Note: Descriptions are shown in the official language in which they were submitted.
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HOMOCYSTEINE IMMUNOASSAY
FIELD OF THE INVENTION
The present invention relates generally among other things to a system for
the detection of homocysteine using an immunoassay. In particular, the
invention
relates among other things to diagnostic tests, methods of use, and kits
related to the
assessment of homocysteine levels in a biological sample. Optionally in such
an
assay the influence of inhibiting substances in the biological sample is
eliminated by
inclusion in the assay of a polyanion.
BACKGROUND OF THE INVENTION
The recognition in recent years of high values of plasma homocysteine as
being one of the dangerous factors for cardiovascular disease has resulted in
an
increased awareness in the clinical field of the necessity of measurement of
homocysteine levels in serum or plasma.
One method for the measurement of homocysteine using an immunological
means has been developed by Erling et al. (Japanese Patent Laid-Open No.
05/513,023). In this competitive immunoassay the homocysteine concentration is
measured after a pretreatment step comprising a first stage where homocysteine
2 0 bound to components in blood by a disulfide bond is dissociated, and a
second stage
where the dissociated homocysteine is reacted with an enzyme (S-
adenosylhomocysteine hydrolase) together with an auxiliary substrate
(adenosine)
so as to convert the homocysteine into a form capable of being measured by
immunoassay.
Other options for measuring the concentration of homocysteine include
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separation and measurement conducted based on physical and chemical properties
of homocysteine using high performance liquid chromatography (HPLC) (Journal
of Chromatography B, volume 779, number 2, November 5, 2002, pages 359 to 363)
or mass spectral analysis (MS). These methods are complicated and not easily
adapted to testing of large numbers of clinical samples.
Thus, to date there has been development of a measuring method for
homocysteine that is superior to an immunoassay in view of simplicity and
convenience. However, when serum or plasma samples are measured by
conventional immunoassays for homocysteine, error of the measurement results.
This is believed to be due to the influence of inhibiting substances present
in blood.
In order to eliminate the influence of such inhibiting substances, a
treatment method has been developed for use in conventional and automated
immunoassays. In this method the biological sample is directly diluted or the
volume of the reaction solution is increased. Such a treatment method is
disadvantageous in that it requires increased labor for the measurement of
homocysteine and results in an unnecessary decrease in sensitivity due to the
dilution. Moreover, in the measurement by a fully automated measuring
apparatus,
the dilution step causes a decrease in the speed of sample processing as well
as an
inability of some of the measuring apparatuses to handle the increased volume
of
the reaction solution.
In other assays and systems separate and apart from a homocysteine
irnmunoassay, polyanions such as heparin have been employed for a variety of
different reasons. In Japanese Patent Laid-Open No. 08/145,998, Mori et al.
added
heparin in an immunoassay of insulin-like growth factor to inhibit the
recombination of insulin-like growth factor with insulin after treatment of
the
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biological sample liberating the factor from insulin. Baker and Ishikawa et
al. use
polyanion as a connecting substance for making antigen or antibody into a
solid
phase (Japanese Patent Laid-Open No. 07/507,871 and Japanese Patent Laid-Open
No. 02/168,162). Yoshimura et al. use polyanion in a chromatographic assay
device for the neutralization of polycation employed for the separation of red
blood
cells (Japanese Patent Laid-Open No. 2002/509,254). Kurokawa et al. teach that
the influence of an interfering substance in an immunoassay using a complete
antibody is eliminated by addition of heparin (Japanese Patent Laid-Open No.
08/029,420). And, in a chromatographic assay system using colloid particles,
Sakamoto et al. report that the non-specific aggregation reaction is inhibited
and the
generation of false positives avoided by the addition of heparin to a blood
sample
(Japanese Patent Laid-Open No 07/151,754). -
Based on the foregoing, there remains a need for the development of a
method whereby the influence of inhibiting substances in an immunoassay of
homocysteine is eliminated. Optimally such a method can be done without
requiring increased labor or sample processing time, without substantially
diluting
the sample, and/or without deteriorating the sensitivity of the assay.
Therefore, it
is an object of the invention to provide among other things diagnostic tests,
methods
of use, and kits for the assessment of homocysteine levels in a biological
sample,
optionally making use of a polyanion. Optimally the tests, methods and kits of
the
invention avoid some of the pitfalls in homocysteine testing which are
inherent in
the currently used methodologies. These and other objects will be apparent
from
the description provided herein.
The foregoing discussion of background information is provided merely to
assist the reader in understanding the invention and is not admitted to
describe or
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constitute prior art to the invention.
SUMMARY OF THE INVENTION
Among other things the present description provides an improvement of a
immunoassay of a biological sample (e.g., an assay of a factor for
cardiovascular
disease, including a homocysteine immunoassay), characterized in that the
sample is
reacted with antibody in the presence of an extrinsic polyanion. Optionally
the
polyanion is selected from the group consisting of heparin, polyacrylic acid,
and
dextran sulfate. In one embodiment as described herein, the polyanion
concentration
ranges from about I p.g/mL to about 100 mg/mL.
Optionally the method of the invention can be employed for an
immunoassay (e.g., an assay of a factor for cardiovascular disease, including
a
homocysteine immunoassay) using any sort of appropriate immunoreaction carried
out on any appropriate instrument. In one embodiment the immunoassay
comprises a competitive immunoassay. In another embodiment the immunoassay
is carried out using an automated measuring apparatus. In yet another
embodiment,
the immunoassay comprises a sandwich assay.
The invention thus provides a method for assaying a biological sample for
an analyte of interest (e.g., a cardiovascular antigen such as homocysteine),
optionally wherein the method comprises:
(a) obtaining a biological sample from a subject (e.g., from a human
subject);
(b) reacting the biological sample with antibody specific for analyte
(e.g., with antibody that reacts with homocysteine) in the presence of
polyanion;
2 5 (c) detecting the binding of analyte (e.g., homocysteine) present in the
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sample with said antibody by any appropriate means; and
(d) quantifying the binding as a measure of the amount of the analyte
(e.g., homocysteine) present in the sample. Optimally the reaction of the
antibody
with analyte in the presence of polyanion is done where the polyanion is
either
added before, during, or after the reaction of the antibody with analyte_ In
one
embodiment, polyanion is added either before or during the reaction of the
antibody
with analyte.
Also provided by the description herein are kits to be used for the
immunoassay according to by the invention (e.g., an assay of a factor for
cardiovascular disease, including a homocysteine immunoassay), wherein the kit
comprises a polyanion.
These and other features, aspects, objects, and embodiments of the
invention will become more apparent in the following detailed description
(including the drawings) which contains information on exemplary features,
aspects,
objects and embodiments of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of changes in signal intensity caused by addition of the
polyanion heparin in a measuring system for homocysteine, as described in
Example
1. The ordinate shows the measured signal whereas the abscissa shows the
concentration of the added heparin in the reaction ( g/mL). Symbols: -~-,
serum being measured in an undiluted state; -s-, serum being measured in high
concentration; -A-, plasma in blood collected with heparin.
FIG. 2 is a graph of the ratio of the concentration of homocysteine in the
absence of the polyanion heparin as compared to the known value measured using
a
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chemiluminescence automated measuring apparatus, as described in Example 2.
The
ordinate shows the measured values for known values (%) whereas the abscissa
shows known homocysteine concentrations ( M).
FIG. 3 is a graph of the ratio of the concentration of homocysteine in the
presence of the polyanion heparin as compared to the known value measured
using a
chemiluminescence automated measuring apparatus, as described in Example 2.
The ordinate shows measured values for known values (%) whereas the abscissa
shows known homocysteine concentrations ( M).
FIG. 4 is a bar chart showing the influence of polyanion on the dissociation
rate where the apparent homocysteine concentration is from a "high"
concentration
sample group and a "normal" concentration sample group, as described in
Example
3. The ordinate shows dissociation rate whereas the abscissa shows the amount
of
polyanion added. Bars (left to right): (a) no polyanion added; (b) 4.2 g/mL
of
heparin; (c) 14 g/mL of heparin; (d) 42 g/mL of heparin; (e) 84 g/mL of
heparin;
(f) 420 g/mL of heparin; (g) 420 g/mL of dextran sulfate; (h) 42 g/mL of
polyacrylic acid; (i) 420 g/mL of polyacrylic acid; (j) 2.8 mg/mL of gelatin;
and
(k) 281 g/mL of bovine y-globulin.
DETAILED DESCRIPTION OF THE INVENTION
The present description relates to a method for improving an immunoassay
for by the addition of a polyanion. Not willing to be bound by any theory,
after
intensive investigation as described herein, it surprisingly has been
discovered that a
substance which is the same as or similar to polyanion in terms of either
structure of
function appears to be causing inhibition of the immunoreaction in a
homocysteine
immunoassay, and that variations in the amount of this polyanion-like
substance
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contained in a sample result in dispersion in immunoassayed values of
homocysteine. Because it typically is not easy to remove a specific substance
existing in blood without deleteriously impacting assay results, the
description
herein provides a method and means for substantially reducing or eliminating
the
influence of the immunoreaction inhibiting substance by adding to the
immunoassay
a sufficient amount of a polyanion.
The present invention thus provides, among other things, diagnostic tests,
methods of use, and kits for the assessment of a cardiovascular factor such as
homocysteine. These and additional embodiments, features, aspects,
illustrations,
and examples of the invention are further described in the sections which
follow.
Definitions
Unless defined otherwise herein, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary skill
in
the art to which this invention belongs.
"Samples" or "biological samples" that can be assayed using the methods
of the present invention include biological fluids, such as whole blood,
serum,
plasma, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid,
bone
marrow aspirate, pleural effusion, urine, as well as tumor tissue or any other
bodily
constituent or any tissue culture supernatant that could contain the analyte
of
2 0 interest. Preferred biological samples in an immunoassay of homocysteine
are
further described below.
"Analyte," as used herein, refers to the substance to be detected, which may
be present in the sample (i.e., the biological sample). The analyte can be any
substance for which there exists a naturally occurring specific binding
partner or for
which a specific binding partner can be prepared. Thus, an analyte is a
substance
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that can bind to one or more specific binding partners in an immunoassay. One
example of an analyte as described herein is an endogenous antigen, including
but
not limited to homocysteine.
A "binding partner," as used herein, is a member of a binding pair, i.e., a
pair of molecules wherein one of the molecules binds to the second molecule.
Binding partners that bind specifically are termed "specific binding
partners." In
addition to the antigen and antibody binding partners commonly used in
immunoassays, other specific binding partners can include biotin and avidin,
carbohydrates and lectins, complementary nucleotide sequences, effector and
receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and
the
like. Furthermore, specific binding partners can include partner(s) that
is/are
analog(s) of the original specific binding partner, for example, an analyte-
analog.
Immunoreactive specific binding partners include antigens, antigen fragments,
antibodies and antibody fragments, both monoclonal and polyclonal, and
complexes
thereof, including those formed by recombinant DNA methods.
As used herein, the term "epitope", "epitopes" or "epitopes of interest"
refer to a site(s) on any molecule that is recognized and is capable of
binding to a
complementary site(s) on its specific binding partner. The molecule and
specific
binding partner are part of a specific binding pair. For example, an epitope
can be
2 0 a polypeptide, protein, hapten, carbohydrate antigen (such as, but not
limited to,
glycolipids, glycoproteins or lipopolysaccharides) or polysaccharide and its
specific
binding partner, can be, but is not limited to, an antibody, e.g., an
autoantibody.
Typically an epitope is contained within a larger antigenic fragment (i.e.,
region or
fragment capable of binding an antibody) and refers to the precise residues
known
to contact the specific binding partner. It is possible for an antigenic
fragment to
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contain more than one epitope.
As used herein, "specific" or "specificity" in the context of an interaction
between members of a specific binding pair (e.g., an antigen and antibody)
refers to
the selective reactivity of the interaction. The phrase "specifically binds
to" and
analogous terms thereof refer to the ability of antibodies to specifically
bind to an
analyte (e.g., an endogeneous antigen such as homocysteine) and not
specifically
bind to other entities. Antibodies or antibody fragments that specifically
bind to an
analyte can be identified, for example, by diagnostic immunoassays (e.g.,
radioimmunoassays ("RIA") and enzyme-linked immunosorbent assays ("ELISAs")
(See, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press,
New
York, pages 332-336 (1989)), BIAcore (Sweden), KinExA (Kinetic Exclusion
Assay, available from Sapidyne Instruments (Boise, Idaho)) or other techniques
known to those of skill in the art. The term "specifically binds" indicates
that the
binding preference (e.g., affinity) for the target molecule/sequence is at
least 2-fold,
more preferably at least 5-fold, and most preferably at least 10- or 20-fold
over a
non-specific target molecule (e.g. a randomly generated molecule lacking the
specifically recognized site(s)).
A "solid phase," as used herein, refers to any material that is insoluble, or
can be made insoluble by a subsequent reaction. The solid phase can be chosen
for
its intrinsic ability to attract and immobilize a capture agent.
Alternatively, the
solid phase can have affixed thereto a linking agent that has the ability to
attract and
immobilize the capture agent. The linking agent can, for example, include a
charged substance that is oppositely charged with respect to the capture agent
itself
or to a charged substance conjugated to the capture agent. In general, the
linking
agent can be any binding partner (preferably specific) that is immobilized on
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(attached to) the solid phase and that has the ability to immobilize the
capture agent
through a binding reaction. The linking agent enables the indirect binding of
the
capture agent to a solid phase material before the performance of the assay or
during
the performance of the assay. The solid phase can, for example, be plastic,
derivatized plastic, magnetic or non-magnetic metal, glass or silicon,
including, for
example, a test tube, microtiter well, sheet, bead, microparticle, chip, and
other
configurations known to those of ordinary skill in the art.
As used herein, term "microparticle" refers to a small particle that is
recoverable by ultracentrifugation. Microparticles typically have an average
diameter on the order of about 1 micron or less.
The term "capture agent" is used herein to refer to a binding partner that
binds to analyte, preferably specifically. Capture agents can be attached to a
solid
phase. As used herein, the binding of a solid phase-affixed capture agent to
analyte forms a "solid phase-affixed complex."
The term "labeled detection agent" is used herein to refer to a binding
partner that binds to analyte, preferably specifically, and is labeled with a
detectable
label or becomes labeled with a detectable label during use in an assay.
A "detectable label" includes a moiety that is detectable or that can be
rendered detectable.
As used with reference to a labeled detection agent, a "direct label" is a
detectable label that is attached, by any means, to the detection agent.
As used with reference to a labeled detection agent, an "indirect label" is a
detectable label that specifically binds the detection agent. Thus, an
indirect label
includes a moiety that is the specific binding partner of a moiety of the
detection
agent. Biotin and avidin are examples of such moieties that are employed, for
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example, by contacting a biotinylated antibody with labeled avidin to produce
an
indirectly labeled antibody.
As used herein, the term "indicator reagent" refers to any agent that is
contacted with a label to produce a detectable signal. Thus, for example, in
conventional enzyme labeling, an antibody labeled with an enzyme can be
contacted
with a substrate (the indicator reagent) to produce a detectable signal, such
as a
colored reaction product.
As used herein, an "antibody" refers to a protein consisting of one or more
polypeptides substantially encoded by imniunoglobulin genes or fragments of
immunoglobulin genes. This term encompasses polyclonal antibodies, monoclonal
antibodies, and fragments thereof, as well as molecules engineered from
immunoglobulin gene sequences. The recognized immunoglobulin genes include
the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as
well as myriad immunoglobulin variable region genes. Light chains are
classified
as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha,
delta,
or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,
IgD
and IgE, respectively_
A typical immunoglobulin (antibody) structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of polypeptide
chains,
each pair having one "light" (about 25 kD) and one "heavy" chain (about 50 -
70
kD). The N-terminus of each chain defines a variable region of about 100 to
110
or more amino acids primarily responsible for antigen recognition. The terms
"variable light chain (VL)" and "variable heavy chain (VH)" refer to these
light and
heavy chains respectively.
Antibodies exist as intact immunoglobulins or as a number of well-
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characterized fragments produced by digestion with various peptidases. Thus,
for
example, pepsin digests an antibody below the disulfide linkages in the hinge
region
to produce F(ab')2, a dimer of Fab which itself is a light chain joined to VH-
CH I by
a disulfide bond. The F(ab')2 may be reduced under mild conditions to break
the
disulfide linkage in the hinge region thereby converting the (Fab')2 dimer
into a
Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge
region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993),
for a more detailed description of other antibody fragments). While various
antibody fragments are defined in terms of the digestion of an intact
antibody, one
of skill will appreciate that such Fab' fragments may be synthesized de novo
either
chemically or by utilizing recombinant DNA methodology.
Thus, the tenn "antibody," as used herein also includes antibody fragments
either produced by the modification of whole antibodies or synthesized de novo
using recombinant DNA methodologies. Preferred antibodies include single chain
antibodies (antibodies that exist as a single polypeptide chain), more
preferably
single chain Fv antibodies (sFv or scFv), in which a variable heavy and a
variable
light chain are joined together (directly or through a peptide linker) to form
a
continuous polypeptide. The single chain Fv antibody is a covalently linked VH-
VL heterodimer which may be expressed from a nucleic acid including VH- and
2 0 VL- encoding sequences either joined directly or joined by a peptide-
encoding
linker. Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While
the VH and VL are connected to each as a single polypeptide chain, the VH and
VL
domains associate non-covalently. The scFv antibodies and a number of other
structures converting the naturally aggregated, but chemically separated,
light and
heavy polypeptide chains from an antibody V region into a molecule that folds
into
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a three dimensional structure substantially similar to the structure of an
antigen-
binding site are known to those of skill in the art (see, e.g., U.S. Patent
Nos.
5,091,513, 5,132,405, and 4,956,778).
As used herein, the singular forms "a", "an" and "the" include plural
references unless the context clearly dictates otherwise.
As used herein, the term "about" refers to approximately a-H-10%
variation from the stated value. It is to be understood that such a variation
is always
included in any given value provided herein, whether or not it is specifically
referred to.
Immunoassay
Thus the present invention provides among other things an immunoassay of
a biological sample, including an imrnunoassay of a cardiovascular factor,
e.g.,
homocysteine, present in the sample. The method comprises inclusion of a
polyanion in the immunoassay (e.g., a homocysteine immunoassay) in the
reaction
of the biological sample with an antibody. When the immunoassay of the present
invention is used, it is possible to reduce or eliminate the influence of
inhibiting
substances existing in a sample without substantial dilution of the sample, or
without using a large amount of an assay buffer solution, at the same time
giving a
highly sensitive and highly reliable measured value of homocysteine in a
simple and
convenient manner. This is particularly advantageous where the immunoassay
(e.g., the homocysteine immunoassay) is fully automated and a large amount of
sample is to be treated within a short time.
The description provided herein pertains among other things to a
homocysteine immunoassay. However, it is expected that the methods described
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herein can be more generally applied, and that the inclusion of exogenous
polyanion
in the reacting step of a biological sample with an antibody against the
antigen of
interest would reduce variance in measurement of other analytes, particularly
other
cardiovascular factors (e.g., endogenous cardiovascular antigens) in
biological
samples.
The immunoassay employed in the method of the present invention can be
any immunoassay so long as it is a detection (e.g., quantitative) method that
utilizes
an antigen-antibody reaction. Accordingly, it can be any of the methods
routinely
employed for immunoassay including but not limited to a competitive method and
a
non-competitive method such as a sandwich method.
Biological Sample Collection and Processing
The assay methods of the invention are generally carried out on samples
derived from an animal, preferably a mammal, and more preferably a human.
These methods can be carried out on samples from asymptomatic subjects or
subjects with one or more symptoms of disease.
The methods of the invention can be carried out using any sample that may
contain analyte of interest, e.g., that may contain homocysteine. Convenient
samples include, for example, blood, serum, and plasma. The biological sample
which is an object for the measurement of homocysteine in the iinmunoassay of
the
present invention can be any biological sample so long as it is a liquid
sample
derived from a living organism such as a body fluid or a tissue extract and,
usually,
it is preferred to use serum, plasma or urine.
The sample may be pretreated, as necessary or desired, by dilution in an
appropriate buffer solution or other solution, or optionally may be
concentrated.
2 5 Any of a number of standard aqueous buffer solutions, employing any of a
variety
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of buffers, such as phosphate, Tris, or the like, optionally at physiological
pH, can
be used.
In regards to a homocysteine immunoassay in particular, since many
homocysteines bind to other thiol or protein such as albumin by means of a
disulfide
bond in a biological sample, it is typical for the measurement of total
homocysteine
in plasma, urine, and other samples to subject the sample to pretreatment with
a
reducing agent such as dithiothreitol (DTT).
Because no antibody which selectively recognizes homocysteine (e.g., as
present in untreated biological sample) is yet available, it further is
typical in an
immunoassay for homocysteine that the homocysteine is subjected to an
enzymatic
treatment or the like to be converted into a molecule which can be recognized
by
antibody. The enzyme and any auxiliary substrate used in such a pretreatment
step
can be any appropriate reagents so long as they are enzyme and auxiliary
substrate
which are able to convert homocysteine into a molecule which is can be
measured
immunologically. For example, it is common to use S-adenosylhomocysteine
hydrolase as an enzyme and adenosine as an auxiliary substrate. In that case,
homocysteine is converted to S-adenosylhomocysteine and is subjected to an
immunoassay.
An auxiliary substrate such as adenosine preferably is charged in a solution
of a reducing agent, but optionally ca be added to any reagent so long as it
is a
reagent which can be added to a pretreated solution or during the first
reaction
without deleteriously impacting the reaction.
Polyanion
A polyanion is "exogenous" in the sense that it typically is added to an
immunoreaction, as described herein. Although the optimum amount of the
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polyanion used according to the present invention varies depending upon the
type of
the polyanion employed, optionally not less than about 1 g/mL of polyanion is
added during the reaction with a sample. Typically the serum amount in the
reaction solution is from about 6% to about 20%, optionally about 10%. Use of
not less than about 1 g/mL of polyanion typically provides that the influence
of the
inhibiting substances is substantially reduced, if not completely eliminated.
Regardless of the magnitude of impact, however, use of not less than about 1
g/mL
of polyanion in a homocysteine immunoassay will allow an effect of the
addition to
be observed. In some circumstances, it may be desirable to include a lesser
amount
of polyanion in the homocysteine assay. For instance, use of too much
polyanion
could result in an increase in manufacturing cost and also cause difficulties
in terms
of mechanical operation (e.g., such as insufficient dispensing amount due to
an
increase in viscosity). Although the upper limit of the amount used varies
depending upon the type of polyanion employed, optionally it is no more than
about
100 mg/mL. Although it depends upon the type of polyanion and the amount of
serum used for the immunoassay, the optimum polyanion concentration is,
therefore,
within a range of from about 1 g/mL to about 100 mg/mL (e.g., from about 1
g/mL to about 100 g/mL, from about 100 g/mL to about 100 mg/mL, or from
about 50 g/mL to about 50 mg/mL) during the reaction of the antibody with the
biological sample, in an immunoassay in which the serum or plasma amount in
the
reaction solution is about 10%. There is a tendency that, when the sample
amount
is a little, the effect is noted with less concentration, whereas when the
sample
amount is high, a significant effect is noted by higher concentration.
For the exogenous (added) polyanion to achieve a desired effect in the
present invention, it is necessary that the polyanion coexists during the
reaction of
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homocysteine in the sample with the antibody. Any route may be employed for
addition of the polyanion. Thus, polyanion may be added to any reagent so long
as
it is a reagent which participates and does not interfere with the reaction of
homocysteine with antibody. For example, exogenous polyanion may be added to
any solution such as solid-phase antibody solution, labeled solution, assay
buffer
solution, pretreatment solution, and the like. Exemplary solutions include but
are
not limited to: Tris buffer; phosphate buffer; borate buffer; Good's buffer;
SSC
buffer; TBE buffer; TAE buffer; and any buffer that is routinely employed in
an
immunoassay.
In the present description, a' polyanion" is a molecule in which the anion is
present in multivalent form, i.e., is in more in one molecule such that there
is a
valence of three or more, and optionally is tetravalent. Where the anion
existing in
one molecule is more than decavalent (i.e., has a valence greater than ten)
and the
molecular weight is several hundred or more, the molecule shows a significant
property as polyanion. Optimally the upper limit of molecular weight of a
polyanion employed as described herein is set within limits such that the
viscosity
does not deleteriously impact the measuring system (i.e., immunoassay).
Typically,
a molecular weight of several hundred thousand to several million is the upper
limit
for the polyanion.
With regard to the polyanion used in the present invention, any type may be
used so long as it meets the aforementioned definition. Examples of suitable
polyanions include but are not limited to: polyacrylic acid where the carboxyl
group
is present in a multivalent state in a polymer chain of carbon, and substances
similar
thereto; dextran sulfate where the polysaccharide is substituted with a
sulfate group;
heparin; heparin sulfate; poly(methyl methacrylate) (PMMA); poly(vinylsulfonic
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acid) (PVSA); poly-L-aspartic acid; and carboxymethyl cellulose (CMC). Other
polyanions that optionally can be employed include but are not limited to
chondroitan- sulfate, hyaluronic acid, dermatan sulfate, and dextran sulfate.
Such a
polyanion optionally can be used in the form of a salt, e.g., sodium salt,
lithium salt,
or other similar salt. The polyanion used can be a sole polyanion, or can be a
mixture of different types of polyanions (e.g., a so-called "plurality"
wherein the
plurality optionally comprises between two and five, added either
simultaneously or
sequentially).
Antibody
The antibody used in the present invention preferably is an antibody that is
able to recognize analyte of interest (e.g., homocysteine). Optionally the
homocysteine is first subjected to a conversion'treatment so as to render it
capable
of being recognized by an antibody. For example, when the sample is previously
treated with S-adenosylhomocysteine hydrolase, an anti-S-adenosylhomocysteine
antibody is used. Such an antibody can be any polyclonal antibody or
monoclonal
antibody. Moreover, the antibody can be not only a complete antibody but also
any
type of an antibody fragment including Fab, Fab', and F(ab')2, or antibody
fragment
where only the active site is taken out by means of genetic recombination, so
long
the antibody provides a specific activity (i.e., a reactivity).
Scoring
The immunoassays according to the invention optimally are scored in
accordance with standard practice and, optionally include the use of positive
and/or
negative controls and/or or standards (calibrators) containing known
concentrations
of antibodies to the analyte of interest. The level of analyte (e.g.,
homocysteine)
.25 optionally is compared with a control level or control range, which can be
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determined when the assay is carried out or, more conveniently, can be
predetermined.
Other Reagents
With regard to the other reagents used in the immunoassay of the present
invention (e.g., reagents such as antibody, labeled substance, reducing sugar
and
enzyme), substances which are routinely used in homocysteine immunoassays can
likewise be employed in the assay as described herein according to their
ordinary
and customary conditions for use. This is further expanded upon below.
Immunoassay Methods - In General
The immunoassay methods of the invention can be carried out in any of a
wide variety of formats. These forn--ats merely are modified as described
above to
include polyanion in the reacting step of analyte antigen (e.g., homocysteine)
with
antibody. For a general review of immunoassays, see Methods in Cell Biology
Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York
(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991),
which is incorporated by reference in its entirety.
In particular embodiments, an inununoassay method of the invention can be
performed by contacting a biological sample suspected of containing an analyte
of
interest, with an antibody reactive therewith in the presence of polyanion,
and under
conditions suffcient for binding of the antibody to any analyte present in the
biological sample. Analyte is detected/quantitated by detecting complex(es)
comprising the analyte antigen bound to the reactive antibody. Such assays can
be
homogeneous or heterogeneous (i.e., employing a solid phase). In heterogeneous
assays, a capture agent that binds to the analyte is typically affixed to a
solid phase.
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Analytes such as homocysteine can be measured in a non-competitive
immunoassay,
wherein the amount of analyte bound to antibody is positively correlated with
the
concentration of analyte present in the biological sample.
In other embodiments, the biological sample is contacted with the antibody
reactive with analyte (and which may, but need not, be affixed to a solid
phase) and
also contacted with another antibody that reacts with analyte so as to form a
"sandwich" where the analyte is bound between two antibody reagents. Analyte
is
detected/quantitated by detecting complex(es) comprising the antigen bound to
the
reactive antibodies.
For example, in one format of a sandwich immunoassay, an embodiment of
the invention, the first antibody is affixed to a solid phase, binding of
analyte
antigen present in the biological sample to the antibody forms a solid phase-
affixed
complex, and detecting comprises detecting a signal from the solid phase-
affixed
complex. In particular embodiments of this format, the solid phase-affixed
complex is detected using a second antibody also reactive with analyte antigen
and
that is directly or indirectly labeled. The bound entities are separated, if
necessary,
from free labeled antibody, typically by washing, and the signal from the
bound
label is detected.
Analyte (e.g., antigen such as homocysteine) can also be measured in
competitive immunoassay, wherein the signal is negatively correlated with the
concentration of analyte present in the biological sample. In an example of a
competitive format, the biological sample is contacted with an antibody (which
may,
but need not, be affixed to a solid phase) and also is contacted with
competing
labeled (directly or indirectly) antigen. This step is carried out under
conditions
sufficient for specific binding of the labeled antigen and analyte antigen to
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antibody. The labeled antigen and analyte antigen compete with each other for
binding to the antibody. Accordingly, the higher the level of analyte antigen
(such
as homocysteine) in a biological sample, the lower is the binding of labeled
antigen
to the antibody. The biological sample may be contacted with the labeled
antigen
and the antibody either simultaneously or sequentially, in any order.
Competitive immunoassays of this type can be conveniently carried out
using a solid phase-affixed antibody. In this case, binding of the analyte
antigen
present in the biological sample to antibody forms a solid phase-affixed
complex,
and detection entails detecting a signal from the solid phase-affixed complex.
The.
bound entities are separated, if necessary, from free labeled antigen,
typically by
washing, and the signal from any bound label (displacing analyte antigen) is
detected.
Capture Agent
Capture agents useful in the immunoassay methods of the invention include
those that bind to analyte antigen (e.g., homocysteine) and can be affixed to
a solid
phase. Convenient capture agents include antibodies specific for the analyte
antigen.
Analyte Antigens
Any endogenous antigen can be used (e.g., assessed as the analyte antigen
or included in a kit as a calibrator or control) in the immunoassay methods of
the
invention.
In particular embodiments, the endogenous antigen is an endogenous
antigen amino acid sequence that can be derived from any organism. Endogenous
antigen amino acid sequences useful in the invention are generally those
derived
from vertebrates, preferably from birds or mammals, more preferably from
animals
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having research or commercial value or value as pets, such as mice, rats,
guinea pigs,
rabbits, cats, dogs, chickens, pigs, sheep, goats, cows, horses, as well as
monkeys
and other primates. In particular embodiments, the endogenous antigen amino
acid
sequence is derived from a human polypeptide.
The methods of the invention can employ full-length endogenous antigens
or one or more fragments thereof. Fragments will generally have at least one
epitope to which an antibody can bind. Such fragments can have a length, e.g.,
of
about 125, 100, 75, 50, 25, or 15 amino acids or a length that falls within a
range
with endpoints defined by any of these values (e.g., 15-125, 25-100, 50-75, 15-
100,
etc.).
The endogenous antigen amino acid sequence can be a wild-type amino
acid sequence or an amino acid sequence variant of the corresponding region of
a
wild-type polypeptide. In certain embodiments, endogenous antigens include a
wild-type endogenous antigen amino acid sequence or an endogenous antigen
amino
acid sequence containing conservative amino acid substitutions, as defined
above.
Endogenous antigens useful in the invention can include other amino acid
sequences, including those from heterologous proteins. Accordingly, the
invention
encompasses fusion polypeptides in which an endogenous antigen amino acid
sequence is fused, at either or both ends, to amino acid sequence(s) from one
or
more heterologous proteins. Examples of additional amino acid sequences often
incorporated into proteins of interest include a signal sequence, which
facilitates
purification of the protein, and an epitope tag, which can be used for
immunological
detection or affinity purification.
Endogenous antigen polypeptides according to the invention can be
synthesized (e.g., for use as calibrators or controls in the kits according to
the
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invention) using methods known in the art, such as for example exclusive solid
phase synthesis, partial solid phase synthesis, fragment condensation, and
classical
solution synthesis. See, e.g., Merrifield, J. Am. Chem. Soc., 85:2149 (1963).
For a
description of solid phase peptide synthesis procedures, see John Morrow
Stewart
and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce
Chemical
Company, 1984).
Endogenous antigen polypeptides can also produced using recombinant
techniques. In certain embodiments, the sequence of an endogenous antigen
coding region is used as a guide to design a synthetic nucleic acid molecule
encoding the endogenous antigen polypeptide that can be incorporated an
expression vector. Methods for constructing synthetic genes are well-known to
those of skill in the art. See, e.g., Dennis, M. S., Carter, P. and Lazarus,
R. A.,
Proteins: Struct. Funct. Genet., 15:312-321 (1993).
The expression vector includes one or more control sequences capable of
effecting and/or enhancing the expression of an operably linked polypeptide
coding
sequence. Control sequences that are suitable for expression in prokaryotes,
for
example, include a promoter sequence, an operator sequence, and a ribosome
binding site. Control sequences for expression in eukaryotic cells include a
promoter, an enhancer, and a transcription termination sequence (i.e., a
polyadenylation signal).
An expression vector according to the invention can also include other
sequences,
such as, for example, nucleic acid sequences encoding a signal sequence or an
amplifiable gene. A signal sequence can direct the secretion of a polypeptide
fused
thereto from a cell expressing the protein. In the expression vector, nucleic
acid
encoding a signal sequence is linked to a polypeptide coding sequence so as to
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preserve the reading frame of the polypeptide coding sequence. The inclusion
in a
vector of a gene complementing an auxotrophic deficiency in the chosen host
cell
allows for the selection of host cells transformed with the vector.
A wide variety of host cells are available for propagation and/or expression
of vectors. Examples include prokaryotic cells (such as E. coli and strains of
Bacillus, Pseudomonas, and other bacteria), yeast or other fungal cells
(including S.
cerevesiae and P. pastoris), insect cells, plant cells, and phage, as well as
higher
eukaryotic cells (such as human embryonic kidney cells and other mammalian
cells).
Vectors expressing endogenous cardiovascular antigen can be introduced
into a host cell by any convenient method, which will vary depending on the
vector-
host system employed. Generally, a vector is introduced into a host cell by
transformation or infection (also known as "transfection") with a virus (e.g.,
phage)
bearing the vector. If the host cell is a prokaryotic cell (or other cell
having a cell
wall), convenient transformation methods include the calcium treatment method
described by Cohen, et al. (1972) Proc. Natl. Acad. Sci., USA, 69:2110-14. If
a
prokaryotic cell is used as the host and the vector is a phagemid vector, the
vector
can be introduced into the host cell by transfection. Yeast cells can be
transformed
using polyethylene glycol, for example, as taught by I-Iinnen (1978) Proc.
Natl.
Acad. Sci, USA, 75:1929-33. Mammalian cells are conveniently transformed
using the calcium phosphate precipitation method described by Graham, et al.
(1978) Virology, 52:546 and by Gorman, et al. (1990) DNA and Prot. Eng. Tech.,
2:3-10. However, other known methods for introducing DNA into host cells, such
as nuclear injection, electroporation, protoplast fusion, and other means also
are
acceptable for use in the invention.
Expression of endogenous antigen from a transformed host cell entails
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culturing the host cell under conditions suitable for cell growth and
expression and
recovering the expressed polypeptides from a cell lysate or, if the
polypeptides are
secreted, from the culture medium. In particular, the culture medium contains
appropriate nutrients and growth factors for the host cell employed. The
nutrients
and growth factors are, in many cases, well known or can be readily determined
empirically by those skilled in the art. Suitable culture conditions for
mammalian
host cells, for instance, are described in Mammalian Cell Culture (Mather ed.,
Plenum Press 1984) and in Barnes and Sato (1980) Cell 22:649.
In addition, the culture conditions should allow transcription, translation,
and protein transport between cellular compartments. Factors that affect these
processes are well-known and include, for example, DNA/RNA copy number;
factors that stabilize DNA; nutrients, supplements, and transcriptional
inducers or
repressors present in the culture medium; temperature, pH and osmolality of
the
culture; and cell density. The adjustment of these factors to promote
expression in
a particular vector-host cell system is within the level of skill in the art.
Principles
and practical techniques for maximizing the productivity of in vitro mammalian
cell
cultures, for example, can be found in Mammalian Cell Biotechnology: a
Practical
Approach (Butler ed., IRL Press (1991).
Any of a number of well-known techniques for large- or small-scale
production of proteins can be employed in producing the polypeptides of the
invention. These include, but are not limited to, the use of a shaken flask, a
fluidized bed bioreactor, a roller bottle culture system, and a stirred tank
bioreactor
system. Cell culture can be carried out in a batch, fed-batch, or continuous
mode.
Methods for recovery of recombinant proteins produced as described above
are well-known and vary depending on the expression system employed. A
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polypeptide including a signal sequence can be recovered from the culture
medium
or the periplasm. Polypeptides can also be expressed intracellularly and
recovered
from cell lysates.
The expressed polypeptides can be purified from culture medium or a cell
lysate by any method capable of separating the polypeptide from one or more
components of the host cell or culture medium. Typically, the polypeptide is
separated from host cell and/or culture medium components that would interfere
with the intended use of the polypeptide. As a first step, the culture medium
or cell
lysate is usually centrifuged or filtered to remove cellular debris. The
supernatant
is then typically concentrated or diluted to a desired volume or diafiltered
into a
suitable buffer to condition the preparation for further purification.
The polypeptide can then be further purified using well-known techniques.
The technique chosen will vary depending on the properties of the expressed
polypeptide. If, for example, the polypeptide is expressed as a fusion protein
containing an epitope tag or other affinity domain, purification typically
includes the
use of an affinity column containing the cognate binding partner. For
instance,
polypeptides fused with green fluorescent protein, hemagglutinin, or FLAG
epitope
tags or with hexahistidine or similar metal affinity tags can be purified by
fractionation on an affinity column.
Antibodies
Antibodies useful in the immunoassay methods of the invention include
polyclonal and monoclonal antibodies. Polyclonal antibodies are raised by
injecting (e.g., subcutaneous or intramuscular injection) an immunogen into a
suitable non-human mammal (e.g., a mouse or a rabbit). Generally, the
immunogen should induce production of high titers of antibody with relatively
high
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affinity for the target antigen.
If desired, the endogenous antigen (i.e., analyte of interest) may be
conjugated to a
carrier protein by conjugation techniques that are well known in the art.
Commonly used carriers include keyhole limpet hemocyanin (KLH), thyroglobulin,
bovine serum albumin (BSA), and tetanus toxoid. The conjugate is then used to
immunize the animal.
The antibodies are then obtained from blood sainples taken from the animal.
The techniques used to produce polyclonal antibodies are extensively described
in
the literature (see, e.g., Methods of Enzymology, "Production of Antisera With
Small Doses of Immunogen: Multiple Intradermal Injections," Langone, et al.
eds.
(Acad. Press, 1981)). Polyclonal antibodies produced by the animals can be
further purified, for example, by binding to and elution from a matrix to
which the
target antigen is bound. Those of skill in the art will know of various
techniques
common in the immunology arts for purification and/or concentration of
polyclonal,
as well as monoclonal, antibodies see, for example, Coligan, et al. (1991)
Unit 9,
Current Protocols in Immunology, Wiley Interscience.
For many applications, monoclonal antibodies (mAbs) are preferred. The
general method used for production of hybridomas secreting mAbs is well known
(Kohler and Milstein (1975) Nature, 256:495). Briefly, as described by Kohler
and
Milstein, the technique entailed isolating lymphocytes from regional draining
lymph
nodes of five separate cancer patients with either melanoma, teratocarcinoma
or
cancer of the cervix, glioma or lung, (where samples were obtained from
surgical
specimens), pooling the cells, and fusing the cells with SI-IFP-1. Hybridomas
were
screened for production of antibody that bound to cancer cell lines.
Confirmation
of specificity among mAbs can be accomplished using routine screening
techniques
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(such as the enzyme-linked immunosorbent assay, or "ELISA") to determine the
elementary reaction pattern of the mAb of interest.
As used herein, the term "antibody" encompasses antigen-binding antibody
fragments, e.g., single chain antibodies (scFv or others), which can be
produced/selected using phage display or yeast display technology. The ability
to
express antibody fragments on the surface of viruses that infect bacteria
(bacteriophage or phage) makes it possible to isolate a single binding
antibody
fragment, e.g., from a library of greater than 1010 nonbinding clones. To
express
antibody fragments on the surface of phage (phage display), an antibody
fragment
gene is inserted into the gene encoding a phage surface protein (e.g., pIII)
and the
antibody fragment-pIII fusion protein is displayed on the phage surface
(McCafferty
et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids
Res.
19: 4133-4137).
Since the antibody fragments on the surface of the phage are functional,
phage-bearing antigen-binding antibody fragments can be separated from non-
binding phage by antigen affinity chromatography (McCafferty et al. (1990)
Nature,
348: 552-554). Depending on the affnity of the antibody fragment, enrichment
factors of 20-fold - 1,000,000-fold are obtained for a single round of
affinity
selection. By infecting bacteria with the eluted phage, however, more phage
can
be grown and subjected to another round of selection. In this way, an
enrichment
of 1000-fold in one round can become 1,000,000-fold in two rounds of selection
(McCafferty et al. (1990) Nature, 348: 552-554). Thus, even when enrichments
are
low (Marks et al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds of
affinity
selection can lead to the isolation of rare phage. Since selection of the
phage
antibody library on antigen results in enrichment, the majority of clones bind
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antigen after as few as three to four rounds of selection. Thus only a
relatively
small number of clones (several hundred) need to be analyzed for binding to
antigen.
Human antibodies can be produced without prior immunization by
displaying very large and diverse V-gene repertoires on phage (Marks et al.
(1991) J.
Mol. Biol. 222: 581-597). In one embodiment, natural VH and VL repertoires
present in human peripheral blood lymphocytes are isolated from unimmunized
donors by PCR. The V-gene repertoires can be spliced together at random using
PCR to create a scFv gene repertoire which can be cloned into a phage vector
to
create a library of 30 million phage antibodies (Id.). From a single "naive"
phage
antibody library, binding antibody fragments have been isolated against more
than
17 different antigens, including haptens, polysaccharides, and proteins (Marks
et al.
(1991) J. Mol. Biol. 222: 581-597; Marks et al. (1993). Bio/Technology. 10:
779-
783; Griffiths et al. (1993) EMBO J. 12: 725-734; Clackson et al. (1991)
Nature.
352: 624-628). Antibodies have been produced against self proteins, including
human thyroglobulin, immunoglobulin, tumor necrosis factor, and CEA (Griffiths
et
aI. (1993) EMBO J. 12: 725-734). The antibody fragments are highly specific
for
the antigen used for selection and have affinities in the I nM to 100 nM range
(Marks et al. (1991) J. Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO
J. 12:
725-734). Larger phage antibody libraries result in the isolation of more
antibodies
2 0 of higher binding affinity to a greater proportion of antigens.
As those of skill in the art readily appreciate, antibodies can be prepared by
any of a number of commercial services (e.g., Berkeley Antibody Laboratories,
Bethyl Laboratories, Anawa, Eurogenetec, etc.).
Solid Phase
For embodiments of the invention that employ a solid phase as a support
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for the capture agent, the solid phase can be any suitable material with
sufficient
surface affinity to bind a capture agent. Useful solid supports include:
natural
polymeric carbohydrates and their synthetically modified, crosslinked, or
substituted
derivatives, such as agar, agarose, cross-linked alginic acid, substituted and
cross-
linked guar gums, cellulose esters, especially with nitric acid and carboxylic
acids,
mixed cellulose esters, and cellulose ethers; natural polymers containing
nitrogen,
such as proteins and derivatives, including cross-linked or modified gelatins;
natural
hydrocarbon polymers, such as latex and rubber; synthetic polymers, such as
vinyl
polymers, including polyethylene, polypropylene, polystyrene,
polyvinylchloride,
polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides,
polymethacrylates, copolymers and terpolyiners of the above polycondensates,
such
as polyesters, polyamides, and other polymers, such as polyurethanes or
polyepoxides; inorganic materials such as sulfates or carbonates of alkaline
earth
metals and magnesium, including barium sulfate, calcium sulfate, calcium
carbonate,
silicates of alkali and alkaline earth metals, aluminum and magnesium; and
aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin,
zeolite,
silica gel, or glass (these materials may be used as filters with the above
polymeric
materials); and mixtures or copolymers of the above classes, such as graft
copolymers obtained by initializing polymerization of synthetic polymers on a
pre-
existing natural polymer. All of these materials may be used in suitable
shapes,
such as films, sheets, tubes, particulates, or plates, or they may be coated
onto,
bonded, or laminated to appropriate inert carriers, such as paper, glass,
plastic films,
fabrics, or the like.
Nitrocellulose has excellent absorption and adsorption qualities for a wide
'variety of reagents including monoclonal antibodies. Nylon also possesses
similar
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characteristics and also is suitable.
Preferred solid phase materials for flow-through assay devices include filter
paper such as a porous fiberglass material or other fiber matrix materials.
The
thickness of such material is not critical and will be a matter of choice,
largely based
upon the properties of the biological sample or analyte being assayed, such as
the
fluidity of the biological sample.
Alternatively, the solid phase can constitute microparticles.
Microparticles useful in the invention can be selected by one skilled in the
art from
any suitable type of particulate material and include those composed of
polystyrene,
polymethylacrylate, polypropylene, latex, polytetrafluoroethylene,
polyacrylonitrile,
polycarbonate, or similar materials. Further, the microparticles can be
magnetic or
paramagnetic microparticles, so as to facilitate manipulation of the
microparticle
within a magnetic field.
Microparticles can be suspended in the mixture of soluble reagents and
biological sample or can be retained and immobilized by a support material. In
the
latter case, the microparticles on or in the support material are not capable
of
substantial movement to positions elsewhere within the support material.
Alternatively, the microparticles can be separated from suspension in the
mixture of
soluble reagents and biological sample by sedimentation or centrifugation.
When the
2 0 microparticles are magnetic or paramagnetic the microparticles can be
separated
from suspension in the mixture of soluble reagents and biological sample by a
magnetic field.
The methods of the present invention can be adapted for use in systems that
utilize microparticle technology including automated and semi-automated
systems
wherein the solid phase comprises a microparticle. Such systems include those
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described in pending U.S. App. No. 425,651 and U.S. Patent No. 5,089,424,
which
correspond to published EPO App. Nos. EP 0 425 633 and EP 0 424 634,
respectively, and U.S. Patent No. 5,006,309.
In particular embodiments, the solid phase includes one or more electrodes.
Capture agent(s) can be affixed, directly or indirectly, to the electrode(s).
In one
embodiment, for example, capture agents can be affixed to magnetic or
paramagnetic microparticles, which are then positioned in the vicinity of the
electrode surface using a magnet. Systems in which one or more electrodes
serve
as the 'solid phase are useful where detection is based on electrochemical
interactions. Exemplary systems of this type are described, for example, in
U.S.
Patent No. 6,887,714 (issued May 3, 2005). The basic method is described
further
below with respect to electrochemical detection.
The capture agent can be attached to the solid phase by adsorption, where it
is retained by hydrophobic forces. Alternatively, the surface of the solid
phase can
be activated by chemical processes that cause covalent linkage of the capture
agent
to the support.
To change or enhance the intrinsic charge of the solid phase, a charged
substance can be coated directly onto the solid phase. Ion capture procedures
for
immobilizing an immobilizable reaction complex with a negatively charged
polymer,
2 0 described in U.S. App. No. 150,278, corresponding to EP Publication No.
0326100,
and U.S.App. No. 375,029 (EP Publication No. 0406473), can be employed
according to the present invention to affect a fast solution-phase
iminunochemical
reaction. In these procedures, an immobilizable immune complex is separated
from the rest of the reaction mixture by ionic interactions between the
negatively
charged polyanion/immune complex and the previously treated, positively
charged
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inatrix and detected by using any of a number of signal-generating systems,
including, e.g., chemiluminescent systems, as described in U.S. App. No.
921,979,
corresponding to EPO Publication No. 0 273,115.
If the solid phase is silicon or glass, the surface must generally be
activated
prior to attaching the specific binding partner. Activated silane compounds
such as
triethoxy amino propyl silane (available from Sigma Chemical Co., St. Louis,
Mo.),
triethoxy vinyl silane (Aldrich Chemical Co., Milwaukee, Wis.), and (3-
mercapto-
propyl)-trimethoxy silane (Sigma Chemical Co., St. Louis, Mo.) can be used to
introduce reactive groups such as amino-, vinyl, and thiol, respectively. Such
activated surfaces can be used to link the capture directly (in the cases of
amino or
thiol), or the activated surface can be further reacted with linkers such as
glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2-
pyridyldithio] propionate), SMCC (succinimidyl-4-[Nrnaleimidomethyl]
cyclohexane- 1 -carboxyl ate), SIAB (succinimidyl [4iodoacetyl]
aminobenzoate), and
SMPB (succinimidyl 4-[ 1 maleimidophenyl] butyrate) to separate the capture
agent
from the surface. Vinyl groups can be oxidized to provide a means for covalent
attachment. Vinyl groups can also be used as an anchor for the polymerization
of
various polymers such as poly-acrylic acid, which can provide multiple
attachment
points for specific capture agents. Amino groups can be reacted with oxidized
2 0 dextrans of various molecular weights to provide hydrophilic linkers of
different
size and capacity. Examples of oxidizable dextrans include Dextran T-40
(molecular weight 40,000 daltons), Dextran T 110 (molecular weight 110,000
daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M
(molecular weight 2,000,000 daltons) (all of which are available from
Pharmacia,
Piscataway, N.J.), or Ficoll (molecular weight 70,000 daltons; available from
Sigma
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Chemical Co., St. Louis, Mo.). Additionally, polyelectrolyte interactions can
be
used to immobilize a specific capture agent on a solid phase using techniques
and
chemistries described U.S. App. No. 150,278, filed Jan. 29, 1988, and U.S.
App. No.
375,029, filed Jul. 7, 1989, each of which is incorporated herein by
reference.
Other considerations affecting the choice of solid phase include the ability
to minimize non-specific binding of labeled entities and compatibility with
the
labeling system employed. For, example, solid phases used with fluorescent
labels
should have sufficiently low background fluorescence to allow signal
detection.
Following attachment of a specific capture agent, the surface of the solid
support
may be further treated with materials such as serum, proteins, or other
blocking
agents to minimize non-specific binding.
Labeling Systems
As discussed above, many immunoassays according to the invention
employ a labeled detection agent, such as a labeled=antibody or a labeled
antigen.
Detectable labels suitable for use in the detection agents of the present
invention include any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical, or chemical means. Useful
labels in the present invention include magnetic beads (e.g., DynabeadsTM),
fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent
protein,
and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA),
chemiluminescent
compounds such as acridinium (e.g., acridinium-9-carboxamide),
phenanthridinium,
dioxetanes, luminol and the like, radiolabels (e.g., 3H, 1251, 35S, 14C, or
32P),
catalysts such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase,
beta-
galactosidase and others commonly used in an ELISA), and colorimetric labels
such
as colloidal gold (e.g., gold particles in the 40-80 nm diameter size range
scatter
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green light with high efficiency) or colored glass or plastic (e.g.,
polystyrene,
polypropylene, latex, etc.) beads. Patents teaching the use of such labels
include
U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149;
and 4,366,241.
The label can be attached to the detection agent prior to, or during, or after
contact with the biological sample. So-called "direct labels" are detectable
labels
that are directly attached to or incorporated into detection agents prior to
use in the
assay. Direct labels can be attached to or incorporated into detection
agents'by any
of a number of means well known to those of skill in the art.
In contrast, so-called "indirect labels" typically bind to the detection agent
at some point during the assay. Often, the indirect label binds to a moiety
that is
attached to or incorporated into the detection agent prior to use. Thus, for
example,
an antibody used as a detection agent (a "detection antibody") can be
biotinylated
before use in an assay. During the assay, an avidin-conjugated fluorophore can
bind the biotin-bearing detection agent, to provide a label that is easily
detected.
In another example of indirect labeling, polypeptides capable of specifically
binding
immunoglobulin constant regions, such as polypeptide A or polypeptide G, can
also
be used as labels for detection antibodies. These polypeptides are normal
constituents of the cell walls of streptococcal bacteria. They exhibit a
strong non-
immunogenic reactivity with immunoglobulin constant regions from a variety of
species (see, generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406,
and
Akerstrom (1985) J. Immunol., 135: 2589-2542). Such polypeptides can thus be
labeled and added to the assay mixture, where they will bind to the detection
antibody, as well as to the species-specific antibody, labeling both and
providing a
composite signal attributable to analyte and autoantibody present in the
biological
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sample.
Some labels useful in the invention may require the use of an indicator
reagent to produce a detectable signal. In an ELISA, for example, an enzyme
label
(e.g., beta-galactosidase) will require the addition of a substrate (e.g., X-
gal) to
produce a detectable signal.
Exemplary Formats
Electrochemical Detection Systems
The present invention is for example applicable (e.g., adaptable) to the
jointly owned commercial Abbott Point of Care (i-STAT ) electrocheinical
immunoassay system which performs sandwich immunoassays for several cardiac
markers, including TnI, CKMB and BNP. Immunosensors and ways of operating
them in single-use test devices are described in jointly owned Publication
Nos. US
20030170881, US 20040018577, US 20050054078, and US 20060160164, each of
which is incorporated herein by reference. Additional background on the
manufacture of electrochemical and other types of immunosensors is found in
jointly owned U.S. Patent No. 5,063,081 which is also incorporated by
reference.
Multiplex Formats (Exemplary Panel)
In particular embodiments, useful, for example, for simultaneously
.20 assaying multiple analytes in one biological sample, the solid phase can
include a
plurality of different capture agents, including one that captures endogenous
antigen
or analyte of interest (e.g., homocysteine). Thus, for example, the solid
phase can
have affixed thereon a plurality of antibodies, wherein each is intended to
test for
the presence of different analytes (e.g., homocysteine and endogenous
analytes) in
the biological sample. In an exemplary embodiment, the solid phase can consist
of
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a plurality of different regions on a surface, wherein each region has a
particular
antibody affixed therein.
Multiplex formats can, but need not, employ a plurality of labels, wherein
each label is used for the detection of a particular antigen. For example,
multiple,
different analytes can be detected without using a plurality of labels where a
plurality of capture agents, such as antibodies having different specificites,
are
affixed to the solid phase at different known locations. Because the
specificity of
the capture agent at each location is known, the detection of a signal at a
particular
location can be associated with the presence of antigen bound at that
location.
Examples of this format include microfluidic devices and capillary arrays,
containing different capture agents at different locations along a channel or
capillary,
respectively, and microarrays, which typically contain different capture
agents
arranged in a matrix of spots ("target elements") on a surface of a solid
support. In
particular embodiments, each different capture agent can be affixed to a
different
electrode, which can, for example, be formed on a surface of a solid support,
in a
channel of a microfluidic device, or in a capillary.
tiutomated Instrumentation
Optionally the immunoassays as described herein can be used in kits for
commercial platform immunoassays (e.g., homocysteine blood screening assays on
Abbott's Prism , AxSYM , ARCHITECT and/or EIA (Bead) platforms, as well
as in other commercial and/or in vitro diagnostic assays.
Test Kits
The invention also provides test kits for assaying biological samples for
analytes such as homocysteine and other endogenous antigens. Test kits
according
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to the invention include one or more reagents useful for practicing one or
more
immunoassays according to the invention. A test kit generally includes a
package
with one or more containers holding the reagents, as one or more separate
compositions or,- optionally, as admixture where the compatibility of the
reagents
will allow. The test kit can also include other material(s), which may be
desirable
from a user standpoint, such as a buffer(s), a diluent(s), a standard(s),
and/or any
other material useful in biological sample processing, washing, or conducting
any
other step of the assay.
In certain embodiments, a test kit includes a polyanion, wherein the
polyanion is employed in the reaction of analyte antigen with antibody. If
desired,
this component can be included in the test kit in multiple concentrations,
and/or by
provision of a variety of different types of polyanions (including mixtures).
Kits according to the invention can include a solid phase and a capture
agent affixed to the solid phase, wherein the capture agent is an antibody
specific
for the analyte being assessed in the biological sample. Where such kits are
to be
employed for conducting sandwich immunoassays, the kits can additionally
include
a labeled detection agent.
In certain embodiments, the test kit includes at least one direct label, such
as acridinium-9-carboxamide. Test kits according to the invention can also
include
2 0 at least one indirect label. If the label employed generally requires an
indicator
reagent to produce a detectable signal, the test kit preferably includes one
or more
suitable indicator reagents.
In exemplary embodiments, the solid phase includes one or more
microparticles or electrodes. Test kits designed for multiplex assays
conveniently
2 5 contain one or more solid phases including a plurality of antibodies that
are specific
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for a plurality of different analytes of interest (e.g., homocysteine or
endogenous
antigens). Thus, for example, a test kit designed for multiplex
electrochemical
immunoassays can contain a solid phase including a plurality of electrodes,
with
each electrode bearing a different antibody.
Test kits according to the invention preferably include instructions for
carrying out one or more of the immunoassays of the invention. Instructions
included in kits of the invention can be affixed to packaging material or can
be
included as a package insert. While the instructions are typically written or
printed
materials they are not limited to such. Any medium capable of storing such
instructions and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to, electronic storage
media
(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD
ROM), and
the like. As used herein, the term "instructions" can include the address of
an
internet site that provides the instructions.
The invention will be better understood through the following Examples
illustrating its use and efficacy. The following Examples are offered to
illustrate,
but not to limit, the scope and essential features of the present invention.
EXAMPLES
The following reagents and methods were employed for measurement of
homocysteine using a fully automated chemiluminescence measuring apparatus
Reagents
An anti-S-adenosylhomocysteine mouse monoclonal antibody (procured
from Abbott Laboratories, U.S.A_) was bonded onto a magnetic fine particles
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modified by the addition of carboxyl group (procured from Abbott Laboratories,
U.S.A.) using EDC (N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide
hydrochloride (manufactured by Sigma Aldrich)) to give fine particles where
the
antibody was made into a solid phase. The antibody in a solid phase was added
to
a BisTris buffer solution containing Tween 20 (manufactured by Kanto Kagaku),
EDTA (sodium ethylenediaminetetraacetate) and sodium chloride to prepare a
solution of fine particles of the antibody in a solid phase.
S-adenosylcysteine labeled with acridinium derivative (procured from
Abbott Laboratories, U.S.A.) was added to an MES buffer solution containing
Triton X 100 (manufactured by Sigma Aldrich) to prepare a tracer solution.
S-adenosylhomocysteine hydrolase (procured from Axis Shield, United
Kingdom) was added to a buffer solution containing 30% (by volume) of glycerol
to
prepare an enzyme solution.
DTT and adenosine were added to an aqueous solution of citric acid to
prepare a reducing agent solution.
Method
The following operations and measurement were carried out using an
Architect fully automated immunoassay analyzer (manufactured by Abbott Japan
Co., Ltd.). A sample (18 ELL) was mixed with 79 L of the enzyme solution, 50
L
of the fine particles solution of antibody in a solid phase, and 10 L of the
reducing
agent solution, and the first reaction was started. In this mixed solution,
the
following reactions were generated: (1) a bonded product of homocysteine in
the
sample was liberated to a free homocysteine; (2) the liberated homocysteine
was
converted to S-adenosylhomocysteine; and (3) the converted S-
2 5 adenosylhomocysteine was bonded to the particles of antibody in a solid
phase.
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After 21 minutes, the tracer solution (50 pL) was further mixed therewith
and the reaction was continued for 4 minutes. As a result of this reaction,
the
aforementioned reaction (3), and a competitive reaction to the fine particles
of
antibody in a solid phase among the tracers resulted and, depending upon the
concentration of homocysteine in the sample, the tracers were competitively
bonded
to the fine particles of antibody in a solid phase. Thus, if the homocysteine
concentration in the sample was low, many tracers were bonded to the fine
particles
of antibody in a solid phase. In contrast, when the homocysteine concentration
in
the sample was high, small numbers of tracers were bonded to the fine
particles of
antibody in a solid phase.
Then, after washing with a washing liquid which was exclusive for this
instrument, an emission signal was observed using an emission trigger reagent
(also
was exclusive for this instrument). A standard curve was prepared by a
logistic 4
para method using an Abbott AxSYM homocysteine calibrator (manufactured by
Abbott Japan Co., Ltd.) as a reference solution, and the homocysteine
concentration
was calculated based on the signal obtained from the sample, whereupon the
concentration of the homocysteine in the sample was determined.
Example 1
The concentration of homocysteine contained in the sample was determined
by prior art methods including a diluting operation using a commercially-
available
AxSYM Homocysteine Assay Reagent and AxSYM Analyzer (both
manufactured by Abbott Japan Co., Ltd.). This measuring method is a
fluorescence polarization immunoassay where a fluorescent substance is
utilized in
a labeled substance and, in order to eliminate the influence of an inhibiting
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substance, the sample was diluted to an extent of about 300-fold upon a
competitive
reaction.
In order to confirm the influence of the inhibiting substance on the
measuring system and the effect of polyanion, the concentration of
homocysteine
was measured similarly for undiluted serum, and also for samples where 4.2, 42
and
420 g/mL of heparin was added to the serum. The results of these tests are
shown
in FIG. 1.
It was confirmed that, in the undiluted sample, serum (0) in which the
concentration of homocysteine was able to be precisely measured, and serum (0)
in
which the signal intensity was detected to be a bit low (whereby the apparent
concentration of homocysteine was measured high).
In those two kinds of undiluted serums, although a big difference was noted
in signal intensity, in neither case was heparin was added. It was found that,
as a
result of addition of heparin, the difference between the assay results became
small,
and that when 42 g/mL of heparin was added, there was almost no difference
between them in terms of assay results.
From the above, it appears that a heparin-like anionic substances derived
from a living organism was contained in the latter serum (0), and that those
substances inhibited the present measuring system. By contrast, in the former
serum (M), apparently no substantial amount of inhibiting substance was
present
and as consequence, the influence of the added heparin was stronger.
Plasma (A) in blood collected with heparin (e.g., heparinized tubes) was
similarly investigated and experimental results were obtained similar to those
for the
serum where the homocysteine concentration was apparently highly measured (0).
Because of that, it is also strongly suggested that a heparin-like polyanionic
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substance derived from a living organism was contained in the serum where
homocysteine concentration was apparently highly measured.
From the aforementioned results, it was confirmed that, when a sufficient
amount of polyanion is added to the sample, it is possible to eliminate the
variation
in measured data caused by the amount of inhibiting substances contained in
each
sample.
In order to confirm whether a rheumatoid factor participates as an
inhibiting substance in a measuring system for homocysteine, the concentration
of
rheumatoid factor was quantified for the sample used in the Examples. As a
result
of the measurement for six samples where homocysteine was able to be precisely
measured and for four samples where it was measured in apparently high
concentrations, only one among the samples where homocysteine was able to be
precisely measured contained the rheumatoid factor in an amount of more than
the
standard value while all other samples were within a range of normal value.
This
suggests that a rheumatoid factor does not participate as a reaction inhibitor
in an
immunoassay of homocysteine.
Example 2
Twelve kinds of serum and two kinds of plasma in blood collected with
heparin were subjected to measurement of homocysteine concentration using a
fully
automated chemiluminescent measuring apparatus, and each concentration was
compared with the homocysteine concentration (known value) determined by an
AxSYM Analyzer in the same manner as in Example 1. The results of these
experiments are shown in FIG. 2.
As can be seen in FIG. 2, out of the twelve kinds of serum, the
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concentrations of seven of the serums (0) were no different from the actual
homocysteine concentration, whereas for five kinds thereof (40), higher
measured
concentrations than the actual concentration were obtained. In the plasma in
blood
collected with heparin, both samples showed higher measured concentrations
than
the actual concentration (AL).
An immunoreaction also was conducted using the same serum and plasma
samples in the presence of 42 g/mL of heparin. The measured result are shown
in
FIG. 3.
As can be seen from FIG: 3, in both cases of the plasma samples, and the
five samples where a higher measured value was obtained than the actual one,
with
addition of heparin the concentrations were then able to be precisely
measured.
The above experimental result confirm that addition of a polyanion such as
heparin is able to eliminate the influence of an inhibiting substance, and
also that the
homocysteine concentration was able to be precisely measured in the presence
of
polyanion.
Example 3
It also was investigated whether the addition of polyanion other than
heparin was similarly able to eliminate the influence of an inhibiting
substance.
FIG. 4 shows rates of dissociation in the higher sample group and the
normal sample group at each of the concentrations of dextran sulfate,
polyacrylic
acid, gelatin and y-globulin. Each 7 and 8 samples were used as the higher and
the
normal sample groups, respectively.
In order to eliminate the dispersion in the measured values among the
samples by the inhibiting substance and to appropriately measure the
homocysteine
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concentration, it is necessary that such values are within about 100% 10%.
It is noted from FIG 4 that, when not less than 4.2 g/mL of heparin is
present in the first reaction, the influence of inhibition by the sample was
able to be
effectively avoided. Further, all of the investigated three kinds of
polyanions
(heparin, dextran sulfate, and polyacrylic acid) showed a significant effect
whereas
in the case of gelatin or y-globulin which are not polyanions, such an effect
was not
achieved. Accordingly, the effect of eliminating the influence of the
inhibiting
substance was found to be common in polyanions.
Typical heparin has one anion per a molecular weight of 150 whereas in
some polyanions, there are molecules in which the anions are more densely
present.
For example, polyacrylic acid has one anion per a molecular weight of 71. When
the presence in the polyanion molecule of anions being densely present is
further
taken into consideration, it is expected that an ability to eliminate or
suppress the
influence of the inhibiting substance is achieved when the polyanion
concentration
is not more than about I g/mL.
The disclosure of all patents, publications, including published patent
applications, and database entries referenced in this specification are
specifically
incorporated by reference in their entirety to the same extent as if each such
individual patent, publication, and database entry were specifically and
individually
indicated to be incorporated by reference.
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Although the invention has been described with reference to certain
specific embodiments, various modifications thereof will be apparent to those
skilled in the art without departing from the spirit and scope of the
invention. All
such modifications as would be apparent to one skilled in the art are intended
to be
included within the scope of the following claims.
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