U.S. patent application number 10/235127 was filed with the patent office on 2003-01-09 for methods and apparatus for improved luminescence assays.
This patent application is currently assigned to Igen, Inc.. Invention is credited to Blackburn, Gary F., J. Massey, Richard, P. Shah, Haresh, Wilkins, Elizabeth W..
Application Number | 20030008339 10/235127 |
Document ID | / |
Family ID | 46252829 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008339 |
Kind Code |
A1 |
J. Massey, Richard ; et
al. |
January 9, 2003 |
Methods and apparatus for improved luminescence assays
Abstract
What is described are methods and apparatus for performing a
binding assay for an analyte of interest present in a sample. The
methods include the steps of: forming a composition containing the
sample, an assay-performance-substance which contains a component
linked to a label compound capable of chemiluminescing when
triggered, and a plurality of particles capable of specifically
binding with the analyte and/or the assay-performance-substance;
incubating the composition to form a complex which includes a
particle and the labeled component; collecting the complex in a
collection zone; introducing into the collection zone a trigger
capable of triggering the label such that the label luminesces; and
measuring the emitted luminescence to measure the presence of the
analyte of interest in the sample.
Inventors: |
J. Massey, Richard;
(Rockville, MD) ; Blackburn, Gary F.;
(Gaithersburg, MD) ; Wilkins, Elizabeth W.;
(Germantown, MD) ; P. Shah, Haresh; (Gaithersburg,
MD) |
Correspondence
Address: |
Barry Evans, Esq.
Kramer Levin Naftalis & Frankel LLP
919 Third Avenue
New York
NY
10022
US
|
Assignee: |
Igen, Inc.
|
Family ID: |
46252829 |
Appl. No.: |
10/235127 |
Filed: |
September 5, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10235127 |
Sep 5, 2002 |
|
|
|
08335183 |
Nov 7, 1994 |
|
|
|
6448091 |
|
|
|
|
08335183 |
Nov 7, 1994 |
|
|
|
07652427 |
Feb 6, 1991 |
|
|
|
07652427 |
Feb 6, 1991 |
|
|
|
07539389 |
Jun 18, 1990 |
|
|
|
07539389 |
Jun 18, 1990 |
|
|
|
07266882 |
Nov 3, 1988 |
|
|
|
10235127 |
Sep 5, 2002 |
|
|
|
08335183 |
Nov 7, 1994 |
|
|
|
6448091 |
|
|
|
|
08335183 |
Nov 7, 1994 |
|
|
|
07539389 |
Jun 18, 1990 |
|
|
|
07539389 |
Jun 18, 1990 |
|
|
|
07266882 |
Nov 3, 1988 |
|
|
|
Current U.S.
Class: |
435/14 |
Current CPC
Class: |
C12Q 1/6816 20130101;
G01N 33/54366 20130101; G01N 33/582 20130101; G01N 33/54313
20130101; C12Q 1/6825 20130101; G01N 33/54326 20130101; C12Q
2563/113 20130101; C12Q 2531/107 20130101; C12Q 2565/628 20130101;
C12Q 2563/113 20130101; C12Q 2563/131 20130101; G01N 21/66
20130101; C12Q 1/6825 20130101; C12Q 1/686 20130101; G01N 21/76
20130101; C12Q 2563/103 20130101; C12Q 2565/628 20130101; C12Q
2563/103 20130101; C12Q 2563/103 20130101; C12Q 2565/628 20130101;
C12Q 2563/103 20130101; C12Q 2563/113 20130101; C12Q 2563/103
20130101; G01N 33/5438 20130101; C12Q 1/686 20130101; G01N 1/40
20130101; C07H 21/00 20130101; C12Q 1/686 20130101; C12Q 1/6825
20130101; C12Q 1/6816 20130101; G01N 21/69 20130101; G01N 2458/30
20130101 |
Class at
Publication: |
435/14 |
International
Class: |
C12Q 001/54 |
Claims
What is claimed is:
1. A method for performing a binding assay for an analyte of
interest present in a sample comprising the steps of: (a) forming a
composition containing (i) said sample (ii) an
assay-performance-substance which contains a component linked to a
label compound capable of chemiluminescing when triggered, and
(iii) a plurality of particles capable of specifically binding with
the analyte and/or said assay-performance-substance; (b) incubating
said composition to form a complex which includes a particle and
said labeled component; (c) collecting said complex in a collection
zone; (d) introducing into said collection zone a trigger capable
of triggering said label such that said label luminesces; and (e)
measuring the emitted luminescence to measure the presence of the
analyte of interest in the sample.
2. A method as recited in claim 1 wherein said trigger is an
oxidant capable of oxidizing said label.
3. A method as recited in claim 1 wherein said particles are
magnetically responsive and said complex is magnetically collected
in said collection zone.
4. A method as recited in claim 2 wherein said oxidant is hydrogen
peroxide or superoxide.
5. A method as recited in claim 1 wherein said
assay-performance-substance further contains an enzyme for
converting a precursor to an oxidant capable of oxidizing said
label, and said trigger is the precursor.
6. A method as recited in claim 5 wherein the enzyme is glucose
oxidase, the precursor is glucose and the oxidant is hydrogen
peroxide.
7. A method as recited in claim 1 wherein said particles further
contain an enzyme for converting a precursor to an oxidant capable
of oxidizing said label, and said trigger is the precursor.
8. A method as recited in claim 7 wherein the enzyme is glucose
oxidase, the precursor is glucose and the oxidant is hydrogen
peroxide.
9. A method as recited in claim 1 conducted as a batch process, the
composition being permitted to reside within said cell for a time
sufficient to permit collection of said particles.
10. A method as recited in claim 1 conducted as a flow process
wherein said composition is flowed through said cell at a
sufficiently low rate to permit collection of at least a portion of
said particles.
11. An assay method as recited in claim 7 wherein said particles
have a density of from 0.1 to 5 g/mL.
12. An assay method as recited in claim 11 wherein said particles
have a density of from 0.5 to 2 g/mL.
13. An assay method as recited in claim 7 wherein the size of said
particles, measured as the mean diameter, ranges from 0.001 to 100
.mu.m.
14. An assay method as recited in claim 13 wherein the size of said
particles ranges from 0.01 to 10 .mu.m
15. An assay method as recited in claim 7 wherein the concentration
of particles in said composition is from 1 to 10,000 .mu.g/mL.
16. An assay method as recited in claim 15 wherein said
concentration of particles is in the range of from 5 to 1000
.mu.g/mL.
17. An assay method as recited in claim 7 wherein said particles
have a magnetic susceptibility of at least 0.001 cgs units.
18. A method as recited in claim 17 wherein the magnetic
susceptibility is at least 0.01 cgs units.
19. An assay method as recited in claim 7 wherein the magnetic
susceptibility, density, size and concentration of said particles
in said composition is such that the settling rate of said
particles is at least 0.5 mm/mim.
20. An apparatus for performing a binding assay for an analyte of
interest present in a sample based upon measurement of
chemiluminescence comprising: (a) a cell defining a sample
containing volume having a vertical columnar zone and having inlet
and outlet means, and further including means for generating a
magnetic field positioned below a substantial volume of said cell
and said columnar zone; and (b) means to measure the
chemiluminescence generated at a collection zone.
21. An apparatus as recited in claim 20 wherein said means for
generating a magnetic field includes a plurality of magnets in
north-south orientation and separated by nonmagnetic material.
Description
[0001] This application is a continuation-in-part of copending
Massey et al. application Ser. No. 07/652,427 filed Feb. 6, 1991,
which is a continuation-in-part of application Ser. No. 07/539,389
filed Jun. 18, 1990, which is a continuation of application Ser.
No. 07/266,882 filed Nov. 3, 1988, now abandoned, entitled
Electrochemiluminescent Assays, and this application is a
continuation-in-part of copending application Ser. No. 07/539,389
filed Jun. 18, 1990, which is a continuation of application Ser.
No. 07/266,882 filed Nov. 3, 1988, now abandoned, entitled
Electrochemiluminescent Assays. The subject matter of these
applications is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This application relates generally to methods and apparatus
for conducting binding assays, more particularly to those which
measure the presence of an analyte of interest by measuring
luminescence emitted by one or more labeled components of the assay
system. More specifically, the invention relates to precise,
reproducible, accurate homogeneous or heterogeneous specific
binding assays of improved sensitivity in which the luminescent
component is concentrated in the assay composition and collected
before being caused to chemiluminescence.
BACKGROUND OF THE INVENTION
[0003] Numerous methods and systems have been developed for the
detection and quantitation of analytes of interest in biochemical
and biological substances. Methods and systems which are capable of
measuring trace amounts of microorganisms, pharmaceuticals,
hormones, viruses, antibodies, nucleic acids and other proteins are
of great value to researchers and clinicians.
[0004] A very substantial body of art has been developed based upon
the well known binding reactions, e.g., antigen-antibody reactions,
nucleic acid hybridization techniques, and protein-ligand systems.
The high degree of specificity in many biochemical and biological
binding systems has led to many assay methods and systems of value
in research and diagnostics. Typically, the existence of an analyte
of interest is indicated by the presence or absence of an
observable "label" attached to one or more of the binding
materials. Of particular interest are labels which can be made to
luminesce through photochemical, chemical, and electrochemical
means. "Photoluminescence" is the process whereby a material is
induced to luminesce when it absorbs electromagnetic radiation.
Fluorescence and phosphorescence are types of photoluminescence.
"Chemiluminescent" processes entail the creation of luminescent
species by chemical transfer of energy. "Electrochemiluminescence"
entails creation of luminescent species electrochemically.
[0005] Chemiluminescent assay techniques where a sample containing
an analyte of interest is mixed with a reactant labeled with a
chemiluminescent label have been developed. The reactive mixture is
incubated and some portion of the labeled reactant binds to the
analyte. After incubation, the bound and unbound fractions of the
mixture are separated and the concentration of the label in either
or both fractions can be determined by chemiluminescent techniques.
The level of chemiluminescence determined in one or both fractions
indicates the amount of analyte of interest in the biological
sample.
[0006] It is desirable to carry out chemiluminescent assays without
the need for a separation step during the assay procedure and to
maximize the signal modulation at different concentrations of
analyte so that precise and sensitive measurements can be made.
[0007] Among prior art methods for separation assays are those such
as described in U.S. Pat. No. 4,141,687 and European Patent
Application 0,030,087 which relate to magnetically separating
particles in a conduit after which the particles are removed to a
separate chamber for analysis of the label.
[0008] Among prior art methods for nonseparation assays are those
which employ microparticulate matter suspended in the assay sample
to bind one or more of the binding components of the assay.
[0009] U.S. Pat. No. 4,305,925 relates to the detection and
determination of clinically relevant proteins and peptides by means
of nephelometric and turbidimetric methods. The methods disclosed
involve binding the antigen or antibody to latex particles which
perform the function of light scattering or adsorption.
[0010] U.S. Pat. No. 4,480,042 relates to techniques employing
particle reagents consisting of shell-core particles. The shell
contains functional groups to which compounds of biological
interest can be covalently bonded, and the high refractive index of
the core results in high sensitivity to light scattering
measurements. The technique is based upon agglutination reactions
which result from the reaction of bivalent antibodies with
multivalent antigens of interest to produce aggregates which can be
detected and/or measured in various ways.
[0011] U.S. Pat. No. 4,419,453 likewise relates to the use of
colored latex agglutination test methods useful for detecting the
presence of immunochemicals such as antibodies and immunogens.
[0012] Based upon this prior art, it would not have appeared
possible to use microparticulate matter in assays wherein a
luminescent phenomenon is measured. One would expect that the
luminescence from free chemiluminescent moieties would be absorbed,
scattered, or otherwise suffer interference from the
microparticulate matter.
[0013] Contrary to that expectation, U.S. Application Serial No.
266,882 (PCT published application U.S. 89/04919) teaches
sensitive, specific binding assay methods based on a luminescent
phenomenon wherein inert microparticulate matter is specifically
bound to one of the binding reactants of the assay system. The
assays may be performed in a heterogeneous (one or more separation
steps) assay format and may be used most advantageously in a
homogeneous (nonseparation) assay format.
[0014] U.S. 89/04919 relates to a composition for an assay based
upon a binding reaction for the measurement of luminescent
phenomenon, which composition includes a plurality of suspended
particles having a surface capable of binding to a component of the
assay mixture. In another aspect, it is directed to a system for
detecting or quantitating an analyte of interest in a sample, which
system is capable of conducting the assay methods using the assay
compositions of the inventions. The system includes means for
inducing the label compound in the assay medium to luminesce, and
means for measuring the luminescence to detect the presence of the
analyte of interest in the sample.
[0015] Thus, U.S. 89/04919 is directed to methods for the detection
of an analyte of interest in a sample, which method includes the
steps of (1) forming a composition comprising (a) a sample
suspected of containing an analyte of interest, (b) an
assay-performance-substance selected from the group consisting of
(i) analyte of interest or analog of the analyte of interest, (ii)
a binding partner of the analyte of interest or its said analog,
and (iii) a reactive component capable of binding with (i) or (ii),
wherein one of said substances is linked to a label compound having
a chemical moiety capable of being induced to luminesce, and (c) a
plurality of suspended particles capable of specifically binding
with the analyte and/or a substance defined in (b)(i), (ii), or
(iii); (2) incubating the composition to form a complex which
includes a particle and said label compound; (3) inducing the label
compound to luminesce; and (4) measuring the luminescence emitted
by the composition to detect the presence of the analyte of
interest in the sample. Those same methods may be used to quantify
the amount of analyte in a sample by comparing the luminescence of
the assay composition to the luminescence of a composition
containing a known amount of analyte.
[0016] Analogs of the analyte of interest, which may be natural or
synthetic, are compounds which have binding properties comparable
to the analyte, but include compounds of higher or lower binding
capability as well. Binding partners suitable for use in the
present invention are well-known. Examples are antibodies, enzymes,
nucleic acids, lectins, cofactors and receptors. The reactive
components capable of binding with the analyte or its analog and/or
with a binding partner thereof may be a second antibody or a
protein such as Protein A or Protein G or may be avidin or biotin
or another component known in the art to enter into binding
reactions.
[0017] Advantageously, the luminescence arises from
electrochemiluminescence (ECL) induced by exposing the label
compound, whether bound or unbound to specific binding partners, to
a voltametric working electrode. The ECL reactive mixture is
controllably triggered to emit light by a voltage impressed on the
working electrode at a particular time and in a particular manner
to generate light. Although the emission of visible light is an
advantageous feature the composition or system may emit other types
of electromagnetic radiation, such as infrared or ultraviolet
light, X-rays, microwaves, etc. Use of the terms
"electrochemiluminescence," "electrochemiluminescent,
"luminescence," "luminescent," and "luminesce" includes the
emission of light and other forms of electromagnetic radiation.
[0018] The methods taught in U.S. Ser. No. 89/04919 permit the
detection and quantitation of extremely small quantities of
analytes in a variety of assays performed in research and clinical
settings. The demands of researchers and clinicians makes it
imperative, however, to lower the detection limits of assays
performed by these methods to increase the sensitivities of those
assays and to increase the speed at which they can be
performed.
[0019] Various methods are known in the art for increasing the
signal from labeled species by concentrating them before subjecting
them to a measurement step. In U.S. Pat. No. 4,652,333, for
example, particles labeled with fluorescent, phosphorescent or
atomic fluorescent labels are concentrated by microfiltration
before a measurement step is performed.
[0020] It is also known in the art to concentrate labeled
immunochemical species prior to a measurement step, by, e.g.,
drawing magnetically responsive labeled particles to the surface of
a measurement vessel. In U.S. Pat. Nos. 4,731,337, 4,777,145, and
4,115,535, for example, such particles are drawn to the vessel wall
and then are irradiated to excite a fluorophoric emission of
light.
[0021] In U.S. Pat. No. 4,945,045, particles are concentrated on a
magnetic electrode. An electrochemical reaction takes place at the
electrode facilitated by a labeled chemical mediator. The
immunochemical binding reaction alters the efficiency of the
mediator resulting in a modulated signal when binding takes
place.
OBJECTS OF THE INVENTION
[0022] It is therefore a primary object of this invention to
provide homogeneous (non-separation) and heterogeneous (separation)
methods, reagents and apparatus, for the conduct of binding
assays.
[0023] It is a further object of this invention to provide
non-separation, specific binding assays, reagents and apparatus,
based upon the measurement of chemiluminescence emitted from an
assay composition containing microparticulate matter.
[0024] It is a further and related object to provide such assays,
reagents and apparatus having improved sensitivity, faster assay
time, greater sensitivity, lower detection limits and greater
precision than has heretofore been achieved.
[0025] DESCRIPTION OF THE INVENTION
Definition of Terms
[0026] Chemiluminescence is defined as a luminescence reaction in
which the energy responsible for generating the high-energy excited
state of a molecule is derived from an energetic chemical reaction.
A chemiluminescent reaction thus involves the direct conversion of
chemical energy to electromagnetic radiation (ultraviolet, visible,
or infrared radiation). Luminescence occurs when the excited-state
molecule returns to its ground-state energy level, emitting a
photon having a particular wavelength which is characteristic of
the molecule and the energy of its excited state relative to its
ground-state.
[0027] Energy is generated by many chemical reactions; such
reactions are called exothermic reactions. In most cases the energy
appears as heat and induces vibrational, rotational, and
translational energy in the molecule. In a chemiluminescence
reaction at least part of this energy is channeled into the
formation in the high-energy excited state. This generally requires
a highly energetic and rapid reaction of two molecules, one of
which is capable of luminescence emission:
A+B.fwdarw.C*+D C*.fwdarw.C+hV
[0028] The quantity hV represents a photon of electromagnetic
radiation. h is Planck's constant and V is the frequency of the
emitted light.
[0029] In some chemiluminescent reactions the electronic energy of
the excited-state molecule C* is transferred to another
molecule,
C+E.fwdarw.C+E*
[0030] which then decays to its ground-state by emitting a photon
of electromagnetic radiation,
E*=E+hV.
[0031] Specific binding assays, e.g. immunoassays, using
chemiluminescent detection use one of the reactants as a label
attached to one of the binding partners. In such assays, the
reactants are generally called the label and the trigger and react
according to the equation:
Label+Trigger.fwdarw.Label*+By-products
Label*.fwdarw.By-products+hV
[0032] Examples of chemiluminescent labels which have been used in
specific binding assays include acridinium esters, luminol,
isoluminol, oxalate esters, dioxetanes, and luciferin. In many
cases, the trigger molecule is an oxidant such as hydrogen peroxide
which is capable of oxidizing the label in a highly energetic
reaction which is capable of generating the excited state of the
label.
[0033] Enhancer molecules are sometimes used in chemiluminescent
reactions as a means of increasing the efficiency of the
chemiluminescence process. Such molecules generally slow the
reaction rate of the reaction and increase the quantum yield of the
light emission.
[0034] Chemiluminescent binding assays have also been demonstrated
in which an enzyme is used as the label. In these cases, the enzyme
catalyzes the chemiluminescent reaction in the presence of a
trigger solution. An example is the use of the enzyme horseradish
peroxidase to catalyze the chemiluminescent reaction of luminol in
the presence of hydrogen peroxide and hydroxide ion.
[0035] The term "chemiluminescent moiety," "label," "label
compound," and "label substance," are used interchangeably. It is
within the scope of the invention for the species termed
"chemiluminescent moiety," "label compound," "label substance" and
"label" to be linked to molecules such as an analyte or an analog
thereof, a binding partner of the analyte or an analog thereof, and
further binding partners of such aforementioned binding partner, or
a reactive component capable of binding with the analyte, an analog
thereof or a binding partner as mentioned above. The
above-mentioned species can also be linked to a combination of one
or more binding partners and/or one or more reactive components.
Additionally, the aforementioned species can also be linked to an
analyte or its analog bound to a binding partner, a reactive
component, or a combination of one or more binding partners and/or
one or more reactive components. It is also within the scope of the
invention for a plurality of the aforementioned species to be bound
directly, or through other molecules as discussed above, to an
analyte or its analog. For purposes of brevity, these ligands are
referred to as an assay-performance-substan- ce.
[0036] The terms detection and quantitation are referred to as
"measurement", it being understood that quantitation may require
preparation of reference compositions and calibrations.
[0037] The terms collection and concentration of complex may be
used interchangeably to describe the concentration of complex
within the assay composition and the collection of complex at,
e.g., a surface of a flow cell.
[0038] Advantageously, the luminescence arises from
chemiluminescence induced by exposing the label compound, whether
bound or unbound to specific binding partners, to a trigger capable
of triggering said label such that the label luminesces.
Preferably, the trigger is an oxidant capable of oxidizing said
label such that the label is oxidized and luminesces. The
chemiluminescent reactive mixture is controllably triggered to emit
light at a particular time and in a particular manner to generate
light. Although the emission of visible light is an advantageous
feature, the composition or system may emit other types of
electromagnetic radiation, such as infrared or ultraviolet light,
X-rays, microwaves, etc. Use of the terms "chemiluminescence" and
"chemiluminescent" includes the emission of light and other forms
of electromagnetic radiation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 is a schematic drawing of a cell including a
permanent magnet for performing the microparticulate-based
nonseparation and separation assays of the invention.
[0040] FIG. 2 is a schematic representation of a direct
incorporation PCR format using chemiluminescent labeled
oligonucleotides and biotin chemiluminescent labeled
oligonucleotides as primers.
[0041] FIG. 3 is a schematic representation of a normal PCR format
using a biotinylated primer to allow the generation of biotinylated
PCR and PRODUCT.
[0042] FIG. 4 is a schematic representation of an asymmetric PCR
assay format generating single-stranded biotinylated DNA for later
hybridization to chemiluminescent labeled oligonucleotides.
[0043] FIG. 5 is a schematic representation of a sedimentation
assay cell which employs an electromagnet to cause the complex to
settle in the collection zone surface.
[0044] FIG. 6 is a schematic representation of the lines of force
in the vicinity of the collection zone as a function of the
orientation of the magnet beneath the surface of the collection
zone.
[0045] FIG. 7 is a schematic representation of a rotary flow cell
wherein the complexes are deposited in the collection zone by
centrifugation.
BRIEF DESCRIPTIONS OF THE INVENTION
[0046] In its broadest embodiment, the invention is in a method for
performing a binding assay for an analyte of interest present in a
sample. The steps include:
[0047] (a) forming a composition containing
[0048] (i) said sample
[0049] (ii) an assay-performance-substance which contains a
component linked to a label compound capable of chemiluminescing
when triggered, and
[0050] (iii) a plurality of particles capable of specifically
binding with the analyte and/or said
assay-performance-substance;
[0051] (b) incubating said composition to form a complex which
includes a particle and said labeled component;
[0052] (c) collecting said complex in a collection zone;
[0053] (d) introducing into said collection zone a trigger capable
of triggering said label such that said label luminesces; and
[0054] (e) measuring the emitted luminescence to measure the
presence of the analyte of interest in the sample.
[0055] Preferably, the trigger is an oxidant capable of oxidizing
the label such that the label is oxidized and chemiluminesces. Also
preferably, the particles are magnetically responsive and the
complex is magnetically collected in the collection zone.
[0056] In another embodiment, the assay-performance-substance or
the particles further contain an enzyme for converting a precursor
to an oxidant capable of oxidizing the label, and the trigger is
the precursor.
[0057] While the invention is carried out by collecting the complex
in a measurement zone, i.e., on a surface at which it can be caused
to luminesce, the invention also embraces methods wherein the
complex is collected in a measurement zone and thereafter means are
brought to that zone or other steps taken to induce and measure
luminescence.
[0058] The collection of the complex may be carried out by several
different methods, including gravity settling, filtration,
centrifugation and magnetic attraction of magnetically responsive
particles which form part of the complex. The several embodiments
are described in further detail below.
[0059] While batch assays can be performed, continuous or
semi-continuous assays can be performed in flow cells. In a flow
cell, the solid-phase remains in the measurement cell while the
solution flows through and exits the cell. If the solid-phase
(e.g., particles) are more dense than water, i.e., have a density
greater than that of water, (more than 1.0 g/mL) the force of
gravity upon the particles causes them to fall to the bottom of the
cell. The cell can be constructed such that the particles settle to
the bottom as the fluid flows through the cell or the cell can be
constructed such that the majority of the sample is contained in
the cell in a columnar compartment above the collection zone.
Sufficient dwell time in the cell must be provided to permit the
particles to settle in the collection zone before inducing
chemiluminescence.
[0060] In another embodiment of the invention, the assay
composition containing suspended particles having a density greater
than the balance of the assay composition may be subjected to
centrifugation in order to remove the particles to a measurement
zone where they are subsequently brought into contact with, e.g., a
trigger to induce chemiluminescence.
[0061] In this embodiment, the measurement cell is provided with
means to rapidly rotate the sample and sample enclosure.
Centrifugal force causes the particles in the sample to move
outward from the axis of rotation of the sample enclosure and to
collect on the outer surface of the sample enclosure.
[0062] In a third embodiment, the particles may be removed by
filtration from the assay composition. In this embodiment the
particles need not have a density greater than the balance of the
assay composition. The invention, the particles are separated from
the solution and concentrated by drawing the solution through a
filter, e.g. pumping and collecting the particles on the surface of
the filter.
[0063] In a preferred embodiment, the suspended particles are
magnetically responsive, e.g. they may be paramagnetic or
ferromagnetic, and are collected in a measurement zone by
imposition of a magnetic field on the particles. The measurement
cell is equipped with a magnet. The magnetic field of the magnet
applies a force on the particles as they reside in a batch cell or
as they flow through a flow cell, causing them to separate from the
bulk of the solution onto the surface of the cell which is in
closest proximity to the magnet.
[0064] Several different heterogeneous and homogeneous formats for
binding assays can be implemented using the methods described above
to collect and concentrate the complex. In a heterogeneous binding
assay the complex is separated from the composition before
measuring luminescence from the label. In homogeneous assays, no
separation of the bound (to the solid phase) and unbound labeled
reagents is made.
[0065] In a homogeneous assay, when the complex is concentrated in
a collection zone, the measured signal from the label is much
greater than it would be in the absence of a concentration step.
The signal from the uncomplexed labeled reagents, in contrast, is
not changed. Hence, despite the presence of the uncomplexed labeled
reagents in the measurement cell, the signal from the collected
complex is stronger than in an assay without collection of complex.
The detection limit for the binding assay is, much improved as a
result of the collection procedure.
[0066] In a preferred embodiment of the invention, an in-situ
separation step is included in the homogeneous binding assay
procedure. After the assay composition, i.e., sample, assay
performance substance and particles have been pumped into the
measurement cell and the complex collected in a collection zone, a
second fluid is pumped through the cell which is free of label or
labeled reagents, thereby performing an in-situ wash or separation
of the complex from unbound components of the assay composition.
This assay procedure is technically a heterogeneous binding assay.
However, the ability to perform the separation inside the
measurement cell is advantageous in that it does not require
additional separation apparatus and the procedure is generally much
faster than external separation methods.
[0067] Heterogeneous binding assays are conducted using the
invention by mixing the components of the assay composition and
allowing them to react for a predetermined length of time. The
assay composition is then subjected to a separation step wherein
the solution is separated from the particles. Chemiluminescence is
then measured from either the complex or the solution. Measuring
the chemiluminescence from the complex after a concentration step
permits measurement of analyte with better accuracy and with a
lower detection limit than is possible without concentration.
DETAILED DESCRIPTION OF THE INVENTION
[0068] The invention, as well as other objects and features
thereof, will be understood more clearly and fully from the
following description of certain preferred embodiments.
[0069] The invention is broadly applicable to analytes of interest
which are capable of entering into binding reactions. These
reactions include, e.g., antigen-antibody, ligand receptor, DNA and
RNA interactions, and other known reactions. The invention relates
to different methods and assays for qualitatively and
quantitatively detecting the presence of such analytes of interest
in a multi-component sample.
[0070] The Samples
[0071] The sample which may contain the analyte of interest, which
may be in solid, emulsion, suspension, liquid, or gas form, may be
derived from, for example, cells and cell-derived products, water,
food, blood, serum, hair, sweat, urine, feces, tissue, saliva,
oils, organic solvents or air. The sample may further comprise, for
example, water, acetonitrile, dimethyl sulfoxide, dimethyl
formamide, n-methyl-pyrrolidone or alcohols or mixtures
thereof.
[0072] The Analytes
[0073] Typical analytes of interest are a whole cell or surface
antigen, subcellular particle, virus, prion, viroid, antibody,
antigen, hapten, fatty acid, nucleic acid, protein, lipoprotein,
polysaccharide, lipopolysaccharide, glycoprotein, peptide,
polypeptide, cellular metabolite, hormone, pharmacological agent,
synthetic organic molecule, organometallic molecule, tranquilizer,
barbiturate, alkaloid, steroid, vitamin, amino acid, sugar, lectin,
recombinant or derived protein, biotin, avidin, streptavidin, or
inorganic molecule present in the sample. Typically, the analyte of
interest is present at a concentration of 10.sup.-3 molar or less,
for example, as low as 10.sup.-12 molar or lower.
[0074] Assay-Performance-Substance
[0075] The assay-performance-substance which is combined with the
sample containing the analyte of interest contains at least one
substance selected from the group consisting of (i) added analyte
of interest or its analog, as defined above, (ii) a binding partner
of the analyte of interest or its said analog, and (iii) a reactive
component, as defined above, capable of binding with (i) or (ii),
wherein one of said substances is linked to a compound or moiety,
e.g. a chemiluminescent moiety capable of being induced to
luminesce. The labeled substance may be a whole cell or surface
antigen, a subcellular particle, virus, prion, viroid, antibody,
antigen, hapten, lipid, fatty acid, nucleic acid, polysaccharide,
protein, lipoprotein, lipopolysaccharide, glycoprotein, peptide,
polypeptide, cellular metabolite, hormone, pharmacological agent,
tranquilizer, barbiturate, alkaloid, steroid, vitamin, amino acid,
sugar, nonbiological polymer (preferably soluble), lectin,
recombinant or derived protein, synthetic organic molecule,
organometallic molecule, inorganic molecule, biotin, avidin or
streptavidin. In one embodiment, the reagent is a chemiluminescent
moiety conjugated to an antibody, antigen, nucleic acid, hapten,
small nucleotide sequence, oligomer, ligand, enzyme, or biotin,
avidin, streptavidin, Protein A, Protein G, or complexes thereof,
or other secondary binding partner capable of binding to a primary
binding partner through protein interactions.
[0076] Analogs of the analyte of interest, which can be natural or
synthetic, are typically compounds which have binding properties
comparable to the analyte, but can also be compounds of higher or
lower binding capability. The reactive components capable of
binding with the analyte or its analog, and/or with a binding
partner thereof, and through which the chemiluminescent moiety can
be linked to the analyte, is suitably a second antibody or a
protein such as Protein A or Protein G, or avidin or biotin or
another component known in the art to enter into binding
reactions.
[0077] The function of the chemiluminescent moieties is to emit
electromagnetic radiation as a result of introduction into the
reaction system of a trigger, particularly an oxidant. In order to
do this, they must be capable of being stimulated to an excited
energy state and also capable of emitting electromagnetic
radiation, such as a photon of light, upon descending from that
excited state.
[0078] The amount of chemiluminescent moiety incorporated in
accordance with the invention will vary from system to system.
Generally, the amount of such moiety utilized is that amount which
is effective to result in the emission of a detectable, and if
desired, quantitatable, emission of electromagnetic energy, from
the aforementioned composition or system. The detection and/or
quantitation of an analyte of interest is typically made from a
comparison of the luminescence from a sample containing an analyte
of interest and a chemiluminescent moiety to the luminescence
emitted by a calibration standard developed with known amounts of
the analyte of interest and chemiluminescent moiety. This assumes a
homogeneous format. In the heterogeneous mode, a separation as
discussed previously is carried out prior to chemiluminescent
analysis.
[0079] As can be appreciated by one of ordinary skill in the art,
the identity and amount of the chemiluminescent moiety will vary
from one system to another, depending upon prevailing conditions.
The appropriate chemiluminescent moiety, and sufficient amount
thereof to obtain the desired result, can be determined empirically
by those of ordinary skill in the art, once equipped with the
teachings herein, without undue experimentation.
[0080] The Particles
[0081] The particles advantageously comprise microparticulate
matter having a diameter of 0.05 um to 200 um, preferably 0.1 um to
100 um, most preferably 0.5 um to 10 um, and a surface component
capable of binding to the analyte and/or one or more of the other
substances defined in subparagraphs (a)(i), (a)(ii), or (a)(iii)
above. For example, the microparticulate matter may be crosslinked
starch, dextrans, cellulose, proteins, organic polymers, styrene
copolymer such as styrene/butadiene copolymer,
acrylonitrile/butadiene/styrene copolymer, vinylacetyl acrylate
copolymer, or vinyl chloride/acrylate copolymer, inert inorganic
particles, chromium dioxide, oxides of iron, silica, silica
mixtures, and proteinaceous matter, or mixtures thereof. Desirably,
the particles are suspended in the chemiluminescent system.
[0082] Apparatus for Measuring Chemiluminescence
[0083] An apparatus for carrying out the assays of the invention is
described in FIG. 1. FIG. 1 discloses an advantageous
chemiluminescent apparatus, but the methods of the present
invention are not limited to application in apparatus 10, but
rather may be employed in other types of chemiluminescent apparatus
which include a means for collecting a labeled component. While the
methods of the invention can be carried out in a static or
flow-through mode, apparatus 10 includes a flow-through cell, which
provides distinct advantages for many types of samples including
binding assay samples. Further details of apparatus for carrying
out the chemiluminescent assays of the invention are disclosed in
commonly assigned published PCT applications U.S. Ser. No. 89/04854
and U.S. Ser. No. 90/01370.
[0084] Apparatus 10 includes a measurement cell 12, a light
detection/measurement device 14, which may advantageously be a
photomultiplier tube (PMT), photodiode, charge coupled device,
photographic film or emulsion or the like, and a pump 16, which is
advantageously a peristaltic pump, to provide for fluid transport
to, through and from cell 12. Alternatively, a positive
displacement pump may be used. A shutter mechanism 18 is provided
between cell 12 and PMT 14 and is controllably operated to open
only so far as to expose PMT 14 to cell 12 during chemiluminescent
measurement periods. The shutter mechanism may be closed, for
example, during maintenance. Also included in apparatus 10 but not
illustrated in FIG. 1 is a lightproof housing intended to mount the
various components therein and to shield PMT 14 from any external
light during the chemiluminescent measurements.
[0085] Cell 12 itself includes a first mounting block 20 through
which passes an inlet tube 22 and an outlet tube 24, which may be
advantageously constructed of Plexiglas. Mounting block 20 has a
first, outer surface 26 and a second, inner surface 28 defining one
side of a sample-holding volume 30 of cell 12 in which cell 12
holds the cleaning and/or conditioning and/or measurement solutions
during corresponding operations of apparatus 10. Inlet and outlet
tubes 22, 24 pass through mounting block 20 from outer surface 26
to inner surface 28 and open into sample-holding volume 30. A
second mounting block 32 is advantageously constructed of a
material which is substantially transparent at the wavelength of
chemiluminescent light emitted by the chemiluminescent moiety.
Mounting block 32 is therefore advantageously formed of glass,
plastic, quartz or the like and has a first, outer surface 34 and a
second, inner surface 36. Second mounting block 32 is separated
from first mounting block 20 by an annular spacer 38,
advantageously constructed of Teflon or other non-contaminable
material. Thus, outer surface 34 of mounting block 30 defines the
second side of the sample-holding volume 30. Spacer 38 has an outer
portion 40 and a central aperture 42 whose inner edge 44 defines
the side wall of sample-holding volume 30. Outer portion 40 seals
the inner surface 28 of first mounting block 20 to outer surface 34
of second mounting block 32 to prevent any solution from passing
out from sample-holding volume 30 between the two surfaces 28,
34.
[0086] Inlet tube 22 intersects sample-holding volume 30 at a first
end 50 thereof adjacent to spacer 38 and outlet tube 24 intersects
sample-holding volume 30 at a second end 52 thereof, adjacent
spacer 38. The combination of inlet tube 22, sample-holding volume
30 and outlet tube 24 thereby provides a continuous flow path for
the narrow, substantially laminar flow of a solution to, through
and from cell 12.
[0087] Pump 16 is advantageously positioned at outlet tube 24 to
"pull" solution from a sample volume in the direction of arrow A
into inlet tube 22. The solution will flow through inlet tube 22,
sample-holding volume 30 and outlet tube 24 and out in the
direction of arrow B. Alternatively, pump 16 may be positioned at
inlet tube 22 to "push" the solution through apparatus 10.
Advantageously, this same flow path through inlet tube 22,
sample-holding volume 30 and outlet tube 24 is used for all
solutions and fluids which pass through cell 12, whereby each fluid
performs a hydrodynamic cleaning action in forcing the previous
fluid out of cell 12. Pump 16 may be controlled to suspend its
operation to hold a particular solution in cell 12 for any period
of time.
[0088] The invention is also directed to reagent compositions.
Broadly, the reagent compositions may be any one of the components
of the assay systems of the invention, i.e., (a) electrolyte, (b)
label compound containing a chemiluminescent moiety, (c) particles,
including magnetically responsive particles, (d) analyte of
interest or an analog of the analyte of interest, (e) a binding
partner of the analyte of interest or of its analog, (f) a reactive
component capable of reacting with (d) or (e), (g) a trigger
precursor molecule, or (h) a chemiluminescence-reaction enhancer.
The reagents may be combined with one another for convenience of
use, i.e., two component, three component, and higher multiple
component mixtures may be prepared, provided that the components
are not reactive with one another during storage so as to impair
their function in the intended assay. Desirably, the reagents are
two-component or multicomponent mixtures which contain particles as
well as one or more other components.
[0089] The invention is also directed to kits. The kits may include
vessels containing one or more of the components (a) to (h) recited
above or the kits may contain vessels containing one or more
reagent compositions as described above comprising mixtures of
those components, all for use in the assay methods and systems of
the invention.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0090] While a wide range of particles can be employed in the
particle-based assays of the invention, generally the particles
have a density of from 1.0 to 5.0 g/mL and preferably have a
density of from 1.1 to 2 g/mL. Choice of the optimum density is
within the skill of the art, the rate of settling in gravity-driven
assays being a trade-off between the speed of the assay and the
desire to create a uniform layer of complex in the collection
zone.
[0091] Particles having a wide range of mean diameters can also be
employed. Particles having a mean diameter of from 0.001 to 100
.mu.m can be used and preferably the particles have a mean diameter
of from 0.01 to 10 .mu.m.
[0092] Wide ranges of concentration of particles in the assay
composition can also be employed. For example, the concentration
can range from 1 to 10,000 .mu.g/mL to preferably from 5 to 1000
.mu.g/mL. Desirably, the density of the particles, their size and
their concentration is selected such that the particles settle at a
rate of at least 0.5 mm/min and preferably at a faster rate.
[0093] In the filtration mode of performing the invention, the
filtration means desirably has a pore size, measured as mean
diameter, from broadly 0.01 to 90% of the mean diameter of the
particles and preferably from 10% to 90% of that diameter.
[0094] The art has described a number of magnetic particles which
can be used in the assays of the invention. For example, U.S. Pat.
Nos. 4,628,037, 4,695,392, 4,695,393, 4,698,302, 4,554,088, U.K.
Patent Application GB 2,005,019A and EP 0,180,384, describe a
variety of magnetic particles which can be used with success. The
particles may be paramagnetic or ferromagnetic and may be coated
with various materials to which binding compounds are coupled so
that the magnetic particle can be used in immunoassays. Desirably
the magnetic particles used in the invention have a susceptibility
of at least 0.001 cgs units and desirably the susceptibility is at
least 0.01 cgs units. The magnetic particles may have a broad range
of densities, i.e. from substantially less than that of water,
0.01, to 5 g/mL and preferably from 0.5 to 2 g/mL. The particle
sizes can range from 0.001 to 100 .mu.m and preferably from 0.01 to
10 .mu.m. The concentration of the particles may range broadly from
1 to 10,000 .mu.g per mL and preferably is from 5 to 1000 .mu.g per
mL.
[0095] Desirably the magnetic particles which are used have a low
magnetic remanence, as described for example EP 0,180,384, so that
after the magnetic field is removed from the collection zone, the
particles demagnetize and can be swept out of the assay cell.
Desirably the density, concentration and particle size of the
magnetic particles is chosen such that the settling time is at
least 0.5 mm/min and desirably it is above that rate.
[0096] Assays
[0097] A variety of assays can be performed using the methods of
the invention.
[0098] An assay is performed as shown in FIG. 2. The PCR products
resulting from the reaction are labeled with biotin and a
chemiluminescent label. Streptavidin beads capture the
bifunctionalized DNA via biotin streptavidin binding and this is
followed by washing. The bead bound product is then subjected to
analysis detecting the chemiluminescent label.
[0099] An assay is performed as shown in FIG. 3. The biotinylated
PCR product is captured on streptavidin beads and the
non-biotinylated strand removed. The bead bound PCR product is then
hybridized with a chemiluminescent labeled oligonucleotide. This is
followed by chemiluminescent analysis to detect the label.
[0100] An assay is conducted as shown in FIG. 4. The hybrids are
captured on streptavidin beads. This is followed by
chemiluminescent analysis without washing.
EXAMPLES
Instrumentation, Materials, and Methods
[0101] (1) Instrumentation
[0102] A flow-through apparatus as described in FIG. 1 was
used.
[0103] Teflon Gasket (0.015" thick)
[0104] Plexiglas Faceplate
[0105] Inlet Tubing=0.042" id polypropylene
[0106] Aspiration Rates:variable from 0.01 to 5 mL/min
[0107] Luminometer using Hamamatsu R374 PMT (low gain red sensitive
tube); PMT Voltage variable 0-1400 V
EXAMPLE 1
Chemiluminescent Apparatus and Method for Deposition of
Microparticles
[0108] Magnetic Collection using a Sedimentation Cell.
[0109] A cell for conduct of an assay using magnetic force to cause
the microparticulate to settle is shown in FIG. 5. Reference
numeral 21 refers to a transparent window, reference numeral 22 to
a gasket, reference numeral 23 to the inlet in the cell block,
reference numeral 25 to the sample outlet, reference numeral 26 to
the cell block itself and reference 27 to an electromagnet.
[0110] The plane of the cell block is oriented horizontally.
Labeled microparticles (Dynal) in buffer are drawn to the cell by
means of a peristaltic pump. The pump is turned off after the
microparticles reach the cell. The microparticles in the cell
chamber are drawn to the collection zone by means of a magnetic
field generated using electromagnet 27 operating at 12 volts and
1.5 amps. By application of the electromagnet, the rate of
deposition of microparticles is greatly increased over that
observed when the microparticles settle solely due to the force of
gravity.
EXAMPLE 2
Chemiluminescent Apparatus and Method for Deposition of
Microparticles
[0111] Magnetic Collection Using a Collection Cell.
[0112] An assay is carried out in a cell as described in FIG. 1.
With reference to FIG. 1, reference numeral 32 refers to
transparent window, reference numeral 38 to a gasket, reference
numeral 22 to an inlet in the cell block, reference numeral 20 to
the cell block itself, reference numeral 24 to the sample outlet
and reference numeral 37 to a permanent magnet.
[0113] The plane of the cell block is oriented horizontally.
Labeled microparticles (Dynal) in buffer are drawn to the cell by
means of a peristaltic pump 11. Prior to the sample introduction,
permanent magnet 37 is positioned immediately below the collection
zone at a distance of 0.035 inches. As the sample is being drawn to
the cell, the microparticles collect in a collection zone, as
defined by the area of the magnet. The pump is turned off. The
longer the collection time, the more particles are collected.
EXAMPLE 3
Use of Magnet for Deposition of Microparticles
[0114] Magnetic Field Orientation.
[0115] Microparticles which are attracted to a magnet whether a
permanent magnet or electromagnet, align with the orientation of
the magnetic field. FIG. 6 depicts magnetic fields and the
resultant particle arrangements which are parallel (A) and
perpendicular (B) to the top surface of cell blocks 8 and 4,
respectively, in the vicinity of that surface. One skilled in the
art will appreciate that the orientation of the particles in the
collection zone will affect the efficiency of subsequent contact
with trigger.
EXAMPLE 4
Particle Collection and Concentration by Filtration
[0116] Microparticles which are magnetically responsive,
non-magnetically responsive, and of a wide range of densities can
advantageously be collected by filtration upon the surface of a
membrane filter. In one embodiment of the invention, the particles
are pumped through a portion of a filter membrane which has pore
sizes which are smaller than the diameter of the particles but
preferably are substantially smaller than the particle diameter and
at a sufficiently high surface density such that the collection of
particles will not cause blockage of the pores. The collected
particles are-then exposed to trigger by pumping the trigger
solution through the filter for the purpose of inducing
chemiluminescence from the particles and measuring the luminescence
to measure the quantity of chemiluminescent label on the
particles.
[0117] In another embodiment, the membrane filter having pore sizes
as described above is attached or placed upon the surface of an
absorbent material such that capillarity or "wicking" will
spontaneously draw fluids containing microparticles through the
membrane filter without requiring any apparatus to induce the flow
of fluid through the filter.
[0118] Such a filter is readily mounted in a flow cell such that
the flow-path for the fluid is through the filter. Particles in the
stream are trapped by the filter and are easily washed in-situ
providing for a rapid and simple means for performing heterogeneous
assays without any external washing apparatus.
EXAMPLE 5
Particle Collection and Concentration by Centrifugal Method
[0119] The rotary flow cell shown in FIG. 7 provides another means
to collect the complex in order to measure luminescence. The assay
solution 61 is pumped into cell 62 through rotary seal 63 while a
rotational motion is imparted to the cell. The denser particles of
the complex are concentrated in the collection zone. While the cell
is still rotating the solution passes out of the cell. The light
output passing through cell window 67 is measured by
photomultiplier tube 65. The light output is directed from the
collection zone and reflected off curved mirror surface 66 located
at the center of the cell. The cell is then flushed and cleaned for
the next cycle. This may be accomplished with the cell stopped or
rotating.
EXAMPLE 6
Coating of Particles With Labeled Non-specific Protein at Moderate
Surface Concentration
[0120] 30 mg (1 ml) of 4.5 um uncoated magnetically responsive,
polystyrene M-450 DYNABEADS (DYNAL, Oslo, Norway) are washed by
magnetic separation with a 150 mM phosphate buffer pH 7.5 solution
using 2 ml/wash. 150 .mu.g of acridinium ester-labeled antibody
(London Diagnostics LumaTag TSH Labeled Antibody) in 1 ml of
phosphate buffer saline (PBS) with 0.05% thimerosal is added to the
particles. This mixture is allowed to incubate overnight at room
temperature with agitation. The solution is then magnetically
separated from the particles and the fluid removed. To block
unreacted sites, 1 ml of 3% BSA/PBS with 0.05% sodium azide is
added to the particles, and the resultant solution is allowed to
incubate 2 hours at room temperature. The particles are washed 5
times (2 ml/wash), and then finally resuspended in 6 ml of the same
buffer for storage.
EXAMPLE 7
Chemiluminescent Measurement Using Magnetically Responsive
Particles
[0121] Magnetically responsive particles (Dynal, Oslo, Norway) are
coated with labeled proteins as described in Example 6. The coated
particles are washed with phosphate buffer three times before
making 2 mL of a 30 ug/mL suspension in 0.1 N HNO.sub.3 and 0.5%
hydrogen peroxide. Using a peristaltic pump, 500 ul of the particle
suspension is drawn into the flow cell (Example 2). As the
particles flow through the cell, they are attracted and
concentrated into the collection zone by a magnet. After the
particles are magnetically collected, a solution of 0.25 N NaOH,
0.5% hydrogen peroxide is drawn through the cell while the
chemiluminescence is measured using a Hamamatsu R374
photomultiplier tube centered above the flow cell where particles
have concentrated in the collection zone
EXAMPLE 8
Preparation of Physically Adsorbed Sheep Anti-Thyroid Stimulating
Hormone (TSH) Coated Dynal Particles
[0122] 1 mL of 4.5 .mu.m uncoated magnetic, polystyrene particles
with --OH residues on their surface (DYNAL, DYNABEADS M-450, DYNAL
A. S. Oslo, Norway) is washed by magnetic separation with a 150 mM
sodium carbonate/bicarbonate pH 9.6 solution using 2 mL/wash. 0.5
mg of purified monoclonal anti-TSH antibody (Catalog No. 5064031,
Ventrex Laboratories, Inc., Portland, Me.) in 1 mL of the
carb/bicarb solution is added to the particles. This mixture is
incubated overnight at room temperature with mixing. The solution
is then magnetically separated from the particles and removed. 1 mL
of 3% BSA/PBS with 0.05% sodium azide is added and incubated 2
hours at room temperature with agitation to block unreacted sites.
The particles are washed 5 times (2 mL/wash) and then finally
resuspended in 1 mL of the same buffer for storage. The final
concentration is 3% by weight.
EXAMPLE 9
One Step Separation Sandwich Assay for Thyroid Stimulating Hormone
(TSH)
[0123] 100 .mu.L serum calibrators (London Diagnostics TSH LumiTAG
Kit), 25 .mu.L LumaTag TSH acridinium ester-labeled antibody
(London Diagnostics) in phosphate buffer and 25 .mu.L
anti-TSH-DYNAL particles (Example 8) in phosphate buffer are
combined and incubated in polypropylene tubes for 15 minutes, at
room temperature, with mixing. The particles are then washed by
magnetic separation and then resuspended in 500 .mu.L of pH 4, 10
mM carbonate/bicarbonate buffer. This wash procedure was repeated
two additional times. The particles are drawn into a flow cell
(Example 2), magnetically collected and a solution to trigger the
chemiluminescent reaction is drawn through the flow cell (0.5%
hydrogen peroxide, 0.25 N NaOH). The chemiluminescence for each
sample is measured as described in Example 2. The chemiluminescent
intensity is directly proportional to the concentration of analyte
present in the sample (increasing intensity as the concentration of
analyte increases).
EXAMPLE 10
One Step Non Separation Sandwich Assay for Thyroid Stimulating
Hormone (TSH)
[0124] 100 .mu.L serum calibrators (London Diagnostics TSH LumiTAG
Kit), 25 .mu.L LumaTag TSH acridinium ester-labeled antibody
(London Diagnostics) in phosphate buffer and 25 .mu.L
anti-TSH-DYNAL particles (Example 8) in phosphate buffer are
combined and incubated in polypropylene tubes for 15 minutes, at
room temperature, with mixing. Prior to reading results, 1 mL of pH
4 100 mM carbonate/bicarbonate buffer is added. The particles are
drawn into a flow cell (Example 2), magnetically collected and a
solution to trigger the chemiluminescent reaction is drawn through
the flow cell (0.5% hydrogen peroxide, 0.25 N NaOH). The
chemiluminescence for each sample is read as described in Example
2. The chemiluminescent intensity is directly proportional to the
concentration of analyte present in the sample (increasing
intensity as the concentration of analyte increases).
EXAMPLE 11
Chemiluminescent TSH Immunoassay Using Enzyme to Generate
Trigger
[0125] Using magnetically responsive microparticles as the solid
phase and acridinium ester as the label, a TSH immunoassay can be
performed using the apparatus described in Example 2. An enzyme,
glucose oxidase is used to convert a precursor of the trigger
(glucose) to the trigger (hydrogen peroxide). The enzyme glucose
oxidase catalyzes the oxidation of glucose to gluconic acid and
hydrogen peroxide. Acridinium ester in the presence of hydrogen
peroxide is oxidized to an excited state. The subsequent return to
ground state of oxidized excited product results in the emission of
light which is quantified. Magnetic microparticles coated with both
specific antibody and enzyme glucose oxidase can be used (prepared
as in Example 8 except that 0.5 mg of both antibody and enzyme
(Sigma Chemical) are added to the particles for coating).
Alternatively, separate particles coated with each reagent
(antibody or enzyme) can be mixed and used in the assay (prepared
separately as described in Example 8). D-Glucose Glucose
Oxidase>D- gluconic acid +H 0 H.sub.2 2 Acridinium Ester
+H.sub.2O.sub.2------------->Light (428 nm)
[0126] The TSH immunoassay is based on a two-site sandwich assay
known in the art. Monoclonal anti-TSH antibody coated magnetic
microparticles are prepared as described in Example 8. Enzyme
glucose oxidase coated magnetic microparticles are prepared by the
same method as antibody coated magnetic microparticles. Acridinium
Ester labeled polyclonal anti-TSH antibody and the TSH standards
are obtained from London Diagnostics. Enzyme substrate solution
consists of 100 mM potassium phosphate buffer containing D-glucose
(100 mg/ml).
[0127] A series of tubes (12.times.75 mm polypropylene ) are set up
and labeled according to standards and samples to be assayed. Into
each tube is added 100 .mu.l of standard or sample or control, 100
.mu.l of acridinium ester-labeled antibody and 100 .mu.l of a
mixture of anti-TSH antibody and enzyme coated microparticles. The
tubes are incubated at room temperature with mixing for 15 min.
Following incubation, 1 ml pH 4, 100 mM carbonate/bicarbonate
buffer is added. The particles are drawn into a flow cell (Example
2), magnetically collected and the glucose substrate solution is
drawn through the flow cell. The chemiluminescence for each sample
is read as described in Example 2. The chemiluminescent intensity
is directly proportional to the concentration of analyte present in
the sample (increasing intensity as the concentration of analyte
increases).
* * * * *