U.S. patent number RE33,581 [Application Number 07/072,699] was granted by the patent office on 1991-04-30 for immunoassay using optical interference detection.
Invention is credited to Virgil B. Elings, David F. Nicoli.
United States Patent |
RE33,581 |
Nicoli , et al. |
April 30, 1991 |
Immunoassay using optical interference detection
Abstract
Apparatus and method for providing an optical detection of a
binding reaction between a ligand and an antiligand, including, a
pattern formed by a spatial array of microscopic dimensions of
antiligand material, ligand material interacting with the
antiligand material to produce a binding reaction between the
ligand and the antiligand in the pattern, a source of optical
radiation including energy at at least one wavelength directed to
the pattern at a particular incidence angle to produce scattering
of the energy from the pattern in accordance with the binding
reaction and with a strong scattering intensity at one or more
Bragg scattering angles, and at least one optical detector located
relative to the pattern and aligned with a Bragg scattering angle
to detect the strong scattering intensity at the Bragg scattering
angle to produce a signal representative of the binding
reaction.
Inventors: |
Nicoli; David F. (Goleta,
CA), Elings; Virgil B. (Santa Barbara, CA) |
Family
ID: |
26753647 |
Appl.
No.: |
07/072,699 |
Filed: |
July 13, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
624460 |
Jun 25, 1984 |
04647544 |
Mar 3, 1987 |
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Current U.S.
Class: |
435/7.2;
250/461.1; 356/244; 356/317; 356/318; 356/337; 356/521; 422/417;
422/82.05; 435/7.21; 435/970; 436/518; 436/519; 436/524; 436/527;
436/528; 436/531; 436/541; 436/805; 436/807; 436/909 |
Current CPC
Class: |
G01N
21/4788 (20130101); G01N 33/54373 (20130101); G01N
2021/4711 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); G01N 21/47 (20060101); C01N
033/53 () |
Field of
Search: |
;435/7,7.21,970
;436/501,518,524,528,531,541,519,527,805,807,909
;356/317,318,337,354,244,336,338 ;422/55,62 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Nucker; Christine
Attorney, Agent or Firm: Walker; William B. Terlizzi;
Laura
Claims
We claim: .[.1. An apparatus for providing an optical detection of
a binding reaction between a ligand and an antiligand,
including,
a predetermined pattern formed by a spatial array having a
plurality of elements of microscopic dimensions of antiligand
material,
1igand material interacting with the spatial array of antiligand
material to produce a binding reaction between the ligand and the
antiligand in the pattern,
a source of optical radiation directed toward the pattern to
illuminate the plurality of elements of the spatial array to
produce an optical interference pattern in accordance with the
modulation of the optical properties of the predetermined
antiligand pattern due to the binding reaction, and
one or more optical detectors to detect the strong scattering
intensity at one or more angles to produce a signal representative
of the binding
reaction..]. .[.2. The apparatus of claim 1 wherein the
predetermined pattern is formed by a plurality of stripes of
antiligand material, each stripe having a width of microscopic
dimensions and separated from each other by a constant microscopic
dimension..]. .[.3. The apparatus of claim 2 wherein the plurality
of stripes of antiligand material is formed by antiligand material
supported on a flat continuous surface..]. .[.4. The apparatus of
claim 1 wherein the spacial array if formed by antiligand material
supported on a grooved surface forming an interference grating..].
.[.5. The apparatus of claim 1 including ligand attached to carrier
particles which compete with the unknown ligand for binding sites
on the antiligand material..]. .[.6. The apparatus of claim 1
including antiligand attached to carrier particles to cause a
binding reaction with the ligand for increasing the signal
representative of the binding reaction..]. .[.7. The apparatus of
claim 1 wherein the optical detector is on the same side of the
pattern as the source of optical radiation to detect backscattered,
or reflected, radiation..]. .[.8. The apparatus of claim 1 wherein
the optical detector is on the opposite side of the pattern as the
source of optical radiation to detect forward scattered, or
transmitted, radiation..]. .[.9. The apparatus of claim 1 wherein
the ligand is attached to carrier particles which are biological
entities, such as cells..]. .[.10. The apparatus of claim 9 wherein
the ligand on the carrier particle is naturally occurring..].
.[.11. The apparatus of claim 1 wherein the pattern is formed by
deactivating microscopic sections of an initially uniform layer of
an antiligand supported on a continuous surface..]. .[.12. The
apparatus of claim 11 in which the deactivated sections consist of
a plurality of stripes each stripe having a width of microscopic
dimensions and separated from each other by a constant microscopic
dimension..]. .[.13. Apparatus for producing an output signal in
accordance with a binding reaction between an antibody and an
antigen, including,
a spatial array of antibody having a plurality of elements of
microscopic dimensions to form a predetermined pattern,
antigen located in solution adjacent to the spatial array of
antibody to interact with the antibody in a binding reaction in the
pattern,
an incident source of optical radiation and with the source being
substantially coherent and monochromatic and directed to the
pattern for illuminating the plurality of elements of the spatial
array for producing an optical interference pattern at particular
scattering angles in accordance with the modulation of the optical
properties of the predetermined antibody pattern due to the binding
reaction, and
one or more detectors located near the pattern at one or more of
the particular scattering angles to intercept the radiation and
produce an output signal in accordance with the binding
reaction..]. .[.14. The apparatus of claim 12 wherein the pattern
is formed by a plurality of stripes of antibody material, each
stripe having a width of microscopic dimensions and separated from
each other by a constant microscopic dimension..]. .[.15. The
apparatus of claim 13 wherein the pattern of antibody material is
formed by antibody material supported on a flat continuous
surface..]. .[.16. The apparatus of claim 13 wherein the spacial
array of antibody is formed by antibody material supported on a
grooved surface forming an interference grating..]. .[.17. The
apparatus of claim 13 including antigen attached to carrier
particles which competes with the unknown antigen for binding sites
on the antibody material..]. .[.18. The apparatus of claim 13
including antibody attached to carrier particles to cause a binding
reaction with the antigen for increasing the signal representative
of the binding reaction..]. .[.19. The apparatus of claim 13
wherein the optical detector is on the same side of the pattern as
the source of optical radiation to detect backscattered, or
reflected, radiation..]. .[.20. The apparatus of claim 13 wherein
the optical detector is on the opposite side of the pattern as the
source of optical radiation to detect forward scattered, or
transmitted radiation..]. .[.21. The apparatus of claim 13 wherein
the antigen is attached to carrier particles which are biological
entities..]. .[.22. The apparatus of claim 21 wherein the antigen
on the carrier particle is naturally occurring..]. .[.23. The
apparatus of claim 13 wherein the pattern is formed by deactivating
microscopic sections of an initially uniform layer of an antibody
supported on a continuous surface..]. .[.24. The apparatus of claim
23 in which the deactivated sections consist of a plurality of
stripes each stripe having a width of microscopic dimensions and
separated from each other by a constant microscopic dimension..].
.[.25. A method for providing an optical detection of a binding
reaction between a ligand and an antiligand, including the
following steps,
forming a predetermined pattern having a spatial array having a
plurality of elements of microscopic dimensions of antiligand
material,
providing ligand material to interact with the spatial array of
antiligand material to produce a binding reaction between the
ligand and the antiligand in the periodic pattern,
directing optical radiation including energy of at least one
wavelength to the pattern for illuminating the plurality of
elements of the spatial array to produce scattering of the energy
from the pattern following the physical principle of constructive
optical interference in accordance with the binding reaction and
with a strong scattering intensity at one or more particular
scattering angles, and
detecting the strong scattering intensity at one or more of the
particular scattering angles to produce a signal representative of
the binding reaction..]. .[.26. The method of claim 25 wherein the
predetermined pattern is formed by a plurality of stripes of
antiligand material, each stripe having a width of microscopic
dimensions and separated from each other by a constant microscopic
dimension..]. .[.27. The method of claim 26 wherein the plurality
of stripes of antiligand material is formed by
antiligand material supported on a flat continuous surface..].
.[.28. The method of claim 25 wherein the the spacial pattern of
antiligand material is formed by antiligand material supported on a
grooved surface forming an interference grating..]. .[.29. The
method of claim 25 including ligand attached to carrier particles
which competes with the unknown ligand for binding sites on the
antiligand material..]. .[.30. The method of claim 25 including
antiligand attached to carrier particles to cause a binding
reaction with the ligand for increasing the signal representative
of the binding reaction..]. .[.31. The method of claim 25 wherein
the optical detection is on the same side of the pattern as the
source of optical radiation to detect backscattered, or reflected,
radiation..]. .[.32. The method of claim 25 wherein the optical
detection is on the opposite side of the pattern as the source of
optical radiation to detect forward scattered, or transmitted,
radiation..]. .[.33. The method of claim 25 wherein the ligand is
attached to carrier particles which are biological entities..].
.[.34. The method of claim 33 wherein the ligand on the carrier
particle is naturally occurring..]. .[.35. The method of claim 25
wherein the pattern is formed by deactivating microscopic sections
of an initially uniform layer of an antiligand..]. .[.36. The
method of claim 35 in which the deactivated sections consist of a
plurality of stripes each stripe having a width of microscopic
dimensions and separated from each other by a constant microscopic
dimension..]. .[.37. A method for producing an output signal in
accordance with a binding reaction between an antibody and an
antigen, including the following steps,
providing a spatial array of antibody having a plurality of
elements of microscopic dimensions to form a predetermined
pattern,
1ocating antigen in solution adjacent to the spatial array of
antibody to interact with the antibody in a binding reaction in the
pattern,
directing substantially coherent and monochromatic optical
radiation to the pattern for illuminating the plurality of elements
of the spatial array for producing scattering from the pattern at
particular scattering angles due to optical interference in
accordance with the binding reaction, and
detecting the scattered radiation to produce an output signal in
accordance with the binding reaction..]. .[.38. The method of claim
37 wherein the regular pattern is formed by a plurality of stripes
of antibody material, each stripe having a width of microscopic
dimensions and separated from each other by a constant microscopic
dimension..]. .[.39. The method of claim 38 wherein the plurality
of stripes of antibody material is formed by antibody material
supported on a flat continuous surface..]. .[.40. The method of
claim 37 wherein the spacial pattern of antibody material is formed
by antibody material supported on a grooved surface forming an
interference grating..]. .[.41. The method of claim 37 including
antigen attached to carrier particles which competes with the
unknown antigen for binding sites on the antigen material..].
.[.42. The method of claim 37 including antibody attached to
carrier particles to cause a binding reaction with the antigen for
increasing the signal representative of the
binding reaction..]. .[.43. The method of claim 37 wherein the
optical detection is on the same side of the pattern as the source
of optical radiation to detect backscattered, or reflected,
radiation..]. .[.44. The method of claim 37 wherein the optical
detection is on the opposite side of the pattern as the source of
optical radiation to detect forward scattered, or transmitted,
radiation..]. .[.45. The method of claim 37 wherein the antigen is
attached to carrier particles which are biological entities..].
.[.46. The method of claim 45 wherein the antigen on the carrier
particle is naturally occurring..]. .[.47. The method of claim 37
wherein the pattern is formed by deactivating microscopic sections
of an initially uniform layer of an antiligand..]. .[.48. The
method of claim 47 in which the deactivated sections consist of a
plurality of stripes each stripe having a width of microscopic
dimensions and separated from each
other by a constant microscopic dimension..]. .Iadd.49. A flat
immunoassay substrate having a uniform coating thereon, the uniform
coating comprising an immunochemical optical interference pattern
of microscopic dimensions of alternating areas of an active and
deactivated member of a binding pair consisting of ligand and
antiligand, the uniform coating having effectively uniform
scattering properties and having a minimum scattering intensity at
Bragg angles .theta..sub.s, the pattern of alternating areas
forming an optical interference grating with a detectable
scattering intensity at Bragg angles .theta..sub.s if the one
member of the binding pair is bound to the other member of the
binding
pair. .Iaddend. .Iadd.50. A flat immunoassay substrate of claim 49
wherein the minimum scattering intensity at Bragg angles
.theta..sub.s is close to zero when ligand is not bound to the
active antiligand. .Iaddend. .Iadd.51. A flat immunoassay substrate
of claim 49 wherein the ligand is an analyte and the antiligand is
a receptor which binds with the analyte. .Iaddend. .Iadd.52. A flat
immunoassay substrate of claim 49 wherein the binding pair consists
of an antigen or hapten, and an antibody which binds therewith.
.Iaddend. .Iadd.53. A flat immunoassay substrate of claim 49
wherein the areas of active member of the binding pair are bound
with the other member of the binding pair. .Iaddend. .Iadd.54. A
flat immunoassay substrate of claim 53 wherein said other member of
the binding pair is attached to a label consisting of additional
scattering mass. .Iaddend. .Iadd.55. A flat immunoassay substrate
of claim 53 wherein said other member of the binding pair is
additionally bound with another binding agent binding specifically
therewith consisting of ligand or antiligand. .Iaddend. .Iadd.56. A
flat immunoassay substrate of claim 55 wherein the other member of
the binding pair is attached to a label consisting of additional
scattering mass. .Iaddend. .Iadd.57. A flat immunoassay substrate
of claim 56 wherein the other member of the binding pair is
attached to a carrier particle. .Iaddend. .Iadd.58. A flat
immunoassay substrate of claim 53 wherein the other member of the
binding pair is
located on the surface of a biological entity..Iaddend. .Iadd.59. A
flat immunoassay substrate of claim 57 wherein the biological
entity is a cell. .Iaddend. .Iadd.60. A flat immunoassay substrate
of claim 53 wherein the other member of the binding pair is
attached to a carrier particle..Iaddend. .Iadd.61. An apparatus for
providing an optical detection of a binding reaction between a
ligand and an antiligand, including,
a. a flat immunoassay substrate having a uniform coating thereon,
the uniform coating comprising an immunochemical optical
interference pattern of microscopic dimensions of alternating areas
of an active and deactivated member of a binding pair consisting of
ligand and antiligand, the uniform coating having effectively
uniform scattering properties and having a minimum scattering
intensity at Bragg angles .theta..sub.s, the pattern of alternating
areas forming an optical interference grating with a detectable
scattering intensity at Bragg angles .theta..sub.s if the one
member of the binding pair is bound to the other member of the
binding pair.
b. a source of optical radiation directed toward the pattern to
illuminate the alternating areas of active and deactivated member
of the binding pair of produce a detectable scattering intensity in
accordance with the predetermined antiligand pattern due to the
binding reaction, and
c. one or more optical detectors to detect the scattering intensity
of the scattering signal at one or more Bragg angles to produce a
signal
representative of the binding reaction. .Iaddend. .Iadd.62. An
apparatus of claim 61 wherein the minimum scattering intensity at
Bragg angles .theta..sub.s is close to zero when ligand is not
bound to the active antiligand. .Iaddend. .Iadd.63. An apparatus of
claim 61 wherein the ligand is an analyte and the antiligand is a
receptor which binds with the analyte. .Iaddend. .Iadd.64. An
apparatus of claim 61 wherein the binding pair consists of an
antigen or hapten, and an antibody which binds therewith. .Iaddend.
.Iadd.65. An apparatus of claim 61 wherein the areas of active
member of the binding pair are bound with the other member of the
binding pair. .Iaddend. .Iadd.66. An apparatus of claim 65 wherein
said other member of the binding pair is attached to a label
consisting of additional scattering mass. .Iaddend. .Iadd.67. An
apparatus of claim 65 wherein said other member of the binding pair
is additionally bound with a another binding agent binding
specifically therewith consisting of ligand or antiligand.
.Iaddend. .Iadd.68. An apparatus of claim 67 wherein the other
member of the binding pair is attached to a label consisting of
additional scattering mass. .Iaddend. .Iadd.69. An apparatus of
claim 68 wherein the other member of the binding pair is attached
to a carrier particle. .Iaddend. .Iadd.70. An apparatus of claim 65
wherein the other member of the binding pair is located on the
surface of a biological entity. .Iaddend. .Iadd.71. An apparatus of
claim 70 wherein the biological entity is a cell. .Iaddend.
.Iadd.72. An apparatus of claim 65 wherein the other member of the
binding pair is attached to a carrier
particle. .Iaddend. .Iadd.73. An apparatus of claim 61 wherein the
optical detector is on the same side of the pattern as the source
of optical radiation to detect backscattered, or reflected,
radiation. .Iaddend. .Iadd.74. An apparatus of claim 61 wherein the
optical detector is on the opposite side of the pattern as the
source of optical radiation to detect forward scattered, or
transmitted, radiation. .Iaddend. .Iadd.75. A method for providing
an optical detection of a binding reaction between a ligand and an
antiligand, including the steps of
a. interacting ligand material with a flat immunoassay substrate
having a uniform coating thereon, the uniform coating comprising an
immunochemical optical interference pattern of microscopic
dimensions of alternating areas of an active and deactivated member
of a binding pair consisting of ligand and antiligand, the uniform
coating having effectively uniform scattering properties and having
a minimum scattering intensity at Bragg angles .theta..sub.s, the
pattern of alternating areas forming an optical interference
grating with a detectable scattering intensity at Bragg angles
.theta..sub.s if the one member of the binding pair is bound to the
other member of the binding pair,
b. directing optical radiation including energy of at least one
wavelength toward the pattern to illuminate the alternating areas
of active and deactivated member of the binding pair to produce a
detectable scattering intensity in accordance with the
predetermined antiligand pattern due to the binding reaction,
and
c. detecting the scattering intensity at one or more of the
particular scattering angles to produce a signal representative of
the binding
reaction. .Iaddend. .Iadd.76. A method of claim 75 wherein the
optical detection is on the same side of the pattern as the source
of optical radiation to detect backscattered, or reflected,
radiation. .Iaddend. .Iadd.77. A method of claim 75 wherein the
optical detection is on the opposite side of the the pattern as the
source of optical radiation to detect forward scattered, or
transmitted, radiation. .Iaddend. .Iadd.78. A method of claim 75
wherein the minimum scattering intensity at Bragg angles
.theta..sub.s is close to zero when ligand is not bound to the
active antiligand. .Iaddend. .Iadd.79. A method of claim 75 wherein
the ligand is an analyte and the antiligand is a receptor which
binds with the analyte. .Iaddend. .Iadd.80. A method of claim 75
wherein the binding pair consists of an antigen or hapten, and an
antibody which binds therewith. .Iaddend. .Iadd.81. A method of
claim 75 wherein the areas of active member of the binding pair are
bound with the other member of the binding pair. .Iaddend.
.Iadd.82. A method of claim 81 wherein said other member of the
binding pair is attached to a label consisting of additional
scattering mass. .Iaddend. .Iadd.83. A method of claim 81 wherein
said other member of the binding pair is additionally bound with a
another binding agent consisting of ligand or antiligand binding
specifically
therewith. .Iaddend. .Iadd.84. A method of claim 83 wherein the
other member of the binding pair is attached to a label consisting
of additional scattering mass. .Iaddend. .Iadd.85. A method of
claim 83 wherein the other member of the binding pair is attached
to a carrier particle. .Iaddend. .Iadd.86. A method of claim 81
wherein the other member of the binding pair is located on the
surface of a biological entity. .Iaddend. .Iadd.87. A method of
claim 86 wherein the biological entity is a cell. .Iaddend.
.Iadd.88. A method of claim 87 wherein the other member of the
binding pair is attached to a carrier particle. .Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention is directed to an immunoassay using optical
interference detection. Specifically, the present invention
provides for an immunoassay including a pattern to create a Bragg
scattering peak which appears or changes when a particular chemical
reaction occurs.
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
artifically. 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 (compound 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 to react with
another binding site on the antigen. 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. Any non-specific binding of
the labeled antibody to the surface will, however, contribute to
the signal.
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, posses 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
florescence 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
be 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 serves 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 florescence-based homogeneous techniques 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 principle 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
contributions 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 sources of
background false signals, interferences and non-specific
effects.
A previous invention by the same inventors as the instant
application is embodied in patent application Ser. No. 463,658
filed Feb. 3, 1983 and entitled "Immunoassay". The previous
invention is directed to a technique for detecting including
scanning of a spatial pattern in order to detect a useful signal
associated with the formation of antigen-antibody complexes. The
prior invention therefore provides for not only producing a spatial
pattern of a binding reaction but also for scanning this spatial
pattern with input optical energy so as to detect an output signal
having amplitude levels including a periodic component representing
the labeled binding reaction.
SUMMARY OF THE INVENTION
The present invention is directed to a measurement technique for
homogeneous immunoassays whereby the presence of analyte is
detected quantitatively using optical interference and specifically
a Bragg scattering peak. In order to simplify the description of
the present invention it will be assumed that the analyte of
interest or ligand consists of antigen molecules and the antiligand
of interest is a specific antibody. In general, the concentration
of the antigen in solution is determined indirectly by measuring
the extent of binding of the antigen molecules to antibody
molecules which are specific to the antigen of interest. As an
example, the antigen and antibody may be members of a homologous
binding pair.
In the present invention, the binding or complexing of the antigen
to antibody is caused to occur preferentially at certain specified
locations within the assay solution and typically on a two
dimensional surface so as to form a spatial array of microscopic
dimensions. The spatial array of antigen-antibody binding locations
or sites is then illuminated from a particular direction by a beam
of optical radiation. In the present invention it is preferable
that the source of radiation be relatively coherent and
monochromatic and therefore the preferred source of radiation is
that produced by a laser. Alternatively, the particular embodiments
of the invention may function using a source of illumination
containing a broad range of wavelengths but with the detection at
particular Bragg scattering angles.
The detection of any changes in the intensity of the scattered or
transmitted or diffracted optical radiation due to changes in the
extent of the antigen-antibody binding is provided by at least one
optical detector. This optical detector is located at a specified
Bragg angle and/or position with respect to the spatial array and
the direction of incident illumination.
The present invention is distinct from the invention described in
copending application Ser. No. 463,658 described above since the
present invention relies on the physical principle of optical
interference and is preferably implemented with the use of a
coherent source of optical radiation. Typically, the coherent
source of optical radiation has a wavelength either in the visible
region or in the near UV or near infrared regions of the spectrum.
In the present invention the spatial array is not scanned in order
to detect the useful signal associated with the formation of
antigen-antibody complexes. Rather, the existence of optical
interference causes a useful signal to exist at particular spatial
locations and/or directions with respect to the spatial array and
the direction of incident illumination. The detection of a useful
output signal is due to the antigen-antibody binding and requires
no scanning of either the illuminating beam, the spatial array or
the detector.
The present invention may be provided by an active antibody coating
forming a regular periodic array of parallel stripes of microscopic
dimensions on a solid flat surface. Both the width and
center-to-center spacing for the stripes are of microscopic
dimensions and are on the order of several wavelengths of the
illuminating radiation. The array therefore forms an interference
array to produce the optical interference so that a Bragg
scattering peak may be detected. The array can also consist of a
regular, periodic array of active antibody spots whose size and
spacing are on the order of several wavelengths of light.
Other embodiments of the invention incorporate depositing an
antibody coating on an already existing optical grating. Either the
entire grating surface may be coated or just the peaks or valleys
of the grating may be coated and with a detection of the Bragg
scattered intensities provided in accordance with the incident
illumination. The grating can function as either a transmission or
a reflection grating.
BRIEF DESCRIPTION OF THE DRAWINGS
A clearer understanding of the invention will be had with reference
to the following description and drawings wherein:
FIG. 1 illustrates a first embodiment of the invention
incorporating a periodic spatial array of stripes of antibody on a
solid substrate;
FIG. 2a illustrates an idealized periodic array of point-like
scattering masses useful in explaining the operations of the
present invention;
FIG. 2b illustrates the first embodiment of the invention with the
incident radiation normal to the spatial array;
FIG. 2c illustrates the first embodiment of the invention with the
detector placed so as to detect transmitted radiation (i.e. on the
opposite side of the spatial array).
FIG. 3 illustrates a periodic immunochemical array consisting of
alternating stripes of active and deactivated antiligand on a solid
substrate.
FIG. 4a illustrates an interference grating formed with a
physically grooved surface;
FIG. 4b illustrates the grating of FIG. 4a with an antibody coating
to form a second embodiment of the invention;
FIG. 4c illustrates the second embodiment of the invention and
further including carrier particles to enhance the detection;
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The first embodiment of the present invention is illustrated in
FIG. 1 and includes a solid flat surface 10. The surface 10 is
typically formed of glass, plastic, plastic coating on a solid
surface, gel or some suitable inert material onto which specific
antibody molecules are attached. This attachment is typically by
means such as covalent binding or physical adsorption. The active
antibody coating attached to the surface 10 is shown in FIG. 1 and
has the form of a regular periodic array of parallel stripes 12.
The antibody stripes 12 are in contact with an assay solution 14
which assay solution contains among other molecular components the
antigen of interest.
The parallel stripes 12 of antibody each has a width w and with a
center-to-center spacing d between each stripe in the array.
Additionally the center-to-center spacing d is greater than the
width w. Both w and d are of microscopic dimensions and as an
example, the center-to-center spacing d is typically on the order
of several wavelengths of the illuminating radiation. The very
small size scale for the parallel array of stripes 12 is a specific
requirement for the successful implementation of a detection device
of the present invention using optical interference. Additionally
the very small size scale is one of the important characteristics
which distinguish the present invention from prior art devices.
A light beam is produced by a coherent source such as a laser 32
and with the light beam defined by incident light rays 16. The
light from the laser 32 has a wavelength defined as .lambda.(in
air). The light rays 16 are directed toward the surface 10 which
contains the array of closely spaced parallel stripes 12 of
antibody. The assay solution 14 contains the analyte plus any
additional needed assay reagents and with the solution in contact
with the surface 10. For the specific example of the invention
represented by the embodiment of FIG. 1, it is assumed that the
portion of the assay solution 14 in contact with the surface 10
consists of a layer of liquid of uniform thickness h. This results
in an air-liquid interface 18 having a flat surface which is
parallel to the antibody bearing surface 10.
The light rays 16 of the illuminating light beam are directed to
the air-liquid interface 18 at an angle of incidence designated by
.theta..sub.i relative to a normal to the interface 18. The
direction of the light rays 16 of the incoming light beam is
aligned with respect to the antibody array 12 so that the plane of
incidence containing the beam propagation vector is normal to the
plane of the surface and perpendicular to the long axis of the
individual antibody coated stripes 12. The incident beam of light
energy is refracted at the air liquid interface 18. The resulting
"internal" angle of incidence designated by .phi..sub.i in FIG. 1
is related to .theta..sub.i by Snell's Law as follows: ##EQU1##
where n is the index of refraction of the assay solution (n.sub.air
=1)
A fraction of the intensity of the incident light rays 16 is
reflected at the air-liquid interface 18 due to the mismatch of the
two indices of refraction. The reflected rays 20 are shown by
dashed lines in FIG. 1 and leave the surface 18 at an angle
.theta..sub.r equal to the angle of incidence .theta..sub.i.
The refracted light beam formed by light rays 22 impinges on the
solid surface 10 carrying the array of antibody stripes 12. In a
similar fashion to the above, reflected rays 24 are produced if the
index of refraction of the material comprising the solid surface
differs from that of the assay solution 14. It is also possible to
get multiple reflections within the layer of assay solution 14. If
the substrate material is chosen to be index matched to the
solution 14, there will be no reflected light rays. Normally,
however, some light rays 24 of some intensity are reflected and
with an internal angle of reflection .phi..sub.r equal to the
incident angle .phi..sub.i. The reflected beam defined by the light
rays 24 after partial reflection at the air-liquid interface 18
emerges from the liquid at the angle .theta..sub.r equal to the
original angle of incident .theta..sub.i.
In addition to all of the various internal reflections and
refractions, the incident radiation is scattered by the antibody
coated array of stripes 12. In general, the polarizability of the
antibody molecules differs from that of either the solid substrate
10 or the surrounding assay solution 14. Since the sources of the
scattering which are the antibody coated stripes form a spatially
periodic array, the antibody segments behave like a reflection
grating.
A coherent source of radiation at a single wavelength or over an
extremely narrow range of wavelengths is used to illuminate this
"immunochemical grating". The physical phenomenon of optical
interference results in a relatively strong scattering intensity at
certain scattering angles .theta..sub.s and relatively low
intensity at all other angles. The strength of the constructive
interference at angle .theta..sub.s depends on the number of
antibody stripes illuminated, the uniformity of their spacing
distance d and their individual scattering efficiency. The
efficiency depends on the extent to which the polarizability of the
stripes differs from that of the surrounding liquid medium and of
the solid substrate. The internal incident light rays 22 produce
internal scattered light rays 26 having internal scattering angles
.phi..sub.s. External scattered light rays 28 are then produced
having the external angles .theta..sub.s and with the internal and
external scattering angles related by Snell's Law as follows:
##EQU2##
The angles .theta..sub.s or their equivalent .phi..sub.s correspond
to preferential scattering by the periodic array and specifically
these scattering angles are .[.well known as the Bragg angles..].
.Iadd.referred to herein as the Bragg angles. The term "Bragg
angles", as used herein, is defined as the angle between the normal
to the solid substrate surface and the angle of the scattered,
diffracted light waves, .theta..sub.s. .Iaddend.The intensity of
the preferentially Bragg scattered radiation, also of wavelength
.lambda., is measured by a detector 30 shown in FIG. 1. The
detector 30 may be of any suitable type such as a solid state
photodiode or photomultiplier tube. The detector 30 is located at a
distance from the scattering/reflecting surfaces, and as well as
known in the art, suitable masks or apertures may be used to insure
that the detector 30 receives only radiation which has been
scattered at one of the preferential Bragg angles
.theta..sub.s.
The use of the immunochemical grating to perform the immunoassay
operates as follows. Antigen molecules which are free in the assay
solution 14 scatter light in all directions. This is true also for
any other randomly positioned molecules in the solution as well as
dirt particles or impurities contained in blood serum, etc.
Therefore, the contribution to the light intensity detected by the
detector 30 at the angle .theta..sub.s, due to any of the sources
of scattering in the solution 14, is relatively small because there
will be no phase coherence between the scattered electric fields
produced by these randomly positioned sources of light scattering.
Such scattering will in general be distributed over all angles.
When a fraction of the antigen molecules becomes bound to the
surface-immobilized antibodies forming the stripes 12, there is an
increase in the scattered light intensity along the preferential
angles .theta..sub.s due to an increase in mass of differing
polarizability at the spatially periodic locations forming the
array of stripes 12. In addition, the set of angles .theta..sub.s
does not change with the addition of scattering mass at the
periodic sites of the array since these angles are a function only
of the parameters d, .lambda. and .phi..sub.i (or .theta..sub.i)
which parameters are predetermined. Therefore, the phenomenon of
binding-enhanced Bragg scattering is ideally dependent only on the
amount of scattering mass which becomes attached to the surface in
a spatially periodic way which of course is the array of antibodies
stripes 12. The binding of antigen can thereby be followed as a
function of time by monitoring the increase in the scattered
intensity at one or more of the preferential Bragg scattering
angles .theta..sub.s.
There is a specific relationship between .theta..sub.i, d,
.theta..sub.s and .lambda. required to produce a Bragg interference
maximum in the scattered radiation from a periodic grating
structure. As shown in the present invention, the assay solution 14
is in contact with the surface 10 and the antibody stripes 12 so
that the specific relationship is expressed in terms of the
internal angles .phi..sub.i and .phi..sub.s as shown in FIG. 2a.
The internal angles define the angles of incidence and scattering
within the liquid assay solution 14 which solution has an index of
refraction n. In order to simplify the analysis the scattering
array is considered to consist of a periodic array of point-like
scattering masses 34 and with the distance between the points 34 or
the periodicity to be d. A two dimensional array of stripes may be
achieved simply by converting each point source 40 to a line source
or a set of parallel line sources. The analysis provided by the
simplified structure of FIG. 2a may be applied generally to all of
the embodiments of the invention.
The incident wavelength of the illuminating radiation in the
solution is defined by .lambda..sub.s and is related to the
incident wavelength in air, .lambda., by the following:
##EQU3##
The requirement for constructive interference at the angle
.phi..sub.s is that the scattered electric fields produced by two
adjacent scattering centers be in phase at a distant observer. The
condition occurs when the difference in optical path lengths in
solution, .DELTA.L.sub.s, of two adjacent rays such as the two rays
36 and 37 shown in FIG. 2a is equal to an integral multiple of
.lambda..sub.s. This may be defined as, ##STR1##
An equivalent expression is obtained which relates to the angles
.theta..sub.i and .theta..sub.s and wavelength .lambda. in air and
using Snell's Law as expressed in equations (1) and (2) as well as
equations (3) becomes, ##STR2##
As seen in FIG. 2a, the refraction of both the incident and
scattered rays at the air-solution interface by the layer of assay
solution 14 in contact with the antibody coated surface 10 does not
change the Bragg equation defined above provided that the angles
and wavelength of the radiation refer either to the fluid solution
as defined by equation (4) or the air outside the solution layer as
defined in equation (5).
A local maximum in intensity for the scattered optical radiation is
obtained whenever the condition given by equation (4) or equation
(5) holds. The scattering produced at the angle .theta..sub.s or
.phi..sub.s corresponding to m=1 is referred to as the first order
Bragg maximum; the scattering produced corresponding to m=2 is
second order, etc. It is desirable to choose a incident angle
.theta..sub.i and the order of interference m so that the angle
.theta..sub.s differs significantly from the angle of reflection
.theta..sub.r. In this way, the change in scattering intensity at
angle .theta..sub.s due to antigen-antibody binding is not obscured
by the potentially large reflected intensity caused by a mismatch
in indices of refraction at the air/solution and solution/surface
interfaces. This is true even though ideally the reflected
intensity is constant in time while the scattered intensity at
angle .theta..sub.s increases with time as the extent of
antigen-antibody binding increases.
In order to simplify further the use of the immunochemical
interference grating of the present invention, FIG. 2b illustrates
a structure wherein the illuminating light beam produced by the
laser 32 is incident along a normal to both the assay solution
surface and the antibody coated surface. In such a structure,
.theta..sub.i =.phi..sub.i =0 in both equations (4) and (5). The
resulting Bragg interference condition which yields a maximum in
the scattering at angle .theta..sub.s or .phi..sub.s reduces
to:
or
In the simplified structure of FIG. 2b the specularly reflected
rays return along a normal directly back to the laser source 32 so
that .theta..sub.r =.phi..sub.r =0.
A representative sample of first order (m=1) scattering angles
.theta..sub.s corresponding to a range of antibody array spacing d
for a plurality of choices of wavelength is shown in Table 1. The
specific wavelengths chosen are =.lambda.0.6328.mu.,
.lambda.=1.15.mu. and .lambda.=3.391.mu.. These particular
wavelengths are available using a He-Ne laser but other wavelengths
which lie either in the visible or near infrared regions of the
spectrum are available from other sources including solid state
lasers. The only requirement for the wavelength is that the
solution be relatively transparent at that chosen wavelength.
As shown in Table 1, the longer the incident wavelength the larger
the angle of the first order scattering maximum, .theta..sub.s, for
an array of given periodicity d.
TABLE 1 ______________________________________ d (microns)
.theta..sub.s *(.lambda. = 0.63.mu.) .theta..sub.s *(.lambda. =
1.15.mu.) .theta..sub.s *(.lambda. = 3.39.mu.)
______________________________________ 1 39.3.degree. -- -- 2
18.4.degree. 35.1.degree. -- 3 12.2.degree. 22.5.degree. -- 4
9.1.degree. 16.7.degree. 57.9.degree. 5 7.3.degree. 13.3.degree.
42.7.degree. 10 3.6.degree. 6.6.degree. 19.8.degree.
______________________________________ *m = 1; 1storder Bragg
interference maxims.
FIG. 2c shows a variation on the first embodiment of the invention
in which the immunochemical grating acts as a transmission grating
rather than a reflection grating, as previously described. In this
version the detector 30 is located on the opposite side of the
substrate as the illuminating light source 32. For simplicity, the
incident light rays 16 are shown inpinging on the substrate 10
along a normal to the surface, analogous to FIG. 2b. In this case,
the substrate 10 is chosen to be transparent, so that the Bragg
scattered light rays 28 can pass through the substrate and be
detected by the detector 30.
The Bragg scattering angles .theta..sub.s which correspond to
constructive interference with local intensity maxima are again
given by Equation (7) for the simplified case of normal incidence
of coherent light of wavelength .lambda. and scattering order m.
For the more general case of illumination at an angle of incidence
.theta..sub.i, the Bragg scattering angles .theta..sub.s are given
by Equation (5). Considerations of experimental geometry (i.e.
placement of light source, coated grating and detector), scattering
efficiency and substrate transparency influences the choice of
whether to use the immunochemical grating in the transmission or
the reflection mode in carrying out an immunoassay measurement.
In order to provide for the structure of FIG. 1 and FIG. 2, the
antibody must be successfully deposited on a solid substrate with a
periodic array of very closely spaced stripes of uniform spacing
and with the various dimensions typically being on the order of
microns. There are a number of procedures by which the precision
antibody coating may be accomplished, and some of these procedures
will now be described.
In a first procedure, a continuous antibody coating is In a first
procedure, a continuous antibody coating is initially provided to
uniformly cover the entire substrate surface. The antibody
molecules may be attached to the substrate surface either by
physical adsorption or covalent binding. For example, when most
plastic surfaces are immersed in a solution of protein molecules of
a given kind, the surface acquires a monolayer coating of that
molecular species. Once a coating is achieved, the next step
consists of removing alternate stripe-like regions of the antibody
coating of the desired width and periodicity. Equivalently,
alternate stripe-like regions of the coating may be rendered
chemically inactive with respect to their ability to bind the
homologous antigen. The production of the strip-like regions may be
accomplished by subjecting the antibody coated surface to a focused
electron beam or ion beam which beam is scanned over the surface in
a spatially periodic pattern to deactivate or denature antibody
molecules in the desired stripe-like areas. To enhance the
formation of the stripe-like regions a shadow mask, such as is
fabricated by photolithographic techniques may be used in
conjunction with or in place of the spatial scanning in order to
render alternate stripes of antibody molecules inactive.
In place of the electron beam or ion beam a powerful focussed laser
beam may be used to denature or otherwise render immunochemically
inactive the antibody molecules in the spatial stripe-like regions.
Additionally, an intense incoherent UV source, such as from a
mercury lamp, together with a shadow mask placed near or in contact
with the coated surface also may be used to accomplish the
selective destruction of antibody molecules to form the stripe-like
regions. In general, sources of ionizing radiation may be directed
to the coated surface to produce the spatial pattern to form the
immunochemical grating.
As an alternative to scanning the coated surface or using a shadow
mask, an interference fringe pattern may be established over an
extended region of the coated surface by intersecting two phase
coherent laser beams. The interfering laser beams produce a fringe
pattern which consists of uniformly spaced ribbons or bands of
light which, if sufficiently intense and of a suitable wavelength
may denature or destroy the antibody molecules in alternating
stripe-like areas. Since certain types of molecules contain a
number of ring-like resonant groups which absorb strongly in the UV
region of the spectrum, it is desirable to use UV laser radiation
to establish the fringe pattern.
FIG. 3 illustrates an immunochemical grating array consisting of
alternating stripes of active and deactivated antibody. It is
produced by first applying a uniform coating of active antibody on
a flat substrate 10. Alternating stripe-like regions of deactivated
antibody 13 are then produced using one of the procedures described
above, leaving alternating stripes of active antibody 12 on the
surface of the substrate. Such a grating array has the advantage
that there exists minimal scattered intensity at the Bragg angles
.theta..sub.s in the absence of antigen binding to the active
portions of the antibody, due to the fact that the active and
deactivated antibody regions ideally posses very similar
polarizabilities (i.e. scattering efficiencies), causing the
grating to effectively "disappear" when the antigen is not bound to
the grating.
The immunochemical technique described herein posses two important
characteristics which potentially give it a very high sensitivity
and a great selectivity against interfering sources of false signal
due to non-specific binding of molecules. In the first place the
technique ideally involves a measurement "at null", in which the
"baseline" signal is ideally zero in the absence of
antigen-antibody binding. This results if the immunochemical
grating consists of stripes of active antibody on the substrate
surface which alternate with stripes of denatured, or otherwise
inactive, antibody molecules. In this case light is scattered
equally strongly from each kind of stripe before antigen (or
labelled, mass-enhanced, antigen) binds preferentially to the
active antibody stripes. Therefore, with respect to its scattering
properties the coated substrate surface is effectively uniform,
resulting in no Bragg interference peaks in the scattered light
before the reaction occurs. The detected intensity at any of the
Bragg angles .sub.s is therefore ideally very close to zero--i.e.
is at null. This property causes the assay method to be very
sensitive, because when a small amount of antigen binds to the
stripes of active antibody, a scattering signal appears at the
preferential Bragg angles .theta..sub.s where previously it was
zero.
If the intensity to be detected at angle .theta..sub.s is extremely
weak, due to a very small extent of binding of antigen (or labelled
antigen, to enhance the scattering) to antibody, lock-in (i.e.
phase-sensitive) detection techniques can be used to improve the
signal/noise ratio of the measurement, as described previously. The
important point is that detection of a small signal starting with
nearly zero signal is inherently more sensitive and reliable, in
practice, than detection of an equivalently small signal which is
superimposed on a relatively large background signal, which in
general will drift and otherwise vary in time, making it difficult
to extract the desired signal.
A second important characteristic of this assay method is its
relative insensitivity to non-specific binding of molecules at the
antibody-coated substrate surface. Non-specific binding refers to
the adsorption onto the coated substrate of molecules other than
the antigen specific to the antibody on the substrate. This
behavior may be a consequence of attraction of the molecules to the
antibody molecules per se or to the underlying substrate (i.e.
through electrostatic interactions). For the ideal immunochemical
grating, consisting of alternating segments of active and
deactivated antibody, non-specific binding of foreign molecules
occurs with roughly equal probability for the active and inactive
antibody. In this case, there is no contribution of such bound
molecules to the intensity at any of the Bragg interference angles.
That is, non-specific binding is not a source of false signal in
any of the measured intensities providing the binding does not
occur preferentially to either the active or inactive antibody
coated stripes.
On the other hand, in the case in which the grating surface carries
alternating stripes of active antibody and a substantially
different material (e.g. polymer-coated antibody), both the
detection-at-null property and the insensitivity to non-specific
binding are compromised to some extent. In this case the
immunochemical grating may give rise to a Bragg scattering pattern
of intensity peaks at the angles .theta..sub.s in the absence of
antigen-antibody binding due to the difference in scattering
efficiencies of the two kinds of molecular species comprising the
substrate coating. The closer the polarizabilities of the two kinds
of coating stripes, the smaller will be the resulting quiescent
Bragg intensities. However, in general the resulting assay is less
sensitive than the one previously described, due to the fact that a
small increase in intensity superimposed on a finite background
signal must be detected. Also, in general non-specific binding of
molecules leads to a false Bragg signal, because it is to be
expected that the efficiency of non-specific binding differs for
the two kinds of materials comprising the substrate coating.
The periodic antibody array may also be produced through the use of
photopolymerization. Specifically, a thin layer of polymer solution
is applied on top of the uniform antibody coating on the substrate
surface. Using either a scanned focussed laser beam, a shadow mask
or a stationary fringe pattern, all of which are described above,
the polymer coating is exposed to sufficient radiation to
polymerize alternate stripes of the polymer solution. The portions
of the coating which remain unpolymerized are then washed away
leaving a grating-like array of active antibody molecules,
alternating with segments of polymer blocked antibody to which
antigen cannot specifically bind.
A further method of fabricating the desired antibody array includes
the use of photolithographic methods to lay down a periodic array
of stripes of a particular substance which differs markedly from
the substrate material in its ability to bind antibody.
Specifically, a substance is chosen to which antibody readily
attaches by physical absorption compared to the surrounding
substrate surface so that the substrate surface will not attract
antibody. Alternately, the deposited substance may be of a type to
which antibody will not readily attach when compared to the
surrounding substrate surface. As a specific example,
macromolecules such as proteins generally do not absorb well onto
glass surfaces. Photolithography is then used to construct a
periodic array of thin polymer segments deposited on the glass. In
this way the antibody is selectively absorbed onto the polymer
coated surface but does not readily absorb onto the glass so that a
periodic array of antibody absorbed onto the polymer segments is
produced.
As described above, another important consideration for the
interference immunoassay of the present invention is the inherent
sensitivity of the immunoassay method. The binding of antigen
molecules along to the antibody coated stripes in general yields a
relatively small change in the overall scattered intensity at the
Bragg angles .theta..sub.s. The antibody which forms the grating
array itself can be expected to produce Bragg scattered intensities
at the same set of angles .theta..sub.s. These intensities may be
increased by the preferential deposition of additional scattering
mass on the array by the binding of antigen to antibody. However,
the total scattering in general is expected to be weak since there
is only a relatively small mismatch between the indices of
refraction or the polarizabilities of typical macromolecules and
the surrounding solvent which is mostly water. As described below,
the immunoassay may be enhanced by the use of a scattering label
such as the use of carrier particles. Such means may therefore be
used advantageously with the present invention.
It is desirable therefore to increase the scattering efficiency of
the immunochemical grating by carrying out the assay using a form
of labeled antigen where the label has the effect of greatly
increasing the polarizability and therefore the scattering
efficiency of the antigen. In one specific example, a competitive
type assay may be performed in which free antigen, which comprises
the unknown, and labeled antigen of known concentration, compete
for binding to a limited number of antibody molecules which make up
the grating array. Alternately, a sandwich type reaction may be
used in the following manner. First, binding of the antigen to the
antibody coated grating array is allowed to go to essential
completion. Second, free labeled antibody is added in excess. The
label consists of additional scattering mass. As the labeled
antibody binds to the surface immobilized antigen, there is a
monotonic increase in each of the Bragg scattering intensities at
the various angles .theta..sub.s.
As a specific example of a system which will increase the
scattering efficiency, metallic ions are attached or otherwise
complexed to the antigen for a competitive type assay or to the
antibody for a sandwich assay so as in increase the scattering
efficiency. A further increase in scattering power is achieved by
making the label more massive than the antigen or antibody molecule
itself. For example, more than one antigen or antibody molecule may
be attached by adsorption or covalent binding to chemically inert
carrier particles which scatter light particularly efficiently. A
particularly good example of such an efficient carrier particle is
a colloidal metal particle (sol) such as colloidal gold. It is
known that most macromolecules absorb strongly onto such gold sols
as well as other metal sols. The typical size range for such sols
is 5 to 100 nm. The intensity of scattering produced by colloidal
metal particles in solution exceeds by several orders of magnitude
that which would be produced by antigen or antibody molecules along
in solution.
Colloidal gold particles which have been coated with a variety of
macromolecules (e.g. Avidin, various lectins, goat anti-rabbit IgG,
etc.) are available commercially from a number of sources. In
addition, carrier particles consisting of polystyrene latex or a
similar material, which are preferably in the size range of 20 to
500 nm, may also be utilized to perform an assay using the
immunochemical grating technique of the present invention.
As an alternative to the use of carrier particles which increase
the scattering efficiency, other carrier particles may be used
which absorb the incident light. In this way, as the absorbing
particles become attached to the periodic array of antibody
stripes, the incident light is scattered primarily from the
uncoated areas of substrate between the stripes due to the index
mismatch between the substrate and the assay solution. The
absorbing carrier particles thereby decrease the scattering from
the antibody segments. The preferential scattering from the
uncoated segments of the surface produces the same net result which
is an increase in the Bragg scattering intensities at the same set
of angles .theta..sub.s or .phi..sub.s given by equations (4) and
(5). The various parameters d, .lambda., n and .theta..sub.i are
unaffected whether scattering occurs predominantly from the grating
array represented by the antibody coated segments or from the
complement to the grating array represented by the uncoated
segments.
The above immunoassay technique may be used to detect the presence
of antigen or antibody molecules which are located on the surface
(i.e. in the membranes) of biological cells such as erythrocytes or
lymphocytes. In this way cell typing may be performed to determine
the concentration of cells which carry a particular type of antigen
or antibody. In particular this immunoassay technique can be used
to perform human red blood cell typing. If the cell-associated
molecule is an antigen, the antibody-coated stripes discussed
previously are used to construct the immunochemical grating. When
the cell-associated molecule to be identified is an antibody,
stripes composed of the homologous antigen molecules are used,
alternating with stripe-like regions of inactive antigen.
In this variation of the assay, whole cells become attached to the
grating surface in the immediate vicinity of the active stripes as
a consequence of antigen-antibody binding. Consequently, the cell
performs the physical function of the inert "carrier" particle
described earlier, having the same effect of increasing the
scattering due to a single binding event. Biological cells are
considerably larger than inert carrier particles which are ideally
chosen as scattering amplifiers which particles posses linear
dimensions on the order of 1-10 microns. Hence for this application
an immunochemical grating is designed with a relatively large
stripe-to-stripe periodicity d, as shown in FIG. 1, which exceeds
the largest cell dimension. In this way, when some cells become
attached to the coated substrate the resulting pattern will
resemble stripe-like regions of cells of periodicity d separated by
gaps in which ideally no cells are attached. Because the cell
dimensions may substantially exceed the wavelength of the exciting
light beam, the efficiency of this assay in producing Bragg
intensity peaks depends considerably on the cell size and shape as
well as the grating parameters (i.e. dimensions w and d, FIG.
1).
Fluorescent labels or fluorescent carrier particles do not in
general form any part of the present invention. Fluorescent sources
behave as incoherent sources of radiation in which the emitted
electric field from each fluorescent molecule does not bear a fixed
phase relationship to the incident electric field. Therefore the
field of fluorescent radiation does not display the sharp angular
dependence exhibited by Bragg scattering regardless of the spatial
locations of the emitting fluorescent labels. The existence of
strongly fluorescent sources either in solution or on the substrate
surface simply adds a background contribution to the intensity
measured at any of the Brigg angles .theta..sub.s. If this
background were troublsome, it could easily be removed using an
optical bandpass filter in front of the detector.
It has been previously shown with reference to Table 1 that it is
preferable to use relatively long wavelength laser light, such as
.lambda..gtorsim.0.6.mu., to illuminate the grating array. This
allows for the choice of the largest periodicity d for the stripes.
In addition, the use of relatively long wavelength light minimizes
the fluorescent background intensity from most serum samples since
naturally fluorescing sources are generally excited at shorter
wavelengths.
The embodiment of the present invention, disclosed in FIG. 1,
requires a periodic array of microscopically spaced stripes of
antibody formed on a flat substrate surface. Another embodiment of
the invention is disclosed with reference to FIGS. 4a through
4c.
In general, this other embodiment of the invention is an actual
interference grating having a physically grooved surface to form
the solid substrate on which antibody is immobilized and to which
antigen-antibody binding occurs. The grating consists of a solid or
semisolid material 40 and is preferably a transparent material,
such as plastic, and with one surface containing a periodic
structure of closely spaced grooves 42 of spacing d as shown in
FIG. 4a. The surface of the substrate 40 which contains the
spatially periodic grooves 42 will scatter light preferentially
along the Bragg angles defined by equation (5). Again, the grating
can be used in either the backscatter (i.e. reflection) mode or the
transmission mode.
The individual scattering sources at each point along the grooved
structure do not scatter light with equal efficiencies due to the
detailed shape and structure of the groove pattern. Therefore, the
scattering along the angles .theta..sub.s given by equation (5) is
enhanced due to constructive interference. The efficiency with
which the grating of FIG. 4a will Bragg scatter coherent light into
the various orders on m values is a function of the angle of
incidence, the wavelength and the polarization of the incident
radiation, the index of refraction of the grating material 40, the
groove density and uniformity and the detailed shape and
orientation of the grooves 42.
The major advantage in the use of a structure such as shown in FIG.
4a to form the solid substrate is that the entire grating surface
may be coated with antibody 44 to produce a second embodiment of
the invention shown in FIG. 4b. This avoids entirely the task of
depositing on a flat surface a periodic array of very closely
spaced stripes of antibody as shown in FIG. 1 or alternating
active/deactivated stripes as shown in FIG. 3. The coating process
is accomplished by immersing the solid grating material 40 into a
solution of antibody in an appropriate buffer, for a period of
time. A monolayer of the antibody molecules 44 forms on the grooved
plastic surface by physical adsorption.
The thickness of the macromolecular monolayer 44 is typically only
25 to 100 angstroms compared to the characteristic groove
dimensions of fractions of a micron, or larger. Therefore, the
surface of the coated grating, as shown in FIG. 4b, mimicks even to
fine detail the features of the original grating surface as shown
in FIG. 4a. As an alternate to the use of physical adsorption, the
plastic surface of the substrate 40 may contain chemically reactive
groups and the antibody molecules 44 may be attached to the plastic
coating by covalent binding using standard chemical procedures.
The plastic grating 40 may be obtained commercially. As an example,
acceptable precision gratings can be obtained from commercial
companies such as Edmund Scientific Company of Barrington, N.J. One
particular grating which may be used is found in a recent catalog
from Edmund Scientific and bearing catalog number 40,267. This
particular grating is formed of a flexible sheet of acetate and
with one surface containing a periodic structure of closely spaced
grooves. This particular grating includes 13,400 grooves/inch which
results in d=1.90.mu.. The thin plastic sheet forming the substrate
is essentially transparent and can thereby function as either a
transmission of reflection grating.
Coherent light is directed to the grating 40 and as a specific
example coherent light may be directed along a normal to the
surface of the grating 40. This is as shown in FIG. 2b which
produces a set of Bragg scattered rays along the angles
.theta..sub.s as given by equation (7). When the structure of FIG.
3b is used in the reflection mode, the scattered beams are located
at angles .theta..sub.s less than 90.degree. as defined in the
above equation and as exemplified in Table 1. When the structure of
FIG. 3b is used in the transmission mode, the scattering is in the
forward direction with .theta..sub.s greater than 90.degree.. This
is because the original values of .theta..sub.s given in equation
(7) are replaced by 180.degree.-.theta..sub.s.
When the particular grating, catalog number 40,278 from Edmund
Scientific is used, the scattering is relatively strong in the
first order m=1 and much weaker for the second and higher order
scattering peaks. Using the red light from a HeNe laser,
.lambda.=6328 angstroms, and with the incident light normal to the
surface, a strong first order Bragg spot of light is achieved in
air at .theta..sub.s .congruent.19.5.degree..
The interference grating 40 as uniformly coated with antibody 44,
is therefore used to detect quantitatively the amount of antigen
which becomes bound to the corrogated or grooved surface. Before
the antigen becomes bound, a set of Bragg scattered intensities may
be measured with the angles .theta..sub.s given by equation (5)
since the physical periodic grating is in contact with the assay
solution 14. Because the measurement of interest is the increase in
one or more of the Bragg scattered intensities due to the addition
of antigen to the grating surface, the amount of scattering which
occurs initially or before antigen binding has occurred should be
minimized.
To achieve this minimum, the index of refraction of the plastic, or
other material forming the interference grating, is closely matched
to the index of refraction of the assay solution. If this is
accomplished then there will be relatively little initial Bragg
scattering. This is because the assay solution fills in the grooved
structure 42 of the grating. This approximately index matches the
grooves 42 to the assay solution so that the grating tends to
disappear. The actual levels of the resulting scattered intensities
at the angles .theta..sub.s depend on the closeness of the match of
the indices of refraction of the solution and the grating material
as well as the polarizability of the antibody coating. In addition,
the coating itself may serve to some extent as an index-matching
layer.
It also may be desirable to add additional chemical compounds, such
as sucrose, ethylene glycol etc, to the assay solution to aid in
the index-matching. These compounds can be added if they do not
adversely affect the antigen-antibody binding reaction. The
addition of these compounds will increase the index of refraction
of the solution thereby promoting a closer match to that of the
antibody-coated grating material. This is because the solution is
primarily water and has an index of refraction of approximately
1.33. The typical plastics would have a higher index of refraction
since most plastics have indices of refraction in the range 1.40 to
1.50.
As an alternative to trying to increase the index of refraction of
the solution, the grating may be fabricated using a plastic polymer
material which has an index of refraction more closely matched to
that of the assay solution. In particular the polymers (e.g.
polymethyl methyacrylates) that are used to construct soft contact
lenses have indices of refraction more closely matched to water.
Additionally, polyacrylamide gel may be used to construct a grating
since typically 90 to 95% of the gel consists of trapped water. The
starting solution for the gel, before the gel is cross linked, is
poured onto a master grating. The master is made of a hard material
such as metal or glass so that the master grating acts as a mold.
After polymerization, the slab of gel is lifted away from the
master grating to yield an accurate impression of the grating.
Antibody molecules may then be covalently bonded to the corrogated
surface of the gel.
Once the basic structure such as shown in FIG. 3b is produced, then
the scattered intensities, which are measured at one or more of the
Bragg angles .theta..sub.s, increase with increased binding of
antigen to the antibody coated surface. This is accomplished since
the addition of antigen at each point on the surface in general
results in increased scattering due to a further mismatch in the
polarizabilities of the grating surface with the antibody coating
and the surrounding solution. Essentially, the addition of the
antigen provides for a poor index matching of the grating to the
solution which results in an increase in scattering. As an
alternative to the above, the addition of the antigen may provide a
decrease in the Bragg scattering intensities if the antigen
provides for a better index matching of the grating to the
solution. This would also be an acceptable type of measurement
since the change of intensities for the Bragg scattering, whether
positive or negative, may form a reliable indication of the
antigen-antibody binding.
Since the change in the Bragg scattered intensities, due to the
binding of antigen alone to the antibody coated grating surface, is
fairly small, this change can be intensified through the use of a
labeled antigen or antibody as described above. The label consists
of additional polarizable material which greatly increases the
scattering due to the binding of a single antigen molecule to the
grating surface. One particular structure, as described above, is
the use of tiny carrier particles such as collodial gold particles
preferably of a dimension from 5 to 20 mn to which are absorbed or
otherwise attached the antigen or antibody molecules.
Specifically, antigen coated collodial gold particles of a known
concentration may be used in a competitive mode assay as the tiny
gold particles leave the surrounding assay solution and become
attached to the grating surface. The grating surface thereby
produces a dramatic increase of its Bragg scattering efficiency.
This occurs when the carrier particles are much smaller than the
characteristic dimensions of the grooves, which make up the grating
surface, so that the deposition of these particles on the surface
resembles a fine metallic coating as shown by the particles 46 in
FIG. 3c. It is well known that an interference grating which has
been coated with a thin metallic coating, such as by vacuum
deposition, produces far stronger Bragg scattering than does a
grating of transparent plastic. Therefore if the antibody coated
grating is initially well indexed matched to the assay solution,
the increase in Bragg scattered intensity at a given angle
.theta..sub.s, due to random deposition of individual metal
particles on the surface, is very pronounced. Non metallic carrier
particles such as polystyrene beads may also be used to enchance
the scattering efficiency of the grating.
The apparatus of the present invention may also have the
sensitivity of the assay enhanced by using particular detection
techniques. For example, in some applications it may be necessary
to detect the Bragg scattered light at very low intensity levels.
In order to provide for the detection, the technique of lock-in or
phase-sensitive detection may be employed. This technique can be
utilized in two ways. In the first place, if the Bragg scattered
light intensity is weak relative to a background light level
unrelated to the laser source, the incident laser radiation may be
periodically chopped or otherwise modulated and a lock-in amplifier
used on the detector at angle .theta..sub.s to separate the desired
intensity signal at angle .theta..sub.s from the ambient light
level. In the second place, the phase-sensitive technique may be
used to advantage when the Bragg scattered intensity at angle
.theta..sub.s is very weak relative to the surrounding scattered
light intensity (i.e. at adjacent angles) which occurs over a broad
range of angles. Such as relatively high level of scattered
intensity may be caused by grating imperfections and background or
from scattering from molecules, dirt, etc. in the sample solution
(which scattering may or may not be isotropic). In this case it may
be desirable to achieve a periodic scan in detection angle
.theta..sub.s within a small range of angles encompassing the
desired angle .theta..sub.s and use phase-sensitive detection to
extract the desired intensity signal due to Bragg scattering. This
may be achieved by translating the detector in periodic fashion so
as to sample a range of angles .theta.. Alternatively, the detector
may be kept stationary and the grating substrate/solution layer
rocked, or rotated, back and forth slightly to cause the desire
Bragg scattering spot to sweep back and forth past the
detector.
It should also be noted that a multi-element detector array may be
used to advantage in conjunction with this invention. Using such an
array one may simultaneously detect scattered light over a wide
range of angles .theta. which encompass more than one of the Bragg
angles .theta..sub.s. Measurement of the scattered intensity at
several of the angles .theta..sub.s, i.e. as a function of the
interference order m, provides an internal cross-check on the Bragg
result and may be used to discriminate against background false
intensity signals and ultimately to improve the signal/noise ratio
and overall reliability of the measurement. Also, use of a high
resolution multi-element detector array has the additional
advantage that precise alignment of the detector with respect to
the grating orientation is no longer required. With many detectors
covering a range of angles .theta., by definition there is no
longer the possibility of misalignment of the detector with
resulting loss of the desired signal at angle .theta..sub.s.
As discussed above, a limitation on the sensitivity of this
immunoassay method is imposed by the existence of background light
at the preferential Bragg angle .theta..sub.s due to nonidealities
in the immunochemical grating as well as scattering by molecules
and particulates in the sample solution. An additional method of
enhancing the signal/noise ratio of the measurement is to determine
the initial rate of the binding reaction. This is, the time rate of
change of the detected intensity at angle .theta..sub.s is
determined early in the course of the binding reaction. Ideally,
this should result in a measured quantity which is relatively
insensitive to the background intensity, which is presumed to be
approximately constant over the time course of the rate
determination. This procedure possesses the additional advantage
that it produces a value relatively quickly--i.e. it is not
necessary to wait for the binding reaction to go to essential
completion. Such a measurement of the initial rate, or velocity, of
binding to the grating surface ideally discriminates against all
sources of background light, both associated with grating
imperfections and solution scattering as well as ambient light
levels unrelated to the laser radiation.
One of the advantages of our invention is that the useful signal is
produced by light scattering from the entire illuminated region,
thus causing an interference peak due to the periodicity of
preferential scattering sites which exist over that region.
Therefore, small random defects in the pattern of scattering sites
do not contribute significantly to the signal. This is quite
different from our previous invention, copending application Ser.
No. 463,658, which utilizes the principle of synchronous detection
of a spatial pattern, in which small defects in the pattern--e.g.
bright fluorescent impurity spots on the pattern--would contribute
to the apparent fluorescent signal. In the present invention,
random, isolated defects such as dirt specks or scratches on the
pattern surface result in scattering in all directions, but will
not in general cause the scattering to be concentrated along any of
the preferential Bragg angles. The resulting background intensity
which occurs at any Bragg angle will be small compared to the
scattered intensity caused by the periodic array.
This result carries an important implication for the requirements
of the production of these periodic arrays of antibody stripes.
That is, the individual stripes which comprise the periodic array
do not need to be manufactured to precise dimensional tolerances.
For example, discontinuities in individual stripes or blurred,
non-uniform edges have little effect on the quality of the Bragg
scattering signal (i.e. on the intensity of the Bragg spot). What
does matter, of course, is that the stripe-to-stripe spacing (i.e.
the periodicity) be maintained over the long range of the
illuminated region of the surface Hence, for example the
dimensional tolerances required for the antibody stripe pattern are
not nearly as stringent as those required for typical electronic
integrated circuits.
We have disclosed the possibility of using two different kinds of
periodic arrays for an immunoassay. One array consisted of
alternating stripes of specific, active antibody molecules and
denatured antibody molecules (in which the molecules are made
inactive by an intense beam of ultraviolet light or ionizing
particles, such as electrons) on a flat surface. The other
consisted of specific antibody attached (e.g. by adsorption)
uniformly to the surface of an actual physical grating. It is
important to point out that these two kinds of grating surfaces act
differently with respect to the problem of nonspecific binding of
tagged molecules to the surface. In both cases it is assumed that
the tagged molecules bind to the surface at random locations. On
the flat surface, in which the "grating" exists only by virtue of
the pattern of active antibody stripes, these particles yield
random scattering in all directions and, therefore, do not
contribute significantly to the intensity of the Bragg peak.
On the physical grating surface, on the other hand, those particles
which bind nonspecifically give the same signal as those which bind
specifically to antibody. That is, the physical grating surface
does not discriminate against nonspecific binding. Hence, in any
assay in which the ultimate sensitivity is limited by non-specific
binding of tagged molecules to the surface, the flat surface with
alternating stripes of active antibody would be the preferable
embodiment.
As described previously, this invention utilizes the physical
principle of optical interference to detect antigen-antibody
binding on a surface. In the description thusfar, a particular kind
of scattering pattern was utilized, a periodic array which
resembles a diffraction grating. The grating consists of either a
parallel set of stripes of active antibody molecules attached to a
flat surface or an actual physical grating (i.e. periodically
ridged surface) which is everywhere coated with active antibody. In
both cases one obtains an interference grating which produces
relatively strong scattering intensities along certain preferential
directions, corresponding to the Bragg scattering angles. The
intensity of each of the Bragg peaks, or "spots", increases with
increasing efficiency of scattering due to additional binding of
tagged molecules to the surface-immobilized antibody.
However, it is important to appreciate that Bragg scattering from a
uniform, grating-like array of parallel, periodic scattering sites
represents only one approach by which optical interference can be
utilized to detect antigen-antibody binding at a surface. A more
general approach consists of using some arbitrary, fixed pattern of
active antibody sites on a surface. One then illuminates this
pattern with coherent light from a laser source at a given angle of
incidence in either the transmission or reflection mode. One can
then use a lens located some distance from the pattern surface to
produce an image a further distance away, on the so-called
"transform" plane. The resulting image on the transform plane,
consisting of some two-dimensional pattern of intensities (i.e.
regions of high intensity and low intensity) is, in fact, the
spatial Fourier transform of the original antibody-coated pattern
of scattering sites (i.e. on the "object" plane). In the previous
case, in which the starting antibody pattern consisted of a simple
array of parallel stripes of uniform spacing, the resulting spatial
Fourier transform is a set of discrete "spots" of light, lying
along a straight line in the transform plane, which are the Bragg
peaks corresponding to a given array periodicity. In the more
general case of an arbitrary starting pattern of antibody coating,
the resulting spatial Fourier transform intensity pattern will be
more complicated (and unique to a given starting pattern of
scattering sites on the object plane).
As an example, the pattern might consist of two sets of parallel,
periodic stripes with one set of stripes intersecting normally to
the other set, thereby creating a rectilinear array resembling a
woven fabric. In effect, the pattern consists of two
perpendicularly-crossed gratings. The resulting intensity pattern
on the transform plane consist of two sets of Bragg scattering
spots. Each set of spots lies along a line which is perpendicular
to the long direction of the set of stripes from which it derives.
The two linear arrays of spots are therefore mutually
perpendicular, forming a cross-like pattern of spots, in which the
spot intensity decreases with increasing distance from the center
of the array (i.e. with increasing order of Bragg interference).
The spacing of the spots which form each linear segment of the
array is determined by the periodicity of the corresponding set of
stripes and is given by the Bragg scattering relation (discussed
previously) and the location of the transform plane.
The point to recognize is that this more complicated pattern of
scattered intensities can as well be detected by using more than
one detector, located at several points on the transform plane
where intensity maxima are expected to occur, or by using a mask
which is designed to "recognize" the expected Fourier transform
pattern and to pass some portion of the intensity distribution to a
single detector. Hence, by using some combination of masks and/or
detectors, one can detect changes in the scattered intensity
pattern which occur as a result of changes in the degree of binding
of tagged molecules to an arbitrary pattern of antibody molecules
attached to a surface. The simple array of periodic, parallel
stripes is our preferred embodiment because of its simplicity.
The embodiments of the present invention have been described with
the use of a coherent source of illuminating radiation such as a
laser. However, it should be appreciated that a source of light
having a relatively broad range of wavelengths may also be used.
This is because the grating, having a given periodicity d, uniquely
disperses the incident radiation into the scattered rays of
different angles, .theta.hd s, corresponding to the differing
wavelengths from the source of light and with each different angle,
.theta..sub.s, in accordance with the equations (4) and (5).
Therefore, one or more detectors located at different angles
.theta..sub.s (or a multi-element detector array) to used to
collect information over the entire dispersed spectrum over a
relatively large range of angles. As the grating scatters more
efficiently with an increase in antigen-antibody binding at the
surface, the intensities over the entire spectrum increase. It is
to be appreciated however, that it is simpler to provide for the
detection using a laser source because in that case all of the
Bragg scattering is concentrated into a small number of spots or
angular positions.
For all of the embodiments of the immunoassay technique described
herein, the intensity of a Bragg scattering maximum (i.e. peak)
located at a particular preferential Bragg angle .theta..sub.s will
in general increase with increased extent of antigen-antibody
binding at the grating surface. That is, the greater the amount of
mass bound to the substrate surface, the greater the Bragg
scattered intensity. However, this relationship between the Bragg
scattering intensity and the amount of antigen (or antibody) bound
to the immunochemical grating will in general not be linear,
because the scattering involves coherent radiation. Therefore the
immunoassay instrument which is designed in accordance with the
method described herein must ultimately be calibrated using
standard samples of antigen (or antibody) of known concentration.
The need to construct such standard calibration curves is a
requirement common to all existing immunoassay methods.
It should be appreciated that only a relatively small sample
solution volume is needed for the normal functioning of this
immunoassay technique. The thickness of the layer of solution in
contact with the grating surface should ideally be kept as thin as
possible, in order to limit the time needed for the molecules
comprising the analyte, plus possibly other reagents, to diffuse to
the vicinity of the grating surface, thereby permitting binding.
Also, the dimensions of the grating can be kept relatively small
(e.g. on the order of 1-10 mm), since only that portion of the
grating which is illuminated by the incident laser radiation can
contribute to the desired Bragg intensity signal. This feature of
small sample volume is generally useful, and is consistent with the
requirement that the method posses a high sensitivity. The fact
that the layer of solution can be quite thin means that samples
which are nominally quite turbid may be employed, permitting the
incident laser light and resulting Bragg scattered light to
traverse the liquid layer with relatively little attenuation.
It is useful to point out that the technology described herein
permits the design of an apparatus which allows more than one
immunoassay to be performed simultaneously on a single small volume
of sample solution. Several immunochemical gratings of different
periodicities, each prepared in accordance with previous
suggestions and containing stripes of a different specific antibody
(let us say), can be located in close proximity on a single
substrate surface, with the entire set of arrays in contact with
the sample solution and all gratings illuminated with the same
laser beam (perhaps expanded in size using suitable optics). In
this case several sets of Bragg "spots" of relatively high
intensity can be detected by a set of detectors (or a multi-element
detector array, as described previously) suitably located at the
angles .theta..sub.s1, .theta..sub.s2, .theta..sub.s3, etc. In this
way one can, in principle simultaneously follow the time course of
binding, and thereby determine the concentration of, more than one
type of molecule contained in the sample solution.
It can be seen therefore that the present invention is directed to
an apparatus and method for providing an immunoassay using optical
interference, and specifically by measuring the binding of the
antigen molecules to antibody molecules at preferential specified
locations on an assay surface, and with these preferential
specified locations forming a spatial array which when illuminated
by incident radiation provide for scattering of the incident
radiation along a particular angular direction characterized as
Bragg scattering. The intensity of the Bragg scattered radiation is
then detected and with the level of the detected Bragg scattered
radiation detected either at a particular point in time or with
changes in the intensity of the detected Bragg scattered radiation
detected over a period of time so as to provide for a measure of
the immunoassay. The spatial array of antigen-antibody binding
locations forms an immunochemical grating and with such grating
having a number of different embodiments as disclosed in the
present invention.
It is useful to summarize those essential features, discussed
earlier, which define this invention. One utilizes a chemically
active, predetermined pattern which is attached to a surface. This
pattern consists of predetermined regions, or areas, of active
ligand or antiligand molecules (e.g. antigen or antibody)
surrounded by regions, or areas, which contain no such material, or
ligand or antiligand which has been rendered chemically inactive
(i.e. with respect to its ability to bind the complement molecule).
The physical basis for the invention is the phenomenon of optical
interference. Optical radiation, preferably of nearly a single
wavelength, is directed onto the immuno-chemical pattern-bearing
surface. The light is scattered in general from all points on the
surface, with the efficiency of scattering varying from one point
to the next due to the spatial modulation of the optical properties
of the surface (e.g. absorbance, scattering, transmission, etc.)
caused by ligand-antiligand binding reactions which occur on the
pattern surface. These reactions may occur in conjunction with
reactions which also occur in the surrounding solution containing
the analyte. The individual scattered light waves undergo optical
interference at all points in the surrounding space, resulting in
general in an enhancement of the scattered light intensity at one
or more locations and/or angles in space, provided that a
particularly effective pattern design is chosen. The pattern need
not be periodic; the preferred embodiment of a Bragg interference
grating discussed at length herein, was chosen for convenience. One
or more optical detectors can then be used at one or more of these
points or angles to detect the enhanced intensity(ies), and thereby
deduce the extent of ligand-antiligand binding which has
occurred.
Although the application has been described with reference to
particular embodiments, it is to be appreciated that various
adaptations and modifications may be made and the invention is only
to be limited by the appended claims.
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