U.S. patent number 5,811,311 [Application Number 08/488,228] was granted by the patent office on 1998-09-22 for metal chelate containing compositions for use in chemiluminescent assays.
This patent grant is currently assigned to Dade Behring Marburg GmbH. Invention is credited to Sharat Singh, Edwin F. Ullman.
United States Patent |
5,811,311 |
Singh , et al. |
September 22, 1998 |
Metal chelate containing compositions for use in chemiluminescent
assays
Abstract
Compositions are disclosed comprising (a) a metal chelate
wherein the metal is selected from the group consisting of
europium, terbium, dysprosium, samarium osmium and ruthenium in at
least a hexacoordinated state and (b) a compound having a double
bond substituted with two aryl groups, an oxygen atom and an atom
selected from the group consisting of oxygen, sulfur and nitrogen
wherein one of the aryl groups is electron donating with respect to
the other. Such composition is preferably incorporated in a latex
particulate material. Methods and kits are also disclosed for
determining an analyte in a medium suspected of containing the
analyte. The methods and kits employ as one component a composition
as described above.
Inventors: |
Singh; Sharat (San Jose,
CA), Ullman; Edwin F. (Atherton, CA) |
Assignee: |
Dade Behring Marburg GmbH
(Marburg, DE)
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Family
ID: |
22558459 |
Appl.
No.: |
08/488,228 |
Filed: |
June 7, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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156181 |
Nov 22, 1993 |
5578498 |
|
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704569 |
May 22, 1991 |
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Current U.S.
Class: |
436/518;
252/301.16; 252/301.35; 252/700; 435/7.1; 435/968; 436/172;
436/533 |
Current CPC
Class: |
C07D
265/30 (20130101); C07D 327/06 (20130101); C07D
413/04 (20130101); G01N 33/533 (20130101); G01N
33/54313 (20130101); G01N 33/5436 (20130101); G01N
33/58 (20130101); G01N 33/582 (20130101); G01N
33/585 (20130101); G01N 33/586 (20130101); C07D
279/12 (20130101); Y10T 436/13 (20150115); Y10S
435/968 (20130101); Y10S 436/80 (20130101); Y10S
436/808 (20130101); Y10S 436/805 (20130101) |
Current International
Class: |
C07D
413/04 (20060101); C07D 413/00 (20060101); C07D
327/06 (20060101); C07D 327/00 (20060101); C07D
265/30 (20060101); C07D 265/00 (20060101); C07D
279/00 (20060101); C07D 279/12 (20060101); G01N
33/543 (20060101); G01N 33/58 (20060101); G01N
033/546 () |
Field of
Search: |
;252/700,301.16,301.33,301.35,31.4R ;435/968,7.1
;436/58,533,35,81,91,92,127,128,135,136,177,800 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 070 685 A2 |
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Jan 1983 |
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EP |
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0 070 687 A2 |
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Jan 1983 |
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EP |
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0 324 323 A1 |
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Jul 1989 |
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EP |
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0 345 776 A2 |
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Dec 1989 |
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EP |
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0 515 194A2 |
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Nov 1992 |
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EP |
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WO 92/16840 |
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Oct 1992 |
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WO |
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Other References
Egston, CA 90: 67574, "Growth Regulators as a Factor In Growth
Management of Apple Trees" In Proc. Plant Growth Regul. Work. Group
(1978) 5, 186-194. .
McCapra, et al., Tetrahedron Letters, vol. 23:49, (1982), pp.
5225-5229, "Metal Catalysed Light Emission from a Dioxetan". .
Handley, et al., Tetrahedron Letters, vol. 26:27, (1985), pp.
3183-3186, "Effects of Heteroatom Substituents on the Properties of
1,2-Dioxetanes". .
McCapra, et al., Tetrahedron letters, (1982) vol. 23:49, pp.
5225-5229 "Metal Catalysed Light Emission from a Dioxetan". .
White, et al., Journal of the American Chemical Society, (Apr.
1973) vol. 95: pp. 7050-7058, "Chemically Produced Excited States.
Energy Transfer, Photochemical Reactions, and Light Emission".
.
Wildes, et al., Journal of the American Chemical Society, (Sep. 17,
1971) vol. 93:23, pp. 6286-6288, "The Dioxetane-Sensitized
Chemiluminescence of Lanthanide Chelates. A Chemical Source of
Monochromatic Light". .
Zaklika, et al., Journal of the American Chemical Society, (Jul.
19, 1978) vol. 100:15, pp. 4916-4918, "Substituent Effects on the
Decomposition of 1,2-Dioxetanes". .
Zaklika, et al., Journal of the American Chemical Society, (1980)
vol. 102, pp. 386-389, "Mechanisms of Photooxygenation. 1.
Substituent Effects on the [2+2] Cycloaddition of Singlet Oxygen to
Vinyl Ethers"..
|
Primary Examiner: Green; Lora M.
Attorney, Agent or Firm: Maiorana; David M. Meyers; Kenneth
J. Leitereg; Theodore J.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a divisional of application Ser. No. 08/156,181, filed Nov.
22, 1993, now U.S. Pat. No. 5,578,498, which in turn is a
continuation-in-part of application Ser. No. 07/704,569, filed May
22, 1991, the disclosures of which are incorporated herein.
Claims
What is claimed is:
1. A composition comprising a latex having incorporated therein a
compound of the formula: ##STR40## wherein X" is O, S, or NR",
wherein R" is alkyl or aryl, n is 1 to 4, and Ar and Ar' are
independently aryl, wherein one of Ar or Ar' is electron donating
with respect to the other and Y is hydrogen or an organic radical
consisting of atoms selected from the group consisting of C, O, N,
S, and P and m is 0 to 2;
wherein the latex has a specific binding pair member bound
thereto.
2. The composition of claim 1 wherein R" is methyl or phenyl.
3. The composition of claim 1 wherein n is 2.
4. The composition of claim 1 wherein Ar is selected from the group
consisting of 5-member and 6-member aromatic and heteroaromatic
rings.
5. The composition of claim 1 wherein Ar is phenyl substituted with
an electron donating group at a position of the phenyl that is meta
or para to the carbon that is bonded to the double bond and Ar' is
phenyl.
6. The composition of claim 1 comprising a metal chelate wherein
said metal is selected from the group consisting of europium,
terbium, dysprosium, samarium, osmium and ruthenium in at least a
hexacoordinated state.
7. A composition comprising a latex having incorporated therein a
compound of the formula: ##STR41## wherein X' is S or NR' wherein
R' is alkyl or aryl and D and D' are independently selected from
the group consisting of alkyl and alkyl radical.
8. The composition of claim 7 wherein R' is methyl or phenyl.
9. A composition comprising a latex having incorporated therein a
compound of the formula: ##STR42## wherein X" is O, S, or NR",
wherein R" is alkyl or aryl, n is 1 to 4, and Ar and Ar' are
independently aryl, wherein one of Ar or Ar' is electron donating
with respect to the other and Y is hydrogen or an organic radical
consisting of atoms selected from the group consisting of C, O, N,
S, and P and m is 0 to 2;
wherein the latex has an specific binding pair member bound thereto
and is porous.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods, compositions and kits for
determining an analyte in a sample. In particular, this invention
relates to compositions that exhibit a high quantum yield
chemiluminescence when activated by singlet oxygen, decay rapidly
and emit at long wavelengths.
The clinical diagnostic field has seen a broad expansion in recent
years, both as to the variety of materials (analytes) that may be
readily and accurately determined, as well as the methods for the
determination. Convenient, reliable and non-hazardous means for
detecting the presence of low concentrations of materials in
liquids is desired. In clinical chemistry these materials may be
present in body fluids in concentrations below 10.sup.-12 molar.
The difficulty of detecting low concentrations of these materials
is enhanced by the relatively small sample sizes that can be
utilized.
In developing an assay there are many considerations. One
consideration is the signal response to changes in the
concentration of analyte. A second consideration is the ease with
which the protocol for the assay may be carried out. A third
consideration is the variation in interference from sample to
sample. Ease of preparation and purification of the reagents,
availability of equipment, ease of automation and interaction with
material of interest are some of the additional considerations in
developing a useful assay.
One broad category of techniques involves the use of a receptor
which can specifically bind to a particular spacial and polar
organization of a labeled ligand as a function of the presence of
an analyte. The observed effect of binding by the receptor will
depend upon the label. In some instances the binding of the
receptor merely provides for a differentiation in molecular weight
between bound and unbound labeled ligand. In other instances the
binding of the receptor will facilitate separation of bound labeled
ligand from free labeled ligand or it may affect the nature of the
signal obtained from the label so that the signal varies with the
amount of receptor bound to labeled ligand. A further variation is
that the receptor is labeled and the ligand unlabeled.
Alternatively, both the receptor and ligand are labeled or
different receptors are labeled with two different labels,
whereupon the labels interact when in close proximity and the
amount of ligand present affects the degree to which the labels of
the receptor may interact.
There is a continuing need for new and accurate techniques that can
be adapted for a wide spectrum of different ligands or be used in
specific cases where other methods may not be readily
adaptable.
Homogeneous immunoassays have previously been described for small
molecules. These assays include SYVA's FRAT.RTM. assay, EMIT.RTM.
assay, enzyme channeling immunoassay, and fluorescence energy
transfer immunoassay (FETI); enzyme inhibitor immunoassays (Hoffman
LaRoche and Abbott Laboratories): fluorescence polarization
immunoassay (Dandlicker), among others. All of these methods have
limited sensitivity, and only a few including FETI and enzyme
channeling, are suitable for large multiepitopic analytes.
Luminescent compounds, such as fluorescent compounds and
chemiluminescent compounds, find wide application in the assay
field because of their ability to emit light. For this reason,
luminescers have been utilized as labels in assays such as nucleic
acid assays and immunoassays. For example, a member of a specific
binding pair is conjugated to a luminescer and various protocols
are employed. The luminescer conjugate can be partitioned between a
solid phase and a liquid phase in relation to the amount of analyte
in a sample suspected of containing the analyte. By measuring the
luminescence of either of the phases, one can relate the level of
luminescence observed to a concentration of the analyte in the
sample.
Particles, such as liposomes and erythrocyte ghosts, have been
utilized as carriers of encapsulated water soluble materials. For
example, liposomes have been employed to encapsulate biologically
active material for a variety of uses, such as drug delivery
systems wherein a medicament is entrapped during liposome
preparation and then administered to the patient to be treated.
Particles, such as latex beads and liposomes, have also been
utilized in assays. For example, in homogeneous assays an enzyme
may be entrapped in the aqueous phase of a liposome labelled with
an antibody or antigen. The liposomes are caused to release the
enzyme in the presence of a sample and complement. Antibody- or
antigen-labelled liposomes, having water soluble fluorescent or
non-fluorescent dyes encapsulated within an aqueous phase or lipid
soluble dyes dissolved in the lipid bilayer of the lipid vesicle or
in latex beads, have also been utilized to assay for analytes
capable of entering into an immunochemical reaction with the
surface bound antibody or antigen. Detergents have been used to
release the dyes from the aqueous phase of the liposomes.
2. Brief Description of the Related Art.
White, et al. (White), discuss "Chemically Produced Excited States.
Energy Transfer, Photochemical Reactions, and Light Emission" in J.
Am. Chem. Soc., 93, 6286 (1971).
McCapra, et al. (McCapra), disclose "Metal Catalysed Light Emission
from a Dioxetan" in Tetrahedron Letters, 23:49, 5225-5228
(1982).
Wildes, et al. (Wildes), discuss "The Dioxetane-Sensitized
Chemiluminescence of Lanthanide Chelates. A Chemical Source of
`Monochromatic` Light" in J. Am. Chem. Soc., 93(23), 6286-6288
(1971).
Handley, et al. (Handley), disclose "Effects of Heteroatom
Substituents on the Properties of 1,2-Dioxetanes" in Tetrahedron
Letters, 26, 3183 (1985).
Zaklika, et al. (Zaklika), discuss "Substituent Effects on the
Decompositon of 1,2-Dioxetanes" in J. Am. Chem. Soc., 100, 4916
(1978).
European Patent Application No. 0,345,776 (McCapra) discloses
specific binding assays that utilize a sensitizer as a label. The
sensitizers include any moiety which, when stimulated by excitation
with radiation of one or more wavelengths or other chemical or
physical stimulus (e.g., electron transfer, electrolysis,
electroluminescence or energy transfer) will achieve an excited
state which (a) upon interaction with molecular oxygen will produce
singlet molecular oxygen, or (b) upon interaction with a leuco dye
will assume a reduced form that can be returned to its original
unexcited state by interaction with molecular oxygen resulting in
the production of hydrogen peroxide. Either interaction with the
excited sensitizer will, with the addition of reagents, produce a
detectible signal.
European Patent Application No. 0,070,685 (Heller, et al. I)
describes a homogeneous nucleic acid hybridization diagnostic by
non-radiative energy transfer.
A light-emitting polynucleotide hybridization diagnostic method is
described in European Patent Application No. 0,070,687 (Heller, et
al. II).
SUMMARY OF THE INVENTION
One aspect of the present invention is directed to compositions
comprising (a) a metal chelate comprising a metal selected from the
group consisting of europium, terbium, dysprosium, samarium, osmium
and ruthenium in at least a hexacoordinated state and (b) a
compound having a structural portion that is a double bond
substituted with two aryl groups, an oxygen atom and an atom
selected from the group consisting of oxygen, sulfur and nitrogen.
The aryl groups are characterized in that one is electron donating
with respect to the other. Preferably, the composition is
incorporated in a latex particulate material.
Another aspect of the present invention is a compound of the
formula: ##STR1## wherein X' is S or NR' wherein R' is alkyl or
aryl and D and D' are independently selected from the group
consisting of alkyl and alkyl radical.
Another aspect of the present invention is a composition comprising
a latex having incorporated therein a compound of the formula:
##STR2## wherein X" is O, S or NR" wherein R" is alkyl or aryl, n
is 1 to 4, and Ar and Ar' are independently aryl wherein one of Ar
or Ar' is electron donating with respect to the other and Y is
hydrogen or an organic radical consisting of atoms selected from
the group consisting of C, O, N, S, and P and m is 0 to 2.
Another aspect of the present invention is a composition comprising
a latex having incorporated therein Compound 1.
Another aspect of the present invention is a method for determining
an analyte which comprises (a) providing in combination (1) a
medium suspected of containing an analyte, (2) a photosensitizer
capable in its excited state of activating oxygen to a singlet
state, where the photosensitizer is associated with a specific
binding pair (sbp) member, and (3) one of the above-mentioned
compositions incorporated into a latex particulate material having
bound thereto an sbp member, (b) treating the combination with
light to excite the photosensitizer, and (c) examining the
combination for the amount of luminescence emitted therefrom. The
amount of luminescence is related to the amount of analyte in the
medium.
Another aspect of the present invention is a kit comprising in
packaged combination: (1) a composition comprising a suspendible
latex particle comprising one of the above-mentioned compounds and
(2) a photosensitizer. The particle has bound thereto a specific
binding pair (sbp) member. The photosensitizer is capable in its
excited state of activating oxygen to its singlet state.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention is directed to chemiluminescent compositions
that upon activation by singlet oxygen exhibit chemiluminescent
emission that rapidly decays, generally having a half life of 0.5
seconds to 30 minutes, preferably 0.5 to 30 seconds, usually less
than twenty seconds. In addition, the present chemiluminescent
compositions can exhibit high chemiluminescent quantum yield upon
activation by singlet oxygen, generally 0.1 to 0.9, usually 0.1 to
0.6, preferably 0.2 to 0.4. The chemiluminescent light emitted by
the metal chelate after activation in the present compositions
generally has a wave length of about 550 to 700 nm, usually greater
than 600 nm. The chemiluminescent compositions of the present
invention are particularly useful in luminescent assays. For
example, the long wavelength emission avoids interference from
serum absorption in assays on blood or serum samples. The high
quantum yield improves detectibility and the short lifetime further
improves detectibility by causing all the light that is emitted to
be delivered in a short pulse rather than over an extended period
of time. This can provide higher light intensity at lower quantum
yields.
The quantum yield of chemiluminescence of the present
chemiluminescent compositions, when activated by singlet oxygen, is
generally about 10 to 100 fold greater, preferably, 10 to 50 fold
greater, than that observed upon irradiation of the components of
the composition separately. Furthermore, the rate of decay of
chemiluminescence is significantly enhanced with some of the
present compositions. These properties render the present
compositions extremely useful in assays for the determination of
analytes.
Before proceeding further with a description of the specific
embodiments of the present invention, a number of terms will be
defined and described in detail.
Metal ligand--a compound in which two or more atoms of the same
molecule can coordinate with a metal to form a metal chelate. The
metal chelates that form part of the compositions of the present
invention comprise a metal selected from the group consisting of
europium, terbium, dysprosium, samarium, osmium and ruthenium. One
of the above metals is coordinated with one or more metal ligands,
which may be, for example, 3-(2-thienoly)-1,1,1-trifluoroacetone
(TTA), 3-benzoyl-1,1,1-trifluoroacetone (BFTA),
3-naphthoyl-1,1,1-trifluoroacetone (NPPTA),
2,2-dimethyl-4-perfluorobutyoyl-3-butanone (fod), is 2,2'-dipyridyl
(bpy), phenanthroline(phen), salicylic acid, phenanthroline
carboxylic acid, bipyridyl carboxylic acid, aza crown ethers
trioctylphosphine oxide, aza cryptands, and so forth. Usually, the
metal in the metal chelate is at least hexacoordinated, but may be
octacoordinated or more highly coordinated depending on the metal
ligands. The metal chelate will be uncharged, thus the number of
acidic groups provided by its ligands will equal the oxidation
state of the metal. Usually, the metal ligands will be relatively
hydrophobic so as to impart solubility of the metal chelate in
non-polar solvents. Rare earth metals will usually have an
oxidation state of three, ruthenium will have an oxidation state of
two and osmium will have an oxidation state of two. Examplary of
such metal chelates, by way of illustration and not limitation, is
as follows: ##STR3##
One TTA in 3(a) or 3(b) can be replaced by one of the following:
##STR4## wherein DPP (Diphenylphenanthroline) in 3(b) can be
replaced by one of the following: ##STR5##
Two TTA's in 3(a) and 3(b) can be independently replaced by
compounds selected from the following: ##STR6##
Three TTA's can be independently replaced by compounds selected
from the following: ##STR7## Many of these metal ligands and metal
chelates are known in the art and many are commercially available.
In general, metal chelates can be prepared from metal ligands by
combining the metal chloride with the desired ratio of metal ligand
molecules in an organic solvent such as, e.g., acetonitrile and
sufficient base, e.g., pyridine, to take up the released
hydrochloric acid. For example, metal chelates can be prepared by a
procedure such as that described by Shinha, A. P., "Fluorescences
and laser action in rare earth chelates," Spectroscopy Inorganic
Chemistry, Vol 2, (1971), 255-288.
Aryl group--an organic radical derived from an aromatic hydrocarbon
by the removal of one atom and containing one or more aromatic
rings, usually one to four aromatic rings, which are generally
five- or six-member rings such as, e.g., phenyl (from benzene),
naphthyl (from naphthalene), biphenylenyl, azulenyl, anthryl,
phenanthrenyl, pyridyl, indolyl, benzofuranyl, benzothiophenyl,
quinolinyl, isoquinolinyl, carbazolyl, acridinyl, imidazolyl,
thiazolyl, pyrazinyl, pyrimidinyl, purinyl, pteridinyl, etc.
Aralkyl--an organic radical having an alkyl group to which is
attached an aryl group, e.g., benzyl, phenethyl, 3-phenylpropyl,
1-naphthylethyl, etc.
Electron donating group--a substituent which when bound to a
molecule is capable of polarizing the molecule such that the
electron donating group becomes electron poor and positively
charged relative to another portion of the molecule, i.e., has
reduced electron density. Such groups may be, by way of
illustration and not limitation, amines, ethers, thioethers,
phosphines, hydroxy, oxyanions, mercaptans and their anions,
sulfides, etc.
Alkyl--a monovalent branched or unbranched radical derived from an
aliphatic hydrocarbon by removal of one H atom; includes both lower
alkyl and upper alkyl.
Alkyl radical--a substituent formed from two or more alkyl groups,
which may be independently lower or upper alkyl groups, linked
together by a functionality such as an ether, including thioether,
an amide, an ester and the like.
Lower Alkyl--alkyl containing from 1 to 5 carbon atoms such as,
e.g., methyl, ethyl, propyl, butyl, isopropyl, isobutyl, pentyl,
isopentyl, etc.
Upper Alkyl--alkyl containing more than 6 carbon atoms, usually 6
to 20 carbon atoms, such as, e.g., hexyl, heptyl, octyl, etc.
Alkylidene--a divalent organic radical derived from an aliphatic
hydrocarbon, such as, for example, ethylidene, in which 2 hydrogen
atoms are taken from the same carbon atom.
Substituted--means that a hydrogen atom of a molecule has been
replaced by another atom, which may be a single atom such as a
halogen, etc., or part of a group of atoms forming a functionality
such as a substituent having from 1 to 50 atoms (other than the
requisite hydrogen atoms necessary to satisfy the valencies of such
atoms), which atoms are independently selected from the group
consisting of carbon, oxygen, nitrogen, sulfur and phosphorus, and
which may or may not be bound to one or more metal atoms.
Analyte--the compound or composition to be detected. The analyte
can be comprised of a member of a specific binding pair (sbp) and
may be a ligand, which is monovalent (monoepitopic) or polyvalent
(polyepitopic), usually antigenic or haptenic, and is a single
compound or plurality of compounds which share at least one common
epitopic or determinant site. The analyte can be a part of a cell
such as bacteria or a cell bearing a blood group antigen such as A,
B, D, etc., or an HLA antigen or a microorganism, e.g., bacterium,
fungus, protozoan, or virus.
The polyvalent ligand analytes will normally be poly(amino acids),
i.e., polypeptides and proteins, polysaccharides, nucleic acids,
and combinations thereof. Such combinations include components of
bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell
membranes and the like.
For the most part, the polyepitopic ligand analytes to which the
subject invention can be applied will have a molecular weight of at
least about 5,000, more usually at least about 10,000. In the
poly(amino acid) category, the is poly(amino acids) of interest
will generally be from about 5,000 to 5,000,000 molecular weight,
more usually from about 20,000 to 1,000,000 molecular weight; among
the hormones of interest, the molecular weights will usually range
from about 5,000 to 60,000 molecular weight.
A wide variety of proteins may be considered as to the family of
proteins having similar structural features, proteins having
particular biological functions, proteins related to specific
microorganisms, particularly disease causing microorganisms, etc.
Such proteins include, for example, immunoglobulins, cytokines,
enzymes, hormones, cancer antigens, nutritional markers, tissue
specific antigens, etc.
The following are classes of proteins related by structure:
protamines, histones, albumins, globulins, scleroproteins,
phosphoproteins, mucoproteins, chromoproteins, lipoproteins,
nucleoproteins, glycoproteins, T-cell receptors, proteoglycans,
HLA, unclassified proteins, e.g., somatotropin, prolactin, insulin,
pepsin, proteins found in the human plasma such as blood clotting
factors, other polymeric materials such as mucopolysaccharides and
polysaccharides, microorganisms such as bacteria, viruses and
fungi.
The monoepitopic ligand analytes will generally be from about 100
to 2,000 molecular weight, more usually from 125 to 1,000 molecular
weight. The analytes include drugs, metabolites, pesticides,
pollutants, and the like. Included among drugs of interest are the
alkaloids, steroids, steroid mimetic substances, lactams,
aminoalkylbenzenes, benzheterocyclics, purines, those derived from
marijuana, hormones, vitamins, prostaglandins, tricyclic
antidepressants, anti-neoplastics, antibiotics, nucleosides and
nucleotides, miscellaneous individual drugs which include
methadone, meprobamate, serotonin, meperidine, lidocaine,
procainamide, acetylprocainamide, propranolol, griseofulvin,
valproic acid, butyrophenones, antihistamines, chloramphenicol,
anticholinergic drugs, such as atropine, metabolites related to
diseased states include spermine, galactose, phenylpyruvic acid,
and porphyrin Type 1, aminoglycosides, polyhalogenated biphenyls,
phosphate esters, thiophosphates, carbamates, polyhalogenated
sulfenamides.
For receptor analytes, the molecular weights will generally range
from 10,000 to 2.times.10.sup.8, more usually from 10,000 to
10.sup.6. For immunoglobulins, IgA, IgG, IgE and IgM, the molecular
weights will generally vary from about 160,000 to about 10.sup.6.
Enzymes will normally range from about 10,000 to 1,000,000 in
molecular weight. Natural receptors vary widely, generally being at
least about 25,000 molecular weight and may be 10.sup.6 or higher
molecular weight, including such materials as avidin, DNA, RNA,
thyroxine binding globulin, thyroxine binding prealbumin,
transcortin, etc.
The term analyte further includes polynucleotide analytes such as
those polynucleotides defined below. These include m-RNA, r-RNA,
t-RNA, DNA, DNA-RNA duplexes, etc. The term analyte also includes
receptors that are polynucleotide binding agents, such as, for
example, restriction enzymes, activators, repressors, nucleases,
polymerases, histones, repair enzymes, chemotherapeutic agents, and
the like.
The analyte may be a molecule found directly in a sample such as a
body fluid from a host. The sample can be examined directly or may
be pretreated to render the analyte more readily detectible.
Furthermore, the analyte of interest may be determined by detecting
an agent probative of the analyte of interest such as a specific
binding pair member complementary to the analyte of interest, whose
presence will be detected only when the analyte of interest is
present in a sample. Thus, the agent probative of the analyte
becomes the analyte that is detected in an assay. The body fluid
can be, for example, urine, blood, plasma, serum, saliva, semen,
stool, sputum, cerebral spinal fluid, tears, mucus, and the
like.
Member of a Specific Binding Pair ("sbp member")--one of two
different molecules, having an area on the surface or in a cavity
which specifically binds to and is thereby defined as complementary
with a particular spatial and polar organization of the other
molecule. The members of the specific binding pair are referred to
as ligand and receptor (antiligand). These will usually be members
of an immunological pair such as antigen-antibody, although other
specific binding pairs such as biotin-avidin, hormones-hormone
receptors, nucleic acid duplexes, IgG-protein A, polynucleotide
pairs such as DNA-DNA, DNA-RNA, and the like are not immunological
pairs but are included in the invention and the definition of sbp
member.
Polynucleotide--a compound or composition which is a polymeric
nucleotide having in the natural state about 50 to 500,000 or more
nucleotides and having in the isolated state about 15 to 50,000 or
more nucleotides, usually about 15 to 20,000 nucleotides, more
frequently 15 to 10,000 nucleotides. The polynucleotide includes
nucleic acids from any source in purified or unpurified form,
naturally occurring or synthetically produced, including DNA (dsDNA
and ssDNA) and RNA, usually DNA, and may be t-RNA, m-RNA, r-RNA,
mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNA
hybrids, or mixtures thereof, genes, chromosomes, plasmids, the
genomes of biological material such as microorganisms, e.g.,
bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals,
humans, and fragments thereof, and the like.
Ligand--any organic compound for which a receptor naturally exists
or can be prepared.
Ligand Analog--a modified ligand, an organic radical or analyte
analog, usually of a molecular weight greater than 100, which can
compete with the analogous ligand for a receptor, the modification
providing means to join a ligand analog to another molecule. The
ligand analog will usually differ from the ligand by more than
replacement of a hydrogen with a bond which links the ligand analog
to a hub or label, but need not. The ligand analog can bind to the
receptor in a manner similar to the ligand. The analog could be,
for example, an antibody directed against the idiotype of an
antibody to the ligand.
Receptor ("antiligand")--any compound or composition capable of
recognizing a particular spatial and polar organization of a
molecule, e.g., epitopic or determinant site. Illustrative
receptors include naturally occurring receptors, e.g., thyroxine
binding globulin, antibodies, enzymes, Fab fragments, lectins,
nucleic acids, protein A, complement component C1q, and the
like.
Specific binding--the specific recognition of one of two different
molecules for the other compared to substantially less recognition
of other molecules. Generally, the molecules have areas on their
surfaces or in cavities giving rise to specific recognition between
the two molecules. Exemplary of specific binding are
antibody-antigen interactions, enzyme-substrate interactions,
polynucleotide interactions, and so forth.
Non-specific binding--non-covalent binding between molecules that
is relatively independent of specific surface structures.
Non-specific binding may result from several factors including
hydrophobic interactions between molecules.
Antibody--an immunoglobulin which specifically binds to and is
thereby defined as complementary with a particular spatial and
polar organization of another molecule. The antibody can be
monoclonal or polyclonal and can be prepared by techniques that are
well known in the art such as immunization of a host and collection
of sera (polyclonal) or by preparing continuous hybrid cell lines
and collecting the secreted protein (monoclonal), or by cloning and
expressing nucleotide sequences or mutagenized versions thereof
coding at least for the amino acid sequences required for specific
binding of natural antibodies. Antibodies may include a complete
immunoglobulin or fragment thereof, which immunoglobulins include
the various classes and isotypes, such as IgA, IgD, IgE, IgG1,
IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab,
Fv and F(ab').sub.2, Fab', and the like. In addition, aggregates,
polymers, and conjugates of immunoglobulins or their fragments can
be used where appropriate so long as binding affinity for a
particular molecule is maintained.
A substituent having from 1 to 50 atoms (other than the requisite
hydrogen atoms necessary to satisfy the valencies of such atoms),
which atoms are independently selected from the group consisting of
carbon, oxygen, nitrogen, sulfur and phosphorus--an organic
radical; the organic radical has 1 to 50 atoms other than the
requisite number of hydrogen atoms necessary to satisfy the
valencies of the atoms in the radical. Generally, the predominant
atom is carbon (C) but may also be oxygen (O), nitrogen (N), sulfur
(S), phosphorus (P), wherein the O, N, S, or P, if present, are
bound to carbon or one or more of each other or to hydrogen or a
metal atom to form various functional groups, such as, for example,
carboxylic acids, alcohols, thiols, carboxamides, carbamates,
carboxylic acid esters, sulfonic acids, sulfonic acid esters,
phosphoric acids, phosphoric acid esters, ureas, carbamates,
phosphoramides, sulfonamides, ethers, sulfides, thioethers,
olefins, acetylenes, amines, ketones, aldehydes, nitrites, and the
like. Illustrative of such organic radicals or groups, by way of
illustration and not limitation, are alkyl, alkylidine, aryl,
aralkyl, and alkyl, aryl, and aralkyl substituted with one or more
of the aforementioned functionalities.
Linking group--the covalent linkage between molecules. The linking
group will vary depending upon the nature of the molecules, i.e.,
photosensitizer, chemiluminescent compound, sbp member or molecule
associated with or part of a particle, being linked. Functional
groups that are normally present or are introduced on a
photosensitizer or chemiluminescent compound will be employed for
linking these materials to an sbp member or a particle such as a
latex particle.
For the most part, carbonyl functionalities will find use, both
oxocarbonyl, e.g., aldehyde and non-oxocarbonyl (including nitrogen
and sulfur analogs) e.g., carboxy, amidine, amidate, thiocarboxy
and thionocarboxy.
Alternative functionalities of oxo include active halogen, diazo,
mercapto, olefin, particularly activated olefin, amino, phosphoro
and the like. A description of linking groups may be found in U.S.
Pat. No. 3,817,837, which disclosure is incorporated herein by
reference.
Common functionalities in forming a covalent bond between the
linking group and the molecule to be conjugated are alkylamine,
amidine, thioamide, ether, urea, thiourea, guanidine, azo,
thioether and carboxylate, sulfonate, and phosphate esters, amides
and thioesters.
For the most part, the photosensitizer and chemilumenescent
compound will have a non-oxocarbonyl group including nitrogen and
sulfur analogs, a phosphate group, an amino group, alkylating agent
such as halo or tosylalkyl, oxy (hydroxyl or the sulfur analog,
mercapto) oxocarbonyl (e.g., aldehyde or ketone), or active olefin
such as a vinyl sulfone or .alpha., .beta.-unsaturated ester. These
functionalities will be linked to amine groups, carboxyl groups,
active olefins, alkylating agents, e.g., bromoacetyl. Where an
amine and carboxylic acid or its nitrogen derivative or phophoric
acid are linked, amides, amidines and phosphoramides will be
formed. Where mercaptan and activated olefin are linked, thioethers
will be formed. Where a mercaptan and an alkylating agent are
linked, thioethers will be formed. Where aldehyde and an amine are
linked under reducing conditions, an alkylamine will be formed.
Where a carboxylic acid or phosphate acid and an alcohol are
linked, esters will be formed.
Photosensitizer--a sensitizer for generation of singlet oxygen
usually by excitation with light. The photosensitizer can be
photoactivatable (e.g., dyes and aromatic compounds) or
chemiactivated (e.g., enzymes and metal salts). When excited by
light the photosensitizer is usually a compound comprised of
covalently bonded atoms, usually with multiple conjugated double or
triple bonds. The compound should absorb light in the wavelength
range of 200-1100 nm, usually 300-1000 nm, preferably 450-950 nm,
with an extinction coefficient at its absorbance maximum greater
than 500 M.sup.-1 cm.sup.-1, preferably at least 5000 M.sup.-1
cm.sup.-1, more preferably at least 50,000 M.sup.-1 cm.sup.-1 at
the excitation wavelength. The lifetime of an excited state
produced following absorption of light in the absence of oxygen
will usually be at least 100 nsec, preferably at least 1 msec. In
general, the lifetime must be sufficiently long to permit energy
transfer to oxygen, which will normally be present at
concentrations in the range of 10.sup.-5 to 10.sup.-3 M depending
on the medium. The sensitizer excited state will usually have a
different spin quantum number (S) than its ground state and will
usually be a triplet (S=1) when, as is usually the case, the ground
state is a singlet (S=0). Preferably, the sensitizer will have a
high intersystem crossing yield. That is, photoexcitation of a
sensitizer will produce the long lived state (usually triplet) with
an efficiency of at least 10%, desirably at least 40%, preferably
greater than 80%. The photosensitizer will usually be at most
weakly fluorescent under the assay conditions (quantum yield
usually less that 0.5, preferably less that 0.1).
Photosensitizers that are to be excited by light will be relatively
photostable and will not react efficiently with singlet oxygen.
Several structural features are present in most useful sensitizers.
Most sensitizers have at least one and frequently three or more
conjugated double or triple bonds held in a rigid, frequently
aromatic structure. They will frequently contain at least one group
that accelerates intersystem crossing such as a carbonyl or imine
group or a heavy atom selected from rows 3-6 of the periodic table,
especially iodine or bromine, or they may have extended aromatic
structures. Typical sensitizers include acetone, benzophenone,
9-thioxanthone, eosin, 9,10-dibromoanthracene, methylene blue,
metallo-porphyrins, such as hematoporphyrin, phthalocyanines,
chlorophylls, rose bengal, buckminsterfullerene, etc., and
derivatives of these compounds having substituents of 1 to 50 atoms
for rendering such compounds more lipophilic or more hydrophilic
and/or as attaching groups for attachment, for example, to an sbp
member. Examples of other photosensitizers that may be utilized in
the present invention are those that have the above properties and
are enumerated in N. J. Turro, "Molecular Photochemistry", page
132, W. A. Benjamin Inc., N.Y. 1965.
The photosensitizers are preferably relatively non-polar to assure
dissolution into a lipophilic member when the photosensitizer is
incorporated in an oil droplet, liposome, latex particle, etc.
The photosensitizers useful in this invention are also intended to
include other substances and compositions that can produce singlet
oxygen with or, less preferably, without activation by an external
light source. Thus, for example, molybdate (MoO.sub.4.sup.=) salts
and chloroperoxidase and myeloperoxidase plus bromide or chloride
ion (Kanofsky, J. Biol. Chem. (1983) 259 5596) have been shown to
catalyze the conversion of hydrogen peroxide to singlet oxygen and
water. Either of these compositions can, for example, be included
in particles to which is bound an sbp member and used in the assay
method wherein hydrogen peroxide is included as an ancillary
reagent, chloroperoxidase is bound to a surface and molybdate is
incorporated in the aqueous phase of a liposome. Also included
within the scope of the invention as photosensitizers are compounds
that are not true sensitizers but which on excitation by heat,
light, or chemical activation will release a molecule of singlet
oxygen. The best known members of this class of compounds includes
the endoperoxides such as 1,4-biscarboxyethyl-1,4-naphthalene
endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide and
5,6,11,12-tetraphenyl naphthalene 5,12-endoperoxide. Heating or
direct absorption of light by these compounds releases singlet
oxygen.
Support or Surface--a surface comprised of a porous or non-porous
water insoluble material. The surface can have any one of a number
of shapes, such as strip, rod, particle, including bead, and the
like. The surface can be hydrophilic or capable of being rendered
hydrophilic and includes inorganic powders such as silica,
magnesium sulfate, and alumina; natural polymeric materials,
particularly cellulosic materials and materials derived from
cellulose, such as fiber containing papers, e.g., filter paper,
chromatographic paper, etc.; synthetic or modified naturally
occurring polymers, such as nitrocellulose, cellulose acetate, poly
(vinyl chloride), polyacrylamide, cross linked dextran, agarose,
polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),
polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,
poly(vinyl butyrate), etc.; either used by themselves or in
conjunction with other materials; glass available as Bioglass,
ceramics, metals, and the like. Natural or synthetic assemblies
such as liposomes, phospholipid vesicles, and cells can also be
employed.
Binding of sbp members to the support or surface may be
accomplished by well-known techniques, commonly available in the
literature. See, for example, "Immobilized Enzymes," Ichiro
Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol.
Chem., 245:3059 (1970).
Particles--particles of at least about 20 nm and not more than
about 20 microns, usually at least about 40 nm and less than about
10 microns, preferably from about 0.10 to 2.0 microns diameter,
normally having a volume of less than 1 picoliter. The particle may
be organic or inorganic, swellable or non-swellable, porous or
non-porous, having any density, but preferably of a density
approximating water, generally from about 0.7 to about 1.5 g/ml,
preferably suspendible in water, and composed of material that can
be transparent, partially transparent, or opaque. The particles may
or may not have a charge, and when they are charged, they are
preferably negative. The particles may be solid (e.g., polymer,
metal, glass, organic and inorganic such as minerals, salts and
diatoms), oil droplets (e.g., hydrocarbon, fluorocarbon, silicon
fluid), or vesicles (e.g., synthetic such as phospholipid or
natural such as cells and organelles). The particles may be latex
particles or other particles comprised of organic or inorganic
polymers; lipid bilayers, e.g., liposomes, phospholipid vesicles;
oil droplets; silicon particles; metal sols; cells; and dye
crystallites.
The organic particles will normally be polymers, either addition or
condensation polymers, which are readily dispersible in the assay
medium. The organic particles will also be adsorptive or
functionalizable so as to bind at their surface, either directly or
indirectly, an sbp member and to bind at their surface or
incorporate within their volume a photosensitizer or a
chemiluminescent compound.
The particles can be derived from naturally occurring materials,
naturally occurring materials which are synthetically modified and
synthetic materials. Natural or synthetic assemblies such as lipid
bilayers, e.g., liposomes and non-phospholipid vesicles, are
preferred. Among organic polymers of particular interest are is
polysaccharides, particularly cross-linked polysaccharides, such as
agarose, which is available as Sepharose, dextran, available as
Sephadex and Sephacryl, cellulose, starch, and the like; addition
polymers, such as polystyrene, polyacrylamide, homopolymers and
copolymers of derivatives of acrylate and methacrylate,
particularly esters and amides having free hydroxyl functionalities
including hydrogels, and the like. Inorganic polymers include
silicones, glasses, available as Bioglas, and the like. Sols
include gold, selenium, and other metals. Particles may also be
dispersed water insoluble dyes such as porphyrins, phthalocyanines,
etc., which may also act as photosensitizers. Particles may also
include diatoms, cells, viral particles, magnetosomes, cell nuclei
and the like.
Where the particles are commercially available, the particle size
may be varied by breaking larger particles into smaller particles
by mechanical means, such as grinding, sonication, agitation,
etc.
The particles will usually be polyfunctional or be capable of being
polyfunctionalized or be capable of being bound to an sbp member,
photosensitizer, or chemiluminescent compound through specific or
non-specific covalent or non-covalent interactions. A wide variety
of functional groups are available or can be incorporated.
Exemplary functional groups include carboxylic acids, aldehydes,
amino groups, cyano groups, ethylene groups, hydroxyl groups,
mercapto groups and the like. When covalent attachment of a sbp
member, chemiluminescent compound or photosensitizer to the
particle is employed, the manner of linking is well known and is
amply illustrated in the literature. See for example Cautrecasas,
J. Biol. Chem., 245:3059 (1970). The length of a linking group may
vary widely, depending upon the nature of the compound being
linked, the nature of the particle, the effect of the distance
between the compound being linked and the particle on the binding
of sbp members and the analyte and the like.
The photosensitizer can be chosen to dissolve in or noncovalently
bind to the surface of the particles. In this case these compounds
will preferably be hydrophobic to reduce their ability to
dissociate from the particle and thereby cause both compounds to
associate with the same particle.
The number of photosensitizer or chemiluminescent molecules
associated with each particle will on the average usually be at
least one and may be sufficiently high that the particle consists
entirely of photosensitizer or chemiluminescer molecules. The
preferred number of molecules will be selected empirically to
provide the highest signal to background in the assay. In some
cases this will be best achieved by associating a multiplicity of
different photosensitizer molecules to particles. Usually, the
photosensitizer or chemiluminescent compound to sbp member ratio in
the particles should be at least 1, preferably at least 100 to 1,
and most preferably over 1,000 to 1.
Latex particles--"Latex" signifies a particulate water suspendible
water insoluble polymeric material usually having particle
dimensions of 20 nm to 20 mm, more preferably 100 to 1000 nm in
diameter. The latex is frequently a substituted polyethylene such
as: polystyrene-butadiene, polyacrylamide polystyrene, polystyrene
with amino groups, poly-acrylic acid, polymethacrylic acid,
acrylonitrile-butadiene, styrene copolymers, polyvinyl
acetate-acrylate, polyvinyl pyrridine, vinyl-chloride acrylate
copolymers, and the like. Non-crosslinked polymers of styrene and
carboxylated styrene or styrene functionalized with other active
groups such as amino, hydroxyl, halo and the like are preferred.
Frequently, copolymers of substituted styrenes with dienes such as
butadiene will be used.
The association of the photosensitizer or chemiluminescent compound
with latex particles utilized in the present invention may involve
incorporation during formation of the particles by polymerization
but will usually involve incorporation into preformed particles,
usually by noncovalent dissolution into the particles. Usually a
solution of the chemiluminescent compound or sensitizer will be
employed. Solvents that may be utilized include alcohols, including
ethanol, ethylene glycol and benzyl alcohol; amides such as
dimethyl formamide, formamide, acetamide and tetramethyl urea and
the like; sulfoxides such as dimethyl sulfoxide and sulfolane; and
ethers such as carbitol, ethyl carbitol, dimethoxy ethane and the
like, and water. The use of solvents having high boiling points in
which the particles are insoluble permits the use of elevated
temperatures to facilitate dissolution of the compounds into the
particles and are particularly suitable. The solvents may be used
singly or in combination. Particularly preferred solvents for
incorporating photosensitizer are those that will not quench the
triplet excited state of the photosensitizer either because of
their intrinsic properties or because they can subsequently be
removed from the particles by virtue of their ability to be
dissolved in a solvent such as water that is insoluble in the
particles. Aromatic solvents are preferred, and generally solvents
that are soluble in the particle. For incorporating
chemiluminescent compounds in particles a solvent should be
selected that does not interfere with the luminescence because of
their intrinsic properties or ability to be removed from the
particles. Frequently, aromatic solvents will also be preferred.
Typical aromatic solvents include dibutylphthalate, benzonitrile,
naphthonitrile, dioctylterephthalate, dichlorobenzene,
diphenylether, dimethoxybenzene, etc.
Except when the photosensitizer or chemiluminescent compound is to
be covalently bound to the particles, it will usually be preferable
to use electronically neutral photosensitizers or chemiluminescent
compounds. It is preferable that the liquid medium selected does
not soften the polymer beads to the point of stickiness. A
preferred technique comprises suspending the selected latex
particles in a liquid medium in which the photosensitizer or
chemiluminescent compound has at least limited solubility.
Preferably, the concentrations of the photosensitizer and
chemiluminescent compound in the liquid media will be selected to
provide particles that have the highest efficiency of singlet
oxygen formation and highest quantum yield of emission from the
chemiluminescent compound in the media but less concentrated
solutions will sometimes be preferred. Distortion or dissolution of
the particles in the solvent can be prevented by adding a miscible
cosolvent in which the particles are insoluble.
Generally, the temperature employed during the procedure will be
chosen to maximize the singlet oxygen formation ability of the
photosensitizer labeled particles and the quantum yield of the
chemiluminescent compound particles with the proviso that the
particles should not melt or become aggregated at the selected
temperature. Elevated temperatures are normally employed. The
temperatures for the procedure will generally range from 20.degree.
C. to 200.degree. C., more usually from 50.degree. C. to
170.degree. C. It has been observed that some compounds that are
nearly insoluble at room temperature, are soluble in, for example,
low molecular weight alcohols, such as ethanol and ethylene glycol
and the like, at elevated temperatures. Carboxylated modified latex
particles have been shown to tolerate low molecular weight alcohols
at such temperatures.
An sbp member may be physically adsorbed on the surface of the
latex particle or may be covalently bonded to the particle. In
cases wherein the sbp member is only weakly bound to the surface of
the latex particle, the binding may in certain cases be unable to
endure particle-to-particle shear forces encountered during
incubation and washings. Therefore, it may be preferable to
covalently bond sbp members to the latex particles under conditions
that will minimize adsorption. This may be accomplished by
chemically activating the surface of the latex. For example, the
N-hydroxysuccinimide ester of surface carboxyl groups can be formed
and the activated particles to reduce nonspecific binding of assay
components to the particle surface, are then contacted with a
linker having amino groups that will react with the ester groups or
directly with an sbp member that has an amino group. The linker
will usually be selected to reduce nonspecific binding of assay
components to the particle surface and will preferably provide
suitable functionality for both attachment to the latex particle
and attachment of the sbp member. Suitable materials include
maleimidated aminodextran (MAD), polylysine, aminosaccharides, and
the like. MAD can be prepared as described by Hubert, et al., Proc.
Natl. Acad. Sci., 75(7), 3143, 1978.
In one method, MAD is first attached to carboxyl-containing latex
particles using a water soluble carbodiimide, for example,
1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. The coated
particles are then equilibrated in reagents to prevent nonspecific
binding. Such reagents include proteins such as bovine gamma
globulin (BGG), and detergent, such as Tween 20, TRITON X-100 and
the like. A sbp member having a sulfhydryl group, or suitably
modified to introduce a sulfhydryl group, is then added to a
suspension of the particles, whereupon a covalent bond is formed
between the sbp member and the MAD on the particles. Any excess
unreacted sbp member can then be removed by washing.
Chemiluminescent compound--compounds that form part of the
compositions of the present invention are enol ethers generally
having the structural portion selected from the group consisting
of: ##STR8## wherein Ar and Ar' are independently aryl wherein one
of Ar or Ar', preferably Ar, is electron donating with respect to
the other. This may be achieved, for example, by the presence of
one or more electron donating groups in one of Ar or Ar'. The part
of the above structures represented by the broken lines are not
critical to the present invention and may be any substituent as
long as such substituent does not interfere with dioxetane
formation and transfer of energy. Generally, the compounds are
those of Compound 2 wherein, preferably, m is 0, and n is 1 to
3.
For the most part the compounds that form part of the present
composition have the structural portion: ##STR9## wherein X is O, S
or N wherein the valency of N is completed with hydrogen or an
organic radical consisting of atoms selected from the group
consisting of C, O, N, S, and P and Ar and Ar' are independently
aryl wherein one of Ar or Ar' is electron donating with respect to
the other.
The broken lines in the above structure signify that the ring can
be independently unsubstituted or substituted with a substituent
having from 1 to 50 atoms. In addition, the substituents may be
taken together to form a ring such as, for example, aryl, which may
in turn be substituted with a substituent having from 1 to 50
atoms.
Exemplary enol ethers, by way of illustration and not limitation,
are set forth in the following chart with reference to the
following structure: ##STR10## wherein Compounds 9-17 have the
following moieties for X, Ar, and Ar'.
______________________________________ X Ar Ar' *
______________________________________ ##STR11## ##STR12## 9 S
##STR13## ##STR14## 10 S ##STR15## ##STR16## 11 S ##STR17##
##STR18## 12 S ##STR19## ##STR20## 13 S ##STR21## ##STR22## 14 S
##STR23## ##STR24## 15 ##STR25## ##STR26## ##STR27## 16 ##STR28##
##STR29## ##STR30## 17 ______________________________________
*Compounds 9-17
The chemiluminescent compounds undergo a chemical reaction with
singlet oxygen to form a metastable intermediate that can decompose
with the simultaneous or subsequent emission of light within the
wavelength range of 250 to 1200 nm. Preferably, the intermediate
decomposes spontaneously without heating or addition of ancillary
reagents following its formation. However, addition of a reagent
after formation of the intermediate or the use of elevated
temperature to accelerate decomposition will be required for some
chemiluminescent compounds. The chemiluminescent compounds are
usually electron rich compounds that react with singlet oxygen,
frequently with formation of dioxetanes or dioxetanones, such as
those represented by the following structure where the substituents
on the carbon (C) atoms are those present on the corresponding
olefin: ##STR31## some of which decompose spontaneously, others by
heating and/or by catalysis usually by an electron rich energy
acceptor, with the emission of light. For some cases the dioxetane
is spontaneously converted to a hydroperoxide whereupon basic pH is
required to reform the dioxetane and permit decomposition and light
emission.
The chemiluminescent compounds of interest will generally emit at
wavelengths above 300 nanometers and usually above 400 nm.
Compounds that alone or together with a fluorescent molecule emit
light at wavelengths beyond the region where serum components
absorb light will be of particular use in the present invention.
The fluorescence of serum drops off rapidly above 500 nm and
becomes relatively unimportant above 550 nm. Therefore, when the
analyte is in serum, chemiluminescent compounds that emit light
above 550 nm, preferably above 600 nm are of particular interest.
In order to avoid autosensitization of the chemiluminescent
compound, it is preferable that the chemiluminescent compounds do
not absorb light used to excite the photosensitizer. Since it will
generally be preferable to excite the sensitizer with light
wavelengths longer than 500 nm, it will therefore be desirable that
light absorption by the chemiluminescent compound be very low above
500 nm.
The chemiluminescent compounds of the present invention can be
prepared in a number of different ways. In one approach a
2-thioethanol derivative is condensed with an appropriate diaryl
substituted alpha-hydroxy ketone (substituted benzoin) where one
aryl is substituted on the ketone carbon and the other is
substituted on the carbon containing the alpha-hydroxy group. The
condensation reaction yields the appropriate enol ether directly.
The above condensation can be carried out in an inert solvent such
as toluene. Usually, the temperature of the reaction is about
90.degree.-130.degree. and the reaction is allowed to proceed for a
period of 5-50 hours. Generally, the reaction is carried out at the
reflux temperature of the combined reagents. The condensation is
carried out in the presence of a Lewis acid, for example, an acyl
chloride, silyl chloride, stannous chloride, etc. The following
reaction scheme is illustrative of the above-described method for
preparing the chemiluminescent compounds of the present invention:
##STR32##
Another reaction scheme for preparing compounds in accordance with
the present invention, particularly those containing an alkyl
radical, is depicted in the following schematic for synthesizing
Compound 13: ##STR33##
In the above synthesis ethyl 5-bromovalerate is condensed with
N-methylaniline to give 22 which is converted by Kilsmeier-Haak
synthesis (DMF/POCl.sub.3) to aldehyde 23. Benzoin condensation of
23 with benzaldehyde yields 24 which is hydrolyzed with potassium
hydroxide and converted to amide 25 with didecylamine and
diphenylphosphoryl azide (DPPA). Conversion to Compound 13 was
carried out by condensation with mercaptoethanol and
trimethylsilylchloride.
Another approach for preparing compounds in accordance with the
present invention, particularly involving regioselective synthesis
is shown in the following schematic for synthesizing Compound 14:
##STR34##
In the above synthesis reaction of p-nitrophenylacetic acid (27)
with decanal in the presence of pd/carbon and hydrogen gas at 100
psi gives didecylamine 28, which is condensed with p-heptylbenzene
to give ketone 29. Bromine and trifluoroacetic acid are used to
brominate 29 and bicarbonate converts the product to benzoin 30.
Conversion to Compound 14 is carried out by condensation with
mercaptoethanol and trimethylsilylchloride.
Ancillary Materials--Various ancillary materials will frequently be
employed in the assay in accordance with the present invention. For
example, buffers will normally be present in the assay medium, as
well as stabilizers for the assay medium and the assay components.
Frequently, in addition to these additives, proteins may be
included, such as albumins, organic solvents such as formamide,
quaternary ammonium salts, polycations such as dextran sulfate,
surfactants, particularly non-ionic surfactants, binding enhancers,
e.g., polyalkylene glycols, or the like. When the photosensitizer
is activated chemically rather than by irradiation, hydrogen
peroxide will often be included as an ancillary reagent. When it is
desired to shift the emission wavelength of the chemiluminescent
compound to longer wavelength or catalyse the decomposition of its
oxygen-activated form, a fluorescent molecule may be employed.
Wholly or Partially Sequentially--when the sample and various
agents utilized in the present invention are combined other than
concomitantly (simultaneously), one or more may be combined with
one or more of the remaining agents to form a subcombination. Each
subcombination can then be subjected to one or more steps of the
present method. Thus, each of the subcombinations can be incubated
under conditions to achieve one or more of the desired results.
One aspect of the present invention is directed to compositions
comprising (a) a metal chelate comprising a metal selected from the
group consisting of europium, terbium, dysprosium, samarium, osmium
and ruthenium in at least a hexacoordinated state and (b) a
compound having a structural portion that is a double bond
substituted with two aryl groups, an oxygen atom and an atom
selected from the group consisting of oxygen, sulfur and nitrogen.
The aryl groups are characterized in that one is electron donating
with respect to the other. The composition of the present invention
comprising a metal chelate and an olefinic compound is generally in
a medium that may be liquid or solid, usually solid particulate.
The liquid medium is usually a high-boiling, water immisinble
liquid such as one from the group comprising toluene, lipids,
fluorocarbons, diphenylether, chlorobenzene, dioctylphthalate,
dimethoxybenzene, mineral oil and triacylglycerides and the solid
particulate medium can be an organic polymer such as polystyrene,
polymethylacrylate, polyacrylate, polyacrylamide, polyvinylchloride
and copolymers thereof, nylon and other polyamides, etc.
Preferably, the composition is incorporated in a latex particulate
material.
The metal chelate is present in an amount to maximize the
chemiluminescent quantum yield and minimize the decay time of
chemiluminescence. Usually, the metal chelate is present at 0.2-500
mM, preferably 2-100 mM. In some circumstances, usually when the
metal chelate is hexacoordinated, reduction in the decay time is
accompanied by a reduction in quantum yield and a balance must be
reached between these two effects. Accordingly, the concentration
of the metal chelate in the composition should be adjusted to
achieve such a balance. The concentration of the chemiluminescent
compound in the composition is usually 0.1-500 mM, preferably 2-100
mM.
Preferred compounds of the present invention have the formula of
Compound 1. Representative of such compounds are Compounds 10-16.
Particularly preferred compounds are those of the formula of
Compound 1 wherein X' is S or NR' wherein R' is lower alkyl or aryl
and D and D' are independently lower alkyl, preferably wherein X'
is S. Particularly preferred compounds within the above are those
wherein D and D' are methyl and R' is methyl or phenyl, and a most
preferred compound is one in which X' is S and D and D' are methyl.
Compound 13 is one of the more preferred of the above
compounds.
One aspect of the present invention is a composition comprising a
latex having incorporated therein Compound 2. Preferred
compositions are those wherein R' is methyl or phenyl and wherein n
is 1 or 2 and m is 0. Preferably, Ar is selected from the group
consisting of 5-member and 6-member aromatic and heteroaromatic
rings. In a preferred embodiment Ar is phenyl substituted with an
electron donating group at a position of the phenyl that is meta or
para to the carbon that is bonded to the double bond and Ar' is
phenyl. Exemplary compositions are those containing a compound
selected from the group consisting of Compounds 9-16. The latex
particles are usually suspendible and have an average diameter of
0.04 to 4000 nanometer. For assays the particle will have an spb
member bound to it and will have an average diameter of 100 to 1000
micrometers.
Another embodiment of the present invention is a method for
determining an analyte. The method comprises (a) providing in
combination (1) a medium suspected of containing an analyte, (2) a
photosensitizer capable in its excited state of activating oxygen
to a singlet state, the photosensitizer associated with a specific
binding pair (sbp) member, and (3) a suspendible latex particulate
material comprising Compound 2. The particulate material has bound
thereto an sbp member. The combination is treated with light,
usually by irradiation, to excite the photosensitizer, and is then
examined for the amount of luminescence emitted. The amount of such
luminescence is related to the amount of analyte in the medium. The
photosensitizer may be incorporated in a second suspendible
particulate material. Particularly useful compositions for
determining an analyte in accordance with the present invention are
those containing Compound 1.
In the assay protocol the components are provided in combination
and the light produced as a function of activation of oxygen by the
sensitizer will be a function of analyte concentration.
Advantageously, the methods of the present invention can be carried
out without heating the medium to produce light. Consequently, the
assay of the present invention can be conducted at a constant
temperature.
The chemiluminescent compound may be bound to a sbp member that is
capable of binding directly or indirectly to the analyte or to an
assay component whose concentration is affected by the presence of
the analyte. The term "capable of binding directly or indirectly"
means that the designated entity can bind specifically to the
entity (directly) or can bind specifically to a specific binding
pair member or to a complex of two or more sbp members which is
capable of binding the other entity (indirectly). Preferably,
assays conducted in accordance with the present invention utilize
one of the above compositions in a latex particle. This latex
particle has an sbp member generally capable of binding directly or
indirectly to the analyte or a receptor for the analyte. When the
sbp members associated with the photosensitizer and the
chemiluminescent compound are both capable of binding to the
analyte, a sandwich assay protocol results. When one of the sbp
members associated with the photosensitizer or chemiluminescent
compound can bind both the analyte and an analyte analog; a
competitive assay protocol can result.
The photosensitizer is usually caused to activate the
chemiluminescent compound by irradiating the medium containing the
above reactants. The medium must be irradiated with light having a
wavelength with energy sufficient to convert the photosensitizer to
an excited state and thereby render it capable of activating
molecular oxygen to singlet oxygen. The excited state for the
photosensitizer capable of exciting molecular oxygen is generally a
triplet state which is more than about 20, usually at least 23,
Kcal/mol more energetic than the photosensitizer ground state.
Preferably, the medium is irradiated with light having a wavelength
of about 450 to 950 nm although shorter wavelengths can be used,
for example, 230-950 nm. The luminescence produced may be measured
in any convenient manner such as photographically, visually or
photometrically to determine the amount thereof, which is related
to the amount of analyte in the medium.
Although it will usually be preferable to excite the
photosensitizer by irradiation with light of a wavelength that is
efficiently absorbed by the photosensitizer, other means of
excitation may be used as for example by energy transfer from an
excited state of an energy donor such as a second photosensitizer.
When a second photosensitizer is used, wavelengths of light can be
used which are inefficiently absorbed by the photosensitizer but
efficiently absorbed by the second photosensitizer. The second
photosensitizer may be bound to an assay component that is
associated, or becomes associated, with the first photosensitizer,
for example, bound to a surface or incorporated in the particle
having the first photosensitizer. When a second photosensitizer is
employed it will usually have a lowest energy singlet state at a
higher energy than the lowest energy singlet state of the first
photosensitizer.
The 632.6 nm emission line of a helium-neon laser is an inexpensive
light source for excitation. Photosensitizers with absorption
maxima in the region of about 620 to about 650 nm are compatible
with the emission line of a helium-neon laser and are, therefore,
particularly useful in the present invention.
The method and compositions of the invention may be adapted to most
assays involving sbp members such as ligand-receptor; e.g.,
antigen-antibody reactions; polynucleotide binding assays, and so
forth. The assays may be homogeneous or heterogeneous, competitive
or noncompetitive. The assay components, chemiluminescent compound
and photosensitizer, can be utilized in a number of ways with (1) a
surface, when employed, (2) nucleic acid or receptor and (3)
nucleic acid or ligand. The association may involve covalent or
non-covalent bonds. Those skilled in the art will be able to choose
appropriate associations depending on the particular assay desired
in view of the foregoing and the following illustrative
discussion.
In a homogeneous assay approach, the sample may be pretreated if
necessary to remove unwanted materials. The reaction for a
noncompetitive sandwich type assay can involve an sbp member,
(e.g., an antibody, nucleic acid probe, receptor or ligand)
complementary to the analyte and associated with a chemiluminescent
compound; a photosensitizer associated with an sbp member, (e.g.,
antibody, nucleic acid probe, receptor or ligand) that is also
complementary to the analyte; the sample of interest; and any
ancillary reagents required. Preferably, at least the
chemiluminescent compound is incorporated in particles to which an
sbp member is attached. The photosensitizer may be directly
attached to an sbp member or it may also be incorporated into
particles. In a competitive protocol one sbp member can be a
derivative of the analyte and the other sbp member can be
complementary to the analyte, e.g., an antibody. In either protocol
the components may be combined either simultaneously or wholly or
partially sequentially. The ability of singlet oxygen produced by
an activated photosensitizer to react with the chemiluminescent
compound is governed by the binding of an sbp member to the
analyte. Hence, the presence or amount of analyte can be determined
by measuring the amount of light emitted upon activation of the
photosensitizer by irradiation, heating or addition of a chemical
reagent, preferably by irradiation. Both the binding reaction and
detection of the extent thereof can be carried out in a homogeneous
solution without separation. This is an advantage of the present
invention over prior art methods utilizing chemiluminescence.
In a heterogeneous assay approach, the assay components comprise a
sample suspected of containing an analyte which is an sbp member;
an sbp member bound to a support, which may be either a
non-dispersible surface or a particle having associated with it one
member of a group consisting of the chemiluminescent compound and
the photosensitizer; and an sbp member having the other member of
the group associated with it wherein the sbp members can
independently, either directly or indirectly, bind the analyte or a
receptor for the analyte. These components are generally combined
either simultaneously or wholly or partially sequentially. The
surface or particles are then separated from the liquid phase and
either the separated phase or the liquid phase is subjected to
conditions for activating the photosensitizer, usually by
irradiating the particular phase in question, and measuring the
amount of light emitted.
The binding reactions in an assay for the analyte will normally be
carried out in an aqueous medium at a moderate pH, generally that
which provides optimum assay sensitivity. Preferably, the
activation of the photosensitizer will also be carried out in an
aqueous medium. However, when a separation step is employed,
non-aqueous media such as, e.g., acetonitrile, acetone, toluene,
benzonitrile, etc. and aqueous media with pH values that are very
high, i.e., greater than 10.0, or very low, i.e., less than 4.0,
usually very high, can be used. As explained above, the assay can
be performed either without separation (homogeneous) or with
separation (heterogeneous) of any of the assay components or
products.
The aqueous medium may be solely water or may include from 0.01 to
80 volume percent of a cosolvent but will usually include less than
40% of a cosolvent when an sbp member is used that is a protein.
The pH for the medium of the binding reaction will usually be in
the range of about 4 to 11, more usually in the range of about 5 to
10, and preferably in the range of about 6.5 to 9.5. When the pH is
not changed during the generation of singlet oxygen the pH will
usually be a compromise between optimum binding of the binding
members and the pH optimum for the production of signal and the
stability of other reagents of the assay. When elevated pH's are
required for signal production, a step involving the addition of an
alkaline reagent can be inserted between the binding reaction and
generation of singlet oxygen and/or signal production. Usually the
elevated pH will be greater than 10, usually 10-14. For
heterogenous assays non-aqueous solvents may also be used as
mentioned above, the main consideration being that the solvent not
react efficiently with singlet oxygen.
Various buffers may be used to achieve the desired pH and maintain
the pH during an assay. Illustrative buffers include borate,
phosphate, carbonate, tris, barbital and the like. The particular
buffer employed is not critical to this invention, but in an
individual assay one or another buffer may be preferred.
Moderate temperatures are normally employed for carrying out the
binding reactions of proteinaceous ligands and receptors in the
assay and usually constant temperature, preferably, 25.degree. to
40.degree., during the period of the measurement. Incubation
temperatures for the binding reaction will normally range from
about 5.degree. to 45.degree. C., usually from about 15.degree. to
40.degree. C., more usually 25.degree. to 40.degree. C. Where
binding of nucleic acids occur in the assay, higher temperatures
will frequently be used, usually 20.degree. to 90.degree., more
usually 35.degree. to 75.degree. C. Temperatures during
measurements, that is, generation of singlet oxygen and light
detection, will generally range from about 20.degree. to
100.degree., more usually from about 25.degree. to 50.degree. C.,
more usually 25.degree. to 40.degree. C.
The concentration of analyte which may be assayed will generally
vary from about 10.sup.-4 to below 10.sup.-16 M, more usually from
about 10.sup.-6 to 10.sup.-14 M. Considerations, such as whether
the assay is qualitative, semiquantitative or quantitative, the
particular detection technique the concentration of the analyte of
interest, and the maximum desired incubation times will normally
determine the concentrations of the various reagents.
In competitive assays, while the concentrations of the various
reagents in the assay medium will generally be determined by the
concentration range of interest of the analyte, the final
concentration of each of the reagents will normally be determined
empirically to optimize the sensitivity of the assay over the
range. That is, a variation in concentration of the analyte which
is of significance should provide an accurately measurable signal
difference.
The concentration of the sbp members will depend on the analyte
concentration, the desired rate of binding, and the degree that the
sbp members bind nonspecifically. Usually, the sbp members will be
present in at least the lowest expected analyte concentration,
preferably at least the highest analyte concentration expected, and
for noncompetitive assays the concentrations may be 10 to 10.sup.6
times the highest analyte concentration but usually less than
10.sup.-4 M, preferably less than 10.sup.-6 M, frequently between
10.sup.-11 and 10.sup.-7 M. The amount of photosensitizer or
chemiluminescent compound associated with a sbp member will usually
be at least one molecule per sbp member and may be as high as
10.sup.5, usually at least 10-10.sup.4 when the photosensitizer or
chemiluminescent molecule is incorporated in a particle.
While the order of addition may be varied widely, there will be
certain preferences depending on the nature of the assay. The
simplest order of addition is to add all the materials
simultaneously. Alternatively, the reagents can be combined wholly
or partially sequentially. When the assay is competitive, it will
often be desirable to add the analyte analog after combining the
sample and an sbp member capable of binding the analyte.
Optionally, an incubation step may be involved after the reagents
are combined, generally ranging from about 30 seconds to 6 hours,
more usually from about 2 minutes to 1 hour before the sensitizer
is caused to generate singlet oxygen and the light emission is
measured.
In a particularly preferred order of addition, a first set of
specific binding pair members that are complementary to and/or
homologous with the analyte are combined with the analyte followed
by the addition of specific binding pair members complementary to
the first specific binding pair members, each associated with a
different member of the group consisting of a photosensitizer and a
composition of the present invention. The assay mixture, or a
separated component thereof, is then irradiated and the light
emission is measured.
In a homogeneous assay after all of the reagents have been
combined, they can be incubated, if desired. Then, the combination
is irradiated and the resulting light emitted is measured. The
emitted light is related to the amount of the analyte in the sample
tested. The amounts of the reagents of the invention employed in a
homogeneous assay depend on the nature of the analyte. Generally,
the homogeneous assay of the present invention exhibits an
increased sensitivity over known assays such as the EMIT.RTM.
assay. This advantage results primarily because of the improved
signal to noise ratio obtained in the present method.
Another aspect of the present invention relates to kits useful for
conveniently performing an assay method of the invention for
determining the presence or amount of an analyte in a sample
suspected of containing the analyte. The kits comprise in packaged
combination: (1) a composition comprising a suspendible latex
particle comprising a compound of the formula of Compound 2,
preferably of Compound 1, where the particle can bind a specific
binding pair (sbp) member, and (2) a photosensitizer capable in its
excited state of activating oxygen to its singlet state. The
photosensitizer can be part of a composition comprising a second
suspendible particle comprising the photosensitizer where the
second particle has bound thereto a sbp member or it may be
directly bound to a sbp member. The kit can further include a
written description of a method in accordance with the present
invention and instructions for using the reagents of the kit in
such method.
To enhance the versatility of the subject invention, the reagents
can be provided in packaged combination, in the same or separate
containers, so that the ratio of the reagents provides for
substantial optimization of the method and assay. The reagents may
each be in separate containers or various reagents can be combined
in one or more containers depending on the cross-reactivity and
stability of the reagents. The kit can further include other
separately packaged reagents for conducting an assay including
ancillary reagents, and so forth.
EXAMPLES
The invention is demonstrated further by the following illustrative
examples. Parts and percentages used herein are by weight unless
otherwise specified. Temperatures are in degrees centigrade
(.degree. C.).
Abbreviations
Ab.sub.F (anti-fluorescein)--Mouse monoclonal antibody to
fluorescein.
Ab.sub..UPSILON.3 (anti-T.sub.3)--mouse monoclonal antibody to
T.sub.3
t-Bu--tert-butyl
TFA--trifluoroacetic acid
T.sub.3 --
.PHI.--chemiluminescence quantum yield
PMT--
EtoAc--ethyl acetate
BSA--Bovine serum albumin
Chl-a--Chlorophyll-a
D-H.sub.2 O--dionized water
DPP--4,7-Diphenylphenanthroline
DPPC--dipalmitoylphosphatidyl choline
DPPG--dipalmitoylphosphatidyl glycerol
DPPE--dipalmitoylphosphatidyl ethanolamine
EDAC--1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide
hydrochloride.
nC.sub.10 --tetra-(n-decyl)phthalocyanin aluminum chloride
complex.
PB--Polystyrene beads
PB/nC.sub.10 --PB containing nC.sub.10
PBS--phosphate buffered saline 0.02M NaPi, 0.14M NaCl/pH 7.2
Pi--Phosphate
Sulfo-NHS--Sulfo-N-hydroxysuccinimide
SATA--S-acetylthioglycolic acid N-hydroxysuccinimide ester
RLU--Relative light units.
NHS--N-hydroxysuccinimide
DMSO--dimethyl sulfoxide
DMF--dimethyl formamide
DCC--dicyclohexylcarbodiimide
TEA--triethylamine
TLC--thin layer chromatography
TNBSA--2,4,6-trinitrobenzenesulfonic acid
BGG--bovine gamma globulin
TMSCl--trimethylsilyl chloride
MeOH--methanol
Biotin-LC.sub.7
-NHS-sulfosuccinimidyl-6-(biotinamido)-hexanoate
.lambda.max ABS--lambda maximum of absorption
.lambda.max EMI--lambda maximum of fluorescence emission
max CH.EM.--lambda maximum of chemiluminescence emission
All monoclonal antibodies were produced by standard hybrid cell
technology. Briefly, the appropriate immunogen was injected into a
host, usually a mouse or other suitable animal, and after a
suitable period of time the spleen cells from the host were
obtained. Alternatively, unsensitized cells from the host were
isolated and directly sensitized with the immunogen in vitro.
Hybrid cells were formed by fusing the above cells with an
appropriate myeloma cell line and culturing the fused cells. The
antibodies produced by the cultured hybrid cells were screened for
their binding affinity to the particular antigen, e.g. TSH or HCG.
A number of screening techniques were employed such as, for
example, ELISA screens. Selected fusions were then recloned.
Example 1
Total Triiodothyronine Assay
I. Bead Preparations
Materials
175 nm Carboxylate modified latex (CML beads) from Bangs
Laboratories.
Ethylene glycol, ethoxy ethanol, benzyl alcohol, chlorophyll-a from
Aldrich.
Europium (III) thienoyl trifluoroacetonate (EuTTA) from Kodak.
Trioctyl phosphine oxide (TOPO) from Aldrich.
Dioxene [1-(4-dimethylaminophenyl)-6-phenyl 1,4 dioxene]:
Prepared by a modification of a procedure described in: Giagnon, S.
D. (1982) University Microfilms International (Ann Arbor,
Mich.)
Procedures
1. Chlorophyll-a Sensitizer Beads
A solution of chlorphyll-a in benzyl alcohol (1.0 mL, 0.6 mM) was
added to 8.0 mL of benzyl alcohol at 105.degree. C. A suspension of
carboxylate modified latex, 175 nm size, in water (10%, 1.0 mL) was
added to the benzyl alcohol solution. The mixture was stirred for 5
min at 105.degree.0 C., and cooled to room temperature. Ethanol
(10.0 mL) was added and the mixture centrifuged. The pellet was
resuspended in a 1:1 ethanol-water mixture (10.0 mL) and the
suspension centrifuged. The same resuspension and centrifugation
procedure was repeated in water (10.0 mL), and the pellet was
resuspended in water (1.8 mL).
Characterization
A. Dye concentration: A solution prepared by adding 10 .mu.L of the
above bead suspension to dioxane (990 .mu.L) was found to have an
absorbance of 0.11 at 660 nm, corresponding to 2.6 .mu.moles of
chlorophyll-a in one gram of beads.
B. Singlet oxygen generation: A mixture of chlorphyll-a beads (200
.mu.g) 2.times.10.sup.-4 moles of anthracene 9,10-dipropionic acid
(ADPA) in two mL of phosphate buffer (50 mM, pH 7.5, containing 100
mM NaCl) was irradiated with a tungsten-halogen lamp equipped with
a 645 nm cut-off filter for 20 min. The beads were removed by
filtration, and the concentration of the oxygenation product was
determined spectrophotometrically at 400 nm. The rate was found to
be 3.0 nmoles of oxygenation product per min. Under the same
conditions, 38 pmoles of a soluble sensitizer, aluminum
phthalocyanin tetrasulfonate generated the same amount of
oxygenation product (the amount of sensitizer in the beads was
200.multidot.10.sup.-6 .multidot.2.6.multidot.10.sup.-6 =520
pmoles).
2. Chlorophyll-a/Tetrabutyl Squarate Sensitizer Beads
A suspension of carboxylated latex beads (175 nm size, 10% solids
in water, 30.0 mL) was centrifuged. The supernatant was discarded
and the pellet was resuspended in ethylene glycol (60.0 mL). The
suspension was heated to 100.degree. C. 9.0 mL of a benzyl alcohol
solution which is 1.67 mM in chlorophyll-a and 3.33 mM in
tetrabutyl squarate [1,3 bis(4-dibutylaminophenyl)squarate] was
added slowly over 3 min to the suspension. The heating was
continued for 7 min, then the suspension was cooled to room
temperature in a water bath. The benzyl alcohol suspension was
added to cold ethanol (120 mL). The mixture was centrifuged and the
supernatant discarded. The pellet was resuspended in 50% ethanol in
water and the suspension was centrifuged. The same resuspension and
centrifugation procedure was repeated in 5% ethanol in water (30
mL).
Characterization
A. Dye concentration. The concentration of the tetrabutyl squarate
in the beads was determined spectrophotometrically as described
above for the chlorophyll-a beads. It was found to be 44 .mu.M dye
in the beads.
B. Singlet oxygen generation. Twenty-five .mu.L of a 5 mM solution
of ADPA in ethanol were added to suspension of beads (100 .mu.g) in
phosphate buffer, pH 7.0 (20 mM, containing 50 mM NaCl). The
mixture was irradiated as above, using a 610 nm long pass filter.
The rate of singlet oxygen formation was calculated from the rate
of the decrease in absorbance (at 400 nm) of the ADPA. It was found
that the beads generated 7.multidot.10.sup.-2 .mu.moles of singlet
oxygen/min.
3. Dioxene/EuTTA/TOPO Acceptor Beads
20 mL of 175 nm carboxylated latex beads (10% suspension in water)
was added to ethoxy ethanol (20.0 mL). The mixture was heated to
90.degree. C. 20 mL of a solution which is 10 mM
2-(p-dimethylaminophenyl)-3-phenyl dioxene, 20 mM EuTTA and 60 mM
TOPO in ethoxy ethanol were added to the mixture. The heating was
continued for 7 min at a temperature up to 97.degree. C. The
mixture was cooled to room temperature. Ethanol (40.0 mL) was added
and the mixture was centrifuged. The pellet was resuspended in 80%
ethanol and the suspension was centrifuged. The resuspension and
centrifugation procedure was repeated in 10% ethanol (36 mL).
Characterization
A. Dye concentration. The concentration of EuTTA in the beads was
determined spectrophotometrically and was found to be 0.07M.
Because the concentration of dioxene cannot be determined in the
presence of EuTTA, it was measured in beads which were dyed with
the dioxene only, 2-(p-dimethylaminophenyl)-3-phenyl dioxene, under
the same conditions. The concentration was found to be 0.016M.
B. Signal generation. A suspension of beads (25 .mu.g) in phosphate
buffer (0.5 mL, 20 mM phosphate, 50 mM NaCl, 0.1% Tween 20, pH 7.0)
was mixed with a solution of 2 .mu.M aluminum phthalocyanine
tetrasulfonate (0.5 mL) in the same buffer. The mixture was
illuminated for one minute with a 125 w tungsten-halogen lamp
equipped with a 610 nm long pass filter. Following illumination,
the mixture was placed in a Turner TD-20e luminometer, and the
luminescence was measured for 20 sec. The intensity was found to be
327 RLU (relative light unit)/sec. The wavelength of the emitted
light was measured using Perkin-Elmer 650-40 scanning
spectrofluorimeter. The major emission peak was centered near 615
nm.
II. Assay Procedure
EDAC/NHS Coupling of Antibody to 40 nm Beads
73.6 mg sulfo-NHS (N-hydroxysulfo-succinimide, Pierce Chemical Co.
#24510 G) was dissolved in 6 mL of a suspension of 4 mg/mL
carboxylate-modified 40 nm polystyrene beads (dyed with
chlorophyll-a and tetrabutyl squarate) in water. 136 uL 0.5M
Na.sub.2 HPO.sub.4 was added. PH was adjusted to 5.2. 136 uL
additional water was added. 130.4 mg EDAC
(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride,
Sigma Chemical Co. #E-6383) in 454 .mu.L water was slowly added to
stirring bead suspension. The suspension was incubated for 20 min
at room temperature. The beads were centrifuged for 20 min. at
15,000 rpm in Sorvall SA-600 rotor at 4.degree. C. The supernatant
was discarded. The beads were then resuspended in 1.2 mL 5 mM
sodium phosphate, pH 5.8, and the suspension was sonicated to
redisperse beads. The beads were slowly added to 4.8 mL of a
stirring solution containing 1.7 mg/mL IgG (mouse monoclonal
anti-fluorescein) and 6.7 mg/mL BSA and 17 mM borax, pH 9.2, and
mixed gently overnight at 4.degree. C. 800 uL 2 M glycine was added
which was then followed by 2.8 mL 50 mg/mL BSA in 0.1M borax to the
bead suspension. The suspension was sonciated and allowed to mix
gently for 3 h at 4.degree. C. The beads were centrifuged for 30
min at 15,000 rpm. The supernatant was discarded. The beads were
resuspended in 3 mL 50 mM sodium phosphate and 150 mM NaCl, pH 7.6,
and the suspension was sonciated. The centrifugation, resuspension
and sonification steps were repeated for a total of three spins.
After the third spin, beads were resuspended in 2.4 mL 50 mM sodium
phosphate and 150 mM NaCl, pH 7.6. The resulting suspension was
sonicated and stored at 4.degree. C.
III. EDAC/NHS Coupling of Avidin-D to 175 nm Beads
4.4 mg sulfo-NHS was dissolved in 0.4 mL of a suspension of 25
mg/mL carboxylate-modified 175 nm polystyrene beads (dyed with
2-(p-dimethylaminophenyl)-3-phenyl dioxene/Eu(TTA)/TOPO) in water.
0.0160 mL 0.25M Na.sub.2 HPO.sub.4 was added. 8 mg EDAC, dissolved
in 0.030 mL water, was added slowly to vortexing bead suspension.
The suspension was incubated for 20 min at room temperature. The
beads were centrifuged 20 min at 15,000 rpm in Sorvall SA-600 rotor
at 4.degree. C. The supernatant was discarded. The beads were
resuspended in 0.6 mL 0.005M sodium phosphate, pH 5.8. The
suspension was sonicated to resuspend beads. The beads were again
slowly added to 3 mL of a stirring solution containing 1.33 mg/mL
avidin-D (Vector) and 17 mM borax, pH 9.2, and mixed gently
overnight at 4.degree. C. 0.004 mL 1M succinic anhydride in DMF was
added. The suspension was incubated for 1 h at 4.degree. C. with
gentle mixing. 0.4 mL 50 mg/mL BSA in 10 mM sodium phosphate and
150 mM NaCl, pH 7.0 was added. The suspension was allowed to mix
gently for 3 h at 4.degree. C. The beads were centrifuged for 30
min at 15,000 rpm. The supernatant was discarded. The beads were
resuspended in 3 mL 50 mM sodium phosphate and 150 mM NaCl, pH 7.6.
The suspension was sonicated. The centrifugation, resuspension and
sonification steps were repeated for a total of three spins. After
the third spin, the beads were resuspended in 2.25 mL 50 mM sodium
phosphate and 150 mM NaCl, pH 7.6. The suspension was sonicated and
stored at 4.degree. C.
IV. Total T.sub.3 Assay
Assay buffer: 0.075M barbital, 0.2M NaCl, 0.4% BSA, 1.25% mouse
IgG, 10 mg/mL dextran sulfate (MW 500,000), 1.0 mg/mL dextran
T-500, 10 .mu.g/mL aggregated IgG.
Beads
Acceptor Beads: Avidin-EDAC, 175 nm, dyed with
2-(p-dimethylaminophenyl)-3-phenyl dioxene/Eu (TTA).sub.3
/TOPO.
Sensitizer Beads: Antifluorescein-EDAC, 40 nm, dyed with
chlorophyll-a/squarate.
Assay Protocol
50 .mu.L of 8-anilino-1-naphthalene sulfonic acid, ammonium salt
(Sigma, A-3125) solution in assay buffer (0.75 mg/mL) was added to
50 .mu.L of T.sub.3 standard or sample. 100 .mu.L of assay buffer
was added. Biotinylated anti-T.sub.3 was prepared according to
standard procedures by reaction of biotin-LC.sub.7 NHS (Pierce
Chemical Company) with monoclonal anti-T.sub.3 followed by
purification by chromatography on a Sephadex column. 50 .mu.L of
biotinylated anti-T.sub.3 (70 ng/mL) in assay buffer was added. The
tracer, T.sub.3 -LC.sub.21 -Fl (1.8 ng/mL) ##STR35## in assay
buffer (50 .mu.L) was added. The mixture was incubated for 15
minutes at 37.degree. C. 500 .mu.L of a suspension of sensitizer
beads (50 .mu.g) and acceptor beads (6.25 .mu.g) in assay buffer
were added, and the mixture was incubated for 15 minutes at
37.degree. C. The "stop solution" (50 .mu.L) (10 .mu.M fluorescein,
0.5 mM biotin) was added.
Signal was read by halogen lamp with a 610 nm cut-off filter, one
minute illumination, 20 sec measurement.
Results
The luminescence signal was plotted as a function of T.sub.3
concentration. Signal modulation was 94% with 8.5 ng/mL T.sub.3. At
0.5 ng/mL the signal modulation was 38%.
Example 2
Chemiluminescence Quantum Yield and Decay Rate Determinations
Preparation of Compound 11
To a stirred solution of 2.55 g of 4-dimethylaminobenzoin (10 mmol)
in 50 mL of dry toluene, 1.2 mL of 2-mercaptoethanol (15 mmol) was
added, followed by 2.5 mL of TMSCl. The reaction mixture was
refluxed under argon for 18 hours, allowed to come to room
temperature and poured in 150 mL of saturated bicarbonate solution.
The two-phase mixture was separated. The organic layer was again
washed with 100 mL of saturated bicarbonate solution. The combined
aqueous layer was extracted with 75 mL of CH.sub.2 CL.sub.2. The
combined organic layers were dried over sodium sulfate (20 g) and
evaporated. The remaining residue was flash chromatographed
(CH.sub.2 Cl.sub.2) to give 2.6 g of Compounds 11 and 20 (4:1
mixture of the 2-regioisomers). The ash colored solid was
recrystallized from CH.sub.2 Cl.sub.2 --MeOH (10:90) mixture to
yield 1.8 g of needle-shaped crystals of a single regioisomer of
compound 11.
M.P. 108.degree.-110.degree. C.; .sup.1 HNMR (CDCl.sub.3, 250MHz):
.delta. 2.85 (s, 6H), 3.22 (t, 2H), 4.5 (t, 2H), 6.55 (d, 2H),
7.1-7.3 (m, 7H).
Mass Spectrum (CI: m/e, relative intensity) Major Peaks: 297
(M.sup.+, 40), 165 (100).
Absorption Spectra (Toluene): 330 nm (.epsilon.13,000).
Photooxygenation Procedure
25 Milligrams of Compound 11 (major regioisomer from above) was
dissolved in 10 mL of CH.sub.2 Cl.sub.2 in a photooxygenation tube.
Approximately 50 mg of polystyrene bound Rose Bengal was added and
oxygen bubbler connected. Oxygen was passed slowly through the
solution while the sample as irradiated with a Dolan-Jenner lamp
equipped with a 500 nm cut-off filter. Progress of the reaction was
monitored by TLC. A spot for the thioester product could be
detected and had a lower R.sub.f (CH.sub.2 Cl.sub.2) than Compound
11. The reaction was judged complete when Compound 13 was
completely consumed. The sensitizer was filtered off and solution
was evaporated on a rotary evaporator to yield 26 mg of thioester
32 as the only product. ##STR36##
1HNMR: (CD.sub.2 CL.sub.2): .delta. 3.05 (s, 6H) , 3.4 (5, 2H),
4.45 (5, 2H), 6.72 (d, 2H), 7.5 (m, 3H), 7.85 (d, 2H), 8.05 (d,
2H).
Mass Spectra (CI, relative intensity) Major Peaks: 329 (M.sup.+,
25), 148 (100).
Absorption Spectrum (CH.sub.2 Cl.sub.2): 342 nm
(.about.30,000).
Fluorescence Spectrum (Toluene): 370 nm.
Fluorescence Measurements
A solution of thioester 32 was taken in four different solvents
(Toluene-dry; CH.sub.2 Cl.sub.2 ; hexane; and acetonitrile) and
placed in a 1-cm square quartz cuvette in the sample compartment of
a Perkin-Elmer 650-40 fluorometer. The sample was excited at the
absorption maxima of each solvent (slit width 2 nm) and emission
spectra (slit width 3 nm) was recorded by scanning from 350 nm to
470 nm. The fluorescence efficiency was determined and tabulated in
Table 1.
TABLE 1 ______________________________________ Efficiency of
Thioester in Different Solvents .lambda. .lambda. ABS EMI Compound
Solvent nM nM .PHI. ______________________________________ Diester*
Toluene 314 360 0.1 400 Thioester 32** Toluene 338 370 0.025
CH.sub.2 Cl.sub.2 340 390 0.07 Hexane 332 370 .about.0.006 CHCN 342
390 .about.0.006 ______________________________________
*2-(p-dimethylaminophenyl)3-phenyl ethyldiester, Giagnon, S. D.
(1982) University Microfilms International (Ann Arbor, Michigan).
**Thioester is rapidly photobleached on excitation at 340 nm in
toluene.
Determination of Quantum Yield of Chemiluminescence.
Preparation of Eu(TTA)Phen:
8.69 g of Eu(TTA).sub.3 .multidot.3H.sub.2 O(10 mmoles, Kodak) and
1.8 g of 1,10-phenanthroline (10 mmoles, Aldrick) in 50 ml of dry
toluene were heated to 95.degree. C. in an oil bath for one 1 hour.
Toluene was removed under reduced pressure. The ash coloured solid
was cystallized from 10 ml of toluene to yeild 10 grams of
Eu(TTA).sub.3 Phen.
Absorption spectrum: 270 nm (20,000), 340 nm (60,000) (Toluene)
1.R(KBr): Cm.sup.-1 : 3440(s), 1600(s), 1540(s), 1400(s),
1300(s)
Energy Transfer to Eu(TTA).sub.3 Phen
A solution of Compound 11 (regioisomers from above) (0.1 mM) (8:2
mixture), aluminum phthalocyanine (0.1 .mu.M), and Eu(TTA).sub.3
Phen from above (0-4.0 mM) in dry toluene was placed in a 1-cm
square quartz cuvette (two sided silvered) in the sample
compartment of a Spex Fluorolog spectrophotometer. The temperature
of the sample holder was maintained by a circulating external water
bath at 25.degree. C. A 640 nm cut-off filter was placed in front
of the excitation beam. The sample solutions were placed in the
sample compartment for at least 3 minutes for thermal equilibrium
to be reached. The emission was recorded in the time drive mode.
Samples were irradiated at 680 nm (slit width 24 nm) until a steady
state of emission at 613 nm (slit width 8 nm) was reached. The
steady state light intensity at various concentrations of
Eu(TTA).sub.3 Phen was recorded and is summarized in Table 2. From
the steady state light intensity quantum yields were determined.
Double reciprocal plots of chemiluminescence intensity against
Eu(TTA).sub.3 Phen concentration were linear.
TABLE 2 ______________________________________ Chemiluminescence
Efficiency as a Function of Eu(TTA).sub.3 Phen Concentration
Compound 11* Eu(TTA.sub.3 Phen nM nM RLU at 613 nm
______________________________________ 0.1 0 -- 0.1 0.05 7.43
.times. 10.sup.4 0.1 0.1 1.8 .times. 10.sup.5 0.1 0.2 2.89 .times.
10.sup.5 0.1 0.5 6.13 .times. 10.sup.5 0.1 1.0 9.45 .times.
10.sup.5 0.1 2.0 1.17 .times. 10.sup.6 0.1 4.0 1.32 .times.
10.sup.6 0.1** 4.0 1.6 .times. 10.sup.6
______________________________________ *Except for last run
Compound 11 used in these experiments contained 20% of its
regioisomer 20 **Compound 11 used in this experiment was greater
than 98% of a single regioisomer.
Chemiluminescence from Dioxene 9
Experiment 1: A solution of dioxene 9 (0.1 mM) and aluminum
phthalocyanine (0.1 .mu.M) in dry toluene was irradiated at 680 nm
as described above. The emission in light intensity at 400 nm (slit
width 8 nm) was recorded as a function of irradiation time. The
light intensity was 8793 RLU's for 180 seconds of irradiation
(average of three experiments).
Experiment 2: Rate of dioxene 9 dioxetane decomposition was
monitored by decay of chemiluminescence of an aerated solution in
dry toluene at 25.degree. C. Rate of decomposition was monitored in
the presence of 1.0.mu.M aluminum phthalocyanine and dioxene (less
than 0.1 mM of dioxene). The chemiluminescence decay was monitored
on Spex Fluorolog spectrophotometer under previously described
conditions. The rate constant of decay at 25.degree. C. was
2.88.times.10.sup.-4 S.sup.-1.
Preparation of Acceptor Beads
Four mL of 20% suspension (400 mg) of washed 175 nm carboxylate
modified latex was diluted with 3 mL of ethoxyethanol in a 25 mL
round bottom (R.B.) flask with a stir bar. The R.B. flask was then
placed in an oil bath at 105.degree. C. and stirred for 10 minutes.
Then, 3.3 mM thioxene 11 and 15.5 mM Eu(TTA).sub.3 DPP was added;
the beads were stirred for 5 minutes more. At this point 1.0 mL of
0.1N NaOH was added slowly over 5 minutes. During all the
additions, the oil bath temperature was maintained at 105.degree.
C. The oil bath temperature was slowly allowed to drop to room
temperature over 2 hours.
After cooling, the mixture was diluted with 20 mL of ethanol and
centrifuged (12,500 rpm, 30 minutes). Supernatants were discarded
and the pellets resuspended in ethanol by sonication.
Centrifugation was repeated, and the pellet was resuspended in
water; and centrifugation was repeated. The pellet was resuspended
in 5 mL of aqueous ethanol to a final volume of 40 mL. The final
concentration of the beads was 10 mg/mL.
The concentration of Eu(TTA).sub.3 DPP was determined
spectrophotometrically. An aliquot of the bead suspension was
reduced to dryness under a stream of dry argon and the residue
dissolved in dioxane. Using a density of 1.06 g/cc for polystyrene,
(.epsilon.340 nm=6.7.times.10.sup.4) for Eu(TTA).sub.3 and
(.epsilon.270 nm=4.0.times.10.sup.4) for DPP, the concentration of
Eu(TTA).sub.3 DPP was determined to be 100 mM. The concentration of
compound 11 in the beads could not be determined because its
absorbance was masked by Eu(TTA).sub.3 DPP.
Chemiluminescence of the beads was measured in an ORIEL luminometer
using water-soluble aluminum phthalocyanine sensitizer. An aliquot
of beads was diluted to 100 .mu.g/mL in phosphate buffer pH 8.0
containing 0.1% Tween-20. 1.0 .mu.M of aluminum phthalocyanine
tetrasulfonic acid was added and chemiluminescent signal was
measured as a function of irradiation time. An identical sample was
also placed in a Spex Fluorolog fluorometer and irradiated at 680
nm (slit width 20 nm; 640 cut-off filter). The chemiluminescence
emission spectra was recorded by scanning from 570 nm to 620 nm.
Chemiluminescence decay and quantum yields is summarized in Table
3.
Determination of Quantum Yields in Beads
Dioxene 9 Beads
A solution of dioxene 9 beads (0.2 mg) and aluminum phthalocyanine
tetrasulfonic acid (2.5 .mu.M) in phosphate buffer (pH 8.2; 50 mM
0.1% Tween-20) was placed in a 1 cm quart cuvette (two sides
silvered) in the sample compartment of a Spex Fluorolog
spectrophotometer. The temperature of the sample holder was
maintained 25.degree. C. A 640 cut-off filter was placed in front
of the excitation beam. The sample solutions were placed in the
sample compartment for at least 3 minutes for thermal equilibrium
to be reached. The light emission at 360 nm was followed in the
time drive mode. Samples were irradiated at 680 nm (slit width 24
nm) for 60 seconds. The emission at 360 nm (slit width 16 nm) was
recorded with time for 5000 seconds. Total light emitted was
determined by the cut-weigh method. Peak shape correction was also
done by the cut and weigh method. The total light emitted at 360 nm
was 8.87.+-.0.2.times.10.sup.4 RLU's/4500 seconds (after peak shape
correction; average of 2 experiments).
Dioxene 9: Eu(TTA).sub.3 TOPO Beads
A solution of dioxene 9 Eu(TTA).sub.3 TOPO beads (0.2 mg) and
aluminum phthalocyanine tetrasulfonic acid (2.5 .mu.M) in phosphate
buffer (pH 8.2, 50 mM 0.1% Tween-20) was placed in a 1-cm quartz
cuvette (two sides silvered) in the sample compartment of a Spex
Fluorolog spectrophotometer. The rest of the experiment was
performed as described for dioxene 9 beads. The light emission from
beads was followed at 613 nm (slit width 16 nm).
Total light emitted was determined by the cut and weigh method. PMT
correction was done as described previously in solution studies.
The total light emitted at 613 nm was 25.0.+-.0.3.times.10.sup.5
RLU's/4500 seconds (after PMT correction; average of 2
experiments)
Steady State Methods
Dioxene 9: Eu(TTA)TOPO Beads. A solution of dioxene 9:
Eu(TTA).sub.3 TOPO beads (0.5 mg) and aluminum phthalocyanine
tetrasulfonic acid (0.05 .mu.M) in phosphate buffer (pH 8.2; 50 mM
0.1% Tween-20) was placed in a 12-75 mM test tube in the sample
compartment of an Oriel chemiluminometer. The temperature of the
sample holder is 37.degree. C. A 610 cut-off filter was placed in
front of the excitation beam. The sample solutions were placed in
the sample compartment for at least 5 minutes for thermal
equilibrium to be reached. The sample was irradiated for 30-second
intervals followed by a 5-second read time until a steady state of
emission is reached. The average intensity at steady state emission
is 21,000.+-.1000 RLU's (3 experiments).
Compound 11: Eu(TTA)DPP Beads. A solution of thioxene 11:
Eu(TTA).sub.3 DPP beads (0.5 mg) and aluminum phthalocyanine
tetrasulfonic acid (0.05 .mu.M) in phosphate buffer (pH 8.2, 50 mM,
0.1% Tween-20) was placed in 12-75 mM test tube in the sample
compartment of an Oriel chemiluminometer. The temperature of the
sample holder is 37.degree. C. A 610 cut-off filter was placed in
front of the excitation beam. The sample solutions were placed in
the sample compartment for at least 5 minutes for thermal
equilibrium to be reached. The sample was irradiated for 6-second
intervals followed by 3 seconds read time until a steady state of
emission was reached. The average intensity at steady state
emission is 32,000.+-.1000 (3 experiments).
TABLE 3 ______________________________________ Chemiluminescent
Properties of Thioxene 11 and Dioxene 9 .lambda.max Compound Medium
(CH.EM) t1/2 .PHI. ______________________________________ 11
Toluene 400 nM 2.1 sec low* (100 .mu.M) 11 + Toluene 613 nM 1.8-2.1
sec 0.20*** Eu(TTA).sub.3 Phen (4 mM) 11 + CML beads 613 nM decay
0.46 Eu(TTA).sub.3 DPP multiphasic (100 mM) (initial t1/2 at
37.degree. C. is -0.5 secs) 9 Toluene 420 nM 3462 sec 0.015 (100
.mu.M) ** CML beads 360 nM decay 0.008 multiphasic 9 + CML beads
613 nM decay 0.31 Eu(TTA).sub.3.TOPO (Major) multiphasic (16 mM)
400 nM ______________________________________ *less than 0.0003
**Control, no compound present ***0.37 for Eu(TTA).sub.3 Phen
concentration extrapolated to infinity
Example 3
Preparation of C-26 Thioxene (Compound 13):
A. 62 g of N-methyl aniline (0.5 mole) and 62 g of ethyl
5-bromovalerate (0.3 mole) were heated to 100.degree. C. in a
sealed tube for 16 hours. The reaction mixture was cooled to room
temperature and poured into 100 ml of ethyl acetate. The ethyl
acetate solution was washed with 20% sodium hydroxide (3.times.100
ml). The aqueous layer was extracted with 50 ml of ethyl acetate.
The combined ethyl acetate solution was dried over sodium sulphate
(50 g) and removed under reduced pressure. The residue was
distilled under high vacuum (130.degree.-137.degree. C.) to yield
60 g of N-methyl N-ethyl valerate aniline.
.sup.1 H NMR (CDCl.sub.3, 250 MHz):.delta. 1.3 (t, 3H), 1.65 (m,
4H), 2.3 (t, 2H), 2.8 (s, 3H), 3.3 (t, 2H), 4.2 (q, 2H), 6.65 (d,
2H), 7.2 (m, 3H) .
B. To a stirred solution of DMF (8.8 g) in an ice bath POCl.sub.3
(5.06 g) was added slowly. After the addition was complete, the
reaction is stirred at 4.degree. C. for 10 minutes. N-methyl
N-ethyl valeroyl aniline from Part A above (3.76 g) was added and
the reaction was heated to 100.degree. C. for 1 hour. The reaction
mixture was poured into ice and neutralized with 20% sodium
hydroxide. The mixture was extracted with ethyl acetate (3.times.50
ml). The combined ethyl acetate solution was dried over sodium
sulphate (50 g) and removed under reduced pressure. The residue was
passed through silica gel (CH.sub.2 Cl.sub.2 .fwdarw.CH.sub.2
Cl.sub.2 :EtOAc 9:2).
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 1.2 (t, 2H), 1.6 (m,
4H), 2.3 (t, 2H), 2.9 (s, 3H) 3.3 (t, 2H), 4.1 (q, 2H), 6.6 (d,
2H), 7.6 (d, 2H), 9.7 (s, 1H).
C. To a refluxing solution of 5.0 g of N-methyl
N-ethyl-.omega.-valeroyl p-formyl aniline from Part B above (20
mmole) and 2 g of potassium cyanide in 60% ethanol under argon was
added 2.15 g of benzaldehyde (20 mmole) in 20 ml of ethanol in 90
minutes. The reaction mixture was refluxed for 15 minutes more and
extracted with ethyl acetate (3.times.50 ml). The combined ethyl
acetate solution was dried over sodium sulphate (50 g) and removed
under reduced pressure. The product was purified on preparative TLC
(hexane: ethyl acetate 5:1) to yield 2.2 g of substituted
benzoin.
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 1.3 (t, 3H), 1.6 (m,
4H), 2.4 (t, 2H), 2.9 (s, 3H), 3.3 (t, 2H), 4.1 (q, 2H), 4.8 (d,
1H), 5.8 (d, 1H), 6.5 (d, 2H), 7.3 (m, 5H), 7.8 (d, 2H).
D. To a stirred solution of the benzoin from Part C above (1.1 g)
in 15 ml of ethanol was added 7 ml of water and 100 mgs of KOH. The
reaction was stirred at room temperature for 3 hours. TLC (silica
gel , CH.sub.2 Cl.sub.2 :EtOAc 9:1) showed no starting material.
The solvent was neutralized and the carboxylic acid product was
extracted with ethyl acetate (5.times.50 ml). The combined ethyl
acetate solution was dried over sodium sulphate (50 g) and removed
under reduced pressure. The carboxylic acid product was used as is
for the next step.
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 1.6 (m, 4H), 2.4 (t,
2H), 2.9 (s, 3H), 3.3 (t, 2H), 5.8 (s, 1H), 6.5 (d, 2H), 7.3 (m,
5H), 7.8 (d, 2H).
E. To a stirred solution of the carboxylic acid from Part D above
(1.7 g, 5 mmole) and didecyl amine (1.9 g, 6.3 mmole) in 80 ml of
DMF at 4.degree. C. was added DPPA (1.8 g, 8 mmole) followed by
addition of triethyl amine (1.25 ml). The reaction mixture was
stirred at 4.degree. C. and then at room temperature for 16 hours.
The solvent was neutralized and the product was extracted with
ethyl acetate (5.times.50 ml). The combined ethyl acetate solution
was dried over sodium sulphate (50 g) and removed under reduced
pressure. The product was purified on preparative TLC (CH.sub.2
Cl.sub.2 : ethyl acetate 9:1) to yield 2.6 g of substituted amide
benzoin.
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 0.8 (t, 6H), 1.3 (m,
36H), 1.6 (m, 12H), 2.3 (t, 2H), 2.7 (m, 4H), 3.0 (s, 3H), 3.3 (m,
6H), 4.8 (d, 1H), 5.8 (d, 1H), 6.5 (d, 2H), 7.3 (m, 5H), 7.8 (d,
2H).
F. To a stirred solution of 1.5 g of substituted benzoin (2.5
mmole) in 50 ml of dry toluene, 1.2 ml of 2-thioethanol (15 mmole)
was added, followed by 2.5 ml of TMSCl. The reaction mixture was
refluxed in an oil bath under argon for 30 hours. The reaction
mixture was allowed to come to room temperature poured into 150 ml
of saturated bicarbonate solution. The organic layer was separated
and washed with 100 ml of saturated bicarbonate solution. The
combined aqueous layer was extracted with 75 ml of CH.sub.2
Cl.sub.2.
The combined organic solution was dried over sodium sulphate (50 g)
and removed under reduced pressure. The product was purified on
silica gel (CH.sub.2 Cl.sub.2 : ethyl acetate 9:1) to yield 1.2 g
of C-26 thioxene as an pale yellow oil.
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 0.8 (t, 6H), 1.3 (m,
36H), 1.6 (m, 12H), 2.3 (t, 2H), 2.8 (s, 3H), 3.3 (m, 9H), 4.5 (t,
2H), 6.5 (d, 2H), 7.1 (d, 2H), 7.3 (m, 5H).
Mass Spectrum (CI: m/e) M.sup.+ 662. Absorption Spectra (Toluene):
330 nm (.epsilon.13,000).
Example 4
Preparation of C-8 Thioxene (Compound 15): ##STR37## A. To a
refluxing solution of 3.0 g of p-dimethylamino-benzaldehyde (20
mmole) and 2 g of potassium cyanide in 60% ethanol under argon was
added 4.4 g of p-octyl benzaldehyde (20 mmole, Kodak) in 20 ml of
ethanol in 90 minutes. The reaction mixture was refluxed for 15
minutes more and extracted with ethyl acetate (3.times.50 ml). The
combined ethyl acetate solution was dried over sodium sulphate (50
g) and removed under reduced pressure. The product was purified on
preparative TLC (hexane: ethyl acetate 5:1) to yield 1.2 g of
substituted benzoin.
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 0.85 (t, 3H), 1.3 (m,
12H), 1.5 (m, 2H), 2.5 (t, 2H), 2.9 (s, 6H), 4.8 (d, 1H), 5.8 (d,
1H), 6.5 (d, 2H), 7.3 (q, 4H), 7.8 (d, 2H).
B. To a stirred solution of 0.94 g of substituted benzoin from Part
A above (2.5 mmole) in 50 ml of dry toluene, 1.2 ml of
2-thioethanol (15 mmole) was added, followed by 2.5 ml of TMSCl.
The reaction mixture was refluxed in an oil bath under argon for 30
hours. The reaction mixture was allowed to come to room temperature
and poured into 150 ml of saturated bicarbonate solution. The
organic layer was separated and washed with 100 ml of saturated
bicarbonate solution. The combined aqueous layer was extracted with
75 nl of CH.sub.2 Cl.sub.2. The combined organic solution was dried
over sodium sulphate (50 g) and removed under reduced pressure. The
product was purified on silica gel (CH.sub.2 Cl.sub.2 : ethyl
acetate 9:1) to yield 0.75 g of C-8 thioxene Compound 15 as pale
yellow solid.
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 0.8 (t, 3H), 1.3 (m,
10H), 1.6 (m, 2H), 2.5 (t, 2H), 2.9 (s, 6H), 3.3 (t, 2H), 4.5 (t,
2H), 6.5 (d, 2H), 7.1 (d, 2H), 7.3 (m, 5H).
Mass Spectrum (CI: m/e, relative intensity) 409 (M.sup.+ 100), 165
(40). Absorption Spectra (Toluene): 330 nm (.epsilon.3,000).
Example 5
Preparation of N-Phenyl Oxazine (Compound 16): ##STR38## A. 5 g of
p-dimethylaminobenzoin was dissolved in 5 ml of CH.sub.2 Cl.sub.2
and stirred in an ice bath. 10 ml of SOCl.sub.2 was added and the
reaction mixture stirred for 1 hour. The solvent was removed under
reduced pressure and the product was crystallized from MeOH.
.sup.1 H NMR (CDCl.sub.3, 250 MHz): .delta. 3.0 (s, 6H), 6.3 (s,
1H), 6.5 (d, 2H), 7.4 (m, 5H) 7.8 (d, 2H).
B. 0.271 g of P-(2-phenyl-2-chloro acetyl) dimethylaminobenzene (1
mmole) and 0.274 g of N-(2 hydroxy ethyl) aniline (2.0 mmole) were
dissolved in 3 ml of dry ethanol and heated in a sealed tube at
80.degree. C. for 8 hours. On cooling the product crystallized out
as pale yellow needles, which was filtered and dried to yield 0.2 g
of N-phenyl oxazine Compound 15.
1H NMR (CDCl.sub.3, 250 MHz) .delta. 3.0 (bs, 6H), 3.7 (bt, 2H),
4.4 (bt, 2H), 6.5 (bd, 2H), 7.4 (m, 12H). Mass Spectrum (CI: m/e,
relative intensity) 356 (M.sup.+, 100), 180 (70).
Example 6
Preparation of N-Phenyl Indole Oxazine (Compound 17): ##STR39##
0.283 g of 3-(2-phenyl-2-chloro acetyl) N-methylindole (1 mmole)
(H. Nakamura and T. Goto, Heterocyles, 10, 167-170 (1978). and
0.274 g of 2-anilino ethanol (2.0 mmole) were dissolved in 3 ml of
dry ethanol and heated in a sealed tube at 80.degree. C. for 8
hours. On cooling the product crystallized out as pale yellow
needles which was filtered and dried to yield 0.21 g of N-phenyl
indole oxazine Compound 17.
1H NMR (CDCl.sub.3, 250 MHz) .delta. 3.7 (bs, 3H), 3.8 (bt, 2H),
4.4 (bt, 2H), 7.2 (bm, 15H). Mass Spectrum (CI: m/e, relative
intensity) 366 (M.sup.+ 100), 180 (70).
Example 7
Table 4 summarizes the properties of Compounds 11, 16, and 17
determined in a manner similar to that described in Example 2.
TABLE 4 ______________________________________ Properties of
Chemiluminescent Compounds And Compositions .lambda.max .lambda.max
.lambda.max Compound** (AbS) (EMI) (CH.EM) t1/2 .PHI.
______________________________________ 11 330 nM 400 nM 400 nM 2.1
sec Low* (b) 11 + 615 nM 1.3 sec 0.0024 Eu(TTA).sub.3 (a) (b) 11 +
615 nM 1.8 sec 0.14 Eu(TTA).sub.3 Phen (a) (b) 16 400 nM 550 nM 120
sec Low* (b) 16 + Eu(TTA).sub.3 615 nM 11 sec 0.005 (1.5 .times.
10.sup.-4 M) 16 + Eu(TTA).sub.3 615 nM 3.5 sec 0.04 (5.0 .times.
10.sup.-4 M) (b) (c) (d) 17 550 nM 120 sec Low* 17 + Eu(TTA).sub.3
615 nM 12 sec 0.04 (0.6 .times. 10.sup.-4 M) 17 + Eu(TTA).sub.3 615
nM 2 sec 0.026 (0.6 .times. 10.sup.-4 M) (b) (c) (d)
______________________________________ *less than 0.0003 **in
toluene (a)R.sup.1 O.sub.2Rate of reaction of singlet oxygen with
thioxene in toluene is 18.9 .times. 10.sup.7 M.sup.-1 sec.sup.-1.
After correction fo regioisomers and rate of reaction, the quantum
yield was determined. (b)Quantum yield determined by steady state
method. (c)Assuming that the rate of reaction of singlet oxygen
with morphilino oxene and dioxene is the same. (d)The rate of
chemiluminescence decay and quantum yield depend on Eu(TTA).sub.3
concentration.
The above discussion includes certain theories as to mechanisms
involved in the present invention. These theories should not be
construed to limit the present invention in any way, since it has
been demonstrated that the present invention achieves the results
described.
The above description and examples disclose the invention including
certain preferred embodiments thereof. Modifications of the methods
described that are obvious to those of ordinary skill in the art
are intended to be within the scope of the following claims and
included within the metes and bounds of the invention.
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