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Description  |
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BACKGROUND OF THE INVENTION
A. Field of the Invention
This invention relates to a novel method, composition, and kit for the
determination of the presence or amount of an analyte in a test sample by
monitoring an analyte-mediated ligand binding event in a test mixture. In
particular, this invention relates to a novel method, composition, and kit
for the determination of the presence or amount of an analyte in a test
sample by monitoring differences and changes in the surface-enhanced Raman
scattering spectrum in a test mixture which comprises the test sample, a
specific binding member, a Raman-active label, and a particulate having a
surface capable of inducing surface-enhanced Raman light scattering.
The affinity of binding displayed by certain molecules (referred to here as
binding molecules) towards other specific molecules (referred to here as
ligands) is used commonly as the basis of assays to measure the quantity
of a particular binding molecule or ligand in a sample.
The two molecules involved in forming a binding molecule-ligand complex are
also referred to as a specific binding pair. One member of a specific
binding pair is referred to as a specific binding member. This invention
includes methods for performing assays using specific binding pairs of
binding molecules and ligands, with surface-enhanced Raman light
scattering as the method of detection. This invention also includes
materials and kits used in performing the assays.
An assay is a test (1) to detect the presence of a substance in a sample,
(2) to identify a substance in a sample, and/or (3) to measure the amount
of a substance in a sample. In the terminology of this art, the substance
that the assay is designed to detect, identify, or measure is called an
"analyte."
Ligand binding assays are especially relevant to medical diagnostics. In
modern medical practice, ligand binding assays are routinely run on
patients' blood, urine, saliva, etc. in order to determine the presence or
levels of antibodies, antigens, hormones, medications, poisons, toxins,
illegal drugs, and others.
New, better, less expensive, and faster assays can advance the level of
health care. Such assays can provide a physician with more and better
information about a patient and do so consistent with reasonable cost. In
addition, by making assays easier and less expensive, a higher level of
health care can be extended to less developed parts of the world. Ligand
binding assays are also being used to monitor groundwater contamination,
toxins and pesticides in foods, industrial biological processes, and in
many areas of biological research.
B. Present Ligand Binding Assays
For many assays it is required that minute quantities of a certain
substance (the analyte) be detected and measured in the presence of much
larger quantities of other substances. This is possible because the high
affinity a binding molecule can have for a ligand can result in a high
degree of specificity of binding for that particular ligand, irrespective
of the presence of other substances. The most common ligand binding assays
are immunoassays.
In an immunoassay, an antibody serves as a binding molecule which
specifically binds an antigen, which serves as the ligand, thereby forming
a specific binding pair. In order to measure the extent of the
antibody/antigen binding, one member of the specific binding pair is
tagged or labeled with a traceable substance. The unique properties of the
traceable substance allow its presence, and hence the presence of the
specific binding member to which it is attached, to be detected or
measured. The labeled member of the specific binding pair is referred to
as the indicator reagent.
In a direct immunoassay, the quantity of indicator reagent bound to the
other member of the specific binding pair is measured. In an indirect
immunoassay, the degree of inhibition of binding of the indicator reagent
to the other member of the specific binding pair by the analyte is
measured.
The individual members of a specific binding pair do not have to be
antigens or antibodies, however. Any two molecules having affinity for
each other may comprise a specific binding pair and may form the basis of
a ligand-binding assay. Other examples of such specific binding pairs are:
lectins and the complex carbohydrates to which they bind, hormones and
their receptors, any effector molecule and its receptor, binding molecules
designed through molecular modeling and synthesized specifically to bind
another molecule, and other molecules with mutual affinity such as avidin
and biotin.
Two commonly-used immunoassay techniques are radioimmunoassay (RIA) and
enzyme immunoassay (EIA), both of which employ a labeled specific binding
member as an indicator reagent. RIA uses a radioactive isotope as the
traceable substance attached to a specific binding member. Because the
radioactive isotope can be detected in very small amounts, it can be used
to detect or quantitate small amounts of analyte. There are, however, a
number of substantial drawbacks associated with RIA. These drawbacks
include the special facilities and extreme caution that are required in
handling radioactive materials, the high costs of such reagents and their
unique disposal requirements.
EIA uses an enzyme as the label attached to a specific binding member which
in the presence of its substrate produces a detectable substance or
signal. This enzyme-labeled specific binding member then serves as the
indicator reagent, and enzymatic activity is used to detect its binding.
While EIA does not have some of the same disadvantages as RIA, EIA
techniques require the addition of substrate materials to elicit the
detectable enzyme reaction. Another disadvantage is that enzyme stability
and rate of substrate turnover are temperature sensitive, the former
decreasing and the latter increasing with temperature.
A drawback common to all of these assay configurations is the necessity of
separating unbound labeled reagent from that bound to the analyte. This
usually entails wash steps which are tedious when the assays are performed
manually and require complicated robotics in automated formats.
Immunoassays may also be performed by automated instruments. Examples of
such instruments are the TDx.RTM., IMx.RTM., and IMx SELECT.TM. analyzers
which are commercially available from Abbott Laboratories, Abbott Park,
Ill. These instruments are used to measure analyte concentrations in
biological fluids such as serum, plasma and whole blood. The IMx.RTM. and
IMx SELECT.TM. analyzers have been described by Charles H. Keller, et al.,
"The Abbott IMx.RTM. and IMx SELECT.TM. System," J. Clin. Immunoassay, 14,
115, 1991; and M. Fiore et al., "The Abbott IMx.TM. Automated Benchtop
Immunochemistry Analyzer System," Clin. Chem., 34, 1726, 1988.
Other types of assays use the so-called "dipstick" and "flowthrough"
methods. With these methods, a test sample is applied to the "dipstick" or
"flowthrough" device, and the presence of the analyte is indicated by a
visually detectable signal generated by a color forming reaction.
Flowthrough devices generally use a porous material with a reagent
immobilized at a capture situs on a matrix layered thereon or incorporated
therein. The test sample is applied to the device and flows through the
porous material. The analyte in the sample then reacts with the reagent(s)
to produce a detectable signal on the porous material. Such devices have
proven useful for the qualitative determination of the presence of an
analyte.
More recently, assay techniques using metallic colloid particles have been
developed. The specific binding member to be labeled is coated onto the
metal or colloid, particles by adsorption and the metal particles become
the label. Localization of these labeled binding members on a solid
support via an immunoreaction can produce a signal that is visually
detectable, as well as measurable by an instrument.
Fluorescent and visible dyes and spin labels have also been used as labels
in ligand binding assays.
All of these binding molecule-ligand assays have inherent drawbacks. The
use of Raman light scattering as a means of detecting or measuring the
presence of a labeled specific binding member, avoids some of these
drawbacks, as explained below.
C. Rayleigh Light Scattering
For many years, it has been known that when certain molecules are
illuminated by a beam of light, for example ultraviolet, visible, or near
infrared, a small fraction of the incident photons are retained
momentarily by some of the molecules, causing a transition of the energy
levels of some of those molecules to higher vibrational levels of the
ground electronic state. These higher vibrational levels are called
virtual states. Most of the time, these are elastic collisions, and the
molecules return to their original vibrational level by releasing photons.
Photons are emitted in all directions at the same wavelength as the
incident beam (i.e., they are scattered). This is called Rayleigh
scattering.
D. Raman Light Scattering
In 1928, C. V. Raman discovered that when certain molecules are
illuminated, a small percentage of the molecules which have retained a
photon do not return to their original vibrational level after remitting
the retained photon, but drop to a different vibrational level of the
ground electronic state. The radiation emitted from these molecules will
therefore be at a different energy and hence a different wavelength. This
is referred to as Raman scattering.
If the molecule drops to a higher vibrational level of the ground
electronic state, the photon emitted is at a lower energy or longer
wavelength than that absorbed. This is referred to as Stokes-shifted Raman
scattering. If a molecule is already at a higher vibrational state before
it absorbs a photon, it can impart this extra energy to the remitted
photon thereby returning to the ground state. In this case, the radiation
emitted is of higher energy (and shorter wavelength) and is called
anti-Stokes-shifted Raman scattering. In any set of molecules under normal
conditions, the number of molecules at ground state is always much greater
than those at an excited state, so the odds of an incident photon
interacting with an excited molecule and being scattered with more energy
than it carried upon collision is very small. Therefore, photon scattering
at frequencies higher than that of the incident photons (anti-Stokes
frequencies) is minor relative to that at frequencies lower than that of
the incident photons (Stokes frequencies). Consequently, it is the Stokes
frequencies that are usually analyzed.
The amount of energy lost to, or gained from, a molecule in this way is
quantized, resulting in the scattered photons having discrete wavelength
shifts. These wavelength shifts can be measured by a spectrometer. Raman
scattering was considered to have the potential to be useful as an
analytical tool to identify certain molecules, and as a means of studying
molecular structure. However, other methods, such as infrared
spectroscopy, proved to be more useful.
E. Resonance Raman Scattering
Interest in Raman spectroscopy was renewed with the advent of the laser as
a light source. Its intense coherent light overcame some of the
sensitivity drawbacks of Raman spectroscopy. Moreover, it was discovered
that when the wavelength of the incident light is at or near the maximum
absorption frequency of the molecule, and hence can cause electronic as
we! l as vibrational transitions in the molecules, resonance Raman
scattering is observed. With resonance Raman scattering, the re-emitted
photons still show the differences in vibrational energy associated with
Raman scattering. However, with resonance Raman scattering, the electronic
vibrational absorption is approximately 1000 times more efficient. Even
with the increased signal from resonance Raman scattering, its usefulness
as an analytic tool was limited due to its still comparatively weak
signal. The relatively recent discovery of the surface enhancement effect,
however, has provided a means to further dramatically enhance Raman
scattering intensity.
F. Surface Enhanced Raman Scattering
A significant increase in the intensity of Raman light scattering can be
observed when molecules are brought into close proximity to (but not
necessarily in contact with) certain metal surfaces. The metal surfaces
need to be "roughened" or coated with minute metal particles. Metal
colloids also show this signal enhancement effect. The increase in
intensity can be on the order of several million-fold or more. In 1974,
Dr. Richard P. Van Duyne was the first to recognize this effect as a
unique phenomenon and coined the term "surface enhanced Raman scattering"
(SERS).
The cause of the SERS effect is not completely understood; however, current
thinking envisions at least two separate factors contributing to SERS.
First, the metal surface contains minute irregularities. These
irregularities can be thought of as spheres (in a colloid, they are
spheroidal or nearly so). Those particles with diameters of approximately
1/10th the wavelength of the incident light are considered to contribute
most to the effect. The incident photons induce a field across the
particles which, being metal, have very mobile electrons.
In certain configurations of metal surfaces or particles, groups of surface
electrons can be made to oscillate in a collective fashion in response to
an applied oscillating electromagnetic field. Such a group of collectively
oscillating electrons is called a "plasmon." The incident photons supply
this oscillating electromagnetic field. The induction of an oscillating
dipole moment in a molecule by incident light is the source of the Raman
scattering. The effect of the resonant oscillation of the surface plasmons
is to cause a large increase in the electromagnetic field strength in the
vicinity of the metal surface. This results in an enhancement of the
oscillating dipole induced in the scattering molecule and hence increases
the intensity of the Raman scattered light. The effect is to increase the
apparent intensity of the incident light in the vicinity of the particles.
A second factor considered to contribute to the SERS effect is molecular
imaging. A molecule with a dipole moment, which is in close proximity to a
metallic surface, will induce an image of itself on that surface of
opposite polarity (i.e., a "shadow" dipole on the plasmon). The proximity
of that image is thought to enhance the power of the molecules to scatter
light. Put another way, this coupling of a molecule having an induced or
distorted dipole moment to the surface plasmons greatly enhances the
excitation probability. The result is a very large increase in the
efficiency of Raman light scattered by the surface-absorbed molecules.
The SERS effect can be enhanced through combination with the resonance
Raman effect. The surface-enhanced Raman scattering effect is even more
intense if the frequency of the excitation light is in resonance with a
major absorption band of the molecule being illuminated. The resultant
Surface Enhanced Resonance Raman Scattering (SERRS) effect can result in
an enhancement in the intensity of the Raman scattering signal of seven
orders of magnitude or more.
G. Application of SERS to Immunoassays
The SERS effect has been used by physical and analytical chemists to follow
chemical reactions on electrode surfaces in order to study molecular
surface structure and dynamics. Recently, the technique has also been
applied to biological molecules containing Raman-active prosthetic groups,
such as hemes.
Up until now, there has been no application of the SERS effect to
immunodiagnostics.
Utilization of this technology in immunodiagnostics offers several unique
advantages. Because of the extraordinary dependence of the SERS signal
upon close association with a suitable surface, only those reporter
molecules which have become immobilized on or near the SERS-active surface
will contribute a significant signal, while the signal contribution of
those remaining in solution will be negligible. Molecules bound in
different environments or different orientations can exhibit differences
in their Raman scattering characteristics.
SUMMARY OF THE INVENTION
According to one feature of the present invention there is provided a
method for assaying, or determining the presence or amount of an analyte
by: Monitoring an analyte-mediated ligand binding event in a test mixture
containing the test sample, specific binding member, Raman-active label
and a particulate by allowing a complex to be formed, in the test mixture,
between an analyte, a specific binding member, a Raman-active label, and a
particulate wherein the particulate is characterized by having a surface
capable of inducing a surface-enhanced Raman light scattering;
illuminating the test mixture with a radiation sufficient to cause the
Raman-active label in the complex to emit a detectable Raman spectrum; and
monitoring differences in the detected surface-enhanced Raman scattering
spectra, the differences being dependent upon the amount of the analyte
present in the test mixture.
According to another feature of the present invention, there is provided a
method for assaying, or determining the presence or amount of an analyte
in a test sample by: Monitoring an analyte-mediated ligand binding event
in a test mixture by forming a test mixture comprising the test sample, a
labeled analyte-analog and a particulate capture reagent comprising the
specific binding member immobilized on a particulate having a surface
capable of inducing surface-enhanced Raman light scattering wherein the
labeled analyte-analog comprises an analyte-analog molecule expressing an
analyte epitope recognized by a specific binding member, said
analyte-analog being attached to a Raman-active label either directly, or
indirectly, through an intervening molecule, then, allowing the labeled
analyte-analog to be bound to the specific binding member on the
particulate, wherein the extent of the binding of the labeled
analyte-analog to the specific binding member on the particulate is
affected by the presence of the analyte; then, illuminating the test
mixture with a radiation sufficient to cause the Raman-active label on the
bound labeled analyte-analog in the test mixture to emit a detectable
Raman spectrum; and then monitoring difference in the detected
surface-enhanced Raman scattering spectra, the differences being dependent
upon the amount of the analyte present in the test mixture.
According to another feature of the present invention, there is provided a
method for assaying, or determining the presence or amount of, an analyte
in a test sample by monitoring an analyte-mediated ligand binding event in
a test mixture by: Forming the test mixture from the test sample
containing the analyte and a particulate capture reagent comprising a
specific binding member conjugated to a particulate having a surface
capable of inducing a surface-enhanced Raman light scattering and also
having associated with it a Raman-active label: then applying the test
mixture onto a chromatographic material having a proximal end and a distal
end, wherein the chromatographic material comprises a capture reagent
immobilized in a capture situs and capable of binding to the analyte; then
allowing the test mixture to travel from the proximal end toward the
distal end by capillary action; then illuminating the capture situs with a
radiation sufficient to cause a detectable Raman spectrum; and, then
monitoring differences in the detected surface-enhanced Raman scattering
spectra, the differences being dependent upon the amount of the analyte
present in the test mixture.
According to yet another feature of the present invention, there is
provided a composition to be used for determining the presence or amount
of an analyte in a test sample by monitoring an analyte-mediated ligand
binding event in a test mixture, the composition comprises a particulate
having a surface capable of inducing a surface-enhanced Raman light
scattering and having been labeled with a Raman-active label.
According to still another feature of the present invention, there is
provided a kit for determining the presence or amount of an analyte in a
test sample by monitoring an analyte-mediated ligand binding event in a
test mixture, the kit comprises: A Raman-active label; a particulate
having a surface capable of inducing a surface-enhanced Raman light
scattering; and a specific binding member for the analyte.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a profilometer tracing of an intact, chemically deposited silver
film surface.
FIG. 2 is Raman spectra of (A) 2,4-dinitrobenzene solution, 10.sup.-3 M, in
the presence of chemically deposited silver film, (B)
2,4-dinitrophenyl-BSA conjugate, 10.sup.-7 M with respect to DNP moieties,
in the presence of a chemically deposited silver film, and (C)
2,4-dinitrobenzene, 10.sup.-3 M, in the absence of a silver film (ordinate
expanded fourfold relative to A and B to enhance features). Spectra
acquisition conditions: acquisition time, 19 sec; power, 41 mW; excitation
wavelength, 457.9 nm.
FIG. 3 is a SERRS spectrum obtained from a chemically deposited silver film
incubated in (A) a 3 mM solution of HABA and (B) a 2.5.times.10.sup.-5 M
solution of avidin subsequently made 0.3 mM in HABA. No discernible
spectrum was observed in this region from surface absorbed avidin in the
absence of HABA (C). Spectra acquisition conditions: acquisition time, 100
sec. power, 50 MW; excitation wavelength, 457.9 nm.
FIG. 4 is a combined plot of typical SERRS spectra obtained in a "sandwich"
immunoassay for TSH antigen using a DAB-anti-TSH antibody conjugate.
Silver electrodes coated with anti-TSH capture antibody were incubated
with various concentrations of TSH antigen and then transferred to a 40
.mu.g/ml solution of DAB-anti-TSH antibody conjugate. (A) SERRS spectrum
of a 40 .mu.g/ml solution of DAB-anti-TSH antibody conjugate in the
absence of a silver surface. Plots (B), (C), (D), (E), and (F) show
spectra obtained by incubating capture antibody-coated electrodes in
solutions containing 0, 4, 10, 25 and 60 .mu.lU of TSH antigen,
respectively, followed by transfer to a 40 .mu.g/ml solution of
DAB-anti-TSH antibody conjugate.
FIG. 5 is a plot of average SERRS intensity at 1410 cm.sup.-1 as a function
of TSH antigen concentration for known TSH standards. Values were obtained
at five different places on the silver electrode and averaged. One
electrode was used for each concentration of TSH antigen measured. Numbers
in parentheses are the coefficients of variation (standard deviation/mean)
for each concentration of analyte measured.
FIG. 6 is absorbance (492 nm) vs. TSH antigen concentration obtained using
reagents from a commercial enzyme immunoassay kit (Abbott Labs No. 6207).
Each data point represents the average of four determinations. The numbers
in parentheses are the coefficients of variations (standard
deviation/mean) for each concentration of TSH antigen measured.
FIG. 7 is a SERS spectra using near IR excitation for (A) spectrum of a
blank silver film determined separately and added to a solution state
spectrum done in the absence of a silver surface, of the
p-dimethylamino-azobenzene bovine serum albumin conjugate at 20 mg/ml, (B)
spectrum obtained by immersing the blank silver film in the aforementioned
solution of the p-dimethylaminoazobenzene bovine serum albumin conjugate.
FIG. 8 shows a no-wash immunoassay of standards of human chorionic
gonadotrophin (HCG), prepared in pig serum, using gold colloid, a cresyl
violet dye or reporter molecule, and a SERRS readout plotted as a function
of HCG concentration.
FIG. 9 shows a no-wash immunoassay of standards of human chorionic
gonadotrophin (HCG), prepared in human serum using gold colloid, a cresyl
violet dye or reporter molecule, and a SERRS readout plotted as a function
of HCG concentration.
FIG. 10 shows a no-wash immunoassay of standards of theophylline, prepared
in citrate buffer, using silver colloid, an
N,N-dimethylanaline-4-azobenzy-4-thiocarbomoyl ethyl aminoethyldisulfide
dye or reporter molecule, and a SERRS readout, plotted as a function of
theophylline concentration.
FIG. 11 shows a no-wash detection of the inhibition of binding by free
biotin, of bovine serum albumin conjugated to both a dye or reporter
molecule [dimethylaminoazobenzene (DAB)], and biotin, [abbreviation of
complete conjugate is biotin-BSA-DAB], to streptavidin-coated silver
colloid, by a SERRS readout plotted as a function of biotin-BSA-DAB
concentration.
FIGS. 12A and 12B show surface-enhanced Raman scattering (SERRS) spectra of
20:1 mixture of methylene blue:oxazine 725 on silver colloid, where the
colloid was made either using (A) hydrogen and (B) citrate as the reducing
agent.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
As previously stated, the present invention involves assay methods,
compositions and kits for the determination of the presence or amount of
an analyte in a test sample by monitoring differences and changes in the
surface-enhanced Raman scattering spectrum of a test mixture which
comprises the test sample, a specific binding member, a Raman-active
label, and a particulate having a surface capable of inducing
surface-enhanced Raman light scattering. It is believed that the presence
of an analyte in a dispersed particulate mixture will affect the Raman
spectrum obtained from the mixture.
Before proceeding further with the description of various embodiments of
the present invention, a number of terms will be defined.
DEFINITIONS
"Analyte," as used herein, is the substance to be detected in the test
sample using the present invention. The analyte can be any substance for
which there exists a naturally occurring specific binding member (e.g., an
antibody) or for which a specific binding member can be prepared, and the
analyte can bind to one or more specific binding members in an assay. 37
Analyte" also includes any antigenic substances, haptens, antibodies, and
combinations thereof. The analyte can include a protein, a peptide, an
amino acid, a carbohydrate, a hormone, asteroid, a vitamin, a drug
including those administered for therapeutic purposes as well as those
administered for illicit purposes, a bacterium, a virus, and metabolites
of or antibodies to any of the above substances.
"Analyte-analog", as used herein, refers to a substance which cross reacts
with an analyte specific binding member although it may do so to a greater
or lesser extent than does the analyte itself. The analyte-analog can
include a modified analyte as well as a fragmented or synthetic portion of
the analyte molecule so long as the analyte analog has at least one
epitopic site in common with the analyte of interest.
"Analyte epitope," as used herein, denotes that part of the analyte which
contacts one member of the specific ligand binding pair during the
specific binding event. That part of the specific binding pair member
which contacts the epitope of the analyte during the specific binding
event is termed the "paratope."
"Analyte-mediated ligand binding event," as used herein, means a specific
binding event between two members of a specific ligand binding pair, the
extent of the binding is influenced by the presence, and the amount
present, of the analyte. This influence usually occurs because the analyte
contains a structure, or epitope, similar to or identical to the structure
or epitode contained by one member of the specific ligand binding pair,
the recognition of which by the other member of the specific ligand
binding pair results in the specific binding event. As a result, the
analyte specifically binds to one member of the specific ligand binding
pair, thereby preventing it from binding to the other member of the
specific ligand binding pair.
"Ancillary Specific binding member," as used herein, is a specific binding
member used in addition to the specific binding members of the captured
reagent and the indicator reagent and becomes a part of the final binding
complex. One or more ancillary specific binding members can be used in an
assay. For example, an ancillary specific binding member can be used in an
assay where the indicator reagent is capable of binding the ancillary
specific binding member which in turn is capable of binding the analyte.
"Agglutination," means a reaction whereby particles suspended in a liquid
collect into clumps.
"Associated," as used herein, is the state of two or more molecules and/or
particulates being held in close proximity to one another.
"Capture reagent," as used herein, is a specific binding member, capable of
binding the analyte or indicator reagent, which can be directly or
indirectly attached to a substantially solid material. The solid phase
capture reagent complex can be used to separate the bound and unbound
components of the assay.
"Conjugate," as used herein, is a substance formed by the chemical coupling
of one moiety to another. An example of such specie | | |
