Note: Descriptions are shown in the official language in which they were submitted.
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PRESSURE-MEDIATED BINDING OF BIOMOLECULAR COMPLEXES
Field of the Invention
The invention is in the general field of analyte
detection, assays, and methods for the separation of
particular compounds from a mixture.
Background of the Invention
Assays of Biomolecules: Assays can be used to
determine whether, and how much of, an analyte is present
in a sample. In some cases, such assays rely on
selective binding or complexation (specific or
nonspecific) of the analyte in the sample with an
exogenously supplied capture reagent or binding partner.
Effects of endogenous binding partners on assays:
Often such samples contain an endogenous component that
forms a complex with the analyte, and the resulting
endogenous complex may interfere with detection of the
analyte. For example, detection by absorbance,
fluorescence, molecular weight, or other analyte
characteristics may be adversely affected by endogenous
complexes. Where detection itself depends on formation
of a complex with an exogenously supplied reagent,
analyte present in endogenous complexes may be unable to
effectively complex with the exogenous binding partner
(as is required for detection in such assays). In that
way, the endogenous complex interferes with the assay's
reliability. For example, the analyte goes undetected or
is incompletely detected -- i.e., it provides a false
negative or non-quantitative result.
This problem can be illustrated with standard
enzyme-linked immunosorbent assays (ELISAs), in which
sample antigen is detected only if it is recognized and
bindable by immobilized antibody. Endogenous sample
antibodies that react with the analyte may prevent at
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least some portion of the analyte from complexing with
one or both of the exogenous assay reagents (antibodies),
thereby reducing the effectiveness of the assay.
Assays for antigens or antibodies that are
characteristic of a pathogen are particularly susceptible
to problems caused by endogenous binding partners. if
the patient being assayed has developed an immune
response to the analyte antigen, a significant portion of
sample antigen may be present in undetectable endogenous
antigen/antibody complexes. Similarly, in serology
assays where the antibody is the analyte to be measured,
some of the sample antibody that the assay is designed to
measure may be complexed with endogenous pathogen
antigen.
In addition to endogenous antibody/antigen
complexes, other endogenous complexes can interfere with
assays, for example, various serum globulins can
interfere with immunoassays for thyroxine, estradiol,
cortisol, and testosterone. See, Thorell, J. I., and
Larson, S. M, in "Radioimmunoassay and Related
Techniques," C.V. Mosby, St. Louis, 1978. Vitamin B12
assays are perturbed by the binding of transcobalamin.
See, Laue et al. Blood 26:202 (1965). Immunoassays for
prostate-specific antigen (PSA) are perturbed by
endogenous complexes with a serine protease inhibitor,
al-antichymotrypsin. See, Lilja et al. C1in. Chem.
37:1618-1625 (1991).
Another area in which endogenous complexes may
seriously affect assay results is the use of tumor
antigens to mark the tumor's presence, e.g. in an
immunoassay. Frequently, these tumor markers may be
masked by endogenous complexes. For example, serum
thyroglobulin autoantibody interferes with detection of
differentiated thyroid carcinoma. Another example of the
difficulty of obtaining accurate quantitation of a serum
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tumor antigen is the epithelial mucin MUC-1. Gorevitch
et al., Br. J. Cancer, 72:934-938 (1995); and Hilgers et
al. Scand. J. C1in. Ob. Invest. Suppl., 221:81-86 (1995).
A particular problem which may be related to
endogenous complex formation has surfaced in HIV assays.
Tsiquaye et al., AIDS, 2:41-45 (1988); McHugh et al.,
J. infect. Dis., 158:1088-1091 (1988); Nishanian et al.,
J. infect. Dis., 162:21-28 (1988); and Carini et al.,
Scand. J. Immuno., 26:1 (1987). Other assays in which
this problem can arise include: epithelial mucin (MUC-1
and PEM) assays (Gorevitch et al., Br. J. Cancer, 72:934-
938 (1995); and Hilgers et al. Scand. J. Clin. ob.
invest. Suppl., 221:81-86, 1995); H1 histones in assays
for systemic lupus (Wesierska-Gadek et al. Arthritis
Rheum, 33:1273-1278, 1990); assays for the tuberculosis
pathogen (Dlugovitzky et al. Braz. J. Med. Biol. Res.
28:331-335, 1995); alpha-fetoprotein assays to detect
hepatocellular carcinoma (Tsai et al., Br. J. Cancer,
72:442-446, 1995); assays for Yersinia enterocolitca and
Yersinia pseudotuberculosis (Didenko et al. J. Basic
Microbiol. 35:163-170, 1995); and assays for the leprosy
pathogen (Sinha et al. Int. J. Lepr. Other Mycobact.
Dis., 60:396-403, 1992).
Various methods have been described to dissociate
endogenous antibody/antigen complexes and thereby to
improve assay sensitivity, including solvent extraction,
heating, protein precipitation, use of competitive
inhibitors, and pH changes. For example, Mosier, U.S.
Patent 4,656,251, and Weil et al., J. Immunology,
134:1185-1191 (1985), disclose pretreating a canine
sample to break up immune complexes before assaying for
heartworm antigens. Mosier '251 discloses a process that
includes acidification to dissociate the complex,
followed by heating to denature dissociated antibodies.
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Weil discloses (p. 1186, right column) a process
including the addition of EDTA followed by heating.
Accelerating High Sensitivity Assays: While
sensitivity may be improved by lengthening incubation
time (e.g., overnight), high throughput and automation
are also important goals that may be inconsistent with
lengthy incubation. As high throughput automated
instruments have become widely utilized, assay results
are needed more quickly (i.e., within a few minutes).
The need to accelerate analyte/binding partner
interactions may be addressed by adding a large excess of
the exogenous binding partner, or by using temperature
conditions above optimum to drive the binding reaction as
far as possible in an acceptable assay time.
Separation of Biomolecules: A widely accepted
method for purification of bioactive compounds is
affinity chromatography. This method is based on the
premise that many bioactive compounds bind to other
molecules with extraordinary specificity. These other
molecules are commonly referred to as "ligands." For
example, ligands that have been identified for binding
specific compounds include, but are not limited to,
nucleic acids, vitamins, carbohydrates, fats, and
proteins (e.g., enzymes, antibodies, and receptors).
The first step in affinity chromatography
typically is identification of a ligand that binds
specifically to the compound of interest. Such ligands
are already known for many enzymes and other compounds.
Once a ligand has been identified and obtained, the
ligand can be attached to a solid support. The solid
support can, for example, be trapped within a porous sack
or, more commonly, immobilized in a porous column. A
solution known to contain, inter alia, the compound of
interest is generally flushed through the column so that
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the solution comes into binding contact with the
immobilized ligand.
The quantity of immobilized ligand required
depends on the amount of the desired compound expected to
be present. Typically, each ligand can bind to a limited
number of (e.g., often one) molecules of the compound.
Numerous complications render this generalization less
valid in practice, however. For example, steric
constraints can limit the number of molecules of the
compound that can exist within a given volume, especially
if the compound is, for example, a relatively large
molecular weight, multi-domain protein. Also, there can
be other, undesirable compounds capable of weakly binding
to the same ligand that the compound of interest binds
tightly to. The latter problem can become especially
acute if the undesired weakly binding compound is present
in excess (i.e., relative to the desired compound).
Therefore, it is desirable to promote high affinity, high
specificity interactions.
In a typical preparative application of affinity
chromatography, an impure solution containing the desired
compound is passed through a porous material (e.g., in a
bed or column) containing the immobilized ligand. The
desired compound becomes bound to the ligand and
therefore is itself immobilized. The remaining
impurities that were in the solution, including other
compounds, are washed away with an additional fresh
buffer or solvent, leaving the immobilized ligand bound
to the desired compound.
Once the impurities have been washed away, the
compound of interest can be released from the binding
relationship with the immobilized ligand. This process
is called "elution." Elution can be effected by making
the compound-ligand complex unstable, for example, with
altered pH, temperature, or ion concentration, or by
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adding a different ligand known to have still greater
affinity for the compound relative to the immobilized
ligand (i.e., to displace the compound). It is desirable
to effect elution with relatively mild conditions to
avoid irreversible damage to either the ligand or the
desired compound. It is also desirable to elute the
desired compounds under conditions that ensure simple
recovery following separation.
Affinity chromatography sometimes uses antibodies,
enzymes, or other binding proteins as the immobilized
member of the binding pair, with the specific ligand or
substrate being the desired compound to be isolated or
purified. The type of affinity chromatography termed
immunoaffinity chromatography uses antibodies as ligands.
Some advantages of immunoaffinity chromatography are
that: 1) the immunological process of antibody diversity
does the work of finding a ligand, and 2) antibodies
exhibit high specificity.
Summary of the Invention
The invention features: (1) pressure-mediated
dissociation of an analyte complexed with an endogenous
binding partner to enable detection of a complex formed
from the analyte and an exogenous binding factor, (2)
pressure-mediated association of an analyte and an
exogenous binding partner to enable more rapid and/or
more sensitive detection of an analyte, and (3) pressure-
mediated association and dissociation of biomolecular
complexes to enable separation of one biomolecule from a
complex mixture.
Pressure can be used to improve assays by
dissociating endogenous analyte complexes and improving
assay speed and sensitivity by associating the analyte
molecules with exogenously supplied binding partners.
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Pressure can also be used to improve the separation of
compounds from contaminated mixtures.
Assays: Pressure can be controlled to dissociate
endogenous analyte complexes and thereby improve
detection of analyte present in samples containing
endogenous components that complex with the analyte. As
described above, such endogenous complexes can interfere
with analyte detection in various ways. One specific
example of such interference involves detection formats
that rely on a determination of complexing between
analyte and an exogenously supplied specific binding
partner for the analyte.
In one aspect of the invention, endogenous analyte
complexes are dissociated under controlled pressure to
improve analyte availability for detection. For example,
pressure-induced dissociation of the endogenous complexes
(weak or strong) can improve analyte detection by
improving binding (i.e., kinetically or
thermodynamically) to the exogenous binding partner.
This aspect of the invention features a dissociation step
in which the sample is subjected to elevated pressure
sufficient to dissociate an endogenous complex formed
from an analyte and an endogenous sample component (e.g.,
preferably at least 15,000 psi, i.e., 105 MPa, and most
preferably at least 30,000 psi, or 210 MPa). This
dissociation step is followed by an analyte detection
step, e.g., in which the exogenous specific binding
partner is reacted with sample analyte. In this format
of the invention, it is believed that increasing the
pressure results in structure disruption (either a
reversible or irreversible change in three-dimensional
conformation) of a component of the endogenous complex
which prompts binding partner dissociation.
More specifically, the dissociation pressure can
(but need not necessarily) be high enough to irreversibly
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dissociate the analyte from endogenous binding component.
If dissociation is irreversible (for example, because one
member of the complex is structurally disrupted in a way
that substantially prevents endogenous complex
reassociation), then the assay step can be performed
without first removing the structurally disrupted binding
component from the analyte. If desired, an agent that
prevents or reduces reassociation can be added, such as a
denaturing agent (e.g., urea); a water miscible solvent;
a chelating agent, such as EDTA, EGTA, or o-
phenanthroline; a detergent; or a chaotrope, such as
dithiothreitol, urea, or thiocyanate. This agent or
agents preferably should be tolerated in subsequent assay
steps, so that it need not be removed prior to those
steps.
For reversible dissociation, the analyte is removed
from the endogenous sample component in a separate step
performed after the dissociation step. For example, the
exogenous analyte binding reagent can be immobilized in a
chamber with the sample; chamber pressure is increased to
dissociate the complex, after which the endogenous
binding component is removed from the chamber while
pressure is maintained. The chamber can be a
semipermeable membrane selected to pass endogenous
analyte, but not endogenous sample component.
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The present invention thus provides a method of
assaying for an analyte, the method comprising: a)
providing a sample, wherein the sample comprises an
endogenous complex between the analyte and an endogenous
sample component; b) subjecting the sample to an elevated
pressure to dissociate the analyte from the endogenous
sample component; and c) detecting or measuring the
dissociated analyte by reacting the dissociated analyte
with an exogenously supplied specific binding reagent and
determining complexation between the analyte and the
binding reagent.
In another embodiment (or in a combination of the
above described embodiments), temperature, pressure, or
both, are controlled (usually increased) to control the
association between a ligand present in a mixture and an
exogenously supplied binding partner. For example, an
assay is performed where pressure and temperature are
maintained to improve association compared to ambient
(1 atm, room temperature) conditions. It is possible to
combine the various aspects of the invention by first
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subject.ing a mixture to a temperature and pressure for
separating a ligand (analyte) in the mixture from
endogenous binding partners in the mixture, and then
changing temperature or pressure, or both, of the
separated ligand to a second temperature or pressure, or
both, selected to enhance ligand complex formation
relative to complex formation at ambient temperature and
pressure. Generally, the second temperature or pressure
is intermediate between ambient conditions and the
temperature and pressure used in the separating step
above. While at the second temperature and pressure, the
separated ligand is reacted with the exogenously supplied
binding partner.
The method can be performed using apparatus
(described in WO 96/27432) in which a valved inlet
connects the chamber to a pressurized supply area, a
valved outlet connects the chamber to a waste collection
area, and controllers operate the valved outlets. Analyte
is flushed out of the chamber and into the collecting
chamber by introducing material from the pressurized
supply area.
The invention can be practiced with a wide
diversity of analytes. In particular, the analyte may be
an antigen, and the method may be used to dissociate an
endogenous antibody that complexes with the antigen.
Where the analyte is an antibody, the method can be used
to dissociate a complex between an antigen and the
antibody. In addition, where the analyte is complexed
with multiple endogenous components, the method can be
used to dissociate the analyte from such one or more of
these complexes.
Such dissociation is particularly useful where
endogenous complexes are known to interfere at least to
some extent with assaying. The invention can
specifically be used to assay: a) HIV antigens (e.g.,
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p24, gp41, gp120, gp160, and p15) where the sample is a
human bodily fluid containing anti-analyte antibody; b)
non-protein analytes, such as thyroxine, estradiol,
cortisol, and testosterone, where the sample is a bodily
fluid comprising serum globulin; c) Vitamin B12 where the
sample contains transcobalamin; d) prostate-specific
antigen where the sample contains al-antichymotrypsin or
a2-macroglobulin; e) an epithelial mucin, where the
sample contains endogenous antibody that complexes with
the analyte; f) antibody to Hi histones in an assay for
systemic lupus, where the sample contains endogenous H1
histones; g) tuberculosis pathogen, where the sample
contains anti-analyte antibodies; h) alpha-fetoprotein
where the method is a diagnostic for hepatocellular
carcinoma; i) an antigen of Yersinia enterocolitca or
Yersinia pseudotuberculosis; j) an antigen of the leprosy
pathogen, Mycobacterium leprae; k) anti-DNA antibodies or
DNA binding thereto; 1) Dirofilaria immitis antigen or
antibody thereto; m) growth hormone or growth hormone-
binding protein; n) cholesterol; o) low density
lipoprotein; p) high density lipoprotein; and q) tumor
antigens that can be used as diagnostic, monitoring, or
prognostic indicators for cancer-related pathological
states.
A specific concern with many tumor markers is that
although they are often detected in benign disease, they
may be absent in early-stage malignancy, due to complex
formation with a patient's antibodies. For example,
serum thyroglobulin autoantibody interferes with
detection of differentiated thyroid carcinoma. With
proper controls and dissociation of complexes, tumor
markers may be used to quantitate tumor burden, e.g., to
monitor clinical progress and patient status. In
particular, tumor antigen burden can be measured serially
over time. The invention also permits separation of
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total analyte levels into dissociated analyte and analyte
present in endogenous complexes. The information thus
derived, e.g., autoantibody levels, can be useful
clinically. Where free antigen and complexed antigen
appear out of balance, the balance can be corrected
extracorporeally to increase both cellular and humoral
cytotoxicity to the tumor.
Not only does the invention improve assays
involving endogenous complexes, it also makes possible a
better understanding of precisely how much and what type
of antigens and antibodies are present, regardless of
whether they are present in complexes. This capability
can improve the ability to track the immune response to,
and the course of, a disease.
The invention may also improve assays in which the
endogenous analyte is not initially complexed with the
analyte, but the assay protocol and reagents induce
formation of such an undesired complex. The term
"endogenous complex" includes such undesired complexes
which are induced by the assay, even complexes which form
with assay components, so long as the complexes are not
the desired complex which results in a detectable event.
Pressure control can also ameliorate such de novo assay-
induced complex formation.
The invention also includes an embodiment wherein
the dissociation of endogenous binding complexes is
accompanied by association of an exogenous binding
partner such as an aptamer.
Separation: Pressure's influence on affinity can
be used to improve affinity chromatography methods.
Contaminated mixtures containing compounds of interest
can be placed in fluid contact with molecules known to
bind to the desired compounds with pressure-dependent
affinity or activity. The pressure in the reaction
vessel can be altered to allow the desired compounds to
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bind more quickly or more tightly to the immobilized
molecules. The contaminants can be flushed away first,
and then the purified compounds can be dissociated from
the immobilized molecules, for example by further
modifying the pressure.
In general, the invention features a method for
separating a desired compound from at least some
contaminants in a mixture containing the desired compound
and one or more contaminants. The method includes
providing binding molecules having pressure-dependent
affinity for the desired compound, providing the mixture
(i.e., in fluid contact with the binding molecules), and
subjecting the molecules and the mixture to a first
pressure that increases the affinity of the binding
molecules for the desired compound, to form a bound
complex. These steps can be performed in any order. The
bound complex is then separated from at least some of the
contaminants, the pressure is changed to a second
pressure (i.e., which decreases the affinity of the
binding molecules for the desired compound), and the
binding molecules are finally separated from the desired
compound.
In the present context, the term "affinity" is
used to describe both the kinetics and thermodynamics of
binding. Thus, when it is said that a set of conditions
"enhances the affinity of an analyte for a reagent," it
is to be understood either that the conditions enhance
the rate of formation of an analyte-reagent complex or
that the conditions drive the equilibrium of the reaction
system toward complex formation, or both. "Enhanced
ligand complex formation" would have the same meaning.
Furthermore, the phrase "molecules that associate
with a compound in a pressure-dependent manner" means
that either the rate of the association (i.e., binding
molecule-desired compound complex formation) is increased
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or the equilibrium of the system is shifted toward
association, or both.
The first and second pressures can each be uniform
throughout the system that includes both the binding
molecules and the mixture in fluid contact with the
molecules.
The binding molecules can be immobilized, for
example by attachment to a solid support (e.g., a polymer
bead, a particle, a strip, a tube, a column, a molded
material, and a polymer matrix) or by using a
semipermeable membrane that passes only one of the
components of the binding pair.
The fluid mixture can include, for example, water,
an aqueous solution, an organic solvent, an organic
solution, a gas, or a supercritical fluid.
The desired compound and the binding molecules can
include, but are not limited to, members of the following
classes which can form bound complexes: polypeptides,
proteins, antigens, haptens, antibodies, prions,
carbohydrates, nucleic acids, steroids, triglycerides,
substrates, enzymes, and hormones. Thus, the bound
complex can be, for example, an enzyme-substrate complex,
a ligand-receptor complex, a glycoprotein-lectin complex,
a protein-cofactor complex, a nucleic acid-cofactor
complex, a hybridized nucleic acid-target complex, a
hapten-antibody complex, or an antigen-antibody complex.
The first pressure, or both the first and second
pressures, can be greater than atmospheric pressure
(e.g., between 500 and 200,000 psi or between 5,000 and
200,000 psi).
The first pressure can be applied prior to or
after providing the mixture, the binding molecules, or
both.
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pH, ionic concentration, fluid composition, or
temperature can be modified to enhance separation of the
compounds of interest from the binding molecules.
Additionally, a reagent can be added to cause
dissociation of the bound complex to yield the free
compound of interest and the unbound molecules. The
reagent can be, for example an acid, a base, a salt, a
metal, a metal-scavenger, a detergent, a dissociating
agent, a chaotropic agent, water, an organic solvent, a
chelating agent, or some other binding partner. More
specifically, such a reagent can be, for example, a
magnesium salt, a lithium salt, sodium dodecyl sulfate,
urea, guanidine hydrochloride, thiocyanate, or dioxane.
The desired compound can be, for example, an
enzyme (wherein the binding molecules can be substrates
for the enzyme); an antibody, such as a monoclonal
antibody (wherein the binding molecules can be antigens
for the antibody); or a cofactor, such as a transcription
cofactor (wherein DNA transcription can be modulated).
The three-dimensional conformation of the desired
compound can, for example, change in the transition from
the first to the second pressure.
In another embodiment, the invention features
another method for separating a compound of interest from
at least some contaminants in a mixture containing the
desired compound and one or more contaminants.
This method includes providing binding molecules
having affinity for the desired compound and further
having pressure-dependent activity for the modification
of the desired compound. This means that the binding
molecules not only bind to the desired compound, but also
cause a modification of the compound. An example of such
a system is provided by an enzyme and its substrate. At
atmospheric pressure, many enzymes bind to their
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substrates, modify the substrates to generate "products,"
then release the products.
The modifying activity of many such enzymes is
attenuated by elevated pressure, while binding affinity
is often enhanced by pressure. Thus, the enzyme's
ability to bind to the substrate can be exploited for
purification, provided that the pressure is not lowered
to a level at which the enzyme exhibits modifying
activity.
The method of this embodiment also includes
providing the aforementioned mixture in fluid contact
with the binding molecules to form a bound complex at a
first pressure that decreases the activity of the binding
molecules for the modification of the compound. These
two steps can be executed in any order. The bound
complex is then separated from at least some of the
contaminants.
In some examples, the pressure is changed to a
second pressure (i.e., that increases the activity of the
binding molecules for the modification of the compound)
and the unbound molecules are finally separated from the
modified compound (i.e., the product).
Alternatively, or additionally, pH, ionic
strength, fluid composition, or temperature can be
modified to enhance separation of the compounds of
interest from the unbound molecule.
In another alternative method, a reagent that
causes dissociation of the bound complex is provided, and
the binding molecules are separated from the desired
compound. Examples of such reagents include acids,
bases, salts, metals, metal-scavengers, detergents,
dissociating agents, chaotropic agents, water, organic
solvents, chelating agents, and other binding partners.
Specifically, the reagent can be, for example, a
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magnesium salt, a lithium salt, sodium dodecyl sulfate,
urea, guanidine hydrochloride, thiocyanate, or dioxane.
The first pressure can be uniform throughout the
system that includes the binding molecules and the
mixture in fluid contact with each other.
The binding molecules can be immobilized, for
example, by compartmentalization within a semipermeable
membrane or by attachment of the binding molecules to a
solid support (e.g., a polymer bead, a particle, a strip,
a tube, a column, a molded material, or a polymer
matrix).
The fluid mixture can be, for example, water, an
aqueous solution, an organic solvent, an organic
solution, a gas, or a supercritical fluid.
The desired compound can be, for example, a
polypeptide, a nucleic acid molecule, an antibody, a
triglyceride, a steroid, a prion, or a carbohydrate.
The binding molecules can be, for example,
enzymes, wherein the bound complex can be an enzyme-
substrate complex.
Alternatively, the desired compound can be, for
example, an enzyme, wherein the binding molecules can be
substrates for the enzyme.
The first pressure can be greater than atmospheric
pressure (e.g., between 500 and 200,000 psi or between
5,000 psi and 200,000 psi). The first pressure can be
applied prior to providing the mixture. The second
pressure can also be greater than atmospheric pressure.
In any embodiment, the method can be repeated at
least once (e.g., 1, 2, 3, or more times) to remove more
of the contaminants from the mixture.
Screening: In another embodiment, the invention
features a method of screening a molecular library for
molecules that bind a target. The method includes the
steps of providing a member of the molecular library in
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fluid contact with the target at atmospheric pressure to
form a complex; using a detection means to monitor the
binding rate and affinity in real-time; subjecting the
complex to an elevated pressure that causes dissociation
of the complex; flushing the dissociated member away from
the immobilized target; repeating these steps for other
members of the library; and then analyzing the results
collected by the detection means to determine which
members of the library bind to the target.
The library can, for example, include proteins,
carbohydrates, antibodies, ribozymes, oligonucleotides,
peptides, or small organic molecules. The target can be,
for instance, a phage display or other immobilized
biomolecules. The detector means can be, for example, a
radioisotopic detector, an infrared spectrometer, a mass
spectrometer, a gas chromatograph, a spectrophotometer, a
spectrafluorometer, an electrochemical detector, a
surface plasmon resonance detector, a nuclear magnetic
resonance spectrometer, a scanning tunneling microscope,
an atomic force microscope, or a chemiluminescence
spectrometer.
Refolding of Denatured Proteins: In still another
embodiment, the invention features a method of refolding
a previously denatured protein. The method includes the
steps of subjecting aggregates of the denatured protein
to elevated pressures sufficient to break up the
aggregates to form dissolved, denatured polypeptide
chains and then rapidly cycling the pressure to cause the
dissolved, denatured polypeptide chains to rapidly sample
numerous conformations until the polypeptide chain has
folded into its lowest energy protein conformation.
In certain cases, the aggregates are mixed with a
pressure-sensitive buffer and contacted with a solid
phase having an ionization volume of opposite charge to
that of the buffer. The pressure cycling is thought to
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disrupt the aggregates by pH fluctuation and disperse the
aggregates by reversible binding to the solid phase. The
buffer itself can be covalently bonded to a solid support
(which may or may not be the same as the solid phase),
and can also serve as a nucleation site for the refolding
of the polypeptide chain. In some cases, reducing and
oxidizing agents can be added to allow reconfiguration of
disulfide bonds within the polypeptide chain.
Unless otherwise defined, 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. Although methods and
materials similar or equivalent to those described herein
can be used in the practice or testing of the present
invention, the preferred methods and materials are
described below. In case of conflict, the present
application, including definitions, will control. In
addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
An advantage of the claimed invention is clean
separation and isolation of the purified compounds. in
certain embodiments, the desired compounds can be eluted
from the immobilized binding molecules without addition
of supplementary reagents. No packing matrix is
necessary either, which means that less of the desired
compound is lost through non-specific adsorption as is
common in the elution or fractionation steps of
traditional chromatographic methods. A packing matrix is
a material which can be packed inside a column either to
provide a solid phase for the immobilized "capture"
reagent or to effect separation by another route such as
size exclusion chromatography. The moderate pressures
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used successfully in the present methods are less likely
than the traditional methods to cause irreversible damage
either to the desired compounds or to the immobilized
binding molecules.
Other features and advantages of the invention
will be apparent from the following detailed description,
and from the claims.
Brief Description of the Drawings
Fig. 1 is a schematic of a reactor that can be
used to apply pressure to assay reagents.
Fig. 2 shows a spring seal and gland washer of the
reactor of Fig. 1.
Fig. 3 is a schematic of the hydraulic system of
the reactor of Fig. 1.
Fig. 4 is a phase diagram.
Fig. 5 is a plot of pressure against time for a
separation process of the invention.
Fig. 6 is a plot of p24:anti-p24 antigen:antibody
binding as a function of time at atmospheric pressure at
ambient temperature.
Fig. 7 is a plot of p24:anti-p24 antigen:antibody
binding as a function of pressure for 10 minutes at
ambient temperature.
Fig. 8 is a.plot of p24:anti-p24 antigen:.antibody
binding as a function of time at 60,000 psi at ambient
temperature.
Detailed Description of the Invention
Assays: Various detailed embodiments are
discussed in greater detail below. As noted, some of
these embodiments involve either irreversible or
reversible separation of endogenous complexes. Other
embodiments improve association of the desired
(exogenous) complex. General considerations regarding
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pressure selection to achieve these various embodiments
are also discussed below.
Irreversible dissociation: Generally,
irreversible separation requires sufficient pressure to
irreversibly alter at least one complexing member, e.g.,
by irreversible structure disruption (e.g., alteration of
the tertiary structure) of a protein. It is usually
preferable to disrupt the structure of the endogenous
sample component, rather than the analyte, so as not to
affect analyte binding to the exogenous binding partner.
In some cases, however, it may be possible to
irreversibly alter the analyte so as to prevent
reassociation of particularly troublesome endogenous
complexes, without affecting the analyte binding
characteristics necessary for the assay. Typically,
where the analyte is a non-protein antigen, and the
endogenous complex includes an endogenous antibody, the
pressure can be used to irreversibly disrupt the
structure of the endogenous antibody without
substantially affecting the antigen. In the case of
serological assays, the endogenous antibody is the
analyte and it would be preferable to irreversibly
disrupt the structure of the antigen resulting from the
host infection.
Pressures over 60,000 psi have been shown to
effect irreversible separation of endogenous complexes,
such as immunocomplexes. Pressure generally will not be
high enough (e.g., not over 150,000 psi, and preferably
not over 80,000 psi) to irreversibly alter the analyte.
In one format of irreversible structure
disruption, the sample is loaded in a small pressure
vessel, such as is disclosed in commonly owned WO 96/27432,
filed March 7, 1996, entitled "Pressure Cycling Reactor".
Pressure is increased to between 30,000 and 60,000 psi
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and thep returned to a pressure that permits or even
enhances the complex formation necessary for assay
determination.
Determination of the optimum pressure may involve
identification of the range in which desired irreversible
structure disruption occurs without adversely affecting
other system components. As described below, analyte
binding generally is related to temperature and pressure.
First, analyte pressure sensitivity can be determined by
simple experiments in which analyte solutions are
subjected to increasing pressures (e.g., 1,000 to 60,000
psi) and then assayed with standard techniques to
determine a structure disruption threshold for the
exogenous complex used in that assay. Then, actual
samples containing endogenous complexes can be subjected
to pressures below that threshold to determine whether
irreversible complex separation occurs.
If necessary, temperature can be modulated to
promote structure disruption. For example, at low
temperature, pressure-induced structure disruption occurs
at lower pressure than at ambient temperature. At high
temperature, higher pressure is required to promote
structure disruption than at ambient temperature.
Further control may be achieved by adding agents such as
chaotropes, water miscible organic solvents, chelating
agents, detergents (e.g., triton X-100TM), urea,
thiocyanates, acids, bases, etc. Preferably, the
concentration of such agents is low enough to neither
cause complex separation at ambient pressure nor affect
subsequent assay steps. High pressure makes the complex
components particularly vulnerable to these agents; thus,
low concentrations can be effective.
Once the sample pressure has been reduced (e.g.,
to ambient pressure), the assay can then be performed
according to any of the many well known formats,
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including competitive and excess reagent immunoassays and
affinity labeling formats. Those skilled in the art will
appreciate that there are many suitable immunoassay
formats, relying on various reagents (e.g., enzymatic
reactions that generate color, fluorescent reactions,
radiolabels, colloidal gold, and many others) to permit
visualization and/or quantitation of analyte.
Those skilled in the art will also understand that
there are many suitable physical formats and devices that
can be used. For example, the pressure treated sample
can simply be removed from the pressure vial and applied
to a standard solid phase capture format (see, e.g.,
David et al., U.S. patents 4,486,530 and 4,376,110).
The reagents used in the above-described formats
can also be varied without altering the spirit of the
invention. Monoclonal antibody-based technology can be
selected for the specific properties at issue, e.g., the
ability to bind an epitope of the sample antigen that is
insensitive to the pressures used to structurally disrupt
other epitopes. Standard monoclonal antibody screening
techniques that can be used to obtain such antibodies
will be apparent from this description.
Reversible Dissociation: In one embodiment, the
pressure dissociates the endogenous antibody sample
complexes, followed by separation of the desired fraction
to allow detection of the partner. There are a number of
ways this can be accomplished. For example, if the aim
is to detect antigen that has been complexed by antibody,
the addition of (or to) an excess of immobilized antigen
will effectively bind up the antibody and remove it from
the system. The completeness of this dissociation will
depend on the amount of excess reagent.
Other separation techniques include physical
fractionation by size (e.g., gel filtration to allow the
large molecular weight component to wash through), the
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use of solvents to extract small hydrophobic antigens, or
removal of freed up antibody by affinity extraction using
an immobilized secondary antibody.
It will be apparent to those skilled in the art
that many physical and immunological separation
techniques are potentially applicable as a means to
separate or otherwise inhibit the newly uncomplexed
component to prevent it from reassociating.
Figs. 1-3, abstracted from WO 96/27432 disclose
an apparatus that can be used to maintain high pressure
on a sample, as described below. Another apparatus is
described therein, that allows reagents to flow through a
high-pressure reactor to remove separated endogenous
sample components from the chamber.
Pressure Cycling Reactor: Referring to Fig. 1, a
reactor 10 includes a reaction module 12 having a wall 13
defining a chamber 15 for containing a sample capsule 17,
made from, e.g., polyethylene. A port 12a in reaction
module 12 permits placement of capsule 17 into chamber
15. A short stroke pressure transmitting pneumatic
cylinder 14 and a pressure transmitting hydraulic
cylinder 16 for applying pressure to chamber 15 are
mounted to a base plate 18. Reaction module 12 is
supported by a truss 20 made of, e.g., stainless steel,
defining a variable volume pressure chamber 23. A piston
22 having, e.g., a 3/16" diameter, supported and guided
by a bearing 24 made from, e.g., a bronze alloy, and
defining a chamber wall 27 communicates between a bore 25
in reaction chamber 12 and cylinders 14 and 16. Piston
22 along with cylinders 14 and 16 form a vessel
pressurizer 21 which controls the pressure in chamber 15.
Reaction module 12 is made from, e.g., stainless steel
and bore 25 has a diameter of about 0.188" and a length
of about 1".
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Referring also to Fig. 2, a seal 36, e.g., a
spring energized seal including a spring 36a and back-up
washers 37, 37a, and 37b available from Bal Seal
Engineering Co. Inc., Santa Ana, CA, supported by, e.g.,
a gland washer 38, seals piston 22 within reaction
chamber 12. Gland washer 38 includes an inclined surface
39 which mates with an inclined surface 39a of back-up
washer 37b. Spring 36a ensures sealing at low pressures
and assists sealing at higher pressures. The back-up
washers and the gland washer act to prevent extrusion of
seal 36 under pressure. Gland washer 38 is supported by
a shelf 40 in truss 20.
Pneumatic cylinder 14 includes a piston 14a
having, e.g., a 2.5" diameter, with an extension rod 26
having a through bore 28. 0-ring 14b forms a seal
between piston 14a and an inner wall 100 of pneumatic
cylinder 14. When pneumatic cylinder 14 is energized,
pressure on piston 14a is transmitted to guide bearing 24
by rod 26 creating an upward force on piston 22. This
force is adjustable by varying the pressure to the
pneumatic cylinder from a source (not shown).attached to
inlet 30. The force applied to piston 22 by pneumatic
cylinder 14 determines the low pressure level within
reaction chamber 12 during pressure pulsing. Pneumatic
cylinder pressure is adjustable up to about 100 psi
(which produces a pressure of about 17,000 psi in
reaction chamber 12).
Hydraulic cylinder 16 includes a piston 16a
having, e.g., a 1" diameter, with an extension rod 32
projecting up through bore 28 of pneumatic cylinder rod
26. The end 34 of hydraulic rod 32 bears against guide
bearing 24. On extension, hydraulic rod 32 drives piston
22 upwards. The pressure generated by hydraulic cylinder
16 plus the pressure generated by pneumatic cylinder 14
determines the high pressure level in the reaction
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chamber. Hydraulic cylinder pressure is adjustable with
an upper limit set at approximately 1,500 psi. The ratio
of the cross-sectional area of hydraulic piston 16a to
the cross-sectional area of piston 22 is 28.4:1.
Reaction module 12 is surrounded by a
thermostatting jacket 42 to control the temperature of
the reaction chamber. Jacket 42 has inlet and outlet
fittings 44, 46, respectively, which permit fluid to be
circulated into chamber 48 surrounding wall 13 from a
temperature controlled heating/refrigeration bath (not
shown). Around the top and bottom of reaction chamber 12
are 0-rings 52, 54 which provide a fluid seal for
thermostatting jacket 42. A thermocouple (not shown) is
mounted to reaction chamber 12 to monitor the temperature
of the chamber. The temperature of chamber 12 is
controlled within a range of about -.15 C to +40 C with an
accuracy of about +/- 1 C. The thickness, e.g., 3/16",
of wall 13 is selected to be thin enough to permit heat
transfer from thermostatting chamber 48 to sample chamber
15 while being thick enough to withstand the pressures
applied to sample chamber 15. The maximum allowable
pressure in reaction module 12 as determined by the
thickness of wall 13 as well as by the performance of
seal 36 is about 40,000 psi.
Mounted at the top of the assembly is a pressure
transducer 56, for example, a 75,000 psi, +/- 0.5%
accuracy strain gauge type transducer available as part
number HP/5651-02-02 from Sensotec, Inc., Columbus, OH.
Pressure transducer 56 is removed from reaction module 12
to allow access to port 12a.
Referring to Fig. 3, hydraulic pressure is
delivered to hydraulic cylinder 16 by an hydraulic pump
62. An hydraulic system 60 includes hydraulic pump 62
driven by a motor 64, a fluid reservoir 63, a relief
valve 67, a check valve 65, a pressure adjustment valve
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66, a pressure gauge 68, an accumulator 70, a manual
valve 71, a four-way directional control valve 72, and
hydraulic cylinder 16.
Motor 64 is, e.g., a 2 HP electric motor with an
output of 0.8 GPM at 3,000 psi max. Actual system
pressure is controlled by pressure adjustment valve 66
and is variable up to the set upper limit of
approximately 1,500 psi.
Directional control valve 72 is, e.g., a three
position spring centered spool valve actuated by dual
electrical solenoids, available as BoschTM part #9810231072
from Pearse-Pearson, Inc., Milford, MA. With both
solenoids de-energized, both hydraulic cylinder ports 19,
19a are connected to a drain line 74, and cylinder piston
16a may be freely moved. In use, energizing one solenoid
pressurizes port 19 and with the other solenoid
deenergized so that it is open to drain line 74, piston
16a is forced to extend thus pressurizing sample chamber
15. To now pulse the pressure in the sample chamber,
both solenoids are deenergized so piston 16a is free to
move and the pressure in chamber 15 forces piston 22 down
releasing the pressure in the chamber (to the level of
pressure applied by pneumatic cylinder 14).
Alternatively, energizing the other solenoid pressurizes
port 19a and with the other solenoid deenergized so that
it is open to drain line 74, piston 16a is forced to
retract. The passive release of pressure from chamber 15
is preferable because it provides for a faster hydraulic
response time and the continual contact between piston 22
and rod 32 avoids producing impact loads between the
piston and the rod. Directional control valve 72 can be
rapidly switched at times down to 20-25 ms to apply
pressure to hydraulic cylinder 16 and to allow release of
pressure from chamber 15.
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Hydraulic accumulator 70 is mounted near
directional control valve 72 to enhance response and
dampen pressure fluctuations. Accumulator 70 has, e.g.,
a one quart capacity and is charged by pump 62 to
hydraulic system line pressure. The presence of check
valve 65 causes accumulator 70 to remain charged after
pump 62 is turned off. Manual valve 71 is used to
discharge the accumulator and depressurize the hydraulic
system. Relief valve 67 limits the maximum delivered
pressure from pump 62.
It is not intended that the present invention be
limited by the nature of the pressure device. A "manual"
instrument system capable of generating pressures of 411
MPa is commercially available; the system uses silicone
oil as the pressurizing medium, and has a 2 ml reaction
vessel (High Pressure Equipment Company, Erie, PA). A
schematic of this system is shown in Fig. 1.
In some experiments, the high pressure apparatus
is a device having the following components: 1 pressure
generator (max. pressure 60,000 psi) cat # 37-5.75-60; 1
pressure gauge cat # 6PG75; 2 valves cat #60-11HF4; 2
tees cat # 60-23HF4; 4, 1/4" x 6" nipples cat #60-8M4-2;
2, 1/4" x 2 3/4" nipples cat # 60-8M4-1 and 1, 2 ml
reaction chamber. A pressure gauge with 5 MPa increments
is also connected to the system.
The solutions to be pressurized are placed in
small deformable polyethylene capsules that are crimp
sealed at the ends. A capsule containing 10 to 50 l of
the enzyme/substrate solution is placed in the reaction
vessel, which is then connected to the system and
pressurized.
It is contemplated that pressure control can be
combined with the use of temperature control. This is
useful for, among other things, controlling the enzyme
during dead time (e.g., time to mix and load the sample
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before pressure treatment, and time to unload and remove
sample for analysis). In some experiments, separate
enzyme and substrate solutions are held on ice. The
reaction chamber can be held in a refrigerator (about
5 C) .
Temperature can be used in conjunction with
pressure to control enzyme activity, as well. For
example, a relatively small decrease in temperature
(i.e., 5 C) can cause significant decreases in the rate
of digestion in some enzyme systems, thus providing a
researcher with greater control over enzyme activity;
this may be especially important during dead time, when
it may be advantageous to prevent (to the extent possible
without harming the enzyme) the digestion taking place
while not under pressure.
Improved association of the desired complex: As
noted, pressure control may enhance analyte binding to
the selected exogenous binding partner, thereby having a
significant effect on assay speed as well as improving
affinity, sensitivity, or specificity. Specifically, a
pressure regime is selected that improves binding by the
pressure selection technique discussed below.
Temperature and other assay conditions can be varied to
further optimize complex formation for a given assay.
These conditions are achieved in a pressure chamber, such
as the one discussed above.
Pressure selection: In general, binding partners
across a range of pressure sensitivities will have a
phase diagram exhibiting both reversible and irreversible
disruption of structure and both can be applied to
improve assay procedures. To optimize use of pressure
(as well as time, temperature and other controls), it is
useful to develop such a phase diagram
(temperature/pressure/association) for the complex being
detected and for any endogenous complexes that may
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interfere with detection. As noted above, pressure can
affect not only the equilibrium of the complex, but also
the rate at which that equilibrium is reached. To
provide the phase diagram and screen candidate binding
partners, one can:
1. define the association rate of the binding
partners under ambient conditions (atmospheric pressure
and constant room temperature);
2. increase pressure to levels below those which
disrupt the structure of the native state of one or both
of the complex binding partners, thereby defining the
effect of pressure on binding rate and equilibrium;
3. increase pressure level still further to
define minimal and maximal pressure for reversible
disruption of the structure of one or both of the binding
partners; and
4. increase pressure still further to define
minimal pressure for irreversible disruption of the
structure of one or both of the binding partners.
Typically, temperature is kept constant (5-40 C; with
thermostable reagents, the temperature can be increased
up to about 95 C) and a constant pressure pulse, ranging
from about 25 milliseconds to about 15 minutes, is
delivered.
Incrementally increasing pressure from atmospheric
up to 150,000 psi defines four domains:
1. a domain at atmospheric pressure;
2. a domain of enhanced rate of association
(typically below 60,000 psi);
3. a domain of reversible dissociation
(reversible structure disruption of one or both binding
partners), typically between about 30,000 and 100,000
psi; and
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4. a domain of irreversible structure disruption
of one or both of the binding partners (typically between
100,000 and 150,000).
The steps required to ascertain the pressure
boundaries of these domains for a given binding pair
include: a) definition of a baseline by determination of
the association properties of the binding system at
atmospheric pressure and room temperature; b)
determination of the association rate in the domain of
enhanced association; c) determination of the range of
reversible structure disruption; and d) determination of
the irreversible disassociation range. Protocols for
developing such phase diagrams are provided below.
Fig. 4 shows a typical phase diagram (see Mozhaev
et al., TIBTECH 12:496, 1994). Based on such diagrams,
it is a routine procedure to determine regions of
dissociation and association. The former (dissociation)
can be further divided into reversible and irreversible.
Once the phase diagram is known, assay pressure may be
used to select a desired regime (reversible dissociation,
irreversible dissociation, improved association) for
operation of the assay.
As a general rule, moderately high pressures
(i.e., up to a point) can accelerate the endogenous
complex formation. The above-described phase diagram can
provide guidance with respect to precise phase boundaries
(changes from association to dissociation) for a specific
system. While the exact phase boundaries of individual
systems may vary, in general, with pH, pressures above
60,000 psi generally will dissociate complexes in
standard buffers. At lower pressures, the complex will
remain associated, in many cases with either faster
kinetics or stronger binding at atmospheric pressure, or
both. Without limiting ourselves to a specific molecular
mechanism, it appears that differential sensitivity of
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analytes, such as proteins, to hyperbaric pressure may be
related to different primary, secondary, tertiary, and
quaternary structures, for example, subunit interactions
of oligomeric proteins. Generally, a protein in solution
can be introduced into a pressure/temperature reaction
chamber. As the pressure is elevated, the protein
initially is able to withstand elevated temperature
without significant structural disruption. However, as
the pressure increases, the solution eventually reaches a
transition point beyond which increasing pressure begins
to considerably disrupt the structure of the protein from
its native state.
The phase diagram also indicates, in general, that
although proteins at slightly elevated temperatures can
be structurally disrupted, the proteins can generally
withstand somewhat higher temperatures without altering
their native states if pressure is applied while
temperature is kept relatively constant. If, however,
the temperature is maintained at a level around 0 C,
increasing pressure may disrupt the structure of the
protein earlier (i.e., at a lower pressure) than would
increasing pressure at ambient temperature; at high
temperatures (e.g., 37 C) the structure is disrupted at
still higher pressure. Typically, a protein (analyte) is
capable of transitioning reversibly through native and
structurally disrupted phases at pressure levels less
than 60,000 psi and at temperatures between about 10 C
and about 40 C.
The protein (analyte) can be denatured by any of
temperature, pH, or pressure acting independently or in
combination. Binder/ligand interactions can be, although
are not limited to, a result of one or more of several
different types of interactions, such as electrostatic,
hydrophobic, aromatic ring stacking, hydrogen bonding,
etc. (e.g., van der Waals interactions). These various
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interactions can interfere with one another
constructively or destructively. For example,
electrostatic interactions can be both increased (e.g.,
hydration of a charged group) and decreased (e.g.,
formation of electrostatic bond) by raising the pressure
of a biosystem.
As the pressure of a solution of binding partners
is increased, the binder/ligand complexation interactions
initially are enhanced. However, as the pressure is
increased further, denaturation of the native bimolecular
state occurs, prompting dissociation of the binder/ligand
complexes. This dissociation may be reversible or
irreversible. The dissociation sensitivity is also
dependent upon solvents and pH. Improvements in
binder/ligand interactions of diagnostic assays are
obtained by increasing the pressure of a reaction
solution containing the binding partners. As described
above, improvement is obtained by two effects, namely: 1)
acceleration of binder/ligand interactions; and 2)
reduction or elimination of endogenous complex
interactions.
Two additional approaches can be used to enhance
assays by separating endogenous complexes. The
separation can be effectively irreversible, in which case
the continued presence of endogenous sample component
does not seriously interfere with the assay.
Alternatively, the separation can be reversible, in which
case the analyte generally should be separated from the
endogenous sample component, if possible, to maximize
assay sensitivity. Kinetic or thermodynamic advantages
may be obtained even without separation, e.g., where the
exogenous binding partner must compete with the
endogenous sample component to bind analyte.
High Throughput Screening: Industrial techniques
utilizing molecular diversity enable the randomized
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generation of large numbers of targets that can, for
example, be directed against antibody epitopes. Rapid
synthesis of combinatorial libraries that include arrays
of novel structures made by the random or directed
synthesis of a combination of smaller molecular building
blocks requires rapid screening procedures. Molecular
diversity can be useful in the discovery of novel
proteins, carbohydrates, antibodies, ribozymes,
oligonucleotides, peptides, antisense, aptamers, DNA
(e.g., single-strand, double-strand, or double-strand
with single-strand overhangs), RNA, and small organic
molecules. For example, in vitro techniques such as
modified recombinant, phage display techniques, which can
be used to generate an immune response against a compound
of interest, generating as many as 105 - 108 antibody
sequence variations, or more. Very large combinatorial
selection processes can then be performed against the
generated antibodies. The binding affinity of the
antibody-antigen complex is modulated by the pressure-
related processes described herein, enabling faster and
more efficient complex selection.
Where it is desirable to monitor the course of the
reaction or the products of the reaction, the pressure
cycling reactor includes a detector connected to detect a
characteristic of a component present in fluid in or
removed from the reaction vessel. The detector can be
computer controlled and can relay information regarding
the analyzed component to the computer. Thus, components
can be analyzed before, during, or after a pressure pulse
while in the reaction vessel; components can also be
analyzed after being removed from the reaction vessel.
Aptamers: One embodiment of the invention
features the dissociation of endogenous antibody analyte
complexes to facilitate subsequent assay of the released
analyte with appropriate immunoassay reagents or
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immunoaffinity purification. If the pressure phase
diagrams of the complexes of the analyte with the
endogenous antibody and the immunoassay reagents are
sufficiently different, dissociation of endogenous
immunocomplexes and association of selected exogenous
binding partners can be enhanced simultaneously under
suitable pressure conditions. Achievement of this goal
requires the identification of specific complexing agents
that are useful in immunoassay or affinity purification
procedures that, for example, have pressure phase
diagrams drastically different from conventional antigen-
antibody complexes. A class of reagents that can meet
this requirement is the family of oligonucleotides that
bind specific analytes with high affinity. These
oligonucleotides are often referred to as aptamers (Annu.
Rev. Biochem., 64:763-797, 1995). The interaction of
oligonucleotides with ligands such as proteins,
polypeptides, and small molecules can occur with very
high affinity and great selectivity. Procedures are
available for selection of such reagents from random
polynucleotide libraries. For example, screening of a
randomized oligonucleotide library for binding to basic
fibroblast growth factor (bFGF) yielded specific 30-mer
sequences that bound to native bFGF with dissociation
constants as low as 0.2 nM, while unable to bind to
denatured bFGF, an indication of high specificity (Proc.
Nat1., Acad. Sci. USA, 90:11227-11231, 1993). Because
the structural principles underlying nucleic acid-ligand
interaction and antibody-antigen interaction are
fundamentally different (cf. pp.789-790 of Gold et al.,
op. cit.), the probability that the pressure phase
diagrams for the binding of a ligand to an aptamer and to
an antibody are different is very high. By determining
the phase diagram for an analyte with an aptamer and
comparing it with the corresponding phase diagram for the
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analyte-antibody immunocomplex, conditions can be found
that will favor the formation of the former over the
latter.
Protein Refolding: A growing number of proteins
are produced using recombinant DNA technologies. A
frequent problem encountered in the commercial and
research-oriented production of recombinant proteins is
that overexpression of the genes encoding for the protein
can lead to aggregated and non-functional protein.
Overproduced proteins are often contained in inclusion
bodies inside of the producing cells. Inclusion bodies
are easily removed from other cell debris by means such
as centrifugation or filtration and provide for rapid
recovery and a powerful purification which becomes useful
only when the protein can be successfully dispersed and
refolded into an active form. Current procedures for
refolding include addition of chaperone proteins,
detergents, organic solvents or chaotropic agents such as
urea or guanidinium chloride followed by dialysis to
remove these agents. These processes can be time
consuming and expensive when performed on a large scale.
The new methods described herein provide a method
that can rapidly disrupt aggregates and allow them to
refold as individual, active proteins without the use of
dialysis. The method utilizes the ability of high
hydrostatic pressure to strongly and rapidly disrupt
aggregation and/or cause reversible protein unfolding.
The refolding reaction mixtures can include, for
example, a pressure sensitive buffer and a solid phase
resin. The pressure effect on the charge of the resin
will be the sum of its own ionization volume and that of
the buffer, allowing large changes in the charge of the
resin. Such a resin and buffer system would constitute
an ion exchange system which can reversibly bind and
release any ionic compound.
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By cycling between high and low hydrostatic
pressure, the following will occur:
1) The pH of the solution will fluctuate, causing
disruption of the aggregated proteins.
2) The charge on the resin will fluctuate,
reversibly binding the protein molecules and catalyzing
the refolding of the protein relative to re-aggregation.
A protein with a multiplicity of acidic groups
would benefit from a resin/buffer system in which the
resin becomes more positively charged at high pressure,
whereas a protein with many basic groups would suggest
the choice of a system in which the solid became more
negatively charged with pressure.
Aggregated proteins that are cross-linked by
disulfide bonds can be refolded, using high pressure and
a pressure sensitive buffer to simultaneously denature
the protein molecules and raise the pH to increase the
activity of a reducing agent such as dithiothreitol.
Upon lowering of the pressure, the protein can refold and
be reoxidized (e.g., by air or other oxidant) to form the
correct disulfide bonds.
The new method has the advantages of increasing
the rate of refolding and facilitating the release of the
protein from the matrix, resulting in a high
concentration of folded protein.
Separation: Hyperbaric pressures can alter
binding rates and affect binding affinity. Binding
partners that have low affinity at atmospheric pressure
can, for example, behave as high affinity partners at
higher pressures. The pressure-mediated variable control
of binding affinity can be used as a purification method.
For instance, contaminated mixtures containing
compounds of interest can be placed in fluid contact with
molecules known to bind the compounds with pressure-
dependent affinity. That is, low affinity at atmospheric
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pressure, higher affinity at elevated pressures
(association enhancement); or vice versa (dissociation
enhancement). Examples are described below. To
facilitate later dissociation of the binding partners and
subsequent collection of the desired compound, the latter
molecules can be immobilized.
In association enhancement, the pressure in the
reaction vessel can be elevated before, during, or
subsequent to the introduction of the desired compounds.
This causes the compounds to bind tightly to the
immobilized binding molecules, as unbound contaminants
are flushed away with vigorous washing with fluidic
liquid (e.g., solvent) or gas. In a final step, the
purified compounds can be dissociated from the
immobilized binding molecules at atmospheric pressure and
collected at atmospheric pressure. .
The association enhancement scenario is
appropriate for describing protein-protein binding
interactions, especially if the affinity of the proteins
for each other is relatively low at atmospheric pressure.
This makes the proteins involved ideal for purification
by the new methods, as the affinity can potentially be
increased drastically by pressure change. An
illustration of a protein-protein interaction is shown in
Fig. 5.
At point A in Fig. 5, one protein is immobilized
on a solid phase support at low pressure. A mixture
containing the other protein is introduced at point B,
also at low pressure. A small amount of complex may form
between the proteins (point C). However, when the
pressure is raised to a higher pressure at point D,
complexation is greatly enhanced. The complexes can be
washed with clean solvent at point E. It is possible to
wash the complexes without losing the complexes
themselves because they are attached to the solid
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support. This step removes at least some of, and
preferably most of, the contaminants. The pressure is
finally lowered (point F), and one protein is separated
from the other protein (point F).
In dissociation enhancement, only the final step
differs. Rather than the purified compounds being
dissociated from the immobilized binding molecules at
atmospheric pressure, the pressure is raised to a second
pressure that is high enough to cause dissociation. As
described above in reference to assays, moderate
pressures tend to increase binding affinity whereas still
higher pressures (e.g., greater than about 30,000, or
even 60,000, psi) can, for example, cause reversible (or
irreversible) structure disruption of one of the binding
partners, which in turn can result in dissociation. The
dissociation enhancement scenario is fitting, for
example, for characterizing the binding of antibodies to
antigens. Antigen-antibody binding constants range from
below 105 mol-1 to above 1012 mol-1, and most typically
are from about 108 to 1010 mol-1.
A number of other scenarios are also plausible.
For example, temperature and pressure can be
simultaneously manipulated to affect association and
dissociation rates, as described above in connection with
assays. Kinetic studies and pressure-temperature
diagrams can also be used to assist in predicting the
association and dissociation characteristics of model
systems.
In still another example, contaminated mixtures
containing the compounds to be isolated can be placed in
fluid contact with binding molecules that bind to and
exhibit pressure-dependent inhibition of enzyme catalytic
activity toward the compounds (i.e., high activity at
atmospheric pressure, essentially no activity at elevated
pressure; high binding affinity at all pressures). In
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this case, the mixture can be introduced to the binding
molecules at high pressures to form non-reactive binding
complexes, the contaminants can be flushed away, and a
reagent can be introduced to cause dissociation of the
binding complex. As used above, enzyme catalytic
activity refers to the ability of the enzyme to
chemically convert the substrate to a product.
The latter procedure can be used, for example, for
purifying the substrate of an enzyme. Enzymes bind to
their substrates with great selectivity and affinity. At
atmospheric pressure, a molecule of an enzyme binds to a
substrate molecule and converts the substrate molecule
into a product molecule. The remarkable ability of
enzymes to bind to their substrates with high selectivity
is generally unaffected by pressure. However, the
catalytic activity of the enzymes is generally rendered
inactive by high pressure conditions.
Thus, although the enzyme will still bind with
great selectivity to its substrate under hyperbaric
conditions, the substrate will not be converted to
product. If the enzyme is immobilized by attachment to a
solid support, for example, the substrate will also be
immobilized, while the contaminants are being flushed
away with a wash solution.
Addition of a reagent that facilitates the
dissociation of the enzyme-substrate complex at the
elevated pressure can allow the purified substrate to be
isolated. Reagents that can be useful for such
dissociation include, but are not limited to: acids;
bases; salts, such as NaCl or MgBr2; metal-scavengers;
detergents, such as sodium dodecyl sulfate (SDS);
dissociating agents; chaotropic agents, such as
thiocyanate; water; organic solvents, such as dioxane,
ethylene glycol, or dimethylsulfoxide; chelating agents;
or other binding partners, such as metal ions. Non-
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chaotropic agents can also be used; examples include AFC
Elution Medium (Sterogene Biochemicals) or InanunopureTM
Gentle Ag/Ab Elution Buffer (Pierce).
Dissociation can also be effected by raising the
pressure of the enzyme-substrate complexes still higher,
as described above in the context of dissociation of
endogenous ligands in preparation for assays. In this.
case, pressures that allow reversible dissociation are
preferred, since the binding molecules ideally will be
reused. The enzymes themselves can also be isolated in a
similar manner, by using immobilized substrates to
capture the enzymes present in a mixture.
Alternatively, if the goal is to isolate a
relatively purified product (i.e., a substance obtained
by enzymatic reaction of the substrate), rather than to
isolate the substrate itself, the same procedure can
again be employed, with the exception that the pressure
is instead lowered after washing. Reducing the pressure
to atmospheric pressure, for example, can allow the
enzyme to regain its activity. The purified substrate
can then be enzymatically converted to a high purity
product. Enzymes typically have much lower affinity for
the products than for the original substrates, so the
products can be easily isolated.
Moderately high or low pressure dissociation is
generally preferable to traditional elution methods
(e.g., chemical methods, such as pH-controlled elution),
as the traditional methods are typically harsher and can
lead, for example, to irreversible structure disruption
of proteins. As mentioned above, very high pressures
(e.g., greater than about 100,000 psi) can also lead to
irreversible structure disruption.
A contaminated mixture in the present context
includes a contaminant, a material that is present but
not desired in the sample. Examples of contaminants
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include byproducts from the manufacture of the desired
compound, degradation products of the desired compound,
catalysts, impurities contained in natural or commercial
materials, and residual high-boiling solvent.
In most cases, there can be two or more binding
components in a mixture, one of which can be of lower
molecular weight and size and can be a peptidic ligand,
an antigen, or a nucleic acid probe, for example. The
other partner can be a receptor, an antibody, or target
DNA or RNA, or other molecules that bind to the first
compound. Either component can play either role; thus,
in the examples given above, either the ligand or the
receptor, the antigen or the antibody, or the probe or
the target can be the desired compound to be purified.
The other component would preferably be present in the
form of a binding molecule immobilized on a solid support
or membrane.
Both the contaminated compound to be purified and
the immobilized binding molecules are dissolved in,
suspended in, or mostly surrounded by a fluid. The fluid
can be a liquid (e.g., acidic, neutral, or basic aqueous
or organic solvents, or solutions thereof), a gas (e.g.,
an inert gas or a noble gas), or even a supercritical
fluid.
Binding molecules can be immobilized to a solid
support, such as a.particle, a solid or hollow polymer
bead, a well, a tube or column, a strip, a molded
material, a polymer matrix, a semipermeable, porous or
nonporous membrane in the form of a filter or bag, or
other support material. Solid supports made of various
polymers in many physical configurations and with
activated or specifically reactive surfaces are
commercially available for use in research, diagnostic,
and bioseparation products. Numerous methods for
covalently immobilizing ligands or binding proteins to
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surfaces are described in "Affinity Chromatography:
Bioselective Adsorption on Inert Supports" by William J.
Scouten, WiZey-Interscience (1981). The binding of the
molecule to the solid support is often achieved through
passive adsorption, where the reactant and surface allow
such manipulation, or by chemical (covalent) linkage to a
specific reactive group on the surface. Non-limiting
examples of contaminants and compatible supports are
provided in Table 1.
TABLE 1
Contaminant Type Example Immobilized
Binder
pyrogens endotoxins, histidine
lipopolysaccharides
proteolytic endoproteases a2-macro-
enzymes globulin,
carrier fixed
detergents triton-X100, or BioBeads SM-2Tm
sodium dodecyl isolation
process
sulfate
lipids lipoproteins Lipidex 1000TM
heavy metals mercury organic thiols
viruses hepatitis B virus octanoic acid
hydrazide
The phrase "bound complex" refers to the actual
combination of the desired compounds and the immobilized
binding molecules. These binding complexes are often
named by their components. For example, if A is a ligand
and B is a receptor, the binding complex can be
represented as [AB]. Preferably, the complexes described
herein are dissociably linked, meaning that the complex
formation is reversible. Generally, the linkage is
electrostatic (e.g., via hydrogen bonds, van der Waals
forces, or ionic bonds), although it can be covalent. In
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most cases, the reverse process (i.e., dissociation) can
be induced by further varying the pressure or by adding a
reagent.
The following examples will illustrate the
invention, but they do not limit it. Examples 1-5 are
prophetic examples; 6-10 are actual examples.
EXAMPLE 1
Pressure modulation and solid-phase immunosorbant used to
prepare serum sample for assay of HIV p24:
Step 1: Immobilizing immunosorbent on the tube
wall.
Into polypropylene tubes, approximately sized for
the pressure device, is dispensed 100 l of a 5 to 10
g/ml dilution of prepurified recombinant HIV-1 p24
(obtained from Immunodiagnostics, Inc., Bedford, MA) in
0.1 M potassium carbonate buffer, pH 9.3. This mixture
is allowed to incubate overnight in a refrigerator at 2-
8 C. The solution is decanted, and 100 l of 2% Bovine
Serum Albumin solution in PBS (0.05 M Phosphate buffered
saline, pH 7.4, PBS) is added. This resulting mixture is
decanted and a second addition of the same solution is
added and allowed to block the walls of the container for
at least two hours at room temperature. The solution is
decanted and the tubes are inverted and allowed to dry at
room temperature. They are stored at 2-8 C in tightly
sealed plastic bags containing added desiccant to assure
dryness.
Step 2: Sample preparation
Into the containers described in Step 1 is added
50 l of serum sample to be tested. An identical volume
of either PBS or PBS containing an appropriate
dissociation accelerant (e.g., glycine-HCL, pH 2.5, urea,
or a water miscible, organic solvent), is added with
mixing. The tubes are sequentially inserted into the
hyperbaric device and the pressure raised to 50,000 psi
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for a suitable period. The tubes are allowed to return
to atmospheric pressure and incubated at room temperature
for at least two hours to assure that the endogenous
binder in the sample will be competitively bound by the
large excess of immobilized antigen on the internal wall
of the tubes. An aliquot of the binder-depleted sample
is now removed for conventional determination of HIV p24.
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EXAMPLE 2
Use of particulate format immunosorbent:
Step 1: Preparation of Latex immunosorbent
A bulk preparation of HIV-1 p24 adsorbed latex
particles is prepared by following the procedure
recommended by Bangs Laboratories (Carmel, IN). The
resultant suspension is stored refrigerated at 2-8 C
until use.
Step 2: Sample preparation
Into polypropylene tubes, approximately sized for
the pressure device, is dispensed 100 l of the Latex
immunosorbent and 50 l of each sample to be tested. The
tubes are sequentially inserted into the hyperbaric
device and the pressure raised to 50,000 psi for an
appropriate period. The tubes are allowed to return to
atmospheric pressure and incubated at room temperature
for at least two hours to assure that the endogenous
binder in the sample will be competitively bound by the
large excess of immobilized antigen on the latex
particles. The tubes are rapidly spun at approximately
2,000 xg to sediment the particles. An aliquot of the
binder-depleted sample is now removed for conventional
testing.
EXAMPLE 3
Pressure modulation and size fractionation used to
prepare serum sample for assay of HIV p24:
Step 1: Size fractionation devices
Micro-sized gel filtration columnar devices from
Pharmacia are prepared just before use by passing
approximately one ml of PBS through.
Step 2: Sample preparation
Polypropylene tubes, approximately sized for the
pressure device, each containing 50 l of a sample to be
tested are subjected to the pressure as in Example 1 and
immediately flash frozen. The sealed base of each tube
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is sequentially cut open and the resultant bottomless
tubes are placed into the gel filtration columnar devices
and allowed to thaw. Without delay, cold (4 C) PBS is
flowed through the tubes into the resin bed at the rate
recommended by the device manufacturer and collection is
initiated. The first fraction, containing the large
antibody portion, is discarded and the second HIV-1 p24
fraction is collected and assayed in the conventional
manner.
EXAMPLE 4
Determination of a Aressure-temperature nhase diagram of
antigen-antibody reactivity for the HIV gag A24:rabbit
anti-p24 pair:
The following procedure illustrates the creation
of data for producing a phase diagram as described above.
The specific illustration involves recombinant HIV (HIV-1
bS) gag p24 and rabbit anti-p24.
Materials
A solid phase immunosorbent for antibody to HIV-1
gag p24 was prepared by coating polystyrene microliter
plates (HiBindTm, Corning/CostarT''', Cambridge, MA) with a one
g/mi suspension of recombinant HIV-1 IIIB p24
(ImmunoDiagnostics, Inc, Bedford, MA) overnight at 4 C in
NaHCO31 pH 9.2. Unreacted sites were blocked with
SuperBloc)Jm in PBS or Tris (Pierce Chemical, Chicago, IL).
Experimental method.
Recombinant HIV-1 IIIB gag p24 and rabbit anti-p24
HIV-1 IIIB IgG were incubated together to form samples of
immune complexes. A sample was placed in a deformable
plastic capsule, and overlaid with melting point bath oil
(Sigma, St. Louis, MO). The capsule was placed in a
device for generating high pressures (HiPTM, Erie, PA).
While pressure was applied to an aliquot of the immune
complex, a parallel sample was subjected to the same
temperature conditions at atmospheric pressure. After
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release of high pressure, the sample and a parallel (no
pressure pre-treatment) control was placed in wells of
the p24 coated microplate. The level of free antibody
(uncomplexed with antigen) in samples was quantified by
reacting samples in the microtiter plates for one hour at
ambient temperature, then detecting the captured antibody
with an HRP-labelled second antibody. The degree of
complexation of the pressure treated and control samples
was determined from a standard curve compiled using
varying amounts of.the preformed immune complex, p24, and
anti-p24 antibody such that the concentration of p24 and
anti-p24 was constant in each measurement. Dissociation
or additional association of the antibody:antigen complex
translates respectively to an increase or decrease in
signal intensity in the assay.
EXAMPLE 5
High Throughput Screening of Phage Display of Mutant
Proteins:
A pressure-modulation apparatus which is capable
of introducing, reacting, and removing fluids from a
reaction chamber while under high pressure has been
described in WO 96/27432. This apparatus is fitted with
a modified reaction chamber that enables real-time
detection and monitoring of biomolecular binding
interactions. An example of this type of chamber
includes, for external spectrometry, an optical window
(e.g., sapphire), or, for internal spectrometry, a fiber
optic bundle in direct contact with internal fluids and
targeted against an internal viewing plane. Another
example of an internal detection sensor is an
electrochemical detection probe.
In an example of a phage display technique, the
phage host (e.g., transformed E. coli cells) expresses
gene III capsid protein of phage and the corresponding
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target protein as a fusion protein on its surface. Each
phage moiety is labeled with a reporter molecule (e.g., a
radioactive dye, etc.) or has a chemical structure that
enables detection, e.g., native fluorescence.
The target protein binds to a previously
immobilized target receptor molecule on a solid support,
which then screens the phages. The screening of the
expressed phage mutants, each with different gene III-
antibody fusion proteins, is based on affinity. Serial
introduction of the phage library into the reaction
chamber, modulation of the pressure, and observation of
the real-time binding rate and affinity of interaction
using laser-induced excitation and emission of
fluorescence spectra allow the determination of phage
binding characteristics.
Following affinity analysis and characterization,
the phage is removed from the reaction chamber by
pressure modulation to dissociate the biomolecular
stereoelectronic interactions. This procedure may either
enhance or perturb binding depending upon the volume
change associated with activation. In either case, a
second phage is then introduced for affinity analysis and
characterization. The pressure-modulated association and
dissociation of complexes enables rapid screening. This
serial process can be modified for parallel screening.
EXAMPLE 6
Use of high hydrostatic pressure to accelerate antigen-
antibody binding:
The use of high hydrostatic pressure to accelerate
the rate of binding of an antibody to an antigen was
demonstrated using recombinant HIV-1 IIIB gag p24 and
rabbit anti-p24 HIV-1 IIIB IgG. A pressure of 60,000 psi
(420 MPa) applied for 10 minutes at ambient temperature
(=-22 C) resulted in a level of binding equivalent to the
binding noted after at least four hours at atmospheric
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pressure. A 10 minute application of 30,000 psi (210
MPa) pressure resulted in binding equivalent to 30
minutes at atmospheric pressure. At 20,000 psi (140
MPa), 10 minutes of pressure application produced only
marginal effects on binding, relative to atmospheric
pressure. The details of the experimental protocol
follow.
125 l of 0.2 ng/ l recombinant HIV-1 IIIB gag p24
antigen (ImmunoDiagnostics, Inc., Bedford, MA) was added
to 125 l of 2 ng/ l rabbit anti-p24 HIV-1 IIIB IgG
antibody (ImmunoDiagnostics, Inc.) in phosphate buffered
saline (PBS), pH 7.4 in a polypropylene microfuge tube,
such that 100 l of the antigen/antibody mixture
contained about 10 ng of antigen and about 100 ng of
antibody. 120 l of the reagent mixture was inserted
into a deformable plastic capsule and overlaid with
melting point bath oil (Sigma, St. Louis, MO). The
capsule was immediately placed in the reaction chamber of
a high pressure apparatus (HiP, Erie, PA), maintained at
ambient temperature and the pressure was then raised to
the desired elevated level (i.e., as indicated in the
previous paragraph) using a manually operated piston.
Control samples, in which either the antibody or
the antigen was omitted, were also subjected to the same
experimental conditions. All experiments were performed
at ambient temperature. A solid phase immunosorbent
ELISA assay for detecting antibody to HIV-1 gag p24 was
developed in-house. Polystyrene microtiter plates
(HiBind, Corning/Costar, Cambridge, MA) were coated with
a 1 g/mi suspension of recombinant HIV-1 gag p24 antigen
overnight at 4 C in aqueous NaHCO31 pH 9.2. Unreacted
sites were blocked with SuperBlock in PBS-Tris (Pierce
Chemical, Chicago, IL).
After elevated pressure had been applied for the
desired time, test samples were measured using the p24-
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coated microwell ELISA assay: A 100 l aliquot of the
test sample was immediately removed from the capsule and
placed in a well of the p24-coated microplate; 100 l of
the nonpressurized test solution was tested in parallel.
Test samples were shaken at ambient temperature in the
microtiter wells for one hour, then washed three times
with PBS-0.05% Tween-20TM (PBS-T). Antibody binding to the
immobilized p24 antigen was detected using goat anti-
rabbit IgG conjugated to horseradish peroxidase (HRP)
(Pierce Chemical), and the HRP substrate 2,2'-azido-
bis(3-ethylbenzothiazoline)-6-sulfonic acid diammonium
salt (ABTS).
The microplates were shaken at ambient temperature
with 100 l of a 1:2,500 dilution of the goat anti-rabbit
HRP conjugate, then the microtiter plate wells were
washed five times with PBS-T. 100 l of ABTS was added,
and the plates were analyzed in a spectrophotometer at
405 nm after a 30 minute incubation.
To select appropriate reagent concentrations for
experiments at high pressure, an initial study was
performed at atmospheric pressure, using various ratios
of antibody to antigen. Antibody and antigen were mixed
in polypropylene microfuge vials, then held overnight at
4-6 C to reach equilibrium binding prior to measurement
in the ELISA assay. This study showed that 100 ng of the
anti-p24 antibody alone (i.e., with no p24 antigen)
resulted in an absorbance value of approximately 1.1 OD
at 405 nm, and (ii) binding of 10 ng of p24 antigen to
the antibody during the overnight incubation resulted in
close to maximal inhibition of the subsequent binding of
the antibody to the p24 antigen immobilized on the
microtiter plate.
Based on the initial data described above, the
kinetics of binding at atmospheric pressure were
determined in mixtures containing 100 ng of antibody and
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ng of antigen per 100 l. The antigen/antibody
mixtures were incubated for different times at ambient
temperature, and then subjected to the ELISA assay. The
absorbance values measured in the ELISA assay were used
5 to calculate the extent of the binding of the p24 antigen
with the anti-p24 antibody using the antibody-only sample
(highest absorbance) as zero binding and the overnight
incubation of the antigen with the antibody (lowest
absorbance) as 100% binding.
10 As shown in Table 2 and Fig. 6, in a typical
experiment conducted at atmospheric pressure and ambient
temperature, approximately 25% binding between antigen
and antibody occurred in one hour; 40% in two hours; and
65% in four hours. Experiments to determine the effect
of high pressure on the binding of anti-p24 antibody to
p24 antigen were then commenced. Preincubation of the
anti-p24 antibody alone (without the p24 antigen) at
60,000 psi for 10 minutes did not result in any
discernible decrease in the absorbance, indicating that
pressures up to 60,000 psi did not affect the ability of
the antibody to subsequently bind to the antigen.
TABLE 2
Time course of the binding of p24 antigen to
anti-p24 antibody at atmospheric pressure
Time (hours) Binding (% max)
1 25
2 40
4 65
16 100
Table 3 and Fig. 7 show binding ( 10%) of antigen
with antibody as a function of pressure applied for 10
minutes. At 10,000 psi, no pressure effect was evident;
approximately 10% binding was observed at 20,000 psi; 20%
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at 30,000 psi, 50% at 40,000 psi; and 80% at 60,000 psi.
TABLE 3
Effect of pressure (10 minutes) on the binding of
p24 antigen to anti-p24 antibody
Pressure (psi) Binding* (% max; 101
14 (1 atm) 0
10,000 0
20,000 10
30,000 20
40,000 50
60,000 80
* max = 100%, observed in overnight incubations at 4-6 C
Table 4 and Fig. 8 show the effect of applying
60,000 psi for different durations on the binding of
antigen to antibody (the solid circles indicate
enhancement). The
TABLE 4
Effect of 60,000 psi on the binding of p24 antigen
to anti-p24 antibody
Binding Time (minutes) Binding* (% max; 10Z
0 0
5 50
10 75
69
25 50 82
* max = 100%, observed in overnight incubations at 4-6 C
data indicate that enhancement of binding occurs rapidly
at 60,000 psi, with 50% binding in 5 minutes. The other
data points correspond to 79% ( 10%) at 10 minutes; 69%
( 10%) at 25 minutes; and 82% ( 10%) at 50 minutes,
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showing that saturation binding was reached in 10
minutes. The data point marked with a solid square shows
binding achieved at atmospheric pressure after 60
minutes.
EXAMPLE 7
Use of high hydrostatic pressure to disrupt an antigen-
antibody complex:
The use of high hydrostatic pressure to disrupt an
antibody:mucin glycoprotein immune complex was
demonstrated in a model system. The antibody was bound
to a mucin glycoprotein immobilized in microtiter plate
wells. High pressure was applied to the microwells in a
custom designed high pressure chamber. Dissociation of
antibody from the immobilized antigen was monitored by
ELISA assays.
A solid phase immunosorbent ELISA assay for
detecting a mouse monoclonal IgM antibody to a mucin
glycoprotein was developed in-house. Polystyrene
microtiter plates (HiBind, Corning/Costar, Cambridge, MA)
were coated with 0.1 ml of epiglycanin at 100 ng/ml
overnight at 4 C in phosphate buffered saline (PBS), pH
7.4. Unreacted sites were blocked for one hour with
SuperBlock in PBS (Pierce Chemical, Chicago, IL).
The mouse monoclonal antibody was then bound to
the immobilized antigen by incubating microwells with 0.1
ml of 100 ng/ml antibody in PBS, pH 7.4 for one hour with
shaking at ambient temperature, (- 22 C). After washing
five times with PBS-0.05% Tween-20 (PBS-T), the bound
antibody was incubated with goat anti-rabbit conjugated
to horseradish peroxidase (HRP) (Pierce Chemical). After
five more washes in PBS-T, 100 l of ABTS was then added,
and the plates were analyzed in a spectrophotometer at
405 nm, after a one hour incubation.
To determine the effect of pressure on the binding
of the antibody, microwells with immobilized antigen and
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bound antibody were overlaid with melting point bath oil
(Sigma, St. Louis, MO), then inserted into the reaction
chamber of a custom designed high pressure apparatus
attached to a manually operated pressure apparatus (HiP,
Erie, PA). Elevated pressure was applied for 20 minutes
to a microwell. Parallel control samples were overlaid
with oil and held at atmospheric pressure during the
application of high pressure to the test sample. All
experiments were performed at ambient temperature.
After the elevated pressure had been applied for
the desired time, the test solutions were immediately
transferred to a second set of microwells to measure the
level of dissociated antibody, using the ELISA assay
described above. The pressurized and parallel control
microwells were also washed with PBS-T, and tested for
retained antibody using the same ELISA procedure.
Pressures of 20,000, 40,000, 60,000, and 80,000
psi resulted in reductions in the absorbance values in
the ELISA assays in which the level of antibody retained
in microwells was measured after the application of high
pressure. Absorbance reductions were in the range 37%
( 15%). Application of 60,000 psi to the immobilized
glycoprotein only (in the absence of antibody) did not
result in any subsequent decrease in absorbance in the
ELISA assay. These data thus indicated that application
of pressures ranging from 20,000 to 80,000 psi (140 to
560 MPa) caused the dissociation of antibodies from the
immobilized mucin glycoprotein.
The effect of the elevated pressure on the
subsequent immunoreactivity of the dissociated antibodies
was revealed by assaying the supernatants in a separate
ELISA assay in which the supernatants of the pressurized
wells were assayed for dissociated antibodies.
Antibodies that had been dissociated at 20,000 psi and
40,000 psi were able to rebind to the immobilized mucin
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glycoprotein, indicating that the effect of these
pressures on the binding of the immune complex was
reversible. In contrast, antibodies that had been
dissociated at 60,000 psi and 80,000 psi did not rebind
to the solid phase, indicating that pressure at these
higher levels had caused irreversible disruption of the
antibody structure.
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EXAMPLE 8
Use of pressure modulation to effect immunosenaration in
flow svstem-
A mucin glycoprotein, epiglycanin (EPGN), was
immobilized to the inner surface of "breakaway" wells of
a conventional microtiter plate in PBS at pH 7.4. The
inner surface of the wells was then blocked with bovine
serum albumin (BSA) in PBS. A small hole was drilled
into the bottom of the microwell, which was then placed
in the reaction chamber of a high pressure flow-through
apparatus, such as that described in WO 96/27432. The
reaction chamber had an internal volume of about 0.1 ml.
A solution of BSA in PBS was pumped through the
reaction chamber at a pressure of about 7,000 psi with
the intent to coat the inner surfaces of the system with
BSA and to prevent subsequent non-specific binding of
other proteins.
An IgM mouse monoclonal antibody (AE3; 1 mg/ml)
with immunospecificity for EPGN was mixed with BSA (1
mg/ml) in PBS. 1 ml of the solution was pumped through
the reaction chamber at a pressure of about 7,000 psi.
The entry and exit valves of the reaction chamber
were closed. The 7,000 psi pressure was applied for 10
minutes to try to achieve enhanced binding of the AE3
antibody to the EPGN immobilized on the solid phase. The
entry and exit valves were then opened, and 1 ml of PBS
was pumped through the reaction chamber, again at 7,000
psi to wash out the system. Antibodies that had been
captured through pressure-enhanced affinity binding to
the EPGN were retained on the solid phase.
Next, a pressure of 20,000 psi was applied to the
PBS solution. The entry and exit valves were closed
again, The 20,000 psi pressure was applied for 10
minutes. The entry and exit valves were then opened once
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again. Additional PBS solution was pumped through the
reaction chamber at 20,000 psi, as 0.1 ml fractions were
collected and assayed by the ELISA assay described in the
previous example. Fractions containing antibodies, which
had been first captured on the solid phase during
application of the 7,000 psi pressure and then
dissociated from the binding partners by application of
the 20,000 psi pressure, were identified by increased
absorbance readings using the ELISA described below.
Control samples were pumped through the high
pressure apparatus at atmospheric pressure. All
experiments were conducted at ambient temperature.
A solid phase immunosorbent ELISA assay for
detecting a mouse monoclonal IgM antibody to a mucin
glycoprotein was developed in-house by standard methods.
Polystyrene microtiter plates (HiBind, Corning/Costar,
Cambridge, MA) were coated with 0.1 ml of epiglycanin at
100 ng/ml overnight at 4 C in phosphate buffered saline
(PBS), pH 7.4. Unreacted sites were blocked for one hour
with SuperBlock in PBS (Pierce Chemical, Chicago, IL).
The mouse monoclonal antibody was then bound to the
immobilized antigen by incubating microwells with 0.1 ml
of 100 ng/ml antibody in PBS, pH 7.4, for one hour with
shaking at ambient temperature. After washing five times
with PBS-0.05% Tween-20 (PBS-T), the bound antibody was
incubated with goat anti-rabbit conjugated to horseradish
peroxidase (HRP) (Pierce Chemical). After five more
washes in PBS-T, 100 l of ABTS was then added. After a
one hour incubation time, the plates were read at 405 nm.
Pressure of 7,000 psi resulted in accelerated
binding of the antibody to the immobilized antigen.
After capture of the antibody at 7,000 psi, subsequent
application of a pressure of 20,000 psi resulted in the
dissociation of antibody from the solid phase. That the
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dissociated antibody was still immunoreactive was shown
by ELISA assay.
EXAMPLE 9
High Pressure Mediated Dissociation of a PSA Immune
Complex:
A solid phase immunosorbant ELISA assay for
detecting antibodies to prostate specific antigen (PSA)
was developed in-house. Polystyrene microtiter plates
(HiBind, Corning/Costar, Cambridge, MA) were coated
overnight at 4 C with 0.1 ml of PSA (Sigma Chemicals, St.
Louis, MO), at concentrations ranging from 625 to 1,265
ng/ml in phosphate buffered saline (PBS), pH 7.4.
Unreacted sites were blocked for one hour with SuperBlock
in PBS (Pierce Chemical, Chicago, IL). An anti-PSA mouse
monoclonal antibody (DRG International, Mountainside, NJ)
was then bound to the immobilized antigen by incubating
the antigen-coated microwells with 0.1 ml of anti-PSA
antibody (78 - 312 ng/ml) in pH 7.4 PBS overnight at 4 C.
The wells were then washed five times with PBS-0.05%
Tween-20 (PBS-T). To determine the level of anti-PSA
antibody bound to immobilized PSA, wells were reacted
with goat anti-mouse IgG (H+L) conjugated to horseradish
peroxidase (HRP) (Pierce Chemical, Chicago, IL). After
five more washes in PBS-T, 100 1 of ABTS was then added,
and the plates were read at 405 nm after one hour
incubation.
To determine the effect of pressure on the binding
of the antibody to the PSA antigen, 0.1 ml of SuperBlock
in PBS (Pierce Chemical, Chicago, IL) was added in
microwells in which anti-PSA antibody had been bound to
immobilized PSA. The microwells were then overlaid with
melting point bath oil (Sigma, St. Louis, MO) and
inserted, at atmospheric pressure, into a custom designed
high pressure chamber attached to a manually operated
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pressure apparatus (HiP, Erie, PA). High pressure was
then applied for 30 minutes to each microwell.
Dissociated antibody was collected into the PBS medium.
Parallel control samples were overlaid with oil and
maintained under ambient conditions during the
application of high pressure to the test sample. After
high pressure had been applied for the desired time, the
test solutions were immediately transferred to a second
set of microwells containing immobilized PSA. The level
of dissociated antibody was measured using the ELISA
assay described above. The pressurized and parallel
control microwells were also washed with PBS-T and tested
for retained antibody using the same ELISA procedure.
The absorbance of a sample pressurized at 60,000
psi and 40 C was 0.937, whereas the absorbance of the
unpressurized control was 1.457. Dissociation of anti-
PSA from PSA was confirmed in the absorbance values of
the supernatant. The absorbance of the supernatant of
the pressurized well was 0.504, while the absorbance of
control held at atmospheric pressure was 0.113. A
decrease in the absorbance of a pressurized microwell
relative to its respective control is consistent with
pressure-induced dissociation of anti-PSA antibody from
immobilized PSA. The dissociation is confirmed if the
supernatant removed from the pressurized well shows an
increase relative to the atmospheric control.
Raising the temperature to 40 C during application
of pressure significantly enhanced dissociation of anti-
PSA from PSA. The combination of 80,000 psi and 21 C
resulted in the absorbance of the pressurized well
decreasing from 1.556 to 1.052. In contrast, the
combination of 80,000 psi and 40 C resulted in the
absorbance decreasing 1.458 to 0.733. The higher level
of absorbance decrease in the later case was consistent
with a higher level of dissociation under those
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conditions. This interpretation of the data was
confirmed by the absorbance values measured in the
supernatant. The absorbance value of the 80,000 psi/400C
pressurized supernatant was 0.629 while the absorbance of
the control was 0.248.
EXAMPLE 10
Procedure for Determining the Phase Diagram Corresponding
to the Binding of an Aptamer to a Protein:
The aptamer derived by in vitro transcription of
Sequence 26B according to the method of Tuerk et al.
(Science, 249:505-510, 1990) was 'labeled with biotin by
including a low level of biotin-16-UTP in the
bacteriophage T7 RNA polymerase reaction, using the
biotin RNA labeling mix manufactured by Boehringer
Mannheim (Indianapolis, IN). A solid phase absorbent for
the aptamer was prepared by incubating polystyrene
microtiter plates (HiBind, Corning/Costar, Cambridge, MA)
with a 1 g/mi solution of bFGF (Bachem, Torrance, CA)
overnight at 4 C in NaHCO31 pH 9.2. Unreacted sites were
blocked with Superblock in PBS (Pierce Chemical, Chicago,
IL).
bFGF and the biotinylated aptamer 26B were
incubated together to form samples of complexes. A
sample was placed in a deformable plastic capsule and
overlaid with melting bath oil (Sigma, St. Louis, MO).
The capsule was placed in a device for generating high
pressures (HiP, Erie, PA). While pressure was applied to
an aliquot of the complex, a parallel sample was
subjected to the same temperature conditions at
atmospheric pressure. After release of the high
pressure, the sample and the parallel control (i.e., no
pressure pretreatment) were placed in wells of bFGF
coated microplate. The level of free aptamer
(uncomplexed with bFGF) was quantified by reacting
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samples in the microtiter plates for one hour at ambient
temperature, then detecting the captured aptamer with
HRP-labeled streptavidin (Sigma, St. Louis, MO). The
degree of complexation of the pressure-treated and
control samples was determined from a standard curve
compiled using varying amounts of the preformed bFGF-
aptamer complex and the biotinylated 26B aptamer such
that the concentration of bFGF-and aptamer was constant
in each measurement. Dissociation or additional
association of the bFGF-aptamer complex translates
respectively into an increase or decrease in signal
intensity in the assay.
Other Embodiments
From the description above, one skilled in the art
can ascertain the essential characteristics of the
invention and without departing from the spirit and scope
thereof, can make various changes and modifications of
the invention to adapt it to various usages and
conditions.
It is to be understood that while the invention
has been described'in conjunction with the detailed
description thereof, that the foregoing description is
intended to illustrate and not to limit the scope of the
appended claims. Other aspects, advantages, and
modifications are within the scope of the following
claims. For example, a well prebound with immobilized
antigen-antibody complex that could then be dissociated
under pressure is also within the scope of the claims.
