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Description  |
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FIELD OF THE INVENTION
This invention relates to immunoassays generally and more particularly
provides a new system for performing virtual homogeneous immunoassaYs
employing colloidal gold.
BACKGROUND OF THE INVENTION
Many human disease states are identified on the basis of immunoassay
techniques which rely upon the specificity between immunoglobulin, whether
monoclonal or polyclonal, and their respective binding partners, which may
be haptens, antigens, or other analytes, all of which may hereafter be
collectively and interchangeably referred to herein as "ligands" and
"ligand binding partners." Furthermore, "ligand" also means any molecule
having an affinity to bind or complex with a "ligand binding partner",
including chelators, immunobinders, nucleic acid strands, bioreceptors,
and hydrophobic binders. Over the past fifteen or so years, there has been
a substantial amount of effort involved in the development of immunoassay
techniques utilizing the so-called sandwich and competitive techniques.
The sandwich technique involves the immobilization of an antigen by one
antibody and then subsequent labeling by attachment of a second antibody
having associated therewith a detectable label. Reverse immunoassays for
the detection of antibody are similar but instead put antigen on the
surface for reaction with the sample antibody. Competitive techniques are
useful for antigens having only a single epitopic site for reaction with
an antibody. Accordingly, and as the name implies, such techniques rely
upon the competition of the antigen with another labeled antigen for a
binding site on an immobilized antibody. The substitutions necessary for
antibody detection tests are obvious and need not be covered here in any
great detail.
Of great importance in the laboratory is the development of highly
sensitive techniques which can be run in either batch random access,
panel, or stat modes. Preferably, such techniques will be homogeneous in
nature, i.e., and as used herein, they will be conducted solely within one
container without any accompanying requirement to physically separate out
components following reactions during the assay.
It is one object of the present invention to provide a new immunoassay
system which is highly sensitive and which is homogeneous in nature.
U.S. Pat. 3,939,350 to Kronick and the Kronick citations therein referenced
describe an immunoassay system which allows for the measurement of
biochemical analytes by fluorescence in a liquid sample. Kronick employs a
physical phenomenon known under the name of total internal reflectance.
This optical phenomenon occurs wherein light, when directed through a high
refractive index material toward the interface of that material with a
second material having a lower refractive index at greater than a critical
angle, all light is reflected from that interface save for a microscopic
evanescent wave which propagates into the second material for only a short
distance. The second material may, for instance, be water or another
aqueous medium in which an assay is being conducted. Kronick noted that
when he brought materials which had been fluorescently labeled down to the
interface and within the field of the evanescent wave, he could energize
the fluorescent molecules and detect fluorescence which then emanated into
the overlying solution. The Kronick system, however, looks at fluorescence
which cannot be readily modified by alteration of the fluorescent labels
in order to suit the system under study. Due to the nature of the
specificity of the fluorescent label with respect to the wavelength of the
excitation frequency, one is limited to a discrete light source providing
the critical excitation frequency. To date, most investigators favor the
He-Ne laser light source due to its reliability and low cost as well as
the low cost of associated optics. Such a light source, however, brings
concomitant difficulties in tailoring fluorescent molecules to be excited
by the He-Ne laser output. The organic, inorganic, and bio-organic
techniques required are especially difficult to control in the immunoassay
arena. Further, Kronick's reliance on fluorescence is accompanied by
additional disadvantages associated with bleaching of the fluorescent
molecules and generally critical matching of fluorescent molecule
excitation wavelength with laser output wavelength necessary to obtain
good quantum efficiency.
It is an object of the present invention to provide a new immunoassay
system which avoids the disadvantages associated with fluorescent labels
and the criticality associated with matching an excitation source.
It is another object of the present invention to employ the principles of
total internal reflection but with far greater flexibility regarding the
choice of illumination sources. U.S. Pat. 4,181,441 to Noller describes a
system similar to that of Kronick. Noller, however, taught that the assay
should be conducted by measurement of light absorption in a liquid sample
which could then be correlated to the presence of biochemical analytes.
Although the Noller system employs different physical principles than the
Kronick system, light absorption measurements are similarly subject to
poorer signal-to-noise ratios due to small differences in large light
signals thereby making such a system inherently less sensitive than
desired. It is another object of the present invention to avoid employing
light absorption measurements while still gaining the advantages to be
provided by the total internal reflectance phenomenon.
U.S. Pat. No. 4,521,522 to Lundstrom teaches yet another immunoassay based
upon reflectance and the use of Brewster s angle. This system relies upon
a different optical phenomenon wherein directing a light beam, polarized
in the plane of incidence, upon an interface, for example that formed
between plastic and liquid, results in the transmission of a strong light
beam into the liquid when such light strikes the interface at the Brewster
angle. At the Brewster angle, substantially no light is reflected.
The Brewster angle is a function of the refractive indices of the two
materials as well as the direction of polarization. Lundstrom noted that
upon the growth of a biochemical layer at the interface, the Brewster
angle condition would be disrupted resulting in increasing light
reflectance, particularly at angles less than the Brewster angle.
Unfortunately, the Lundstrom assay only works effictively with a wash step
since the transmission of the beam into the liquid also results in the
generation of light scatter and thus a spurious signal.
It is another object of the present invention to utilize light scatter but
to avoid light scatter generated by the transmission of light into the
liquid which occurs naturally when light is directed at an interface at
the Brewster angle. Accordingly, it is yet another object of the present
invention to avoid employing a Brewster angle condition.
SUMMARY OF THE INVENTION
In accordance with various aspects and the principles of the present
invention, there is provided an immunoassay system which utilizes
scattered total internal reflectance (STIR) as a measure of the presence
of particular ligands to be determined in an aqueous solution. The
invention relies in part upon the identification of the critical angle
associated with total internal reflectance. The angle is largely a
function of the refractive index of the material through which an incident
light wave is directed, e.g. plastic, and the relatively lower refractive
index of the material in which the immunoassay is being conducted, e.g. an
aqueous solution. It is measured from a line perpendicular to the
interface between the two materials, and thus at its maximum, 90.degree. ,
will lie in the plane of the interface.
Light directed through the plastic toward the interface formed by the
aqueous sample and plastic materials at the critical angle will result in
total internal reflectance of the light within the plastic. It is
recognized that no materials in the real world are perfect and
accordingly, it is preferred that the incident light be directed toward
the interface at an angle several degrees greater than the critical angle,
most preferably in the range of approximately 6.degree. greater in order
to ensure that the basic conditions of total internal reflectance are met.
At such an angle, the incident collimated light, preferably from a laser,
is totally internally reflected within the plastic save for the
propagation of the evanescent wave parallel to the surface of the plastic
and approximately 1/4.lambda. from the surface. Similarly, smooth surfaces
at the interface are preferred for optimum signal quality. Unlike
conventional fluorescent techniques including those of Kronick, the
present assay system is flexible with respect to light wavelength since
particle size may be readily adjusted to match the available light source
(or vice versa) to provide acceptable light scatter. Fluorescent molecules
are not readily adjustable with respect to excitation wavelength.
Most ideally, the light source will be a He-Ne light source, however, other
lasers with different wavelength outputs have been used and still other
sources suggest themselves including light emitting diodes and other
nonlaser light sources.
Applicants' immunoassay system further relies upon conventional immunoassay
techniques. However, applicants' immunoassay system also employs a
particulate label having a higher refractive index than that of the
solution, and most preferably also higher than the first light
transmissive material, e.g. plastic in the foregoing example. Such
Particles would include, for instance, red blood cells, other materials
having a highly reflective surface such as metallic particles, and
nonmetallic substances such as glass or plastics, e.g. latex particles,
and the like. Most preferably, colloidal gold is used as a label for the
solution phase immunologically active component. While the use of
colloidal gold as a label is known, see for example U.S. Pat. No.
4,313,734 Leuvering, almost no nonagglutination related uses of the label
have been made to date due to the difficulties associated with its
detection, particularly in homogeneous type systems. It was surprisingly
discovered by the inventors hereof that the unique combination of STIR
with colloidal gold has resulted in an extremely efficient and sensitive
homogeneous assay system. It is believed, but not known for certain that
this is due primarily to the interaction of the colloidal gold particles
with the evanescent wave. Indeed, experience implies that particles having
an increasingly higher index of refraction than that of the underlying
solid generally increasingly scatter light. While particles with indices
of refraction less than the underlying solid, providing they are also not
equal to that of the aqueous medium, would also scatter light, such are
less preferred.
Assuming for the moment a conventional sandwich technique, one
immunoglobulin or ligand binding partner is immobilized on the surface and
binds antigen or other ligand to be determined. Thereafter, (or
simultaneously, or if not previously) a second immunoglobulin, directed at
a second epitopic site on the ligand, and labeled directly or indirectly
with colloidal gold, binds to the ligand creating the so-called
"sandwich". In this arrangement, the presence of the colloidal gold
disrupts the propagation of the evanescent wave resulting in scattered
light which may be detected by a photomultiplier or other light sensor to
provide a responsive signal. Another important aspect of the present
invention involves the physical location of the detector. The detector is
ideally placed at an angle greater than the critical angle and in a
location whereby only light scattered backward toward the light source is
detected. This location thereby ideally avoids the detection of spurious
scattered light within the bulk liquid medium.
Another feature of the instant invention is that the immunoassays are
diffusion rate controlled and not particularly temperature dependent. This
is in strong contrast to ELISA and various other immunoassay techniques
wherein temperature control is critical since small changes in temperature
in such systems results in wide variations in assay results per unit of
time.
It was surprisingly found by the inventors hereof, that as a result of the
combination of these elements, rapid, sensitive results could be obtained
in a homogeneous environment without requiring the complicated equipment
previously associated with colloidal gold assay techniques.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts an optically transparent member 53 with refractive index
n.sub.2 having a fluid contacting surface 52 in contact with fluid 54
having a refractive index n.sub.1 which is less than n.sub.2. The total
internal reflectance critical angle 55 measured from a line 50
perpendicular to the plane of the fluid contacting surface 51 is the
minimum angle of illumination required to provide total internal
reflectance at surface 52. This angle is defined by the equation
##EQU1##
The critical angle, .theta..sub.c, is only defined in the higher
refractive index medium and can range from 0.degree. to 90.degree. . Light
propagating from a point on the fluid contacting surface 58 at the total
internal reflectance critical angle 55 would follow the path depicted as
57. All light propagating through the optically transparent member 53 from
a point on the fluid contacting surface 58 between the plane of the sample
fluid contacting surface 51 and the total internal reflectance critical
angle 55 of the fluid contacting surface, will propagate in the range
depicted as 56.
FIG. 2 is a simplified elevation view of the cuvette and the rotating
optics mechanism used to illuminate and read it.
FIG. 3 is cross-section of a cuvette.
FIG. 4 is a perspective view of a cuvette and a paraboloidal reflector
which is the first component of the receiving optics.
FIG. 5 depicts an apparatus with laser illumination above critical angle.
FIG. 6 depicts illumination and detection light paths used when
illumination is above the critical angle.
FIG. 7 shows data obtained with the apparatus of FIG. 5.
FIG. 8 depicts an apparatus with light emitting diode illumination above
the critical angle.
FIG. 9 shows data obtained with the apparatus of FIG. 8.
DETAILED DESCRIPTION OF THE INVENTION AND BEST MODE
The present invention provides an apparatus for detecting the presence of
an analyte of interest in a sample. This apparatus comprises a light
source; housing means for receiving an optically transparent member having
a sample contacting surface, said member in said housing means being
disposed such that the sample contacting surface is illuminated with light
emitted from said light source; and photodetection means which excludes
the detection of light which propagates in a geometric optical path from
the light source, said photodetection means being capable of detecting
elastically-scattered light which propagates through the optically
transparent member from the illuminated sample contacting surface between
the plane of the sample contacting surface and the total internal
reflectance critical angle of the sample contacting surface. Within this
application, "photodetection means" is defined as a system for detecting
photons having a wavelength equal to the wavelength of the illuminating
light, and includes combinations of photon detectors (e.g. photomultiplier
tubes), lenses, mirrors, light filters, optical fibers, prisms, apertures,
and masks. A geometric optical path is the path that a family of light
rays will follow based on first order reflection and refraction of
idealized surfaces (imperfection free) and ignoring the effects of surface
and bulk material imperfections, diffraction, interference, scatter, and
partial reflection at surfaces. Further, within this application,
"elastically-scattered light" (also referrred to herein as "scatter" and
"scattered light") means incident light which has been redirected by an
object without changing the wavelength of the light, by means other than
dopler shifting, due to the difference in the refractive index of the
object and its surrounding medium. Fluorescence, also known as inelastic
scatter, is the light emitted by a light absorbing molecule after the
molecule has absorbed a photon of light. The wavelength of the absorbed
light is less than the wavelength of the emitted light. Flouorescent light
is always of a wavelength different from the light incident on the light
absorbing molecule.
"Critical angle", also referred to herein as "total internal reflectance
critical angle", is the angle (less than 90.degree. ) measured from the
line perpendicular to an interface between materials of different
refractive indexes, beyond which total internal reflection can occur, and
is defined by the equation
##EQU2##
wherein n.sub.1 is the lower refractive index and n.sub.2 is the higher
refractive index of the two mediums forming the interface. The critical
angle can only exist in the higher refractive index medium. Light which
illuminates the interface from the lower refractive index material at any
angle (0.degree. to 90.degree. ) cannot be refracted into the higher
refractive index medium at an angle greater than or equal to the critical
angle. Total internal reflection occurs exclusively when an interface
between materials of different refractive indexes is illuminated from the
higher refractive index medium beyond the critical angel, causing all the
incident illumination to be reflected at the interface unless it is
perturbed by diffraction, scatter, or absorption.
The present invention also provides another apparatus for detecting the
presence of an analyte of interest in a sample. This apparatus comprises a
light source; housing means for receiving an optically transparent member
having a sample contacting surface, said member in said housing means
being disposed such that the sample contacting surface is illuminated with
light emitted from said light source which propagates through the
optically transparent member at angles between the plane of the sample
contacting surface and the critical angle for total internal reflectance;
and photodetection means which excludes the detection of light which
propagates in a geometric optical path from the light source, said
photodetection means being capable of detecting elastically-scattered
light which propagates through the optically transparent member from the
illuminated sample contacting surface between the plane of the sample
contacting surface and the total internal reflectance critical angle.
Suitable light sources for the apparatuses of the present invention provide
collimated or uncollimated light, polarized or unpolarized light, or
monochromatic or polychromatic light. Preferred light sources include
lasers (e.g., He-Ne lasers), light emitting diodes (LEDs), flash lamps,
arc lamps, incandescent lamps, and fluorescent discharge lamps.
Suitable optically transparent members, e.g., cuvettes, are comprised of
glass, quartz, silicon, plastics such as polycarbonate, acrylic, or
polystyrene, or oils comprising silicone or high molecular weight
hydrocarbons.
Suitable photodetection means comprise photon detectors such as
photomultiplier tubes, photodiodes (e.g., PIN diodes and
gallium-aluminum-arsenide diodes), cadmium sulfide photoresistive cells,
phototubes, and pyrolytic detectors. Also provided is a method for
detecting the presence of a light scattering molecule on the surface of an
optically transparent material. This method comprises illuminating said
light scattering molecule, detecting light scattered elastically by said
light scattering molecule which propagates through said optically
transparent material between the plane of the surface of the optically
transparent material on which the light scattering molecule is located and
the total internal reflectance critical angle of the surface on which the
light scattering molecule is located, and correlating detected,
elastically-scattered light to the presence of the light scattering
molecule on the surface of the optically transparent material. Within this
application, an "evanescent wave" means a nonpropagating light wave such
as a wave in the region of a surface on the side of the surface opposite
the side of illumination, produced when the illuminating light undergoes
total internal reflection. Also within this application a "light
scattering molecule" means a molecule which causes incident light to be
elastically scattered. "Molecule" includes, in the case of crystalline and
elemental materials, two or more atoms.
Still further, the present invention provides a method for detecting the
presence of a light scattering molecule on the surface of an optically
transparent material. This method comprises illuminating said light
scattering molecule with an evanescent wave resulting from a light wave
which propagates through said optically transparent material, detecting
light scattered elastically by said light scattering molecule which
propagates through said optically transparent material between the plane
of the surface of the optically transparent material on which the light
scattering molecule is located and the total internal reflectance critical
angle of the surface on which the light scattering molecule is located,
and correlating detected, elastically-scattered light to the presence of
the light scattering molecule on the surface of the optically transparent
material.
Further provided is a method for detecting an analyte in a fluid sample
wherein said analyte is a ligand of a ligand - ligand binding partner
pair. This method comprises the steps of:
a) providing an optically transparent material having a refractive index
greater than the refractive index of said fluid sample, said optically
transparent material having a sample contacting surface to which a
plurality of ligand binding partners of said ligand - ligand binding
partner pair are immobilized;
b) further providing light scattering particle-labeled ligands capable of
forming complexes with said immobilized ligand binding partners;
c) contacting said fluid sample and said light scattering particle-labeled
ligands with said sample contacting surface under conditions such that
said analyte and said light scattering particle-labeled ligands each form
complexes with said immobilized ligand binding partners;
d) illuminating said complexes with an evanescent wave resulting from a
light wave which propagates through said optically transparent material;
e) detecting light scattered elastically by said light scattering particles
of said complexes;
f) correlating elastically-scattered light to the presence of complexes on
said sample contacting surface; and
g) comparing the presence of complexes on the sample contacting surface
with the presence of complexes on a sample contacting surface for a
standard control, thereby detecting the analyte in the fluid sample.
Within this application, "particle" means one or more molecules. "Labeled"
means directly linked, e.g., conjugated, cross-linked, or adsorbed, or
indirectly linked, e.g., linked via an antibody.
Further yet is provided a method for detecting an analyte in a fluid sample
wherein said analyte is a ligand of a ligand - ligand binding partner
pair. This method comprises the steps of:
a) providing an optically transparent material having a refractive index
greater than the refractive index of said fluid sample, said optically
transparent material having a sample contacting surface to which a
plurality of ligand binding partners of said ligand - ligand binding
partner pair are immobilized;
b) further providing light scattering ligands caPable of forming complexes
with said immobilized ligand binding partners;
c) contacting said fluid sample and said light scattering ligands with said
sample contacting surface under conditions such that said analyte and said
light scattering ligands each form complexes with said immobilized ligand
binding partners;
d) illuminating said complexes;
e) detecting light scattered elastically by light scattering ligands of
said complexes and which propagates through said optically transparent
material from the sample contacting surface between the plane of the
sample contacting surface and the total internal reflectance critical
angle of the sample contacting surface;
f) correlating elastically-scattered light to the presence of complexes on
said sample contacting surface; and
g) comparing the presence of complexes on the sample contacting surface
with the presence of complexes on a sample contacting surface for a
standard control, thereby detecting the analyte in the fluid sample.
Within this application, "light scattering ligands" means ligands or light
scattering particle-labeled ligands which cause incident light to be
elastically scattered.
Further still is provided a method for detecting an analyte in a fluid
sample wherein said analyte is a ligand of a ligand - ligand binding
partner pair. This method comprises
a) providing an optically transparent material having a refractive index
greater than the refractive index of said fluid sample, said optically
transparent material having a sample contacting surface to which a
plurality of ligand binding partners of said ligand - ligand binding
partner pair are immobilized;
b) further providing light scattering ligands capable of forming complexes
with said immobilized ligand binding partners;
c) contacting said fluid sample and said light scattering ligands with said
sample contacting surface under conditions such that said analyte and said
light scattering ligands each form complexes with said immobilized ligand
binding partners;
d) illuminating said complexes with an evanescent wave resulting from a
light wave which propagates through said optically transparent material;
e) detecting light scattered elastically by light scattering ligands of
said complexes and which propagates through said optically transparent
material from the sample contacting surface between the plane of the
sample contacting surface and the total internal reflectance critical
angle of the sample contacting surface;
f) correlating elastically-scattered light to the presence of complexes on
said sample contacting surface; and
g) comparing the presence of complexes on the sample contacting surface
with the presence of complexes on a sample contacting surface for a
standard control, thereby detecting the analyte in the fluid sample.
In one embodiment of the invention, the methods provided herein may be
performed wherein the sample contacting surface is contacted .with the
fluid sample before being contacted with the ligands<In another embodiment
of the invention, the sample contacting surface is contacted with the
ligands before being contacted with the fluid sample. In yet a further
embodiment of the invention, the sample contacting surface is
simultaneously contacted with said fluid sample and the ligands. In still
another embodiment of the invention, the sample is mixed with the ligands
so as to form a mixture, and the mixture is contacted with the sample
contacting surface.
Furthermore, in a preferred embodiment of the invention, light scattering
particle-labeled ligands comprise ligands labeled with colloidal gold
particles.
In still another embodiment of the invention, a method is provided for
detecting an analyte in a fluid sample. In this method the analyte is a
ligand having an epitope for which a first ligand binding partner is
specific and an epitope for which a second ligand binding partner is
specific. The method comprises:
a) providing an optically transparent material having a refractive index
greater than the refractive index of said fluid sample, said optically
transparent material having a sample contacting surface to which a
plurality of first ligand binding partners are immobilized;
b) further providing light scattering particle-labeled second ligand
binding partners;
c) contacting said fluid sample and said light scattering particle-labeled
second ligand binding partners with said sample contacting surface under
conditions such that immobilized first ligand binding partner : analyte :
light scattering particle-labeled second ligand binding partner complexes
are formed;
d) illuminating said complexes with an evanescent wave resulting from a
light wave which propagates through said optically transparent material;
e) detecting light scattered elastically by said light scattering particles
of said complexes;
f) correlating elastically-scattered light to the presence of complexes on
said sample contacting surface; and
g) comparing the presence of complexes on the sample contacting surface
with the presence of complexes on a sample contacting surface for a
standard control, thereby detecting the analyte in the fluid sample.
Still another method is provided for detecting an analyte in a fluid
sample, wherein said analyte is a ligand having an epitope for which a
first ligand binding partner is specific and an epitope for which a second
ligand binding partner is specific. This method comprises:
a) providing an optically transparent material having a refractive index
greater than the refractive index of said fluid sample, said optically
transparent material having a sample contacting surface to which a
plurality of first ligand binding partners are immobilized;
b) further providing light scattering second ligand binding partners;
c) contacting said fluid sample and said light scattering second ligand
binding partners with said sample contacting surface under conditions such
that immobilized first ligand binding partner : analyte : light scattering
second ligand binding partner complexes are formed;
d) illuminating said complexes;
e) detecting light scattered elastically by said light scattering second
ligand binding partners of said complexes and which propagates through
said optically transparent material from the sample contacting surface
between the plane of the sample contacting surface and the total internal
reflectance critical angle of the sample contacting surface;
f) correlating elastically-scattered light to the presence of complexes on
said sample contacting surface; and
g) comparing the presence of complexes on the sample contacting surface
with the presence of complexes on a sample contacting surface for a
standard control, thereby detecting the analyte in the fluid sample.
Within this application, "light scattering second ligand binding partners"
means second ligand binding partners or particle labeled second ligand
binding partners which cause incident light to be elastically scattered.
Still further is provided a method for detecting an analyte in a fluid
sample wherein said analyte is a ligand having an epitope for which a
first ligand binding partner is specific and an epitope for which a second
ligand binding partner is specific. This method comprises:
a) providing an optically transparent material having a refractive index
greater than the refractive index of said fluid sample, said optically
transparent material having a sample contacting surface to which a
plurality of first ligand binding partners are immobilized;
b) further providing light scattering second ligand binding partners;
c) contacting said fluid sample and said light scattering second ligand
binding Partners with said sample contacting surface under conditions such
that immobilized first ligand binding partner : analyte: light scattering
second ligand binding Partner complexes are formed;
d) illuminating said complexes with an evanescent wave | | |
