fvr antibody bind:~ng
sites. Since.binding of the antibody to the enzyme-
bound-ligand alters its enzymatic activity, the
concentration of ligand present can be estimated by
measuring the rate at which such a mixture converts
substrate to product.
Immunoassays can be further characterized as
homogeneous and heterogeneous. In a treterogeneous
method, the label is equally detectable in bound and
unbound states. To obtain any meaningful assay
results physical separation of the bound versus
unbound antibody is required. A common strategy for
accomplishing this separation entails associating the
label to a solid phase which can be physically
separated from the liquid phase prior to the detection
step. A typical heterogeneous assay is the Tandem~IA
from Hybritech, Inc.
In a homogeneous method, the detectable
property of the label is inherently different
depending on whether bound or unbound. In its bound
state, the label will have greater or lesser signal
CA 02114440 2003-04-10
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intensity. Usually, binding of antibody to the
labeled ligand causes a decrease in signal intensity,
e.g., when the label fs an enzyme. Typical products
in this category include the EMIT~ine of ~zyme
3 immunoassays from Syva Company and the TDX line of
fluorescence polarization immunoassays from Abbott
Diagnostics.
Two further characteristics of immunoassays
are particularly noteworthy. These are the minimal
i0 concentration of analyte that can be detected, and the
dynamic range of detection. The dynamic range is the
range of analyte concentrations over which signal from
a label changes from zero to maximum. The order in
which the sample, the antibody, and a labeled
15 component are combined can significantly affect both
of these key parameters by affecting the degree of
binding of the labeled component, which in turn
affects detection of the label.
In certain known assay methods, the antibody
20 and the analyte are combined prior to addition of the
labeled component. In others, the analyte and labeled
component are combined prior to addition of the
antibody. Each of these cases requires providing two
separate reagents that are combined with the sample
Z5 containing the analyte. The need for two such
separate reagents can be inconvenient and result in a
more cumbersome, complex method. Moreover, because
precise volumetric measurement of each reagent is
critical to good assay performance, the necessity of
30 two measuring steps can cause errors which may lead to
distorted results.
One method to improve assay precision and
thereby enhance assay sensitivity is to provide a
premixed complex of the antibody and labeled
95 component. This is problematic, however, because the
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binding reaction is generally found to be irreversible.
Thus, when a complex of the labeled analyte and antibody
are combined with a solution containing the analyte, no
appreciable displacement of bound label occurs in a
meaningful time frame (seconds to minutes).
The present invention relates to assay
methodology that employs a complex of receptor and a
ligand, wherein the receptor-ligand complex dissociates
in the presence of an analyte and a stable receptor-
analyte complex is formed. Dissociation of the receptor-
ligand complex in the presence of analyte is detectable
thereby positively indicating the presence of analyte in
a test sample. Methods for designing, preparing, using,
and stabilizing such, complexes are taught. The
methodology is applicable both to homogeneous assays and
heterogeneous assays for analytes encompassing a broad
range of types and sizes.
3. SUMMARY OF THE INVENTION
The present invention provides for
heterogeneous and homogeneous release assay methods for
detecting the presence of an analyte in a sample. A kit
for performing the assay method of the invention is also
provided.
In accordance with one embodiment of the
invention there is a method for the detection of an
analyte in a sample comprising: (a) contacting a test
sample with a receptor-ligand complex, comprising a
receptor bound to a release ligand, wherein the receptor
binds to an analyte, wherein the release ligand binds to
the receptor with an association constant of 1~ or less
of the association constant of the analyte for the
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receptor, and wherein the release ligand does not
detectably compete with analyte for binding to the
receptor; and (b) detecting the dissociation of the
release ligand from the receptor as a measure of the
presence or amount of analyte in the sample.
In accordance with another embodiment of the
invention there is a method for the detection of an
analyte in a sample comprising: (a) forming a receptor-
ligand complex; (b) contacting the sample with the
complex; and (c) detecting dissociation of the receptor-
ligand complex and binding of the receptor with the
analyte which positively correlates with the presence of
the analyte in the sample, wherein the release ligand
binds to the receptor with an association constant of 1~
or less of the association constant of the analyte for
the receptor, and wherein the receptor-analyte complex is
substantially unaffected by the presence of free ligand.
In accordance with yet another embodiment of
the invention there is a kit for detecting the presence
of an analyte in a sample comprising a preformed
receptor-release ligand complex, comprising a receptor
bound to a release ligand, wherein the receptor binds to
the analyte, wherein the release ligand binds to the
receptor with an association constant of 1~ or less of
the association constant of the analyte for the receptor,
and wherein the release ligand does not detectably
compete with analyte for binding to the receptor, and
means for detecting dissociation of the complex and
binding of receptor with analyte in the presence of
analyte in the sample.
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In accordance with yet a further embodiment of
the invention there is a kit for detecting the presence
of an analyte in a sample comprising receptor and release
ligand for forming a receptor-release ligand complex,
wherein the receptor binds to the analyte, where in the
release ligand binds to the receptor with an association
constant of 1~ or less of the association constant of the
analyte for the receptor, and wherein the release ligand
does not detectably compete with analyte for binding to
the receptor, and means for detecting dissociation of the
complex and release of receptor which positively
correlates with the presence of analyte in the sample.
According to the present method, a test sample
is contacted with a receptor-ligand complex. The receptor
has much a higher association constant for analyte than
ligand. When the receptor-ligand complex dissociates into
its receptor and ligand components in the presence of
free analyte in the test sample, receptor binds the free
analyte to form a stable receptor-analyte complex, which
is substantially unaffected by the presence of free
WO 93/0336? PCT/US92/06249
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2
ligand. In accordance with the present method,
dissociation of the receptor-ligand complex, i.e.,
release of the receptor and ligand, is a detectable
event indicating the presence of analyte in the test
sample. Detection of either dissociated ligand or
receptor is contemplated. Means for detection are
detailed infra in sections 5.2, 5.2.1 and 5.2.2.
In one embodiment of the invention a kit for
carrying out the present method is provided. The kit
includes either preformed receptor-ligand complex or
receptor and ligand which are mixed to form the
complex prior to conducting the assay. As can be
appreciated, when the pre-formed complex is provided,
apart from test sample, only this single reagent may
be required to carry out the assay.
As shown in the Examples, infra, the present
invention provides surprisingly greater sensitivity,
specificity, accuracy and range of detection than
conventional association assays or competitive
dissociation assays.
3.1. DEFINITIONS
Analyte -- molecule of interest in an assay.
Ligand -- molecule capable of binding to a
receptor specific for analyte with substantially lower
affinity than that of analyte binding to the receptor.
' Receptor -- molecule capable of specifically
binding to analyte or ligand. In each case, a stable
complex is formed, but the association constant of
receptor for analyte is higher than that for ligand.
Dissociation of the receptor-ligand complex into its
receptor and ligand components in the presence of free
analyte results in release of the receptor which binds
to free analyte, thereby forming a stable receptor-
WO 93/03367 PGT/US92/06249
l;. ~ ~ ~~ _ 6
analyte complex substantially unaffected by the
presence of free ligand.
4. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Release of anti-cotinine antibody
in the presence of cotinine in a urine matrix sample.
Anti-cotinine was labeled with peroxidase and
complexed with solid phase cotinine ligand. Free
cotinine was added and released anti-cotinine was
i0 detected by measuring enzyme activity in the
supernatant after 2 min incubation. Ligands were
cotinine conjugated to BGG, using the linker
aminocaproic acid (open squares) or p-aminobenzoic
acid (solid squares)
Figure 2. Release of anti-cotinine antibody
after a 10 minute incubation in a complex with
immobilized ligand. A synthetic urine matrix sample
was spiked with cotinine. Ligands are the same as for
Figure 1.
Figure 3. Release of anti-cotinine antibody
after a 2 minute incubation in urine and synthetic
urine matrix samples. Release of labeled anti-
cotinine was detected from cotinine-aminocaproyl-BGG
in urine (solid squares) and synthetic urine (open
squares) and from cotinine-benzoyl-BGG in urine
(asterisks) and synthetic urine (solid triangles).
Figure 4. Deactivation and inhibition of a
glucose-6-phosphate dehydrogenase-hydroxycotinine
conjugate. Normal enzyme activity (solid squares),
conjugate activity (open squares), and conjugate
activity in the presence of antibody (asterisks) are
shown.
Figure 5. Inhibition and release of a
hydroxycotinine-glucose-6-phosphate dehydrogenase
conjugate. The enzyme activity (A~o vs time) of enzyme
WO 93/03367 PCTlUS92/06249
_ ~ ~~.i~~~~~
conjugate (solid squares), enzyme conjugate plus
antibody to cotinine (solid triangles), and enzyme
conjugate + antibody + free cotinine (open squares)
are shown.
Figure 6. Automated release assay for
cotinine. Release in the presence of cotinine was
measured on an automated analyzer after <1 (dots), 18
(crosses), and 22 (asterisks) hours of complex
incubation.
Figure 7. Efficiency of various low ,
affinity analogs of cotinine conjugated to glucose-6-
phosphate dehydrogenase. Conjugates of traps-hydroxy
cotinine (extensively conjugated (dots), or partially
conjugated (vertical lines, asterisks)), cis-
i5 hydroxycotinine (crosses) and carboxycotinine (open '
squares) to glucose-6-phosphate dehydrogenase were
tested as release ligands in a solid phase assay.
Figure 8. Structure of (a.) nicotine, (b.)
cotinine, (c.) N-isopropyl-4-carboxy-norcotinine and
(d.) N-propyl-4-carboxy-norcotinine.
Figure 9. ELISA format relewse assay.
Microtiter plates were coated with cis hydroxycotinine
G-6-PDH (asterisks); N-isopropyl-norcotinine G-6-PDH
(plus signs); N-propyl-norcotinine G-6-PDH [open
stippled squares].
Figure I0. Comparison of the dpse response
curves of an associative homogenous assay for cotinine
(NiMA) with the release assay. Associative NiMA assay
(plus signs); release assay (open stippled square).
Figure.il. Standard curve for the release
assaylof cotinine by the homogeneous method.
Figure 12. Benzoylecgonine release assay
using a microtiter plate format. Matrix effects were
observed depending on the liquid medium, which was
WO 93/03367 PGT/US92/06249
R. ~1
;, ~~ ~y ''G '~ _ g
?~~. ~.
either water (solid squares) or urine (crosses). BE
is benzoylecgonic.
Figure 13. Effect of incubation time on
benzoylecgonine release assay using a microtiter plate
format. Supernatants were obtained 0 (solid squares),
2 (open squares) and 10 (asterisks) minutes after .
addition of free benzoylecgonine (BE) and checked for
release of labeled anti-benzoylecgonine.
Figure 14. Low affinity chromatography of
anti-beta hCG. Rabbit antiserum to human chorionic
gonadotropin (hCG) was applied to a sheep gonadotropin
affinity column and eluted with 13 ml PBS, 20 ml of pH
4 buffer, and 10 ml of pH 8 buffer. Protein (light
cross-hatch); anti-hCG (dark cross-hatch); IgG (double
i5 cross-hatch). '
5. QIETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention a
method for detecting the presence of analyte in a
. 20 sample and kits therefore are provided. A test sample
can be any body fluid, such as urine~v blood, serum,
saliva, bodily exudate, etc., suspected o.f containing
the target analyte. The release assay method of the
invention involves contacting a test sample with a
25 receptor-ligand complex and, if analyte is present,
detecting dissociation of that complex into its
receptor and ligand components. Dissociation of the
complex in the presence of analyte results in release
of receptor and ligand. Upon release, receptor binds
30 with free analyt~, forming a receptor-analyte complex
that remains stable in the presence of free ligand.
Either dissociated ligand or receptor can be detected
to indicate the presence of analyte in the sample.
The assay method of the invention may be performed in
35 either homogeneous or heterogeneous formats. Specific
WO 93/03367 PGT/US92/06249
- 9 - ~~i~:~~:~~
details of each format are provided infra in Section
5.2.
. A release assay of the present invention
allows preparation of a complex in which receptor
5. binding sites and ligand are present in approximately
equal concentration, i.e., quantitative complex
formation, although this is not essential. Having
each element present in equal concentration enhances
sensitivity, since when receptor is present in excess,
it can bind analyte without releasing from receptor-
ligand complex.
In order to achieve a receptor-ligand
complex in which the number of binding sites of each
element are present in substantially equimolar
i5 amounts, the receptor and ligand are incubated for at '
least ten seconds prior to exposure to sample.
Preferably the incubation time is greater than about
. one hour. A long incubation time allows formation of
the most stable complexes during repeated release and
binding reactions when one element, either receptor or
ligand, is present in excess. After the low affinity
binding reaction reaches equilibrium, the receptor-
ligand complex is isolated from the excess element.
Isolation of the complex may be either by size
exclusion chromatography; density gradient
centrifugation, low affinity chromatography, or other
' techniques for separating complexes from their
components. Alternatively, the receptor and ligand
may be mixed in equal binding site concentrations and
incubated to allow substantially quantitative binding
to create stable complexes. Formation of the reagent-
ligand complex is discussed more fully in Section
5.1., 'infra.
The kit of the present invention preferably
includes preformed receptor-ligand complex, thereby
WO 93/03367 PGT/L~S92/06249
~ y ~E~.'=~ - io -
eliminating the need to prepare reagents prior to
conducting an assay. However, providing both receptor
and ligand separately, and mixing them prior to the
assay, is within the scope of the invention.
The present invention is based on the
principle that dissociation of a complex of receptor
and ligand in the presence of analyte will result in
association of receptor and analyte rather than
reassociation of receptor and ligand when the
association constant of receptor and analyte is
greater than the association constant of receptor and
ligand. The dissociation of a receptor-ligand
complex, release of receptor and ligand, and binding
of receptor to analyte to form a stable complex is
termed herein the release reaction. The corollary of
a release reaction is that free ligand will not affect
the stability of the receptor-analyte complex, so
that the receptor will not bind to free ligand, i.e.,
the release reaction is not reversible. Similarly a
stable receptor-ligand complex will not dissociate and
release receptor to bind a second low affinity cross
reactive ligand because there is no kinetic or the
thermodynamic gain. The dissociation constant of even
a low affinity receptor-ligand complex is relatively
low, and binding to another ligand with an equivalent
affinity constant does not result in a free energy
change. This results in a sensitive, highly specific,
highly accurate (minimally cross reactive) assay.
However, when analyte binds receptor with
30' high affinity and the association constant is high,
dissociation of the receptor from the receptor ligand
complex and binding to the analyte will occur readily.
The high association rate will result in fast "pick-
up" of dissociated receptor. The release reaction is
thermodynamically favorable, since a higher affinity
WO 93/03367 PGT/US92/06249
- 1i -
constant will give a negative net free energy change.
The thermodynamics and kinetics will drive a release
reaction in the presence of analyte, and will result
in no change in the absence of analyte.
Preferably the affinity constant of binding
of receptor to ligand will be about 10%, and more
preferably about 1%, of the affinity constant of
binding of receptor to analyte. This can be observed
qualitatively as relative binding, e.g., by apparent
io activity in an assay.
Because the thermodynamics and kinetics of
the release reaction favor binding of receptor to
analyte over binding of receptor to ligand,
macroscopically analyte appears to induce the release
reaction, i.e., dissociation of the receptor-ligand '
complex.
Since the release reaction depends on a high
affinity association of receptor and analyte, it is
sensitive and specific. That is, receptor will bind
low concentrations of analyte. Dependence of the
release reaction on the differential affinity binding
further increases specificity. Receptor will not
dissociate and bind cross-reactive analogs of the
analyte unless the binding constant is much higher
Z5 than the binding constant of receptor and ligand.
It is important to emphasize that a
significant portion of the complex must dissociate, or
the background "noise" in the system will be too
great. For example, if only 1% of the receptor-ligand
complex is dissociated, 99% of the system is
unaffected. If the standard deviation of measurement
is 1% (equivalent to 99 ~ 1%), which represents an
excellent coefficient of variation in immunoassay
systems, then the effect of 1% release would be 1% ~
1%, nullifying any significance. By taking advantage
WO 93103367 PCT/US92/06249
Pxtv' _ 12 --
r
of the thermodynamic and kinetic impetus of the
release reaction, the present assay provides for
significant dissociation of receptor-ligand ~:omplex,
i.e., release above the baseline levels. Moreover,
the release assay format provide for a broad
concentration range over which analytes can be
detected.
The receptor-ligand complex need not be
fully dissociated for effectiveness, however. For
io example, when a solid phase preparation of
hydroxycotinine linked to a carrier protein (e. g.,
glucose-6-phosphate dehydrogenase) is reacted with
sample, only 5-10% of total antibody labeled with the
enzyme horseradish peroxidase is displaced by analyte.
The exact percentage is difficult to ascertain since '
enzyme activity on a solid phase is known to be less
than that of enzyme in liquid phase. Nevertheless,
the amount of release (greater than 5%) provides a
significant signal over background. In a homogeneous
release assay, the sample containing cotinine induces
100% reversal of enzyme inhibition, which indicates
100% release.
5:1. THE RECEPTOR-LIGAND COMPLEX
An effective release assay requires
formation of a stable low affinity receptor-ligand
complex at equilibrium. This complex usually forms
more slowly than conventional complexes, e.g.,
receptor-analyte, or antibody-antigen complexes, etc.
Dissociation occurs readily during initial receptor-
ligand complex formation. However, after a suitable
incubation period, the receptor-ligand complex becomes
stable. The stability of the complex is in part a
function of the design of the ligand, as well as
incubation time of ligand and receptor. The
WO 93/03367 PCT/US92/06249
-
appropriate incubation time is readily determined for
each receptor and ligand combination. Generally,
~ however, receptor and ligand should be incubated at
least 10 seconds, preferably at least 10 minutes, and
- 5 more preferably longer than 1 hour, prior to exposure
to analyte. In cases where the receptor-ligand
complex is isolated prior to performing the assay,
both receptor and ligand binding sites are present in
substantially equimolar amounts and the incubation
time is not important (see Section 5, sutar_a_). Under
appropriate conditions, e.g., the presence of salts
such as sodium chloride, or stabilizing agents such as
glucose, or both, the stable receptor-ligand complex
will remain releasable over a long time period --
i5 days, weeks or longer. According to the present '
invention, a stable receptor-ligand complex may be in
solution, or it may be dried. In an Example, ~ ra, a
release assay could be run six days after antibody-
ligand complex formation in the presence of 5% sodium
chloride. In another Example, infra, the presence of
glucose stabilized a dry antibody-ligand complex.
Although the present invention is not bound
by any theory or hypothesis, it is believed that
formation of a stable receptor-ligand complex supports
a model of molecular accommodation between the ligand
and the receptor. The equilibrium of this binding
favors a configuration of the complex which stabilizes
it, meaning that the effective affinity of the complex
may, and probably must be, higher in the mature
complex than initially. only by creating a stable,
complex can one assure that dissociation is specific,
and is driven by the much higher affinity of the
receptor for the analyte.
Unless a release type assay is formatted
with differential binding affinities, and preferably
WO 93/03367 PCf/US92/06249
14 _
includes the use of stabilizing agents, undesirable
irreversible receptor-ligand complex formation can
occur. For example, in a conventional homogeneous
immunoassay, if the complex between antibody and
ligand labeled with enzyme is allowed to remain in
solution overnight, the enzyme is gradually denatured.
Evidently molecular accommodation of the antibody-
ligand complex generally occurs in such a way as to
denature the enzyme rather than producing only
reversible inhibition.
The present invention overcomes these
limitations. Continuing with the example of an enzyme
label, in the release system, incubating a complex in
the presence of high salt to prevent formation of an
unreleaseable receptor-ligand complex, in which ligand '
or receptor is labeled with enzyme, preserves the
enzyme activity as well as the ability to dissociate
the receptor-ligand complex. When ligand is labeled
with enzyme, using a molecular linker helps position
the receptor such that formation of the complex does
not denature the label, e.g., enzyme, for at least
several days.
S.l.I.
One element of the receptor-ligand complex
is a receptor having one or more binding sites capable
of specifically binding to analyte, in which the
association constant of binding is high. The receptor
is also capable of binding to ligand with an
association constant of binding relatively low
compared to that of receptor binding to analyte.
Suitable receptors for use in release assays of the
invention include nucleic acid molecules (RNA or DNA),
antibodies (or a fragment of the antibody that
contains the analyte binding site for analyte and
WO 93/03367 PCT/US92/06249
- 15 -
2~i~~~~
ligand), cell surface receptors (or a fragment of a
cell surface receptor that contains the binding site
for analyte and ligand), enzymes (or the substrate
binding site of an enzyme), lectins or any other
molecule or macromolecule capable of specifically
binding to and forming a stable low affinity complex
with a ligand and a stable high affinity complex with
an analyte. Antibodies and cell surface receptors are
preferred, with antibodies more preferred. zn a
l0 preferred embodiment, receptor is generated or
selected to be specific for the most unique epitope on
the analyte.
Various procedures known in the art may be
used for the production of antibodies to analytes of
i5 interest. Such antibodies include but are not limited
to polyclonal, monoclonal, chimeric, single chain, Fab
fragments and an Fab expression library. For the
production of antibodies, various host animals may be
immunized by injection with a particular analyte or
20 analyte conjugated to an immunogenic carrier,
including but not limited to rabbits, mice, rats, etc.
Various adjuvants may be used to increase the
immunological response, depending on the host species,
including but not limited to Freund's (complete and
25 incomplete), mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions,
keyhole limpet hemocyanin, dinitrophenol, and
potentially useful human adjuvants such as BCG
30 (bacilli Calmette-Guerin) and Corynebacterium parvum.
Monoclonal antibodies to analytes may be
prepared by using any technique which provides for the
production of antibody molecules by continuous cell
lines in culture or in vivo. These include but are
35 not limited to the hybridoma technique originally
WO 93/03367 PCT/US92/t16249
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~~ ~~~~~ r
described by Kohler and Milstein, (Nature, 1975,
256:495-497), the more recent human B-cell hybridoma
technique (Kosbor et al., 1983, Immunology Today,
4:72) and the EBV-hybridoma technique (Cole et al.,
1985, Monoclonal Antibodies and Cancer Therapy, Alan
R. Liss, Inc., pp. 77-96). In~an additional
embodiment of the invention monoclonal antibodies
specific to analytes may be produced in germ-free
animals utilizing recent technology (PCT/US90/02545).
l0 According to the invention, human antibodies may be
used and can be obtained by using human hybridomas
(Cote at al., 1983, Proc. Natl. Acad. Sci., 80:2026-
2030) or by transforming human 8 cells with EBV virus
in vitro (Cole et al., 1985, in, Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, pp. 77-96). In
fact, according to the invention, techniques developed
for the production of "chimeric antibodies" (Morrison
et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855;
Neuberger et al., 1984, Nature, 312:604-608; Takeda et
al., 1985, Nature, 3i~:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen
specificity together with genes from .a human antibody
molecule of appropriate biological activity can be
used; such antibodies are within the scope of this
invention.
According to the invention, techniques
described for the production of single chain
antibodies (U.S. Patent 4,946,778) can be adapted to
produce analyte-specific single chain antibodies. An
additional embodiment of the invention utilizes the
techniques described for the construction of Fab
expression libraries (Ruse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification
of monoclonal Fab fragments with the desired
specificity to analytes.
WO 93/03367 PCT/US92/O6Z49
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v '' / I! ~ va
z~~~~.~~
Antibody fragments which contain sites
specific for analytes may be generated by known
techniques. For example, such fragments include but
are not limited to: the F(ab')Z fragments which can be
produced by pepsin digestion of the antibody molecule
and the Fab fragments which can be generated by
reducing the disulfide bridges of the F(ab')2
fragments.
Alternatively, polyclonal or monoclonal
antibody specific for an analyte of interest may be
obtained from commercial sources.
Receptor for binding analyte may be
_ purified, e.g., by affinity chromatography or low
affinity chromatography. Monoclonal antibody may also
be purified by protein A or anti-Ig chromatography. '
Techniques for purifying polyclonal and monoclonal
antibodies are well known in the art. A heterogeneous
receptor preparation, such as polyclonal antibody, may
also be absorbed with a low concentration (e.g., 1% of
the receptor concentration) of ligand to remove any
receptors capable of binding ligand with high
affinity.
5.1.2. IL GAN_D_
As used herein, the term "ligand" includes
molecules with limited cross-reactivity with analyte
for receptor. binding. The term "reland" is used
herein interchangably with ligand. Reland is a term
coined by the co-inventors hereof to refer to a
release ligand. The ligand or reland binds receptor
with a low association constant relative to analyte,
e.g., preferably less than about 10%, more preferabl~r
less than about 1%, and does not affect the stability
of an analyte-receptor complex. Ligand comprises an
analog of the analyte, including an epitope of the
WO 93/03367 ,~ ~~~~~ PCT/US92/06249
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analyte, a derivative of the analyte, a modified
analyte, or an isomer of the analyte. Preferably the
ligand differs from analyte at or near the receptor
binding epitope. These differences may include
stearic, configurational, conformational or ionic
changes. In another embodiment in which the receptor
is a nucleic acid, ligand is a nucleic acid
complementary to the "receptor", provided that the
degree of complementarity is not as high as the
complementarity of the receptor and the nucleic acid
analyte.
Analogs of analyte include the analogous
molecule from a related species of animal, where such
exists, e.g., sheep luteinizing hormone is an analog
of human chorionic gonadotropin (see Section 16., '
,'n~ fray. Molecules prepared to structurally mimic the
analyte are also analogs for use as ligands. Such
structural mimics may be, but need not be, of the same
chemical nature as the analyte so long as the epitope
is chemically similar. Thus, for example, a peptide
may be an analog of a protein. A suitable analog will
simply share a receptor binding epitope or part of an
epitope of the analyte.
Derivatives of analyte may be prepared by
adding or deleting functional groups to the molecule.
Derivatives may also be natural metabolic products of
the analyte. One of ordinary skill will readily know
how to prepare or identify derivatives of analytes for
use in the invention. Preferably changes in molecular
structure of the analyte will alter the receptor
binding epitope composition or conformation in order
to decrease the binding affinity of receptor for
ligand.
Modified analyte includes analyte conjugated
to a carrier protein. Addition of bulky groups or
WO 93/03367 PCT/US92/06249
- 19 -
ionic groups, such as aliphatic, aromatic or cyclic
molecules, to the analyte or to the protein can result
in decreased binding affinity for receptor due to
stearic and/or charge interference. Alternatively,
. 5 conjugation with a bulky group may cause a
conformational change in the receptor binding site of
ligand that decreases binding affinity. Chemical
modifications of organic molecules are well known in
the art and may be used to modify ligand. In a
specific Example, 'n a, an analyte, norcotinine,
modified by the presence of N-isopropyl or N-propyl
groups is a preferred ligand in an assay for cotinine.
Isomers of analytes are molecules with the
same composition but different configuration.
Typically, isomers will have a different configuration '
at a particular carbon center, e.g., cis versus traps,
D versus L. Isomers include diastereomers, which have
the opposite configuration at one or more chiral
centers, and include enantiomers of analytes. Since
. 20 biological binding interactions depend on configura-
tion as well as conformation and composition, use of
an isomer as ligand can result in much lower binding
affinity for receptor.
Where the analyte is a protein, the ligand
may be prepared by recombinant DNA methods using site
directed or other mutagenesis techniques to alter the
amino acid sequence of the protein. These techniques
are well known to those skilled in the art (e.g., see
Zoller and Smith, 1984, DNA 3:479-488; Oliphant et
al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc,
Nat~l. Acad. Sci. U.S.A. 83:710). Polymerase chain
reaction (PCR) techniques are preferred for site
directed mutagenesis (see Higuchi, 1989, "Using PCR to
Engineer DNA," in PCR Technology: Principles and
WO 93/03367 PCT/US92/06249
a, ~. ~=~~ 2 0 -
:. _
Applications for DNA Amplification, H. Erlich, ed.
Stockton Press, Chapter 6, pp. 61-70).
The ligand may further comprise a carrier
protein. The carrier protein may be an essentially
inert protein such as albumin or keyhole limpet
hemocyanin, but a carrier protein should not be the
same carrier. used, if any, for immunization to produce
antibody to the analyte. An enzyme label may function
as a carrier protein.
Ligand may also comprise a linking group. A
linking group may space Iigand from an enzyme label,
thereby preventing or slowing deactivation of the
enzyme after a long incubation with receptor. An
example of a spacer linking group is E-aminocaproic
acid. Also, bulky linking groups may be used to
sterically hinder binding of receptor to ligand. An
example of a bulky linking group is p-aminobenzoic
acid.
Ligands may be evaluated to show their
suitability for use in a release assay. Ligand
evaluation assays include competition assays, binding
enhancement assays, direct assays, low affinity
chromatography, and microtiter plate release assays,
as well as the contemplated release assay itself.
Competitive assays useful for evaluating
potential ligands can be run in an ELISA format, but
' any competitive assay technique may be used. As shown
in Example 16.,, 'infra, a ligand is much less effective
than analyte for inhibiting binding of receptor to
solid phase analyte. A molecule that is a poor
competitive inhibitor relative to analyte can be a
good choice for use as a ligand.
A low concentration of a suitable release
ligand may actually appear to enhance binding of
receptor to analyte in a competition assay. Thus in a
WO 93/03367 PCTlUS92/06249
- 21 -
.~. ~~ ~ p r.~
competition type assay with polyclonal antibody,
increased absolute signal in the presence of the
molecule being tested indicates that molecule may be a
suitable release ligand.
When a direct assay is used to evaluate a
potential ligand, binding of receptor and ligand is
compared to binding of receptor and analyte. An ELISA
format is well suited for this type of assay, but
other assay formats can also be used. A ligand will
i0 demonstrate no more than about 10%, and preferably
less than about 1%, of the binding activity of
analyte. Typically, in an ELISA, binding activity is
represented by titer, i.e., dilution or concentration
of receptor with a particular binding activity.
Alternatively, specific activity at the same receptor '
concentration can be compared.
If an analyte-specific receptor elutes under
mild conditions from an affinity chromatography column
prepared with ligand, that ligand may be a useful for
a release assay.
In the microtiter plate method, ligand or
receptor is coated on the plate. When the microtiter
plate is coated with ligand, the ligand will
preferably comprise a protein to enhance binding to
the plate. The complementary binding partner
(receptor or ligand, respectively is added and a
complex allowed to form. If the receptor-ligand pair
is suitable, release of receptor or ligand can be
detected in a sample supernatant when sample
containing analyte is added. As demonstrated in
Example 11., infra, a ligand-enzyme conjugate designed
for use in a homogeneous assay can be tested in a
microtiter plate assay.
WO 93/03367 PGT/US92/06249
n'i; -22-
,n ,~ a ~.'y a
01 ~, ~~,~1~ 1. ~'
5.2. RELEASE ASSAY FORMAT
.The release assay comprising receptor,
ligand and a means for detection of released, i.e.,
dissociated, receptor-ligand complex, has particular
utility for analyzing a sample for the presence of a
small molecule such as a drug metabolite. Detecting
the presence of drugs or drug metabolites in a sample
of body fluid from a patient is important for many
applications. The presence of drugs or drug
l0 metabolites is useful in determining appropriate
medical treatment, especially under emergency
conditions. Detection of drugs or drug metabolites in
a person is important in law enforcement, employment,
schools and athletics. Samples may be from any
source, but preferably are from an animal, and more
preferably from a human. Samples may include but are
not limited to be body fluid such as blood, plasma,
serum, urine, saliva, bodily exudate, etc.
Analytes may be any antigen, but small
analytes (MW of 100 to 1000 Daltons) are of primary
interest. Such analytes include therapeutic drugs and
metabolites thereof, illicit drugs and metabolites
thereof, steroids, and peptide hormones.
Nevertheless, release assays for larger molecules such
as protein hormones, e.g., insulin, viral antigens,
bacterial antigens, serum proteins, antibodies or any
antigen of interest where detection of presence (or
absence) of the analyte in a rapid, specific,
sensitive assay is desirable are also contemplated.
rn specific Examples '_~nfra,, release assays
for cotinine (a nicotine metabolite associated with
smoking), benzoylecgonine (a cocaine metabolite),
tetrahydrocannabinol (the narcotic agent of
marijuana), thiazides (a class of diuretics), and
beta-~blockers are shown. Moreover, it will be
WO 93/03367 PGT/US92/06249
''
_ 23 _
"l,
apparent that release assays are suitable to detect
any analyte of interest.
For example, a release assay may be prepared
for HIV antibodies. A format for a release HIV assay
follows:
1. Generate antibodies to an HIV peptide
modified in such a way as to make it a release ligand,
e.g., by substitution of amino acids. This antibody
is then analogous to a ligand for purposes of the
assay since it is altered relative to the analyte
(anti-HIV antibody) of interest.
2. Conjugate the native HIV peptide
sequence to a protein such as albumin, and coat the
conjugate on plastic beads or wells of microtiter
plates. The peptide-protein conjugate acts as the
receptor for analyte for purposes of this assay.
3. Mix the receptor (conjugate) and the
ligand (antibody) so that complex is formed. The
ligand should be labeled with a marker e.g., a
fluorescent tag. Wash the plate after complex forms.
4. Add sample containing analyte (anti=YIV
antibodies) to the solid phase. The anti-HIV
antibodies displace the labeled antibody. Measure the
amount of released label.
Preferably the ligand antibody for an HIV
assay is a monoclonal antibody.
The present invention provides for
positively detecting dissociation of a receptor-ligand
complex resulting in release of the labeled component.
Positive detection means that signal positively
correlates with the amount of analyte in a sample. A
positive correlation is advantageous because it is
psychologically satisfying that presence of signal or
increase in signal intensity indicates presence of
analyte in a sample. Results obtained by assay
WO 93/03367 PCT/US92/06249
_ 24 _
:'
methods in which detection of signal decreases when
analyte is present are susceptible to
misinterpretation. It is desirable to employ an
assay, such as that of the present invention, in which
only a positive result generates signal. Assays of
the invention are equally suitable for use both in a
laboratory by technical personnel as well as outside a
laboratory by both technical and non-technical
personnel.
Accordingly, the invention provides for
detecting release of a component of a receptor-ligand
complex. In a heterogeneous release assay (Section
5.2.2, in ra), either receptor or ligand may be
labeled so that upon release in the presence of
analyte, the label signal is separated from reaction
products and is detected. In a homogeneous release
assay (Section 5.2.1, infra), it is preferable to
label ligand, but under certain circumstances, when
using a nonenzymatic label, receptor can be labeled.
For example, when a fluorescent label is used, ligand
may contain a fluorescence quencher. In the receptor-
,F
ligand complex, the fluorescent signal is quenched,
and there is no detection of fluorescence until
dissociation occurs in the presence of analyte.
Suitable labels include enzymes, fluorophores,
chromophores, latex particles, colloidal gold, dyes
and chemiluminescent agents.
The means for detecting dissociation of a
receptor-ligand complex depends in part on whether the
assay;is homogeneous or heterogeneous, as described,
below in sections 5.2.1. and 5.2.2.
Once suitable binding receptor, release
ligand, and means for detecting a particular analyte
are chosen, the assay system must be optimized for use
with a particular sample matrix. A urine sample will
WO 93/03367 PCT/US92/06249
25 2~~ ~~~:t~
have different intrinsic characteristics than a sample
in aqueous buffer. The same is true for sample from
saliva, blood, plasma or serum, or any body fluid.
The assay may be optimized by varying reagent
concentration, buffer composition, release time,
detection time, baseline controls, and other
variables. These variables are well known in the art,
and it will be readily understood how to adjust them
for optimum assay specificity and sensitivity with a
i0 particular assay matrix.
5.2.1. HOMOGENEOUS RELEASE ASSAYS
_ The release assay may be performed in
homogeneous liquid phase. Such an assay is preferred
because it can be performed in a single reaction
vessel, and thus is well suited for use in automated
analyzers.
In one embodiment, the ligand may be
conjugated to an enzyme label that retains a
detectable level of enzyme activity. A ligand-enzyme
conjugate is selected such that upon binding of
receptor to ligand, enzyme activity decreases. To
increase sensitivity, preferably one receptor binding
site is present on the enzyme, but more than one is
also acceptable.
When analyte is added to the receptor-ligand
complex, the complex dissociates into its receptor and
ligand components and released receptor binds analyte.
Upon release of the ligand-enzyme conjugate, enzyme
catalytic activity increases. This increase is
detected by measuring the rate of product formation.
Any enzyme-substrate system can be used, with the
proviso that no endogenous enzyme present in sample
will artificially increase the rate of product
formation. Preferably the enzyme is glucose-6-
WO 93/03367 ' ''~ ~1~.~ hr;;y PCT/US92/06249
.~- _ 26 _
phosphate dehydrogenase, and the reaction product is
reduced nicotine-adenine dinucleotide (NADH), which
can be detected by absorbance at 340 nm.
Alternatively, a receptor-ligand complex in
a homogeneous release assay may comprise a fluorescent
label or chemiluminescent label attached to ligand and
a fluorescence quencher attached to the receptor. The
receptor may itself quench fluorescence. In the
complex, the fluorescence or luminescence will be
quenched and no signal will be observed. However,
upon dissociation of the receptor-ligand complex and
release of receptor and ligand in the presence of
analyte, quenching will diminish, and signal will be
observed. Other proximity dependent signal
attenuators, such as fluorescence polarization, are '
known in the art, and can be adapted for use in a
release assay. It will further be appreciated that
the label may be on receptor and the quencher on
ligand.
5.2.2. HETEROGENEOUS REL3ASE ASSAYS
In another embodiment, a heterogeneous
solid-phase/liquid-phase release assay is provided.
In such an assay, either receptor or ligand is
irreversibly absorbed to a solid phase support. As
used herein, the term "irreversibly absorbed" includes
covalent, non-covalent and ionic association. Solid
phase supports include plastic, polymer beads, glass
beads, glass, silica gel, and membranes. In Examples
3o infra solid phase supports are plastic microtiter
plate wells and nitrocellulose membranes. However,
the release assay is not limited to a particular
choice of solid phase support and any solid phase
support known in the art may be used.
WO 93/03367 '~ ~ ~ ~ ~ ~ ~ PGT/US92/06249
- 27 -
The binding partner of the receptor or
ligand absorbed to the solid phase support, i.e., the
ligand or receptor respectively, is labeled, and a
stable complex comprising the labeled element and the
solid phase element is formed. Once a stable
receptor-ligand complex is formed, it can be exposed
to sample. If the analyte of interest is present in
the sample, the release reaction occurs and signal
from the label is detected in the liquid phase. The
extent of release, and thus the signal intensity in
the liquid phase, positively correlates with the
amount of analyte in the sample. The signal intensity
in the solid phase decreases inversely with the amount
of analyte in the sample (see Table 6).
Many labels can be used in the heterogeneous
release assay. Enzyme labels are practical, even for
a single vessel assay, because enzymes bound to a
solid phase can have 10-to 20-fold less catalytic
activity than the same enzyme in solution. Moreover,
as with homogeneous release, binding of receptor to
ligand-enzyme conjugate may reduce enzyme activity.
Other labels such as chromophores, fluorophores,
chemiluminescent agents, radioisotopes, chelating
complexes, dyes, colloidal gold and latex particles
can be detected in the liquid phase after release
reaction as increased optical density, fluorescence,
luminescence, radioactivity, color (for dyes), and
turbidity (for colloidal gold and latex particles),
respectively. Where the signal from label that
remains bound in the receptor-ligand complex cannot be
detected, the assay may be performed in a single
vessel.
In a particular embodiment preferred for
non-laboratory settings, the presence of an analyte is
indicated by the appearance of a shape, i.e., a
WO 93/03367 ~ , ~ ~~ ~ ~'p'~s PCTlUS92/06249
- 28 -
letter, in a reaction field on a solid phase support.
Accordingly, a reaction field comprising an indicator
zone and a control zone is prepared on a solid phase
support. The indicator zone comprises either
immobilized receptor or ligand, as provided by the
heterogeneous assay format. A receptor-ligand complex
in the indicator zone is sensitive to the release
reaction. The control zone comprises a different
receptor-ligand complex. The receptor-ligand complex
in the control zone is not susceptible to the specific
release reaction, but will indicate non-specific
release if conditions are such as to cause non-
_ specific release.
In practice, contacting sample containing
the analyte of interest to the reaction field will
result in a detectable release reaction in the
indicator zone, and no reaction in the control zone.
The release reaction is detected as formation of a
contrasting zone corresponding to the indicator zone.
To accomplish this, label for both the release complex
and the control complex is chosen to contrast with the
solid support.
If there is no development of a contrast
zone, the sample is negative. "Fade" of both the
indicator zone and control zone, i.e., release of
label from both complexes, indicates a false positive
reaction, inappropriate reaction conditions, and
possible adulteration of the sample. In this way, the
control zone provides a control for accurate assay
results.
Preferably different letters or symbols are
used as the indicator depending on the analyte of
interest. For example, indicator zone specific fox
cocaine use may be shaped like the letter "C"; an
indicator zone for marijuana use shaped like the
WO 93103367 PCT/US92/06249
29 -
letter "M" (or "T" for tetrahydrocannabinol), and a
zone to indicate nicotine use shaped like the letter
nNa
The control zone comprising a complex
immobilized control receptor or ligand and labeled
ligand or receptor, respectively, is analogous to the
indicator zone, with the proviso that the control
complex is insensitive to the presence of analyte,
while the complex in the indicator zone is released in
l0 the presence of analyte. For example, if the analyte
of interest is cotinine, the immobilized ligand is
cotinine coupled to bovine gamma globulin via an
aminocaproic acid linker, and the receptor is anti-
carboxy-cotinine labeled with blue latex, a suitable
control ligand is immobilized trinitrophenol (TNP)
conjugated to bovine serum albumin, and a suitable
control receptor is anti-TNP antibody, also labeled
with blue latex. In the presence of cotinine, labeled
anti-carboxycotinine would be released and a
contrasting zone develop. The TNP-anti-TNP complex is
unaffected by cotinine.
It is clear that other receptor ligand
combinations will work equally well as control
complexes. For example, in an assay to detect
cotinine, a control complex could be immobilized
ecgonine-anti-benzoylecgonine. It is further
envisioned that a single solid phase support can
contain more than one detection field, since each
detection field is specific for a particular analyte
and insensitive for any other analyte. Thus, the
invention provides an assay for multiple analytes,
e.g., tetrahydrocannabinol, benzoylecgonine, and
cotinine, in a single format.
Suitable labels for use in this assay
include but are not limited to colored latex
WO 93/03367 t , ~ ;'~,~ PGT/US92/06249
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- 30 -
particles, dyes, colloidal gold, and enzymes that
catalyze production of an insoluble colored reaction
product when a white or light colored solid phase
support is used, and white latex when a dark solid
phase support is used. Preferably, colored latex
particles are used as labels. Also, any solid phase
support can be used in this embodiment, but plastic
and membranes are preferred.
In a preferred embodiment, such as described
in Section 15., 'ni fro, the solid phase support
comprising receptor-ligand complex and control complex
is provided dry.
The invention will be further clarified by
the following Examples, which are intended to be
purely exemplary of the invention and not as
limitations of the invention. Indeed, various
modifications of the invention in addition to those
shown and described herein will become apparent to
those skilled in the art from the description and
accompanying drawings. Such modifications are
intended to fall within the scope of the appended
claims.
6. EXAMPLE: SOLID PHASE RELEASE ASSAY FOR COTININE
Cotinine and traps-3'-hydroxycotinine are
the major metabolites of nicotine (Langone et al.,
1973, BiOChem. 12:5025-30; Jacob et al., 1991, J.
Chromatography 222:61-70; Neurath et al., 1987, Int.
3~ Arch. Occup. Environ. Flealth 59:199-201). They appear
in urine in a 1:3 ratio (Newrath et al., s_upra_,). The
detection of cotinine in urine, serum or saliva is the
most commonly used biochemical method to determine
levels of exposure to nicotine (Fitzpatrick, 1991,
C.N.N. 11). Unlike other drugs of abuse, cotinine is
WO 93/03367 PCT/US92/06249
2
J
found in bodily fluids of non users due to passive
smoking. The range of interest for a cotinine assay
is from 0.010 ~Cg/ml necessary for saliva and blood
testing, to <l0~cg/ml for urine of tobacco users
. 5 (Greenberg et al., 1984, N. Engl. J. Med. 310:1075-78;
Matsukura et al., 1984, N. Engl. J.Med. 311:828-31;
Sepkovicet al., 1985, Am. J. Public Health 75:663-6;
Sepkovic et al., 1986, J.A.M.A. 256:863; Jarvis et
al., 1987, Am. J. Public Health 77:1435-8; Schepers
i0 and Walk, 1988, Arch. Toxicol. 62:395-7; Langone et
al., 1988, J.I.M. 114:73-8).
This example reports the use of three
_ different cotinine ligands in a heterogeneous solid
phase release assay. One ligand comprises a cotinine
15 metabolite directly bound to an inert carrier protein
(hydroxy-cotinine-BGG), another ligand comprises the
cotinine metabolite conjugated to the protein via a
bulky linker (hydroxycotinine-aminobenzyl-BGG) and a
third comprises the cotinine metabolite conjugated to
20 the protein via a spacer linker (hydroxycotinine-
aminocaproyl-BGG).
6.1. MATERIALS AND METHODS
Trans-3'-hydroxycotinine was obtained
25 according to a published method (Jacob et al., 1990,
J. Med. Chem. 33:1888). Succinic anhydride and
dimethyl-formamide (DMF) were obtained from Aldrich
Chemical Co. Pyridine, N-hydroxysuccinimide, 1-ethyl-
3-(3-dimethylaminopropyl) carbodiiumide (EDC), p-
30 aminobenzoic acid (PABA), E-aminocaproic acid (ACA);
and bovine gamma globulin (BGG) were obtained from
Sigma.
WO 93/03367 PCT/US92/06249
°. f~1 ~r,'u - 3 2 -
6.1.1. PREPARATION OF HYDROXYCOTININE-
BGG ~IGAND
Step A. Hydroxycotinine hemisuccinate was
prepared as follows. In a glass tube, 19 mg of trans-
3'-hydroxycotinine were dissolved in 1 ml of DMF. In
this solution 21 mg of succinic anhydride were
dissolved. After adding 20 ~cl of pyridine, the tube
was covered (PARAFILM~) and incubated overnight at
37°C.
i0 Step B. After equilibration at room.
temperature (r.t.), 14 mg of N-hydroxysuccinimide and
23 mg of EDC were dissolved in the cotinine solution.
The solution was covered (parafilm) and incubated 3
hours at room temperature to activate the cotinine
hemisuccinate. '
Step C. Activated cotinine hemisuccinate
(400 ~1 of the solution from step B.) was added to the
solution of 6 mg BGG dissolved in 1 ml of PBS. The
mixture was gently mixed and incubated 1 hour at room
temperature, then overnight at 4°C. The resulting
conjugate was dialyzed against 6 changes of PBS for 48
hours.
6.1.2. PREPARATION OF HYDROXYCOTININE-~
AMINOBENZOYL-BGG LIGAND
Step D. In 0.6 ml of PBS were dissolved 6
' mg of PABA. To this solution were added 400 ~C1 of
activated cotinine hemisuccinate prepared according to
step B., supra. The resulting solution was mixed well
and.incubated for,l hour at room temperature and
overnight at 4°C.
Step E. The hydroxycotinine-hemisuccinate-
PABA was brought to room temperature and activated by
addition of 0.5 ml of 14 mg/ml N-hydroxysuccinimide in
DMF and 0.5 ml of 24 mg/ml EDC in DMF. The mixture
was incubated 3 hours at room temperature. To 6 mg of
WO 93/03367 PCT/US92/06Z49
33 _
BGG in 2 ml of PBS were added 400 ~1 of activated
cotinine-PABA. The resulting solution was mixed
gently and incubated 1 hour at room temperature and
overnight at 4°C. The resulting conjugate was
dialyzed against 6 changes of PBS over 48 hours.
6.1.3. PREPARATION OF HYDROXYCOTININE
AMINOCAPROYL-BGG LIGAND
Step F. To a solution of 4 mg ACA in 0.6
ml of PBS were added 400 ~1 of the activated cotinine
succinate solution prepared according to step B.,
,us pra. The resulting solution was mixed very well and
incubated 1 hour at room temperature and overnight at
4°C.
Step G. The hydroxycotininehemisuccinate- ,
ACA solution was brought to room temperature and
activated by addition of 0.5 ml of 14 mg/ml N-
hydroxysuccinimide in DMF and 0:5 ml of 24 mg/ml EDC
in D~IF. The resulting mixture was incubated for 3
hours at room temperature. To 6 mg of BGG in 2 ml of
PBS were added 400 girl of activated cotinine-ACA. The
resulting solution was mixed gently and incubated at
room temperature for 1 hour, then overnight at 4°C.
The resulting conjugate-was dialyzed against six
2S changes of PBS over 48 hours.
' 6.1.4. ANTISERUM BINDING REAGENT
Antiserum to carboxycotinine was obtained as
follows: 320 mg of keyhole limpet hemocyanin (KLH)
Protein (Sigma) were dissolved in 40 ml of deionized
water. To this were added 300 mg of
w trans-4'-carboxycotinine (Aldrich) with mixing until
it was dissolved. Then 300 mg of EDC were added to
the reaction mixture with stirring, which was
continued overnight at room temperature. The KLH-
carboxycotinine conjugate was dialyzed for 8 hours at
WO 93/03367 ~,, ~~ PGT/US92/06249
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- 34 -
2-8° against phosphate buffered saline. The dialysis
fluid was changed once after 4 hours.
Rabbits were immunized with the immunogen in
Freund's adjuvant, with multiple injections over
several months according to standard protocols. Test
bleedings were made at defined intervals, and
increases in antibody titer measured using an enzyme
immunoassay for cotinine. Measurements of antibody
affinity and cross-reactivity were also performed.
When these assays indicated satisfactory antibody
performance, rabbits were bled and sera were isolated
and pooled. Antiserum was stored at -40°C.
The IgG fraction was separated from serum by
ammonium sulfate precipitation. An immunoaffinity
chromatography column was prepared by coupling '
succinylated hydroxycotinine through its carboxyl
group to amino-sepharose 48 (see section 9., in a).
The affinity purified antibody was labeled with
horseradish peroxidase using the sodium-m-periodate
method.
Antisera prepared against 4'-
carboxycotinine, which is conjugated to carrier
protein at the 4' position, were expected to bind
cotinine conjugated to protein at the 3' position with
Z5 lower offinity.
6.1.5. ASSAY
Microtiter plates were coated overnight with
100 ~cl of each ligand at 1 ~cg protein per ml of PBS.
Dried;plates were incubated for 1 hr with 100 ~cl of;
enzyme-labeled antibody. Excess antibody was washed
out and 90 ~1 of distilled water and 10 ~cl of urine
sample or standard were added to each well. After
2 minutes of shaking, the supernatant was transferred
to uncoated wells. Released label in the supernatant
WO 93/03367 PCT/US92/06249
was quantitated after reaction with TMB substrate for
15 minutes by measurement of O.D. at 450 nm.
6.2. RESULTS
Release of labeled antibody complexed to
solid phase ligand was detected in supernatant when
free cotinine was present in sample. A comparison of
release from solid phase hydroxycotinine-PABA-BGG and
solid phase hydroxycotinine-ACA-BGG indicates that
after 2 minutes of exposure free cotinine induces the
release of anti-cotinine antibody bound to both
antigens in a concentration dependent manner (Figure
_ 1). The results indicate that release from
' hydroxycotinine-ACA-BGG is slightly more efficient.
Enhanced release reactions were observed for '
release when the release portion of the assay is
extended to 10 minutes (Figure 2). Increases in A,m
could be observed at as low as 0.5 ;cg/ml cotinine.
Again, release from hydroxycotinine-ACA-BGG was more
efficient. A comparison of release after 2 minutes of
incubation with sample in urine and synthetic matrices
(Figure 3) indicates that (i) assay sensitivity is
greater in synthetic matrix than in urine matrix; and
(ii) that hydroxycotinine-ACA-BGG acts more
efficiently for release of anti-cotinine antibody.
6.3. DISCUSSION
These results show that a release assay
indicates the presence of free cotinine in a sample of
urine.or an artificial urine matrix. As$ay plates
prepared by coating the microwells with cotinine
ligand conjugated at the 3' position to a protein and
forming a complex with antibody specific for cotinine
conjugated to the immunogenic carrier protein at the
~4' position, the detection of free cotinine in sample
WO 93/03367 r p E' PGT/US92/06249
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can be completed using this assay in under 30 minutes.
Comparison of Figures 2 and 3 shows that release can
be observed as soon as 2 minutes after sample is
added.
Subtle differences in binding affinity
affect the sensitivity of a release assay. Release
from the hydroxycotinine-aminocaproic acid-bovine
gamma globulin ligand gave slightly more sensitive
readings than release from hydroxycotinine-
aminobenzoyl-BGG. These results demonstrate how
exploitation of differences in binding affinity allows
development of sensitive immunoassays.
7. EXAMPLE: MICROTITER PLATE METHOD
OF ASSAYING LIGAND-PROTEIN CONJUGATES ,
A solid phase assay provides a method to
compare ligand-protein conjugate preparations for use
in release assays, and to determine whether a
particular.ligand binds analyte-specific antibody with
sufficiently low affinity. The. present example
reports results with a cotinine ligand.
7.1. MATERIALS AND METHODS
Reagents, hydroxycotinine-p-amino-benzoic
acid-bovine gamma globulin (BGG) and hydroxycotinine-
e-aminoncaproic acid-BGG were prepared as described in
SeCtl.On 6.1., Sugra.
7.1.1. ASSAY PROTOCOL
Microtiter plates were coated with cotinine-
ligand as described in Section 6.1.5., supra. The
wells were washed twice with wash buffer and 100 ul of
anti-cotinine-peroxidase conjugate (Section 6.1.4.,
supra) were added to each well. The plate was
incubated 60 min at r.t. and washed twice with wash
WO 93/03367 PGT/US92/OE249
buffer. To each well were added 100 ~1 of wash buffer
and 10 ~l of sample. Samples consisted of 0, 0.5, 2.0
and 10 ug/ml cotinine in urine, and two negative and
two positive urine samples. The plate was incubated
10 minutes at r.t. with shaking, and 100 ~C1 of
supernatant were removed and transferred to replicate
wells of on uncoated microtiter plate. To the wells
containing supernatant were added 100 ~1 of 2x TMB
substrate solution. The plate was incubated 3 min at
l0 r.t., and then the reaction stopped by addition.of 50
~1 2N sulfuric acid to each well. Absorbance at 450
nm was measured:
7.2. RESULTS
Presence of cotinine in sample caused
release of labeled anti-cotinine antibody from the
cotinine-ligand solid phase. The A,SO value of
supernatant correlated positively with the amount of
free cotinine present in cotinine-spiked urine
samples. Furthermore, the re:-.ults with negative and
positive urine samples demonstrate the usefulness of
the assay to detect cotinine in urine. The results
are summarized in Table 1.
30
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a. 3 8 _
TABLE 1. COTININE-INDUCED RELEASE FROM
COTININE-BGG-CONJUGATES.
A..S,o
COTININE CONC
COTININE- COTININE-
( ~Cg / m 1 ) PABA-BGG ACA-BGG
0 0.63? 0.589
0.5 0.658 0.721
2'0 0.946 0.986
10.0 2.247 2.251
NEG. URINE 1 0.515 0.585
NEG. URINE 2 0.368 0.420
POS. URINE 1 1.161 1.269
POS. URINE 2 1.077 1.198
7.3 DISCUSSION
These results show that a microtiter plate
assay provides a convenient support for demonstrating
the usefulness of a particular ligand-protein
conjugate for a release assay. In this assay use of a
bulky or spacer linkers in the ligand made little
difference in the results.
8. EXAMPLE: IiOMOGENEOUS RELEASE
ASSAY FOR COTININE
The release system provides improved
sensitivity and specificity over previous assays by
taking advantage of the specificity of dissociation
reactions. Dissociation reactions offer the advantage
that they are less subject to interfering substances.
The present example demonstrates
(1) preparation of cotinine ligands labeled with the
enzyme glucose-6-phosphate dehydrogenase;
(2) inhibition of enzymatic-activity of the enzyme-
cotinine conjugate in the presence of anti-cotinine;
WO 93/03367 PCTlUS92/06249
(3) displacement of anti-cotinine from the enzyme-
cotinine conjugate in the presence of cotinine; (4) an
automated assay for release of antibody from ligand,
particularly after to 22 hrs of complex incubation;
and (5) stabilization of an antibody-ligand complex
for up to 6 days.
8.1. MATERIALS AND METHODS
The following reagents were used: traps-4'-
i~ carboxycotinine, N-hydroxysuccinimide, 1,3-
dicyclocarbodiimide, and dimethylformamide (DMF), all
from Aldrich Chemical Co.; glucose-6-phosphate
dehydrogenase (Beckman, 367 IU/mg protein); glucose-6-
phosphate and beta-nicotinamide adenine dinucleotide
i5 reduced form (NAD), both from Sigma Chemical Co.; and
carbinol [2-(2-ethoxyethoxy)ethanol], from Aldrich
Chemical Co.
Preparation of cis-hydroxycotinine-glucose-
6-phosphate dehydrogenase is described in Section
20 10.1.3., 'n~ fra.
8.1.1. ASSAY PROCEDURE
Assay buffer was 0.05 M Tris-HC1, pH 7.8,
equilibrated at 37°C. To 150 gel of assay buffer were
25 added 10 ~1 of cotinine-enzyme conjugate and 1 ul of
anti-cotinine -L.-antibody (Section 6.1.4., supra), and
the solution incubated 10 minutes at 37°C. To this
solution were added 10 ~1 of sample containing
cotinine. The reaction was started by addition of
30 20 ~cl,of glucose-6-phosphate/NAD, and absorbance at,
340 nm read every 1 minute.
A control reaction in the absence of
antibody was prepared by adding 10 ~1 of enzyme-
cotinine conjugate to 160 gel of assay buffer, and
35 equilibrating at 37°C for 10 min. To start the
CA 02114440 2003-04-10
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reaction, 20 ~.1 of glucose-6-phosphate/NAD were added,
and Ago was measured at intervals of 1 minute.
Cotinine-enzyme conjugate was prepared at
1 mg/ml of protein and diluted 500-fold to a final
concentration of 2.0 ~g/ml. The anti-cotinine
antibody was present in stock solution at an
approximate concentration of 15 mg/ml. The glucose-6-
phosphate-NAD solution was prepared by mixing 3
volumes of 0.11 M glucose-6-phosphate with 2 volumes
of 0.1 M NAD.
8.1.2. AUTOMATED ASSAY PROCEDURE
Reagent A was prepared with an anti-cotinine
anti-serum (1:150 dilution), cotinine-glucose-6-
phosphate dehydrogenase conjugate, and 0.0015% Kathon ~
CG in 0.05 M Tris-HC1, 0.005 M MgCl2, pH 7.8. Reagent
B contained 6.2 mg/ml glucose-6-phosphate and 13.3
mg/ml NAD.
Reagent reservoirs on an EPOS automated
analyzer (Eppendorf) were filled with reagents A and
B. Reagents A (0.2 ml) was incubated for 0, 18 or 22
hrs prior to addition of sample (0.01 ml). Samples,
including standards, were pipetted into the mixture of
reagents A. Reagent H (U.05 ml) was added, and
absorbance at 340" was measured at 0 and 5 min.
8.2.
8.2.1. REDUCTION AND INHIBITION OF
ENZYME ACTIVITY
Enzyme activity of cis-hydroxycotinine-
enzyme conjugate was assayed in the presence and
absence of anti-cotinine antibody. The results are
summarized in Figures 5 and 6.
Conjugation of cotinine to enzyme results in
decreased enzyme activity. Addition of anti-cotinine
antibody to the enzyme-cotinine conjugate further
WO 93/03367 PCT/US92/06249
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reduced (inhibited) enzyme activity (Figure 4).
Inhibition of enzyme activity by antibody binding is
completely reversed by the addition of 10 ~l sample of
4 ~g/ml cotinine (Figure 5).
8.2.2. AUTOMATED ENZYME ASSAY
Samples spiked with known amounts of
cotinine, or which were known to be positive for
cotinine, were assayed 0, 18 and 22 hrs after mixing
the antibody/ligand-enzyme conjugate. The assay was
performed as described in Section 8.1.2. The results
indicate that although long incubation times result in
somewhat decreased absolute rate of change, presence
of cotinine in the sample could be detected at even
the lowest concentration. These results are shown in
Table 2.
TABLE 2: ~UTOMATEO COTININE RELEASE ASSAY.
'
COTININE CONC. OD Rate Change
(mAU per
ml min)
TIME OF PREINCUBATION
OF REAGENT
A PRIOR
TO ADDITION
OF STANDARDS
OR SAMPLES
0 HOURS 18 HOURS 22 HOURS
102:1 101.1 102.4
~'S 116.3 108.5 109.1
2 5 1.0 123.0 111.0 111.3
s 2.p 127.3 114.5 114.8
_ 132.5 118.2 118.2
4.0
g.0 138.0 121.8 121.7
POSITIVE ~'1 131.8 118.5 117.7
3 0 POSITIVE ~2 130.0 117.7 117.9
POSITIVE ~3 110.6 105.? 104.6
NEGATIVE ,~l (T. 103.0 104.7 101.2
D.)
These data indicate that the mixed reagent A
35 remains stable for as long as 22 hours. A statistical
PGT/US92/06249
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,~, r ~.
r 'S . ~~ - -
42
analysis of the estimated amount of cotinine in the
positive and negative urine samples bears out this
conclusion (Table 3).
TABLE 3. STATISTICAL ANALYSIS OF DATA
FROM THEAUTOMATED ASSAY.
REAGENTS A STANDARD ESTIMATED
PRE-INCUBATIONCURVES CONGENTRATION
TIME OF
COTININE
IN
SAMPLES
(Ng/ml)
SLOPE INTERCEP ,1~1 #2 ~'3 T.D.*
ZERO TIME 0.9939 0.00838 3.55 2.80 0.221 0.08
18 HOURS 1.0583 -0.127 4.27 3.63 0.309 0.25
22 HOURS 1.0649 -0.142 3.57 3.72 0.211 0.101
* Negative sample.
Table 3 shows that after 0, 18, or 22 hours
of preincubation, agreement as to the amount of
cotinine present in a sample is reasonably good. For
example, the estimated concentration of cotinine in
positive sample ,~l ranges between 3.6 and 4.3 ~cg/ml.
Similar data are obtained for all four samples. Thus
although the absolute absorbance change is greater
after <1 hours of incubation time, all three curves
provide good data for estimating concentration by
extrapolation from the standard curve (Figure 6).
' 8.2.3. STABILIZATION OF A RECEPTOR-LIGAND
COMPLEX IN HIGH SALT SOLUTION
Antibody-ligand complex was formed in 0.5 M
Tris-HCl, pH 7.9, containing 3 mM magnesium chloride
and O or 5% sodium chloride. The assay was run using
the format described in Section 8.1.1. The
homogeneous release assay for cotinine was tested for
receptor-ligand complex stability fox up to six days.
gesults are shown in Table 4.
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.;
~~ f L~ ~;~!
TABLE 4. RELEASE ASSAY AFTER LONG TERM INCUBATION
OF AN ANTTBODY-LIGAND COMPLEX.
Cotinine conc. Rate of OD Chan a mAu
S~.cqJml) 0% NACL 5% NACL
0 Day 0 12.09 16.6
0.5 13.15 17.63
1 13.43 18.96
2 13.85 18.3
Delta* 1.76 1.7
0 Day 1 7.02 9.67
0.5 7.5 10
1 7.7 11
2 7.8 11
Delta* 0.78 1.33
0 Day 6 7.3 9.1
0.5 7.8 9.3
1 7.6 9.6
2 7.9 10.7
Delta* 0.6 1.6
* Delta is the presence
enzyme of
activity
in
the
2 ~eg/ml cotinine (activity
with in
the
baseline
the abse nce cotinine) subtracted .
of
The data show that 5% sodium chloride
stabilized both enzyme activity and the antibody-
ligand complex in releasable form. Although the
magnitude of the maximum enzyme activity decreased
with time even in the presence of NaCl from 18.3
(day 0), to il (day 1), to 10.7 (day 6}, the enzyme
activity with baseline subtracted remains fairly
constant at 1.7, 1.33 and 1.6 for days 0, 1 and 6,
respectively. The samples incubated in the absence of
NaCl did not remain stable.
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9. EXAMPLE: PREPARATION OF LOW AFFINITY
ANTIBODIESFOR USE IN RELEAS~~ASSAYS
Low affinity chromatography provides a
useful method to purify antibodies or other receptors,
s and may also indicate suitable ligands. In
particular, receptor elution under mild conditions
results in much higher yields than conventional
affinity chromatography, e.g., close to 100%. The
mild conditions preserve antibody from irreversible
denaturation and extend the column life. An affinity
column prepared with a potential ligand indicates that
the ligand can be used in a release assay with that
receptor. This Example demonstrates purification of
anti-cotinine antibodies by a ligand, trans-3'-
hydroxycotinine, affinity column.
9.1. MATERIALS AND METHO~S_
9.1.1. gREPARATION OF IMMUNOABSORBENT
Cotinine hemisuccinate was prepared by
dissolving 19 mg of trans-3'-hydroxycotinine in 1 ml
of dimethylformamide (Aldrich). 21 mg of succinic
anhydride (Aldrich) were added, followed by 15 ~1 of
pyridine (Aldrich). The mixture was incubated
overnight at 45°C.
Z3 Iminodipropylamine-Sepharose~aas prepared as
followed. Cyanogen bromide (CNBr) activated Sepharose
4B (Sigma), 1 gm, was hydrated and washed several
times with 0.1 M carbonate buffer, pH 9.5. Three ml
of 0.1 M 3,3'-iminobispropylamine (Sigma) were added
to 3 ml of CNBr-Sepharose gel and the mixture mixed
overnight at room temperature. The amino Sepharose
was washed with a buffer consisting of 0.1 M sodium
carbonate, pH 8.5, 0.1 M sodium bicarbonate, pH 8.5,
and 0.5 M sodium chloride until no reactivity with
trinitrobenzene-sulfonic acid with free amino groups
was detected.
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Hydroxycotinine hemisuccinate succinic ester
was prepared by adding to 1 ml of cotinine
hemisuccinate 14 mg of N-hydroxysuccinimide (Sigma)
and 23 mg of 1-ethyl-3-(dimethylaminopropyl)
carbodiimide (Sigma) and incubating at room
temperature for 3 hours.
Hydroxycotinine-Sepharose was prepared by
mixing 0.45 ml of hydroxycotinine-succinyl ester with
1 ml of iminodipropylaminosepharose and 0.6 ml of 0.2
M sodium bicarbonate. The mixture was gently shaken
for 3 hours at room temperature and kept at 4°C
overnight.
9.1.2. AFFINITY CHROMATOGRAPHY
A small column was packed with the '
hydroxycotinine-Sepharose gel, and the column washed 3
times with 1 ml of 0.5 M sodium chloride, and then
with phosphate buffered saline (PBS).
A portion of anti-cotinine antiserum
produced by injecting rabbits with carboxycotinine
(section 6.1.4., supra) was diluted 1:1 with PBS, and
the whole mixture (2.1 ml) applied to the column. The
material was allowed to equilibrate for 10 minutes,
and then the column was eluted sequentially with PBS,
0.1 M acetate buffer, pH 4.0, and 0.1 M citrate
buffer, pH 2.3. The column was then washed with 1 M
sodium chloride followed by PBS. Protein
concentration was determined on fractions using Pierce
Coomassie blue reagent, and antibody activity by
determining reactivity on plates coated with cotinine
in an ELISA assay. Orthophenylene-diamine (OPD) and
peroxide were used as indicator and substrate
respectively for peroxidase.
WO 93/03367 PCT/US92/06249
.x.~ -46-
r
i '-,F
9.2. RESULTS
Although most protein eluted in early
fractions (void volume) from the affinity column,
antibody activity was found in later fractions.
Fraction 14 contains antibodies with high affinity for
cotinine. The antibody yield was greater than 80%,
which is very high. Furthermore, the specific
activity of the low affinity purified antibody as
demonstrated by the number of tests performed was
better than with whole sera or the IgG fraction,
thereof (data not shown).
9.3. DISCUSSION
Low affinity chromatography provides a
superior way to obtain low affinity antibodies for use
in release assays with that ligand. Low affinity
antibodies elute from an immuno-affinity
chromatography column earlier, under gentler
conditions, than high affinity antibodies. Less
stringent conditions are sufficient to elute low
affinity antibodies from a solid phase chromatographic
support only if the antibody-ligand interaction is
itself of sufficiently low affinity.
Elution under neutral or mild conditions
provides the additional benefit of reducing or
preventing antibody denaturation.
10. PREPARATION OF LOW AFFINITY ANALOGS OF
COTININE AND THEIR EFFICACY IN A
RELEASE ASSAY
Ligands can be prepared by using analogs or
stereoisomers of analogs of the analyte, thus
introducing a configurational or constitutional
difference that can result in lower affinity binding
to analyte-specific antibody.
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In the present example, anti-cotinine
antibody prepared from rabbits immunized with
carboxycotinine conjugated to KLH (see Section 6.1.4.,
supra) is shown to have useful release properties from
traps-3'-hydroxy-cotinine conjugates of horseradish
peroxidase in the presence of free cotinine. The
optical isomer of traps-3'-hydroxycotinine is cis-3'-
hydroxycotinine, and this is also shown to be a
superior ligand.
l0
10.1. MATERIALS AND METHODS
10.1.1. EXTENSIVE MODIFICATION OF GLUCOSE-6-
PHOSPHATE DEHYDROGENASE WITH TRAMS-3'-
HYDROXYCOTININE TO FORM A LIGAND
Eleven mg of traps-3'-hydroxycotinine were
dissolved in 0.29 ml of dimethylformamide; 12 mg of
succinic anhydride were added to the mixture. When
all solids had dissolved, 9 ~cl of pyridine were added,
and the tube was capped tightly and incubated at 3?°C
overnight. To the hydroxycotinine succinate mixture
were added 7 mg of N-hydroxysuccinimide and 12 mg of
N,N'-dicyclohexylcarbodiimide. This mixture was
incubated for 2 hours at room temperature.
Three tubes were prepared, each containing 1
ml of 0.1 M carbonate buffer, pH 9.0 and 2.8 mg
glucose-6-phosphate dehydrogenase. To the first tube
were added 35 ~cl of the activated trans-
hydroxycotinine over a period of 1 hour, in aliquots
of 5 ~cl at 10 minute intervals. This preparation was
designated as RL~,24. To the second tube were added 70
~l of.activated traps-hydroxycotinine, in the same
regimen as above. This preparation was designated as
RL-25. To the third tube were added 105 ~Cl of
activated traps-hydroxycotinine, in the same regimen
as above, in 15 ~C1 aliquots. This preparation was
designated as RL-26.
WO 93103367 PCT/US92/06249
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~;
'J
~', ~.,c
Fifteen minutes after the last addition of
activated traps-hydroxycotinine, samples were
transferred to dialysis bags and dialyzed at 2-8°C
against 0.05 M Tris buffer, pH 7.8. Dialysis was
continued for approximately 44 hours with several
changes of fluid.
10.1.2. LESS EXTENSIVE MODIFICATION OF GLUCOSE-
6-PHOSPHATE DEHYDROGENASE WITH TRANS-
HYDROX~fCOTININE TO FORM A LIGAND
l0 g,5 mg of traps-3'-hydroxycotinine were
dissolved in 0.25 ml of dimethylformamide. Then 6 mg
of succinic anhydride were added to the mixture and
mixed until dissolved. 7.5 ~cl of pyridine were added,
and the tube was capped tightly and incubated at 37°C
overnight.
To this mixture were added 6 mg of N-
hydroxysuccinimide and 10 mg of N,N'dicyclohexyl-
carbodiimide. The mixture was incubated for 45 min at
room temperature with stirring.
Two tubes w~:re prepared, each containing 1
ml of O.1 M carbonate buffer, pH 9.0, and 2.8 mg
glucose-6-phosphate dehydrogenase. To the first tube
were added 25 gel of the activated trans-
hydroxycotinine over a period of 1 hour, in aliquots
of 5 ~C1 at 10 minute intervals. This preparation was
designated as R_L-29. To the second tube were added 70
~l of activated traps-hydroxycotinine in the same
regimen as above, in 10 ~cl aliquots. This preparation
was designated as ~tL~,30.
~ The preparations were dialyzed overnight in
the cold against 0.05 M Tris buffer, pH ?.8.
10.1.3. A CIS-3'-HYDROXYCOTININE LIGAND
47.5 mg of cis-3'-hydroxycotinine were
35. dissolved in 0.4 ml of pyridine. To this solution
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2
were added 10 mg of succinic anhydride and the mixture
stirred until dissolved. The mixture was incubated
overnight at 37°C and then held at room temperature.
Cis-hydroxycotinine hemisuccinate was
separated from the reaction mixture with preparative
thin layer chromatography on silica gel using a
solvent system composed of benzene: methanol: ammonium
hydroxide, 10:3:0.5. The cis-hydroxycotinine was
visualized with a W lamp. The desired product was
isolated as follows: the area on the chromatogram
containing the cis-hydroxycotinine hemisuccinate was
scraped off and placed in a tube. One ml of methanol.
was added and the tube shaken by hand while the
contents were mixed with a glass spatula. The tube
was centrifuged briefly at 1500 rpm. The supernatant '
was collected, and the silica gel washed three times
with 0.5 ml of methanol.. Following centrifugation,
the supernatants were collected and combined with the
original supernatant. The combined supernatants were
evaporated to dryness under a stream of nitrogen. The
cis-hydroxycotinine hemisuccinate was reconstituted in
0.6 ml of methanol. After spectrophotometry it was
determined that 5.28 mg of product had been obtained.
The material was then evaporated to dryness again.
The cis-hydroxycotinine hemisuccinate was
dissolved in 280 ~cl of dimethylformamide. Eight mg of
N-hydroxysuccinimide and 12 mg of dicyclohexylcarbo-
diimide were added, and the mixture stirred until all
solids had dissolved. The mixture was incubated for 1
hour at room temperature with stirring.
A tube was prepared containing 1 ml of 0.1 M
carbonate buffer pH 9.0 and 2.8 mg glucose-6-phosphate
dehydrogenase. To this tube were added seven 10 ~1
aliquots of the cis-hydroxycotinine hemisuccinate over
a period of 1 hour, and mixing continued for 15
WO 93/03367 PGT/US92/06249
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k l~.a~~l~~~u
minutes after the last addition. The mixture was
transferred to a dialysis bag and dialyzed overnight
in the cold against 0.05 M Tris-HC1 pH 7.8. Dialysis
was continued for an additional 26 hours with several
changes of buffer. This conjugate was designated as
RL-34.
10.1.4. PREPARATION OF GLUCOSE-6-PHOSPHATE
DEHYDROGENASE-CARBOXYCOTININE LIGAND
Carboxycotinine was conjugated to glucose-6-
phosphate dehydrogenase according to the following
procedure:
To 1 ml of 0.1 M sodium carbonate buffer, pH
9.0, were added 0.43 ml of glucose-6-phosphate
dehydrogenase (2.8 mg), 20 mg of NADH (disodium salt), '
10 mg of glucose-6-phosphate, and 300 ~1 of carbinol.
The solution ("enzyme solution") was stored at 4°C to
chill.
To an empty test tube were added 22 mg of
carboxycotinine, 11.5 mg of N-hydroxysuccinimide, 20.6
mg of dicyclohexylcarbodiimide, and 1.0 ml of
dimethylformamide. This mixture was left at room
temperature for 1 hour to allow the activated cotinine
ester to form. After 1 hour, 10 ~cl of the reaction
mixture were added to the cold enzyme solution at 15
minute intervals until a total of 70 ~1 were added (90
minutes total). Fifteen minutes after the final
addition of reaction mixture, the modified enzyme was
dialyzed against five changes of 1 liter each of
0.055,M Tris-HC1 buffer, pH 7.9, for at,least three
hours each.
WO 93/03367 PCT/US92/06249
51 ~~
~~t~~~~~
10.1.5. ASSAY IN MICROTITER PLATES
Strip microtiter plates were coated by
addition of 100 ul of each of the conjugates at a
protein concentration of 1 ~g/ml to each well. The
strips were covered with parafilm and incubated
overnight at room temperature. The well contents were
discarded, and wells washed twice with wash buffer.
The wells were then incubated with 100 ~cl of antibody-
peroxidase conjugate for 1 hour at r.t. Unbound
conjugate was discarded and the wells washed twice
with wash buffer. To each well were added 100 ~1 of
wash buffer and 10 ~1 of cotinine standards, and the
plates were mixed for 2 minutes. After mixing, 100 gel
' of liquid from each well was transferred to wells in
uncoated plates, and 100 ~cl of tetramethylbenzidine '
(TMB) reagent were added. The plates were incubated
for 7 minutes at room temperature, after which the
reaction was stopped by addition of 50 ~cl of 2N
sulfuric acid to each well. Absorbance was measured
at 450 nm.
Residual peroxidase-antibody conjugate
remaining bound to wells after release was measured by
adding 200 ~C1 of TMB to each well. After a 7 minute
incubation, the reaction was stopped by addition of 50
~Cl of 2N sulfuric acid, and absorbance at 450 nm was
measured.
A previously made conjugate of
carboxycotinine-glucose-6-phosphate dehydrogenase was
also assayed by this method.
10.2. RESULTS
The results of assays with various
hydroxycotinine-glucose-6-phosphate dehydrogenase
conjugates are shown in Tables 5, 6 and 7.
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i
TABLE 5. RELEASED ANTIBODY-PEROXIDASE CONJUGATE (A~s~
CONJUGATE 24 25 26 29 30 CARBOXY
,~
COTININE COTININE
STANDARD CONJU-
I
(pg/ml) GATE
0 0.971 1.049 1.030 0.442 0.662 0.498
0.5 1.911 2.071 1.990 0.753 1.051 0.505
2.0 2.529 2.739 2.635 0.948 1_.4360.526 i
2.804 >3 >3 1.355 1.974 0.728
10 TABLE 6. ANTIBODY-PEROXIDASE CONJUGATE (A4~)
REMAINING ON WELLS.
CONJUGATE 24 25 26 29 30 CARBOXY
,1 COTININE
COTININE CONJUGATE
STANDARD
ml
0 2.474 2.514 2.513 1.893 2.097 2.707
0.5 2.403 2.368 2.440 1.776 1.937 2.746
2.0 2.213 2.347 2.360 1.739 1.879 2.730
10 2.051 2.253 2.298 1.695 1.790 2.786
TABLE 7. RELEASE ASSAY WITH CIS-HYDROXYCOTININE.
Absarbance at 450
nm
COTININE RELEASED REMAINING
CONC. (~tg/ml) CONJUGATE CONJUGATE
0 0.475 2.055
--
0.5 1.591 1.921
2.0 2.268 1.730
8.0 2.683 1.210
Release of enzyme-antibody conjugate
activity is also shown graphically in Figure 7. These
results clearly indicate that use of modified analog
as ligand provides the necessary difference in
affinity for a release-type assay.
I
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The cis-hydroxycotinine-glucose-6-phosphate
dehydrogenase ligand was also tested in a homogeneous
release assay such as described in Section 8.1.2.,
supra. After a 10 minute incubation of 10 ~sl of
antibody binding reagent with a 10 gel of ligand in 140
~1 of Tris buffer, 10 ~cl of sample were added. NAD
and glucose-6-phosphate substrate (20 ~1) were added
after an additional 5 minute period, and Ago read at 0,
2, 4, 8, 12, 16 and 20 minutes. Samples containing
cotinine showed a more rapid increase in absorbance
than sample with no cotinine. The results are shown
in Table 8.
TABLE 8. HOMOGENEOUS RELEASE WITH CIS-HYDROXYCOTININE.
Cotinine conc. OD Rate Change
(h /ml) (mA/min)
0 8.8
0.5 17.5
4.0 20.0
These results indicate that a microtiter
plate format is ideal for screening for suitable low
Z5 affinity ligands and ligand-enzyme conjugates. A
ligand found to be suitable in a microtiter plate
s release assay can be used in a homogeneous release
assay as well.
11. EXAMPLE' RELEASE IMMUNOASSAY FOR COTININE
In this Example 11, cotinine release assays
using N-propylcarboxynorcotinine and N-
isopropylcarboxynorcotinine as very low affinity
relands is described. Cis 3'-hydroxycotinine was also
used as a reland. The release assays were performed
CA 02114440 2003-04-10
- 54 -
in both homogeneous (which can be compared to, but
which is demonstrated to be superior than, the EMITTM
System (Rubenstein et al., 1972, Biochem. Biopys. Res.
Comm. 47: 846)j and heterogeneous (microtiter plate,
ELISA format) formats and compared to a conventional
associative assay for cotinine. The results
demonstrated that the release assays of the invention,
based on dissociation, are more precise and exhibit
less interference from cross-reactivity than known
l0 assays based on association. Moreover, the release
assay of the invention has a standard curve that is
linear, r 0.999, over a >10,000 fold range.
The following materials and methods sections
set forth general descriptions of the reagents
prepared and used in the assays, as well as the '
methods employed. The specific ligands used in each
assay are identified in the results sections, and
unless otherwise specified is N-
isopropylcarboxynorcotinine.
ao
11.1. MATERIALS ~;ND METHODS
Instrumentation included an SLT Lab
instruments 340ATTC Microtiter Plate Reader, COBAS
MIRA, and Varion~XR-200. Urine samples were from a
Z5 general population previously analyzed for cotinine.
Samples were stored at -20 degrees.
All chemicals were from Sigma Aldxich unless
otherwise stated. Cis and traps-hydroxycotinine were
purchased from the laboratory of George Neurath (See
30 Neurath et al., supra). Glucose-6-phosphate
dehydrogenase was from Beckman. The Nicotine
Metabolite Assay Kit, NiMA AutoMates''", and the ELISA
Kits, Tobacco Screen~, and the Cotinine Trace
Quantities, CotiTraq~, TMB chromogen system, anti-
35 Cotinine antisera, peroxidase labelled anti-Cotinine,
WO 93/03367 PCT/L)S92/06249
- 55 -
and Cotinine urine standards are commercially
available from Serex, Inc. (Maywood, NJ). Preparation
of anti-cotinine antisera is described in Section
6.1.4., Supra.
11.1.1. PREPARATION OF RELANDS
1-Isopropyl-4-carboxy-5-(3-pyridyl)-2-
pyrrolidinone, (hereafter, N-isopropyl-4-carboxy-
norcotinine) and 1-propyl-4-carboxy-5-(3-pyridyl)-2-
pyrrolidinone (hereafter, N-propyl-4-carboxy-
norcotinine) (Figure 8) were prepared according-to the
method of Cushman & Castagnoli (1972, J.Org. Chem.
37:1268) . Brief ly, to a solution of 17 g of pyridine-
3-carboxy-aldehyde in 50 ml of benzene was added a
benzene solution of 8 g isopropyl amine (or 8 g propyl
amine) and 12 g molecular sieve pellets. The mixture
was stirred at 20°C overnight in a flask. The
solution was filtered through two layers of Whatman
No: 2 filter paper and evaporated under reduced
pressure to give the imine as a yellow oil. The
structure of the products was confirmed by 'H NMR.
N-isopropylcarboxynorcotinine and N-
propylnorcotinine were prepared as follows. Twelve g
of N-3-pyridylidene isopropyl imine or N-3
pyridylidene propyl imine and 15 g succinic anhydride
were refluxed for 24 hours in 100 ml xylene. After
' the mixture cooled, the top layer was decanted and
discarded. The residue brown oil was dissolved in 300
ml of 5% sodium bicarbonate solution, washed with two
30. 250.m1 portions of chloroform, and decolorized by
absorption with 1 g activated charcoal. The
suspension was filtered and the yellow filtrate heated
on a steam bath to remove traces of chloroform. The
pH was adjusted to 4.7 with phosphoric acid to
precipitate the product. The crude carboxylic acid
WO 93/03367 PCT/US92/06249
,, 1 ~: :~ - 5 6 -
~r
!~ ~.
r
was collected by filtration and recrystallized from a
boiling ethanol to give 4 g white crystal. The
structures of the compounds were confirmed by 'H NMR.
11.1.2. PREPARATION OF GLUCOSE-6-PHOSPHATE
DEHYDROGENASE CONJUGATES
Conjugates of N-isopropyl-4-carboxy-
norcotinine, N-propyl-4-carboxy-norcotinine and cis
3'-hydroxycotinine to glucose-6-phosphate
dehydrogenase were prepared according to the methods
described by Rubenstein and Ullman (1975, U.S. Patent
No. 3,875,011). Briefly, to 1 ml of 0.1 M sodium
carbonate buffer, pH 9.0, were added 0.43 ml of
- glucose-6-phosphate dehydrogenase (2.8 mg), 20 mg of
NCH (disodium salt), l0 mg of glucose-6-phosphate, ,
and 300 gel of carbinol. The solution ("enzyme
solution") was stored at 4°C to chill.
To an empty test tube were added 26 mg of N-
propyl or N-isopropylcarboxynorcotinine, 11.5 mg of N-
hydroxysuccinimide, 20.6 mg of dicyclohexyl-
carbodiimide, and l.0 m1 of dimethylformamide. This
mixture was left at room temperature for 1 hour to
allow the activated cotinine ester to form. After 1
hour, 10 ~ul of the reaction mixture were added to the
cold enzyme solution at 15 minute intervals until a
total of 70 ~cl were added (90 minutes total). Fifteen
minutes after the final addition of reaction mixture,
the modified enzyme was dialyzed against five changes
of 1 liter each of 0.055 M Tris-HC1 buffer, pH 7.9,
30 for,at least three hours each.
11.1.3. REAGENTS FOR THE HOMOGENEOUS RELEASE ASSAY
Reagent solutions for the homogeneous assay
of cotini»e were prepared as three separate solutions,
35 reagents A, A+, and B. Reagent A consisted of
glucose-6-phosphate dehydrogenase conjugate at a
WO 93/03367 PGT/US92/06249
- 57 -
2i ~~~:~~._~
~s
protein concentration of 0.74 ~Cg/ml, 0.05 M Tris
buffer, 5 mM MgCl2, 0.5 mM EDTA, 1.75 mg/ml glucose-6-
phosphate, 0.5% BSA, and preservatives at pH 7.9.
Reagent A+ consisted of antisera in reagent A buffer.
Reagents A and A+ were mixed prior to use to form
working solution A, which is stable for one week at
4°C.
Reagent B consisted of NAD at 3.3 mg/ml in
0.02 M Tris buffer, pH 7Ø
Cross-reactivity was tested with cotinine
and/or traps-3'-hydroxycotinine solutions prepared as
follows. To 10 ml of a negative urine pool were added
100 ~g of cotinine or traps-3'-hydroxycotinine. The
mixture was vortexed and serially diluted into the
same negative urine standard to make solutions of 5, '
2:5, 1.25, 0.62, 0.31 and 0.16 ~g/ml of cotinine or
traps-3'-hydroxcotinine.
To prepare the 1:3, cotinine:trans-3'-
hydroxycotinine, solution, a 10 m1 aliquot of negative
urine standard was spiked with 100 ~g of continine and
300 ~g of traps-3'-hydroxycotinine. This solution was
vortexed and serially diluted into the same negative
urine standard to form dilutions of 5 (i5), 2.5 (7.5),
1:25 (3.75), 0.62 (1.87). 0.31 (0.94), 0.16 (0.48)
~eg/ml of cotinine (hydroxycotinine).
Cross-reactivity was calculated using the
' following formula;
concentration found (,ug/ml)
____-________________________________________ x 100%
concentration of cross reactant
(~,g/ml) in sample
11.1.4. ASSAY FORMATS
ELISA format. Corning microtiter plates
were coated overnight with 100 ~Cl of either glucose-6-
Phosphate dehydrogenase conjugated to cis-
hydroxycotine, N-propyl-norcotinine or N-isopropyl-
WO 93/03367 PCT/US92/06Z49
rr'w~ -58-
, ::F
.~_4 1 ~t, =~
v r
norcotinine at 1 ;cg protein per ml of PBS; the wells
were emptied, dried and stored with dessicant until
use. To activate for release, the plate was incubated
<1 hour with 100 ;el of peroxidase labelled affinity
purified anti-cotinine antibody. Excess antibody was
removed by 2 washes with PBS in 0.05% Tween 20.
To a microtiter plate coated with a release
ligand-antibody Ireland) complex, 10 ;el of
urine/standard and 90 ~1 of distilled water were added
i0 to each well. After 2 minutes, 50 ~cl of the
supernatant were transferred to uncoated wells
containing 100 ~1 of TMB and incubated for 10 minutes.
The reaction was stopped with 100 mh of 1N HZS04 and
A,~ was read.
i5 Homocreneous Release Assav. The homogeneous
assay of the present invention, was performed in the
AutoMatest" format, utilizing the same enzyme system
and the same reagents as the conventional homogeneous
associative assay, but modified as follows to become a
Z0 dissociative reaction.
Ab:ligand conjugate + analyte ---> Ab:analyte + ligand
conjugate
25 Before use, reagent A (enzyme conjugate in
buffer) and reagent A+ (antisera in buffer) were mixed
' for a minimum of one hour. The reaction was started
by addition of sample and NAD. As in the associative
assay, enzyme activity was measured by monitoring the
30 formation of NADH at Ago nm, and,enzyme activity is
directly related to the concentration of analyte in
the sample.
The homogenous assay of the invention using
AutoMates~" was performed on the COBAS MIRA according
35 to the application sheet parameters. Two hundred ~1
WO 93/03367 PGT/US92/06249
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of reagent A were incubated with 10 ~1 of reagent B
(NAD) and the mixture was incubated for 25 seconds.
The absorbance was read over the final 200 seconds.
Total time of the assay was 5.0 minutes. Greater
sensitivity of a sample containing 10 ng/ml of analyte
was observed with a 25 ~1 sample.
Associative Homogeneous Assav. The NiMA
AutoMates'~ format is a homogeneous, associative,
competitive assay, like that described by Rubenstein,
Schneider, and Ullman (1972, s_u~ra). There are two
steps to the immune reaction:
Ab + analyte ---> Ab:analyte + Ab
Ab:analyte + Ab + conjugate --->Ab:conjugate
i5 +Ab:analyte
Briefly, sample was pre-incubated with
antisera for several minutes. Into this reaction
mixture was added glucose-6-phosphate dehydrogenase
conjugated with cotinine. Antibody that has not
interacted with cotinine in the sample binds to
cotinine on the glucose-6-phosphate dehydrogenase.
The binding of antibody to the enzyme-linked ligand
inhibits enzyme activity, thus the enzyme activity is
directly related to the concentration of analyte in
sample. Enzyme activity of glucose-6-phosphate
dehydrogenase was measured by monitoring at A~,o nm the
formation of NADH, which forms as the enzyme oxidizes
glucose-6-phosphate to glucono-d-lactone-6-phosphate
and reduces NAD to NADH.
NiMA AutoMates'~' was performed on the COBAS
MIRA according to the application sheet parameters.
Two hundred ~cl of reagent A were incubated with 10 ~cl
of sample at 37 degrees for 75 seconds. Fifty ~1 of
reagent B were added and the mixture was incubated for
WO 93/03367 PCT/US92/06249
,~~c..:,
li Ly
y'~ j1 , ~~ .._
25 seconds. The absorbance was read over the final
250 seconds. Total time of the assay was 5.83
minutes.
11.2. RESULTS
11.2.1. HETEROGENEOUS RELEASE ASSAY (ELISA FORMAT)
Release of labelled antibody complexed to
solid phase was detected in supernatant when free
cotinine was present in sample (Figure 9). Unlike
i0 conventional competitive immunoassays, absorbance or
signal was directly proportional to analyte
concentration.
The cis-hydroxycotinine conjugate bound and
released the antibody most efficiently, but had the
highest background.
11.2.2. HOMOGENEOUS FORMAT
Dose response curves for the associative
(NiMA) and release homogeneous assays demonstrate the
greatly increased range of the release assay (Figure
10). Comparison of the associative and release
formats is presented in Table 9.
TABLE 9. COMPARISON OF A HOMOGENEOUS DISSOCIATIVE
ASSAY FOR COTININE AND A HOMOGENEOUS
SA~SOCIATIVE ASSAY FOR COTININE (NiMA).
RELEASE NiMA
Final Antisera Dilution 4.8 x 10'3 2.4 x 10'3
Final Conjugate Concentra- 0.51 ~g/mL 0.03 ~g/mL
tion
5~ Antis~era Dilution/~Cg 9 . 6 x 10'380 x 10'3
' Conjugate
Lower Limit 0.01 ~g/mL 0.05 ~Cg/mL
Upper Limit 1000 ~g/mL 2 ~g/mL
Time 5 min 5.8 min
WO 93/03367 PCT/US92/06249
Table 9 shows that the release assay
utilizes 17-fold more enzyme and two-fold more
antibody than the associative assay. But the
increased enzyme and antibody do not result in
decreased sensitivity as they do in associative
immunoassays: smaller amounts of all reactants can be
utilized in release, but this limits upper range of
the assay. Figure 11 shows the range of the assay,
0.01-100 ~g/ml. In the example shown, a seventeen-
fold increase in reactants yields a greater than
1,000-fold increase in range of the assay, with no
loss of sensitivity at the low end of the curve. This
formulation is sensitive to 10 ng/ml and can be used
for quantitating saliva samples. The antibody to
enzyme ratio of the release to associative assays is
10:80; that is the associative assay uses 8-fold more
antibody per enzyme molecule than the release. In the
release assay all antibody molecules can be bound to
ligand conjugated to enzymes and are capable of being
released by analyte. In the associative assay there
is a large excess of antibody, which decreases
reaction time. The ability of the release assay to
monitor the activity of a much larger percentage of
the antibody in the reaction mixture increases
sensitivity and decreases background noise and
reaction time.
11.2.3. CROSS-REACTIVITY: TRANS-HYDROXYCOTININE
The specificity of the release assay
relative to the associative assay was confirmed by
comparing the release and association formats for
cross-reactivity with traps-3'-hydroxycotinine (Table
10) .
WO 93/03367 PCT/US92/06249
.,~ r'; ~t ~~G~'' - 62 -
TABLE 10. INTERFERENCE OF TRANS-HYDROXYCOTININE
IN RELEASE AND ASSOCIATIVE (NiMA)
HOMOGENEOUS ASSAYS FOR COTININE.
RELEASE ASSAY
COTININE SPIKE TRANS-HYDROX YCOTININE
.
SPIK E
SPIKE CONCENTRATION % CONCENTRATTON%
CONC. FOUND (ug/mL) RECOVERY FOUND (~g/mL)RECOVERY
/mL
14 9.03 90 1.69 16.9
5 4.22 84 0.72 14.4
2.5 2.15 86 0.29 11.6
1.25 1.15 92 0 0
0.62 0.63 102 0 0
0.31 0.25 81 0 0
0.16 0.14 88 0 0
A~RAGE 89 6.1
NiMA
COTININE TRANS-HYDROXYCOTININE
SPIKE ~ SPIKE
SPIKE CONCENTRATION% CONCENTRATION %
FOUND (~g/mL)RECOVERY FOUND (Ng/mL) RECOVERY
10 11.85 119 1.87 18.7
5 5.6 112 0.85 17
2.5 2.03 81 0.44 17.6
1.25 1.59 127 0.21 16.8
2 5 0.62 0.59 95 0.1? 27.4
0.31 0.49 158 0.11 35.5
' 0.16 0.13 81 0.08 37.5
AVERAGE 110 24.4
The release assay showed one-fourth the
cross-reactivity with hydroxycotinine of the
associative assay. In the release assay there was no
cross-reactivity at the low end of the curve. The
greatest amount of interference in the associative
assay was seen at the low end of the curve. To
evaluate the actual effect of traps-3'-hydroxy
WO 93/03367 PGT/US92/06249
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cotinine on the assay of urine samples, samples were
spiked with cotinine:trans-3'-hydroxycotinine in a 1:3
ratio (this is the ratio reported to exist in smokers'
urine (Engvall et al., 1971, Immunochemistry 8:871))
and assayed in both release and associative
homogeneous assays (Table 11).
TABLE I1. CROSS REACTIVITY OF TRANS-HYDROXCOTININE IN
THE PRESENCE OF COTININE IN THE RELEASE AND
THE ASSOCIATIVE (NiMA) HOMOGENEOUS ASSAYS
FOR COTININE.
RELEASE NiMA
COTWINE(1'RANSCOT1NWECONC. 96COTWPIECOTWOVECONC.96 COT~fU4E
HYDROXYCOTU~WE)FOUND (pt/mL)RECOVERY FOUND (p8lmL)RECOVERY
~/mL
'
10 (!D) 16.82 168 >20 >200
S (IS) 7.SS iSi 14.21 2S4
2.5 (!.S) 3.69 148 4.38 17S
1.25 (3.?S) 1.45 116 2.29 183
0.62 (1.9) 0.92 1S5 1.82 294
0.31 (0.93) 0.4 129 0.54 174
0.16(0.48) 0.23 144 0.37 231
AVERAGE 144 220
Trans-3'-hydroxycotinine contributed to
35 cotinine quantitation in both the release and the
associative assays. The contribution is rather
constant across the range of the assay in both formats
but results in an average 220% recovery in the
associative assay but only 144% in the release assay,
30 indicating that the release assay is less subject to
interference by cross-reactivity, therefore reducing
the potential for false positive results in an assay
for cotinine.
The results for cross-reactivity in Table 10
35 suggest that trans-hydroxycotinine present in a sample
WO 93/03367 PCTlUS92l06249
c ' a', a'~l
.r1 f.; ~ i. :.:. _ 6 4 _
at the expected 3:1 ratio to cotinine would increase
the apparent detection of cotinine by about 45% (3
times about 15% cotinine recovery). Thus the expected
recovery of cotinine in the release assay reported in
Table 11 is about 145%. In fact, the average percent
cotinine recovery was about 144%. A similar analysis
for the associative (NiMAj format indicates that the
same 3:1 traps-hydroxycotinine to cotinine ratio would
increase the apparent cotinine recovery by about 60%
(3 x 20%). Thus the expected recovery of cotinine in
the associative (NiMA) assay reported in Table 11 is
160%. However, the average % cotinine recovery is
_ 220%, and ranged as high as 294%. Thus there appears
to be a "synergystic" effect of traps-hydroxycotinine
on cotinine detection, so that the presence of traps- '
hydroxycotinine skews results in the associative assay
for cotinine in an unexpected, and therefore perhaps
uncorrectable, way.
The release assay exhibits cross-reactivity
as predicted, but the associative format shows average
recoveries of 220%, almost double that predicted by
the simple cross-reactivity data. This demonstrates
that the associative assay is more subject to
interference than release.
11.2.4. CROSS-REACTIVITY OF
N-ISOPROPYL-4-CARBOXY
NORCOTININF
To further test the stability and
releasability of the antibody-ligand complex we
characterized the ability of N-isopropyl-4-carboxy-
norcotinine to interfere in the various release
assays, all of which used the same antibody. Results
are shown in Table 12.
WO 93/03367 PGT/US92/06249
s5
TABLE 12. CROSS REACTIVITY FOR N-ISOPROPYL-4-CARBOXY-
NORCOTININE IN ASSOCIATIVE AND RELEASE
ASSAYS.
%CROSS REACTIVITY
SPIKES
URINE(~Cg/mL) CotiTraq~ Release NiMA
0.24 0% <0
0.5 0% 0% <0
2 0% 0% <0
4 0% 0% 0.05%
10 0.3% 0% 0.11%
100 0.3% 0.4% 0.24%
i5 The ligand N-isopropyl-4-carboxy cotinine
showed less than 0.4% cross-reactivity, even in-
concentrations as high as 100 ~cg/ml. The ligand shows
no~cross-reactivity with the antibody complexed to it
on G-6-P-DH until 100 ~Cg/ml, at which point cross-
2~ reactivity of 0.4% was detected. N-isopropyl-4-
carboxy-cotinine interfered more in the conventional
NiMA and CotiTraq~ Assay format than in the release
assay, indicating that it is not a competitor for the
. antibody in the release format.
25 The release homogeneous assay and
conventional homogeneous and conventional ELISA
formats were also compared. The release heterogeneous
assay, NiMA AutoMates"" and Tobacco Screen~ (ELISA)
assays for cotinine were compared using a cotinine
30 equivalent cutoff of 0.5 ~Cg/ml (0.5~Cg/ml is considered
equivalent to urine composition of 0.25 ~g/ml
cotinine, 0.75 ~g/ml hydroxycotinine in Tobacco
Screen~ and NiMA, and 0.35 ~g/ml cotinine in the
release assay of cotinine). Table 13 shows that
35 release correlates 100% with Tobacco Screen~. Tobacco
WO 93/03367 PGT/US92/06249
- 66
~4 zt~
a. ~ ~ ~y
V ~ ~f~~r~
A~~
Screens correlates 100a with HPLC results using an
HPLC cutoff of 200 ng/ml and a Tobacco Screen~ cutoff
of 400 ng/ml.
TABLE 13. COMPARISON OF RELEASE HOMOGENEOUS COTININE
ASSAYS WITH~THE ASSOCIATIVE ASSAYS, TOBACCO
SCREEN (ELISA) AND NiMA (HOMOGENEOUS),
USING 0.5 uq~mL CUTOFF FOR ALL ASSAYS.
TOBACCO SCREENS NiMA
+ - + -
+ 106 0 + 106 0
Release Release
0 34 - 0 34
n = 140 n = 140
11.2.5. ASSAY PRECISION
The release and NiMA homogeneous assays for
cotinine were evaluated for precision on a COBAS MIRA
using negative, 0.5 ~g/ml and~2 ,ug/ml urine samples
(Table 14). Precision is used to indicate the
coefficient of variation of repetitive tests on the
same sample. The release assay showed more than a
two-fold improvement in precision over the associative
assay. Evew though reactant concentrations are 17-
fold higher than NiMA, the release assay had better
precision.
35
WO 93/03367 FCT/US92/06249
- 67 -
2~.~~~~~
TABLE 14. PRECISION OF THE RELEASE AND THE
ASSOCIATIVE NiMA HOMOGENEOUS ASSAYS FOR
COTININE. RESULTS STATED ARE REACTION RATE
IN mA~IMIN.
RELEASE
Negative Control Cutoff Control Positive Control
0.0 u9ymL 0.5 ~agymL 2.0 ucx~mL
n = 15 n = 15 ~ n = 15
avg = 177.79 avg = 185.4 avg = 194.56
SD = 0.88 SD = 0.89 SD 0.81
=
l0 CV 0.5% CV = 0.5% CV 0.4%
= =
NiMA
Negative Control Cutoff Control Potitive Control
0.0 uq,/mL 0.5 ug~/mL 2.0 uq,~mL
i5 n = 15 n = 15 n = 15 .
avg= 49.52 avg= 55.86 avg = 58.27
SD = 0.69 SD = 0.75 SD = 0.78
CV = 1.3% CV = 1.3% CV = I.3%
20 11.3. DISCUSSION
11.3.1. HETEROGENEOUS FORMAT
A release ELISA assay well contains less
than 1/5 the antibody utilized in conventional ELISA
assays. Sensitivity is enhanced as a result because
25 non-specific activity decreases: Sensitivity is also
enhanced by the almost ten-fold higher enzyme activity
' of the released peroxidase-labelled antibody.
In the ELISA format this end point release
assay reduces the time for the assay from 1.5 hour to
30 under 15 minutes (2 minutes for the release reaction,
and 10 minutes for TMB color development). Time could
be further shortened, for example, by automating the
assay steps such as by running a rate reaction assay
on an automated instrument. The release assay also
35 reduces the number of assay steps by at least half. A
,,
.k. ;.
,:
4,,. ~ t . ~.."
F'-.T ,..,.i
,~ 7
i ~;s
:,. .. . . . .. a . , , ,.
, , a
X3'2 -:,. ~.. ... ..~., . ', _~........_.~c. ,cr . . .. .........,.n~...k
.x.,... ..... . t ~ s ...i, ...,.~,1r. ....n... . .~n .~ .. .V... .,.a
.,.,::~~.., -:.... ,
WO 93/03367 PGT/US92/06249
a ~~~ ~~ - 6 8
~~ Gb y
r
further advantage is the release gives a positive
signal in the presence of analyte.
11.3.2. HOMOGENEOUS FORMAT
In the homogeneous assay format, sensitivity
is further enhanced by the fact that released
peroxidase- labelled antibody has much higher activity
than enzyme on the plate. That is, if one measures
released peroxidase conjugated antibody and that
remaining on the plate, one can see that a 0.1 O.D.
drop in plate absorbance can result in generation of
2.0 O.D. units in the supernatant. This 10- to 20-
fold enhancement of free enzyme activity is probably
in large part due to a decrease in the effects of
diffusion on the released enzyme reaction rate. '
The range of the release assay is extended
because the release assay is a system that starts in
equilibrium. It is therefore possible to use higher
starting concentrations of enzyme complex without
increasing noise or losing low-end sensitivity. In
conventional immunoassays, addition of more reagents
changes the sensitivity of the assay by shifting final
equilibrium conditions. Enzyme concentration
demonstrating release assay were 0.50 ~cg/ml as
compared to 0.03 ~g/ml for conventional homogeneous
(associative) assays. However, the conventional type
assay had about 8 times as much antibody:enzyme as the
release assay. The use of monoclonals will further
improve the ratio of antibody to enzyme in the release
format. The ratio of antibody to enzyme determines
assay sensitivity.
While other assay systems that involve
dissociation of preformed antibody-ligand complexes
(Cocola et al., 1979, Analytical Biochem. 99:121-8-
Hinds et al., 1984, Chin.Chem. 30:1174-8;
a _."
r.. .. c.~-...,.., E,-";
r
.r-~.-r~~~~- a . ","O,." ,
--.c.
~t.:'
>., ~.i ,.~. .~,
Z ,
..d"~.,,. ~ 4
s--,-; .
4,. ,
<~ ..'
>' a ~::...
'1 V
l'J....~f..t , _ .. .4..a~ ..._.w. ~ '.. , ,:.a'.cG.l: L'~~.5.. . .. ' '~A .,
. ~_, ,.. . ... ..,...v,.,.~i.~l~..-,i. .. .a . .~,i.".~..,".vr.... . . .
_,..':...~. ... :.. ~y ,, . . . . -, 1~., o",.~. . .e. . ., ." .~, .~ ,
WO 93/03367 PCT/US92/06249
59
x J~
Hinds et al., 1985, Chin.Chem.Acta 149:105-15) have
utilized competitive ligands as binding partners, the
release assay is a non-competitive system, This is
demonstrated in Table 12, supra, where release ligand
(or reland) cannot compete the antibody off the
antibody-ligand complex. It is also noteworthy that
unlike the release assay of the present invention, the
competitive dissociation assays described by Cocola et
al., Hinds et al., 1984 and Hinds et al., 1985, supra,
have not shown significant improvement over
associative methods of immunoassay. Although the
present invention is not bound by any particular
theory, we hypothesize that antibody binds to the
release ligand via very low affinity interaction, and
in the absence of higher affinity binding partners, '
antibody undergoes a conformational change to a
metastable complex that is releasable and has a time
stable affinity constant for release. The complex may
become too stable, as observed with conventional
ligand conjugates. N-isopropyl-norcotinine was
designed to provide a bulky group at a non-
immunologically critical site to allow dissociation
after equilibrium..
The rapid dissociation of the reland complex
in the presence of ligand may be analogous to turn-
over in antibody catalysis (Benkovic et al., 1990,
Science 250:1135-8) in which antibody prepared to a
transition state substrate conformation binds a
substrate, induces the transition state conformation
in the substrate,, and then releases the cleaved
substrate rapidly since the products no longer appear
in the transition state.
WO 93/03367 PCT/US92/06249
- 70 -
11.3.3. CROSS-REACTIVITY
The release assay is less subject to cross-
reactivity. More importantly, it was not subject to
the synergistic type of cross-reactivity that was
observed with conventional immunoassay. We have
observed this synergistic enhancement of interference
in other conventional immunoassay formats. This may
be a generalized phenomenon and perhaps all cross-
reactivity ought to be reported as % cross-reactivity
seen in the presence of analyte at 50% binding, so
that the true contribution to the assay can be
assessed.
11.3.4. ASSAY PRECISION
The two-fold improvement in precision seen '
with the release assay of the invention is probably
multifactorial: the starting system is in equilibrium
and only one reaction -- dissociation -- occurs, and
the matrix, as evidenced by lowered cross-reactivity,
probably has less effect on the reaction.
11.4. CONCLUSION
We have developed an assay method utilizing
the ability of antibodies to assume an induced fit
with a binding partner for which it has very low
affinity, the release ligand, or reland. The release
assay provides a preformed receptor-reland complex,
which can be rapidly dissociated in the presence of
analyte. The release system can be used in all
immunoassay formats. The release assay has inherent
advantages over conventional or associative assays:
1. By eliminating one step in the immune reaction,
release saves time and steps and possible sources
of error, thereby shortening assay time and
simplifying assay techniques.
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2. Release, i.e., dissociation, is inherently less .
subject to interference making it more accurate.
3. The ability to monitor all antibody in the assay
reduces noise and makes a 1000-10,000-fold
sensitivity range possible. This methodology,
using more sensitive markers, extends the
theoretical range both up and down from that
available in conventional assay formats.
Addition of more reactants does not lower
sensitivity as in conventional immunoassays, but
extends the upper range of sensitivity.
4. An important advantage of release is the mild
conditions under which the dissociation occurs.
This allows the solid phase or complex to be
regenerated. This should advance the
possibilities for biosensors. A major problem
with biosensors is that the solid phase is
usually a disposable; with the release assay the
solid phase can be continually regenerated.
5. The large range, the positive correlation with
presence of analyte, and the low noise of the
system indicates that the release assay format
can be used to screen for many analytes in one
reaction mixture.
12. EXAMPLE: RELEASE ASSAY FOR
A COCAINE METABOLITE
Benzoylecgonine is a metabolite of cocaine.
Release immunoassays that allow detection of soluble
benzoylecgonine in a sample are described below. In
particular, release of an enzyme-labeled anti-
benzoylecgonine antibody can be detected in assay
solution supernatant when free benzoyl.ecgonine is
present. The following example demonstrates an
effective release assay for benzoylecgonine. Two
assay parameters, the effect of the relative amount of
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,'.\
antibody-label conjugate incubated on a ligand-protein
coated plate and incubation time after addition of
sample on detection of released label, were
investigated.
12.1. MATERIALS AND METHODS
Using standard techniques, ecgonine was
conjugated to BGG via a bulky linker (p-aminobenzoic
acid) . Anti-benzoylecgonine-peroxidase conjugate was
prepared using standard techniques.
12.1.1. ~'ITRATION OF CONJUGATE
Microtitre plates were coated with
benzoylecgonine-BGG ligand. After washing, 100 ~,l of
antibody-peroxidase conjugate were added to each well
at either 1:500 or 1:1000 dilution and incubated at
r.t. for 60 min and washed twice with wash buffer.
Benzoylecgonine (BE) standard solutions (100 ~cl) were
added to individual wells. Benzoylecgonine was
present at the fol2owing concentrations in distilled
water or urine: 0, 0.025, 0.3, and 5.0 ~,1/ml. After
adding sample, plates were incubated 10 min with
shaking at r.t., arid 100 ~cl of supernatant were added
to replicate wells of an uncoated microtitre plate.
To each well of assay supernatant were added 100 ~cl of
2x TMB substrate solution. The plates were incubated
6 min at r.t., and the reaction stopped by addition of
50 ~,1 of 2 N H2S04 to .each well. Absorbance at 450 nm
was measured in a plate reader.
.
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12.1.2. ASSAY FOR INFLUENCE OF INCUBATION
TIME AND MATRIX COMPOSITION
The procedure described above (Section
12.1.1.) was followed. To detect the effect of matrix
composition, standards in a synthetic urine matrix
were diluted in deionized water. To assay the effect
of incubation time, i.e., time needed to detect
release of labeled antibody, peroxidase labeled anti-
benzoylecgonine ecgonine-BGG-coated plates were
incubated 0, 2 and 10 minutes after addition of free
benzoylecgonine before transfer of supernatant to a
clean microtiter plate.
12.2. RESULTS
Free benzoylecgonine in sample resulted in
release of antibody-peroxidase conjugate. The amount
of conjugate released correlated with the amount of
conjugate added to the benzoylecgonine-BGG coated
plate as well as with the amount of analyte present in
2o the sample (Table 15). Thus more conjugate is
released from incubation with a 1:500 than a 1:1000
diluted solution.
ZS
s
35
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TABLE 15. RELEASE OF ANTI-BENZOYLECGONINE-PEROXIDASE
FROM ECGONINE-PABA-BGG-COATED PLATE.
Ecgonine conc. A4so of Released
(~cg/mI) Anticotinine-Peroxidase
Stds in Water 1:500 1:1000
0 0.881 0.574
0.025 1.541 0.944
0.30 2.465 1.630
5.0 2.859 ~ 2.218
Stds in Urine
0 0.234 0.112
0.025 0.493 0.255
0.30 1.182 0.630
5.0 1.901 1.010
Matrix effects are also evident from the
Za data in Table 15. In particular, release appears to
be more effective in water than in urine. These
results are expected since matrix is known to affect
antibody antigen reactions. Nevertheless, the
relative release of antibody-enzyme conjugate is not
affected by matrix composition. This relationship can
be seen by comparing the ratio of A4so when free
benzoylecgonine is present to A4so blank in each matrix
(Figure 12). Thus, the matrix effect appears to
result from inhibition of enzyme by components in
urine and does not appear to affect the amount of
antibody-enzyme conjugate released.
The time of incubation appears to have
minimal effect on enzyme activity in supernatant as
shown graphically (Figure 13).
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12.3. DISCUSSION
These results indicate that release of
labeled antibody from a ligand coated plate is
proportional to the amount of labeled antibody added
to the plate.
The results also indicate that while sample
matrix affects the absolute enzyme label activity in
release supernatant, this effect can be corrected for
by normalization over baseline control activity.
Generally, normalization of various samples allows
comparison and quantification of free ligand, in this
case free benzoylecgonine, present in the sample.
Finally, the results in Figure 13 indicate
that release is nearly complete almost immediately
after addition of free ligand, demonstrating that
release assays may be run in short period of time.
13. EXAMPLE: RELEASE ASSAY ON
ANTIBODY-COATED MEMBRANES
In a particular embodiment, presence of
- soluble analyte may be detected by release of labeled
ligand from an antibody-coated solid phase support,
e.g., a membrane. This example demonstrates release
of labeled ecgonine from a complex with solid phase
2S antibody specific for benzoylecgonine.
13.1. MATERIALS AND METHOD
13.1.1. LABELED LIGAND
Ecgonine was linked to alkaline phosphatase
30 via a spacer linker. In 20 ml,of DMSO (Sigma) were
dissolved 40 mg ecgonine (Alltech), 32 mg
N-hydroxysuccinimide (Sigma) and 56 mg of 1-ethyl-
(3-aminopropyl)carbodiimide (Pierce) at r.t. for
4 hrs. A solution of 25 mg N-(4-aminobenzyl)-6-
35 aminocaproic acid (Aldrich) in 0.5 ml DMSO was added
to 2.O m1 of 0.2 M sodium bicarbonate, pN ?.8, and
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this mixture was added to the activated ecgonine
solution. The resulting solution was mixed for 1 hr.
at r.t. and held at 4°C overnight. To 1.0 ml of
activated ecgonine-linker solution were added o.3 ml
of 10 mg/ml N-hydroxysuccinimide and 16.7 mg/ml 1-
ethyl-3-(3-dimethylamino)carbodiimide (Sigma) in DMSO,
and the resulting mixture shaken for 4 hrs at r.t. To
1 ml of a solution of 1 mg/ml alkaline phosphatase
(Biozyme ALPI IIG, 1650 U/mg) were added 0.65 ml of
i0 activated ecgonine-linker, and the mixture incubated 2
hrs at r.t., then held at 4°C overnight. The product
was dialyzed in the cold against 0.5 M carbonate
buffer, pH 7.8, for 8 hours, with dialysis fluid
changed every 4 hours.
' '
13.1.2. ANTIBODY-COATED LATEX
,~fEMBR_ANE ASSAYS
A suspension of 0.5 ml of polystyrene latex
particles, 0.776 microns (IDC) mixed with 3.5 ml of
Phosphate buffer pH 7.5 was prepared. Goat anti-
rabbit IgG, Fc fraction, was diluted to 0.5 mg/ml in
phosphate buffer (0.2 ml to 4 ml). The~two were mixed
by inversion and incubated at room temperature for 1
hour, and than stored at 4°C. 1 ml of this latex
preparation was centrifuged for 5 minutes at 3000 RPM,
washed with 0.01 M phosphate buffer, pH ?.5, and
recentrifuged. The latex particles ware resuspended
in 5 ml of phosphate buffer containing 0.1% Tween 20.
55 ~l of rabbit antibody against benzoylecgonine were
diluted in 5 ml of the Tween~hosphate and added to
the latex suspension. The mixture was gently mixed at
room temperature for 60 minutes. It was then
centrifuged for 5 minutes at 3000 RPM, washed twice
with Twee phosphate with centrifugation and
resuspension. The preparation was resuspended in
Tween~hosphate buffer containing 0.1% sodium azide.
WO 93/03367 PCT/US92/06249
Membranes were prepared by soaking 0.45
filtration membranes in 80 ~C1 of Tween/phosphate
buffer, and 20 ~1 of anti-benzyolecgonine-coated latex
were added and allowed to absorb. Then 10 ~ul of
labeled ecgonine were added to each latex spot. The
membranes were incubated 3 hrs at r.t. to allow stable
receptor-ligand complex to form and subsequently
washed three times with phosphate buffer containing
0.1% Tween 20. Sample or standard was added (100 ~ul),
to and the membrane incubated to min. at r.t. with
shaking. The test was generally run as a visual test
by adding substrate directly to membranes where it is
washed away released ligand-enzyme samples. Decreased
color-intensity was proportioned to the
benzoylecgonine concentration in the sample.
For quantitating release the liquid phase
(supernatant) was collected by filtration and 50 ~cl
aliquots were added to wells of microtiter plates. To
each well were added 100 ~cl of p-nitrophenylphosphate
(Sigma) in diethanolamine buffer, pH 9.7, and the
plates were incubated 30 min at room temperature.
Absorbance at 405 nm was measured.
13.2. RESULTS
The results of this assay are shown in
Table 16.
TABLE 16. Enzyme Activity of Liquid Phase
BENZOYLECGONINE CONC.
(~,g/ml) A4a5
0 0.214 I
5 0.352
These results demonstrate release of
ecgonine-linker-alkaline phosphatase from the latex-
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antibody preparation when free benzoylecgonine is
present in sample.
14. EXAMPLE: RELEASE ASSAY FOR
TETRAHYDRO~,AN ABINOL (T~iC)
A solid phase membrane assay, similar to
that described in Section 13, supra, has been prepared
to allow detection of tetrahydrocannabinol, the major
active component of marijuana.
14.1. MATERIALS AND METHODS
14.1.1. PREPARATION OF MEMBRANES
Polyclonal antibbdies (sheep) to THC were
used. Ammonium sulfate purified antibody, 13 mg/ml in
0~5 M carbonate buffer pH 9.3 in volume of 3 ~1, were
absorbed to 5 mm disks of Gelman KV30o0 ultrabind
membranes. The disks were allowed to dry for 30
minutes at room temperature, and then were treated
with 0.1% (w/v) bovine serum albumin in carbonate
Z0 buffer fox 15 minutes at room temperature. Excess
albumin was removed by aspiration, and membranes were
washed for l0 minutes at room temperature with a
buffer consisting of 2% (w/v) glucose and 0.01% (w/v)
2,6-di-tart butyl-4-methylphenol (BHT) (Aldrich) in
Z5 0.5 M carbonate buffer, pH 9.3. Disks were allowed to .
dry at room temperature and stored at 4°C.
14.1.2. PREPARATION OF LIGAND
Two mg of 9-carboxy-11-nor-delta-9
30 tetrahydrocannabinol (C-THC) were dissolved in 0.2 ml
of dimethylsulfoxide, and 0.74 mg of n-hydroxysuccini-
mide were added. After reaction at room temperature
for 2 hours, 0.5 ml of this mixture were added to 5 mg
of alkaline phosphatase in 3 ml of phosphate buffered
35 saline (PBS). The entire reaction mixture was mixed
overnight at room temperature.
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14.1.3. ASSAY PROCEDURE
Disks containing 3 ~C1 of anti-THC antibody
at 1:10 dilution were placed in wells of microtiter
plates. To these disks were added 100 ~1 of the THC-
alkaline phosphatase ligand at a 1:2000 dilution.
Plates were incubated for 30 minutes at room
temperature. To assess the effect of various
components on the stabilization of dried receptor-
ligand complex, various formulations were used to wash
the complex prior to drying. Wells were washed with
Tris buffer containing 0.1% Tween 20 or with Tris
buffer containing glucose. The disks were then
allowed to dry and 200 ~1 of standards containing 0 or
10 ~Cg/ml THC were added to the disks in wells. The
assay could then be read visually by adding substrate
to the membrane and noting a definite fading of color
when THC effected release. The reaction could also be
quantitated as described further. The plates were
incubated for 30 min at room temperature and 100 ~1 of
supernatant from each well were transferred to wells
in a clean replicate microtiter plate.
To each well of the replicate microtiter
plate were added 100 ~cl of p-nitrophenylphosphate
(Sigma). The plates were incubated for 15 minutes at
room temperature, and absorbance read at 405 nm.
The remaining incubation mixture of solid
phase antibody and conjugate was incubated for 30 min
at 37°C and 100 ~cl of this supernatant was transferred
to replicate wells in clean microtiter plates. To
each well were added 100 ~1 of p-nitrophenylphosphate
and the plates were incubated for 15 min at room
temperature, and absorbance measured at 405 nm.
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t.
~;.~(~I~;~w
r
14.2. RESULTS
The results of this assay are shown in
Table 17.
TABLE 17. RELEASE OF LABELED THC-ALKALINE
PHOSPHATASE CONJUGATE AS A FUNCTION
OF STABILIZATION OF RELEASE COMPLEX.
30' INCUBATION 30' INCUBATION,
ROOM TEMP ROOM
TEMP. PLUS
30' AT 37C
S~pLE TRIS/TWEEN TRIS/TWEEN/ TRIS/TWEEN TRIS/TWEEN
WASH GLUCOSE WASHWASH GLUGOSE
WASH
0 pg/ml 0.101 0.111 0.129 0.212
THC
10 Ng/ml 0.134 0.152 0.260 0.484
THC
These results show the optimization of
incubation times and formulation of washing buffer on
the release of THC:alkaline phosphatase conjugate from
antibody bound to membranes. In particular, the
. 20 presence of glucose in the washing buffer enhances
released enzyme activity in the supernatant. Most
importantly, these results indicate preference of the
antibody for the analyte in the sample, thus
demonstrating an effective release assay for THC.
~ It should be noted that the release reaction
visualized on membranes was much more dramatic.
15. EXAMPLE: RELEASE ASSAY FOR BETA-BLOCKERS
A homogeneous release assay for beta-
blockers is presented: Detection of beta-blockers:is
useful for monitoring patients on these drugs. Tn
particular, the stability of antibody complexes with
ligand was tested.
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15.1. MATERIALS AND METHODS
15.1.1. LIGAND: ANALYTE-ENZYME CONJUGATE
Atenolol acid (6.5 mg) (Imperial Chemical
Industries) was dissolved in 0.125 ml of 0.1 M sodium
carbonate with the aid of 0.015 ml of 1 N HC1. After
dissolution, 0.1 ml of deionized water and 0.25 ml of
dimethylsulfoxide were added, followed by 6 mg of N-
hydroxysuccinimide and 10 mg of EDC. The reaction
mixture was kept at room temperature for 2 hours. To
a glass tube were added 1 ml of 0.1 sodium carbonate
buffer, pH 9.0, and 2.8 mg of glucose-6-phosphate
dehydrogenase; after dissolving, 20 mg of reduced
nicotine adenine dinucleotide (NADH), 10 mg of
glucose-6-phosphate and 0.3 ml of carbitol [2-
ethoxyethoxy) ethanol] were added. This mixture was
kept at 4°C for 1 hour.
One hundred forty ~L of the activated
atenolol were added to the glucose-6-phosphate
dehydrogenase reaction vessel over a period of 90
minutes, in aliquots of 10 ~1. Fifteen minutes after
the last addition the material was transferred to a
dialysis bag and dialyzed overnight in the cold
against 2 1 of 0.05 M Tris-HCl, pH 7.9. The next day
dialysis wa.s continued for 8 hours with three changes
of f luid .
15.1.2. ASSAY SYSTEM
Reagent A (160 ~C1) was prepared with 1 gel of
antiserum (rabbit) against 4-hydroxypropranolol, and
12 u1 of O.11M glucose-6-phosphate in 0.05 M Tris
buffer, pH 7.9, 3.3 mM magnesium chloride.
Reagent B (30 ~l) was prepared with 20 ~cl of
conjugate of 1:500 dilution, and 8 ~,1 of 0.1 M
nicotine adenine dinucleotide inØ05 M Tris buffer,
pH 7.9.
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4, 1; :,.y:w~
y
A and B were mixed for 10 seconds and 10 ~cl
of sample or standard were added, followed by mixing
for 10 seconds. The mixture was incubated for the
indicated times at 37°C and absorbance at 340 nm
measured.
15.2. RESULTS
15.2.1. STABILITY OF ANTIBODY-CONJUGATE COMPLEX
These experiments were designed to determine
the stability of the antibody/beta-blocker-enzyme
complex.
The complex was made up with 1.41 ml of
0.05 M Tris-HC1, pH 7.8, 10 ~1 of undiluted antibody,
80 ~1 of 0.1 M NAD and, and 100 ~1 of conjugate at 2
mg/ml and kept at room temperature for the indicated
time periods. At specified times, samples containing
free 4-hydroxpropanolol were added to aliquots of the
complex and the mixture was incubated for 5 min at
room temperature. The reagent containing glucose-6-
phosphate was then added, and absorbance at 340 nm
measure over a 20 minute time period.
30
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~_s ':~
TABLE 18. CHANGE IN OD AFTER DIFFERENT INCUBATION
TIMES OF THE RECEPTOR-LIGAND COMPLEX.
(mA/min)
4-HYDROXY-
PROPRANOLOL 1' 10'
SAMPLE 30' 60'
120'
0.1 ~g/ml 3.918 2.256 2.083 1.034 4.197
1.0 ~g/ml 7.913 4.456 5.427 3.171 6.243
10 ~Cg/ml 12.875 12.855 13.720 10.210 12.759
SLOPE 2 1.097 1.619 1.425 1.555 1.540
I INTERCEPT ~ =0.120 I -0.580I -0.43309.533 -0.522
2 I I ,
In this assay the release time was 5 min. and
measurement time was 20 min.
'
Slope and intercept were calculated according to
the least squares method.
The similarity of the reaction statistics in
Table 18 indicates that the antibody/ligand-enzyme
complex was stable for at least 2 hours at room
_ temperature, and that effective release could occur in
the presence of free analyte during that time.
15.2.2. OPTIMIZING RELEASE TIME AND MEASUREMENT TIME
In addition to showing that the
antibody/ligand-enzyme conjugate was stable for at
' least two hours, the assay was optimized for release
time, i.e., the time after adding sample and NAD
substrate before measuring the change in OD, and
optimal measurement time, i.e., the time from start to
finish for measuring the change in OD. The results of
these optimization assays are shown in Tables 19 and
20.
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~~ ~ i, ;J
TA-BLE 19. OPTIMIZING RELEASE TIME.
mOD/MIN AFTER
4-HYDROXY-
PROPANOLOL 1 MINUTE 3 MINUTES 5 MINUTES
CONCENTRATION
O.1 ug/ml 1.167 0.859 2.895
1.0 3.928 11.382 6.007
10.0 18.334 18.816 9.,852
SLOPE2 1.743 0.857 1.0947
INTERCEPT2 -0.665 0.213 -0.117
Antibody/ligand-enzyme complexes were incubated
10 sec. prior to adding sample; mOD/MIN (rate of
absorbance change) was measured for 10 min.
Slope and intercept were calculated according to
the least squares method.
TABLE 20. OPTIMIZING READING TIME.
mOD/MIN AFTER
4-HYDROXY-
PROPANOLOL
CONCENTRATION 7 MINUTE 10 MINUTES 20
MINUTES
O.1 ug/ml 0.4?6 6.141 3.099
I.0 2.059 14.347 11.364
10.0 6.485 24.554 22.533
SLOPE2 1.481 1.0978 1.1362
INTERCEPT2 -0.4771 -0.1203 -0.1633
' Antibody/ligand-enzyme
complexes
were
incubated
10 min of sample; the release
prior
to
addition
time min.
in
this
assay
was
5
Slope intercept calculated according
and were to
the squares method.
least
WO 93/03367 PCf/US92/06249
Under the condition of this assay, a release
time of 5 min and a measurement time of NAD reduction
for 10 min provide optimum results, with the best
slope and intercept values.
5
16. EXAMPLE: HOMOGENEOUS RELEASE ASSAY FOR THIAZIDES
Thiazides are a class of diuretics, the most
well known of which is hydrochlorothiazide. Detection
of thiazides is important for medical treatment,
l0 particularly in emergency situations.
16.1. MATERIALS AND METHODS
16.1.1. LIGAND: THIAZIDE-GLUCOSE-6-PHOSPHATE
CONJUGATE
15 '
The ligand-enzyme conjugate was prepared as
follows: 15 mg of hydrochlorothiazide were dissolved
in 0.5 ml of 5 N sodium hydroxide, and placed in
boiling water for 30 minutes. The solution was cooled
30 to room temperature and acidified with 1 ml of 6 N
HC1: The tube was placed into an ice bath and 0.5 ml
of 0.2 M sodium nitrite added. The mixture was
allowed to remain at 0°C for 10 minutes, and then 0.5
ml of 7% ammonium sulfate were added.
25 The pH of the mixture was raised to between
5 and 6 using 100 ~,1 of 0.5 M sodium carbonate. In an
S ice bath 280 ~ul of the mixture were mixed immediately
with 2.8 mg of glucose-6-phosphate dehydrogenase in
1 ml of 0.1 M carbonate buffer, pH 9.0, containing
30 20 mg of NADH, 10 mg of glucose-6-phosphate and 300 ~ul
of carbinol, and allowed to remain at 0°C for 30
minutes. The reaction mixture was then dialyzed
against 0.05 M Tris buffer, pH 7.8, for 24 hours at
- 2-8°C with four changes of buffer.
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16.1.2. ASSAY
Reagent A (160 ~1) was anti-thiazide
antibody (rabbit), 1 ~1, in 147 dal Tris-HC1, pH 7.8,
with 12 ~l of 0.11 M glucose-6-phosphate. Reagent B,
(22 ~1) was 10 ~1 of conjugate at 1:250 dilution in
12 ~cl of Tris-HCl buffer, pH 7.8, containing 3 mM
magnesium. Reagents A and B were mixed and incubated
for 5 minutes at room temperature. Samples were added
in volumes of 10 ~C1. Incubation was continued at room
temperature for 3 minutes, and then 8 ~1 of 0.1 M NAD
were added. Absorbance at 340 nm was measured for a
10 min period..
16.2. RESULTS
i5 Free hydrochlorothiazide in sample caused
release of antibody from the ligand-enzyme conjugate,
resulting in a dramatic shift in the rate of
absorbance change. These results are summarized in
Table 21.
TABLE 21'. THIAZIDE RELEASE ASSAY.
HYDROCHLOROTHIAZIDE RATE OF
CONCENTRATION ABSORBANCE CHANGE
_(,juq~ m 1 ) ( mAU MIN )
0.1 0.030
1.0 9.501
10 15.399
17. EXAMPLE: IDENTIFICATION OF A LOW
AFFINITY ANALOG OF CONTINTNE BY
ITS ABILTTY TO COMPETE
The second principal urinary metabolite of
nicotine, trans-3-hydroxycotinine, was tested for its
ability to compete in the enzyme immunoassay for
cotinine, which utilizes an antibody raised against
carboxycotinine.
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_ s7 _ ~1 ~!~~~y~3
17.1. MATERIALS AND METHODS
Traps-hydroxycotinine or cotinine were added
to a synthetic urine matrix in levels from 0.1 to 5
~Cg/ml, and assayed in an ELISA for cotinine a mixture
of cotinine and traps-hydroxycotinine in a 1:3 ratio
were also assayed.
17.2. RESULTS
Traps-hydroxycotine was only 10% as
effective as cotinine at inhibiting binding in a
cotinine ELISA. The enhanced binding (greater
absolute OD values) in the presence of hydroxycotinine
is an effect seen before in inhibition assays with low
affinity ligands.
The ineffectiveness of hydroxycotinine as an
inhibitor can also be seen in the inhibition curve of
a 1:3 ratio of cotinine to hydroxycotinine. Although
present in 3-fold excess, the hydroxycotinine cotinine
combination is less inhibitory than cotinine alone.
There is no observable additive inhibition by
hydroxycotinine.
17.3. DTSCUSSION
The cotinine metabolite traps-hydroxycoti-
nine; which differs from cotinine only by the presence
of a hydroxyl group at C-3' in transorientation, binds
antibody to cotinine with much lower affinity than
cotinine. This is the characteristic necessary for a
useful release ligand, and in fact, ligands prepared
with traps-hydroxycatinine are.useful in assays fob
cotinine (see Sections 6.-11., supra).
The phenomenon of enhanced binding, i.e.,
greater absolute O.D. values, in the presence of a low
affinity analog is not well understood. It may be due
to inhibition of cross-reactive antibodies.
WO 93/03367 PCTJUS92/06249
~L~;~ _ ss _
Nevertheless, enhancement of binding in the presence
of low concentrations of a potential inhibition
indicates useful low affinity ligand for release
assays.
18. USE OF LOW AFFINITY CHROMATOGRAPHY
FOR ISOLATION OF ANTIBODIES
Use of low of f inity 1 igands f or
chromatography to purify antisera is advantageous
because elution under mild conditions yields higher
antibody recovery. Such antibodies find particular
use in release assays.
Isolation of antibodies to human chorionic
gonadotropin (hCG) was accomplished using a column to
which a crude extract of sheep gonadotropic hormones
had been covalently bound. While these sheep hormones
are not biologically active in humans, nor do they
crossreact with anti-hCG, there remains enough
sequence conservation, especially with sheep
luteinizing hormone (sLH), to be able interact with
antibodies to hCG under controlled conditions.
18.1. ~iPrTERIALS AND METHODS
18.1.1 . PREPARATION O~ AFFINITY COLUMN
A crude extract of sheep pituitary
gonadotropins was attached to sepharose using 1 g of
cyanogen bromide activated sepharose 4B. The gel was
placed in 1.6 ml of 0.1 M carbonate buffer, pH 8.0,
containing 0.5 M sodium chloride. 24 mg of the crude
extract were added and the material mixed for 3 hours
at room temperature. The gel was centrifuged,
supernatant discarded, and the gel resuspended in
0.01 M Tris buffer, pH 8.0, and kept at 4°C overnight.
The gel was centrifuged again, supernatant
discarded, and the gel suspended in 10 ml of 0.01 M
~..._. ~.:. _. R~ ,rr s ,~. .~ .=x..~~. :,r,..~. r. , ,.,.. .., ~.,..~.. .,~.
~ ",~ .z~...~.~,r..~ ,._ _. .-..T., .~ ... ~..z..~..~. . r_ _. .
WO 93/03367 PGT/US92/06249
-
Tris Buffer, pH 8.0, containing 0.2 M glutamic acid.
This suspension was incubated for 1 hour with mixing.
Then the gel was packed 'into a 1 x 10 cm column and
washed sequentially with 0.1 M acetate buffer, pH 4.0,
0.5 M sodium chloride, and 0.1 M sodium carbonate
buffer, pH 8.0, containing 0.5 M sodium chloride. The
column was then equilibrated with phosphate buffer, pH
7.0, containing 0.5 M sodium chloride and 0.01% sodium
azide.
18.1.2. AFFINITY CHROMATOGRAPHY
The column was primed with 1 ml of normal
rabbit serum overnight at 4°C the serum was eluted
with phosphate buffered saline (PBS) followed by
rinses with the buffers of pH 4 and 8. 1 ml of rabbit
antiserum to hCG with a titer of 1:100,000 was applied
to the column and allowed to sit 10 min. The column
was sequentially eluted with PBS, pH 4 buffer and pH 8
buffer. Protein content of collected fractions was
determined by the Lowry method, and anti-hCG activity
by a solid phase competitive enzyme immunoassay.
18.2. RESULTS
The chromatogram of protein content and
antibody activity is shown in Figure 14. The bulk of
the protein eluted in the void volume of the column,
while the majority of the antibody activity eluted
just after the void volume in protein-poor fractions.
A smaller protein peak eluted in pH 4 buffer, no
grotein eluted in pH $ buffer.
The titer of the pooled fractions of
antiserum was 1:91,200 and the protein content 2.7 mg,
showing an activity recovery of 91%, far higher than
that reported previously in the literature for
affinity-purified antibodies. The specific activity
WO 93/03367 PCT/US92/06249
t
~',~~~ i~~j - 90 -
~i
of the starting antiserum was 2200 U/mg, while that of
the pooled purified fractions was 37,000 U/mg, a 17-
fold purification.
In order to show that the recovery of
antibody by this method was not due to the properties
of the Sepharose itself, a column of Sepharose 48 was
prepared and normal rabbit serum and antiserum applied
and eluted as described previously. All antibody
activity eluted in the void volume with the protein;
none was retained on the column to be eluted
of terward .
The antisera used in the experiment showed a
10% cross-reactivity with sLH after the affinity
chromatography this level of cross-reactivity did not
change.
18.3: DISCUSSION
The purification of antibodies using low
affinity analogs attached to affinity chromatography
gels appears to permit efficient recovery of
antibodies without denaturation and with enhanced
specific activity for antigen.
Affinity purification schemes also provide a
method to identify a low affinity analog by its
ability to retard elution of specific antibody in a
mild eluent. According to the above example, in a
release assay for hCG, sheep gonadotropins may serve
as ligands.
The present invention is not to be limited
in scope by the specific. embodiments described herein.
Indeed, various modifications of the invention in .
addition to those described herein will become
apparent to those skilled in the art from the
foregoing description and accompanying figures. Such
WO 93/03367 PCT/US92/06249
- 91 - ~ '~ ' /~ ~ ,~l
.,
modifications are intended to fall within the scope of
the appended claims.
10
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