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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to a new type of immunoassay, which
includes a sensitive technique for the quantitative detection of low
concentrations of molecules of a particular type such as molecules in
solution.
2. Description of the Prior Art
It is desirable in certain circumstances to measure very low concentrations
of certain organic compounds. In medicine, for example, it is very useful
to determine the concentration of a given kind of molecule, usually in
solution, which either exists naturally in physiological fluids (e.g.
blood or urine) or which has been introduced into the living system (e.g.
drugs or contaminants). Because of the rapidly advancing state of
understanding of the molecular basis of both the normal and diseased
states of living systems, there is an increasing need for methods of
detection which are quantitative, specific to the molecule of interest,
highly sensitive and relatively simple to implement. Examples of molecules
of interest in a medical and/or biological context include, but are not
limited to, drugs, sex and adrenal hormones, biologically active peptides,
circulating hormones and excreted antigens associated with tumors. In the
case of drugs, for example, it is often the case that the safe and
efficacious use of a particular drug requires that its concentration in
the circulatory system be held to within relatively narrow bounds,
referred to as the therapeutic range.
One broad approach used to detect the presence of a particular compound,
referred to as the analyte, is the immunoassay, in which detection of a
given molecular species, referred to generally as the ligand, is
accomplished through the use of a second molecular species, often called
the antiligand, or the receptor, which specifically binds to the first
compound of interest. The presence of the ligand of interest is detected
by measuring, or inferring, either directly or indirectly, the extent of
binding of ligand to antiligand. The ligand may be either monoepitopic or
polyepitopic and is generally defined to be any organic molecule for which
there exists another molecule (i.e. the antiligand) which specifically
binds to said ligand, owing to the recognition of some portion of said
ligand. Examples of ligands include macromolecular antigens and haptens
(e.g. drugs). The antiligand, or receptor, is usually an antibody, which
either exists naturally or can be prepared artificially. The ligand and
antiligand together form a homologous pair. Throughout the text the terms
antigen and antibody, which represent typical examples, are used
interchangeably with the terms ligand and antiligand, respectively, but
such usage does not signify any loss of generality. In some cases, the
antibody would be the ligand and the antigen the antiligand, if it was the
presence of the antibody that was to be detected.
Implementation of a successful immunoassay requires a detectable signal
which is related to the extent of antigen-antibody binding which occurs
upon the reaction of the analyte with various assay reagents. Usually that
signal is provided for by a label which is conjugated to either the ligand
or the antiligand, depending on the mode of operation of the immunoassay.
Any label which provides a stable, conveniently detectable signal is an
acceptable candidate. Physical or chemical effects which produce
detectable signals, and for which suitable labels exist, include
radioactivity, fluorescence, chemiluminescence, phosphorescence and
enzymatic activity, to name a few.
Broadly speaking, immunoassays fall into two general
categories--heterogeneous and homogeneous. In heterogeneous assays, the
purpose of the label is simply to establish the location of the molecule
to which it is conjugated--i.e. to establish whether the labeled molecule
is free in solution or is part of a bound complex. Heterogeneous assays
generally function by explicitly separating bound antigen-antibody
complexes from the remaining free antigen and/or antibody. A method which
is frequently employed consists of attaching one of the members of the
homologous pair to a solid surface by covalent binding, physical
absorption, or some other means. When antigen-antibody binding occurs, the
resulting bound complexes remain attached to this solid surface (composed
of any suitably inert material such as plastic, paper, glass, metal,
polymer gel, etc.), allowing for separation of free antigen and/or
antibody in the surrounding solution by a wash step. A variation on this
method consists of using small (typically 0.05 to 20 microns) suspendable
particles to provide the solid surface onto which either antigen or
antibody is immobilized. Separation is effected by centrifugation of the
solution of sample, reagents and suspendable beads at an appropriate
speed, resulting in selective sedimentation of the support particles
together with the bound complexes.
Notwithstanding the successful application of heterogeneous assay
procedures, it is generally desirable to eliminate separation steps, since
the latter are time-consuming, labor-intensive and sometimes the source of
errors in the signal measurement. Furthermore, the more complicated
protocols associated with heterogeneous assays make them less suitable for
automated instrumentation of the kind needed for large-scale clinical
applications. Consequently, homogeneous assays are more desirable. In the
homogeneous format, the signal obtained from the labeled ligand or
antiligand is modified, or modulated, in some systematic, recognizable way
when ligand-antiligand binding occurs. Consequently, separation of the
labeled bound complexes from the free labeled molecules is no longer
required.
There exist a number of ways in which immunoassays can be carried out. For
clarity a heterogeneous format is assumed, although each approach can be
utilized (with varying degrees of success) in a homogeneous format, given
a suitable label which is modulated by the binding reaction.
In the competitive mode, the analyte, assumed to be antigen, is allowed to
compete with a known concentration of labeled antigen (provided in reagent
form in the assay kit) for binding to a limited number of antibody
molecules which are attached to a solid matrix. Following an appropriate
incubation period, the reacting solution is washed away, ideally leaving
just labeled antigen-antibody complexes attached to the binding surface,
thereby permitting the signal from the labels to be quantitated.
In another method, called the sandwich mode, the analyte, again assumed to
be antigen, reacts with an excess of surface-immobilized antibody
molecules. After a suitable incubation period, an excess of
label-conjugated antibody is added to the system. After this reaction has
gone to essential completion, a wash step removes unbound labeled antibody
and other sources of contamination, permitting measurement of the signal
produced by labels which are attached to antibody-antigen-antibody
complexes.
In yet another approach, called the indirect mode, the analyte, this time
assumed to consist of specific antibody, is allowed to bind to
surface-immobilized antigen which is in excess. The binding surface is
then washed and allowed to react with label-conjugated antibody. After a
suitable incubation period the surface is washed again, removing free
labeled antibody and permitting measurement of the signal due to labeled
antibody. The resulting signal strength varies inversely with the
concentration of the starting (unknown) antibody, since labeled antibody
can bind only to those immobilized antigen molecules which have not
already complexed to the analyte.
One of the most sensitive immunoassays developed thusfar is the
radioimmunoassay (RIA), in which the label is a radionuclide, such as
I.sup.125, conjugated to either member of the homologous (binding) pair.
This assay, which is necessarily heterogeneous, has achieved extremely
high sensitivities, extending down to the vicinity of 10.sup.-17 molar for
certain analytes. The obvious advantage of radioactive labeling, and the
reason for the extremely high sensitivity of RIA-type assays, is that
there exists negligible natural background radioactivity in the samples to
be analyzed. Also, RIA is relatively insensitive to variations in the
overall chemical composition of the unknown sample solution. However, the
radioactive reagents are expensive, possess relatively short shelf lives
and require the use of sophisticated, expensive instrumentation as well as
elaborate safety measures for both their use and disposal. Hence, there is
an increasing motivation to develop non-isotopic assays.
Fluorescence provides a potentially attractive alternative to radioactivity
as a suitable label for immunoassays. For example, fluorescein (usually in
the form of fluorescein isothiocyanate, or "FITC") and a variety of other
fluorescent dye molecules can be attached to most ligands and receptors
without significantly impairing their binding properties. Fluorescent
molecules have the property that they absorb light over a certain range of
wavelengths and (after a delay ranging from 10.sup.-9 to 10.sup.-4
seconds) emit light over a range of longer wavelengths. Hence, through the
use of a suitable light source, detector and optics, including excitation
and emission filters, the fluorescence intensity originating from labeled
molecules can be determined.
Several heterogeneous fluorescence-based immunoassays (FIA) have been
developed, including the FIAX/StiQ.TM. method (IDT Corp., Santa Clara,
CA.) and the Fluoromatic.TM. method (Bio-Rad Corp., Richmond, CA.). In the
former case, antigen is immobilized on an absorbant surface consisting of
a cellulose-like polymer mounted on the end of a portable "dipstick",
which is manually inserted into sample, reagent and wash solutions and
ultimately into the fluorescence measuring instrument. A competitive
reaction utilizing FITC-labeled monospecific antibody is typically
employed. In the Bio-Rad assay kit, the solid surface is replaced by
suspendable polyacrylamide gel microbeads which carry covalently-bound
specific antibody. A sandwich mode is typically employed, with centrifugal
sedimentation, followed by resuspension, of the beads for separation and
measurement. Photon-counting techniques can be used to extend the
sensitivity of the fluorescence intensity measurement.
Use of an enzyme as a label has produced a variety of useful enzyme
immunoassays (EIA), the most popular of which is known as ELISA. In the
typical heterogeneous format a sandwich-type reaction is employed, in
which the ligand of interest, assumed here to be antigen, binds to
surface-immobilized specific antibody and then to an enzyme-antibody
conjugate. After suitable incubation, any remaining free enzyme conjugate
is eliminated by a wash or centrifugation step. A suitable substrate for
the enzyme is then brought into contact with the surface containing the
bound complexes. The enzyme-substrate pair is chosen to provide a reaction
product which yields a readily detectable signal, such as a color change
or a fluorescence emission. The use of an enzyme as a label services to
effectively amplify the contribution of a single labeled bound complex to
the measured signal, because many substrate molecules can be converted by
a single enzyme molecule.
As discussed previously, it is generally desirable to eliminate the
separation steps associated with typical heterogeneous assays and,
instead, use homogeneous techniques. One of the first homogeneous assays
to be developed was the fluorescence polarization immunoassay. Here, the
polarization of the emission of the fluorescent dye label is modulated to
an extent which depends on the rate of rotational diffusion, or tumbling,
of the label in solution. Free labeled molecules which rotate rapidly
relative to the lifetime of their excited states emit light of relatively
random polarization (assuming a linearly polarized exciting beam, for
example). However, when the label becomes attached to a relatively large
bound complex, the rate of tumbling becomes relatively slow, resulting in
fluorescence emission of substantially linear polarization (i.e.
essentially unchanged). Unfortunately, this technique is limited in
practice to the detection of low molecular weight ligands, e.g. drugs,
whose rate of tumbling is sufficiently rapid to produce a measurable
change in fluorescence polarization upon binding to the antiligand. The
extent of modulation of the signal, in any case, is quite small.
Another useful fluorescence-based homogeneous technique is the fluorescence
excitation transfer immunoassay (FETI), also known simply as fluorescence
quenching. Here, two different dye labels, termed the donor and the
acceptor, or quencher are used. The pair has the property that when the
labels are brought close together, i.e. to within distances characteristic
of the dimensions of antigen-antibody complexes, there is non-radiative
energy transfer between the electronically excited donor molecule and the
acceptor. That is, the acceptor quenches the fluorescence emission of the
donor, resulting in a decreased intensity of the latter. In a typical
competitive mode, the donor label is attached to the ligand of interest
and the acceptor label fixed to the specific antibody. When ligand is
present in the unknown sample, some fraction of the acceptor-labeled
antibody binds to the free ligand, leaving a fraction of the labeled
ligand unquenched and therefore able to emit fluorescence radiation. The
intensity of the latter increases with increasing analyte concentration.
The principal drawback of the FETI technique is the requirement that the
donor-labeled ligand be relatively pure. Substantial concentrations of
labeled impurities produce a large background signal, making detection of
a small change due to complexing all the more difficult. Along these
lines, U.S. Pat. No. 4,261,968 describes an assay in which the quantum
efficiency of a fluorescent label is decreased when the labeled antigen
becomes bound to the antibody, resulting in a decrease in the total
fluorescence emission of the sample solution.
One of the main factors which limits the sensitivity and reproducibility of
all non-isotopic assays to varying degrees is the presence of background
false signals. For example, in fluorescence-based assays the use of
untreated blood serum may yield relatively high and variable background
fluorescence levels due to the presence of proteins, bilirubin and drugs.
In addition, there may exist variations in the absolute fluorescence
intensity from one sample to the next due to fluorescence from sample cell
surfaces, light scattering from impurities in solution, aberrations on
optical surfaces, temperature dependent effects, etc. Problems related to
impurities are particularly troublesome in homogeneous assays. However,
the background false signal contributions are often relatively constant in
time for any given sample measurement. Hence, a very useful technique for
reducing the background contribution without the necessity of making
additional control measurements is to determine the time rate of change of
the signal. Such a rate determination in the early stages of the
antigen-antibody binding reaction (i.e. when the rate is largest) should,
in principle, be independent of the (constant) background level.
In principle, then, the rate determining procedure can be applied to any
homogeneous assay technique, with the added advantage that the binding
reaction need not be taken to essential completion, thereby resulting in a
faster assay measurement. However, this approach becomes less feasible or
advantageous the smaller the total signal change due to binding, relative
to the background level. Hence, there are invariably practical limitations
to the sensitivity which can be achieved using any of the existing
homogeneous non-isotopic immunoassays, given the typical courses of
background false signals, interferences and nonspecific effects.
SUMMARY OF THE INVENTION
The present invention is directed to an immunoassay technique in which the
contributions to the measured signal due to free labeled ligand or
antiligand as well as contaminants and other sources of "background"
signal are effectively suppressed, thereby permitting measurement of the
desired signal due to labeled bound molecules at very low concentrations.
The resulting assay, therefore, possesses a higher sensitivity than those
currently in use. This requirement of insensitivity to free labeled
molecules plus background contamination holds regardless of the type of
labeling and/or detection scheme employed.
The underlying principle of the present invention is that labeled bound
ligand-antiligand complexes are caused to reside preferentially in a
predetermined spatial pattern. The useful signal due to these labels
therefore is forced to exist at or be associated with only certain
predetermined locations within the sample solution volume or on a surface.
This behavior is in sharp contrast to the origin of the signal due to
unbound labeled ligand or labeled antiligand molecules as well as
background contamination sources of signal which can be expected to be
spatially random. The spatial pattern is scanned spatially while the
antigen-antibody binding reaction is proceeding or, if desired, after the
reaction has run to completion. Using signal enhancement techniques such
as filtering, phase-sensitive detection or autocorrelation, the desired
signal level can be greatly enhanced with respect to the contribution from
free labeled molecules and background contamination sources, thereby
yielding a more sensitive assay. It is to be appreciated that the present
invention is not limited to homogeneous assays although such homogeneous
assays are desirable. Also, the general principle of having the bound
complexes reside in a known spatial pattern is not limited to the use of
fluorescent labels. The detected signal can be related, for example, to
changes in optical density (either at a specific wavelength or over a
broad range of wavelengths), light scattering, color, reflectance,
birefringence, magnetism or any other physical variable which can be
detected with suitable sensitivity and spatial resolution.
The present invention may be accomplished with a number of different
embodiments, but the common principle in each case is to force the bound
complexes produced by the ligand-antiligand reaction to occur
predominantly in a predetermined spatial (or geometric) pattern and then
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