# Patent

## Title
Non-sugar sweeteners for use in test devices

## Abstract

The present invention provides methods, compositions, and kits for rapid detection of analytes in a sample. Surprisingly, it has been found that bulking materials having non-sugar sweeteners effectively enhance chromatographic detection methods by providing component stability and controlled release. Thus, the novel components and methods disclosed herein provide a completely new modality of chromatographic analyte detection.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/564,768, filed Apr. 22, 2004, and U.S. Provisional Application No. 60/568,805, filed May 5, 2004, both of which are herein incorporated by reference in their entirety for all purposes.


BACKGROUND OF THE INVENTION
Numerous analytical methods have been developed for determining the presence or absence and/or quantifying the amount of various analytes in tissues and fluids of organisms, such as blood, urine, fecal material, or tissue biopsy. Lateral flow chromatography is, perhaps, one of the more common of these analytical methods.
Lateral flow chromatography assays and devices are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,569,608, 5,120,643, 5,656,503, 4,855,240, and 5,591,645, British Patent GB 2204398A, and European patent EP 0323605 B1) and such assays are commercially available on a retail or OEM basis for numerous analytes.
Lateral flow immunoassays typically involve the application of a liquid test sample suspected of containing an analyte to be detected to an application zone of a lateral flow (immunochromatographic) test strip. The strip is comprised of a matrix material (e.g., paper, nitrocellulose, etc., see, e.g., U.S. Pat. No. 5,569,608) through which the test fluid and analyte suspended or dissolved therein can flow by capillarity from the application zone to a detection zone where a visible signal, or absence of such, reveals the presence or absence of the analyte.
Typically, the strip will include means for immunospecifically binding the analyte to be detected with its specific binding partner (e.g., where the analyte is an antigen, the binding partner is an antibody or antibody fragment, and vice versa) which bears a detectable label. In one such scheme, as disclosed in U.S. Pat. No. 4,446,232, the strip contains an enzyme labeled, mobile binding partner for the analyte which is in a zone downstream from the sample application zone. If the analyte is present in the test sample, it will combine with its labeled binding partner to form a complex which will flow along the strip to a zone which contains a substrate for the enzyme label capable of providing a signal (e.g., a colored response) in the presence of the enzyme label.
The strip typically contains a zone in which an analyte is immobilized, so that the labeled binding partner which does not combine with analyte, due to absence of analyte in the sample, will be captured and thereby inhibited from reaching a zone downstream. There have been published various modifications of this technique, many of which involve some competitive specific binding system in which the presence or absence of analyte in the test sample is determined by the detection or lack thereof of labeled binding partner in a particular device zone. In U.S. Pat. No. 4,868,108 there is disclosed a similar scheme with the addition of an immobilized capture reagent for the enzyme labeled binding partner in a particular zone to concentrate the enzyme label and enhance its ability to react with the enzyme substrate and thereby render the assay more sensitive.
Not all of the schemes for immunochromatography rely on an enzyme labeled binding partner/enzyme substrate as providing the signal for detection of the analyte. In U.S. Pat. No. 4,806,311 there is disclosed a multizone test device for the specific binding assay determination of an analyte and an immobilized binding partner together with a downstream zone for receiving labeled reagent which migrates thereto from an upstream zone. The downstream zone contains an immobilized form of a binding substance for the labeled reagent. The labeled reagent bears a detectable chemical group having a detectable physical property such as a luminescent group (e.g. a fluorescent or phosphorescent moiety), radioisotopes and electroactive moieties. U.S. Pat. No. 4,313,734 describes the use of gold sols as labels for antibodies which are detectable.
Many lateral flow immunochromatography systems utilize particulate (microparticle) markers (e.g., gelatin, dyed latex, or colloidal gold) which are labeled with a binding partner (e.g., antibody or antigen) that binds the analyte of interest.
The microparticles or other detectable moieties attached to an analyte binding moiety (e.g., an antibody or antigen) are dried onto (or otherwise localized in) either a lateral flow chromatographic strip or onto a sample application pad (typically glass fiber) which in turn is affixed to one end of a strip of chromatographic medium such as nitrocellulose. Another material binding to the analyte of interest is affixed to the chromatographic medium at or near the end opposite to the end having the application pad.
The liquid sample to be analyzed is placed on the pad, causing the suspension of the microparticles into the liquid and allowing any analyte in the liquid sample to bind to the analyte-binding material attached to the microparticles. The liquid sample leaves the application pad by diffusion and capillary action and begins to migrate along the nitrocellulose strip carrying the microparticles down the strip along with the liquid. When the liquid containing the suspended microparticles arrives at the region of the chromatographic strip bearing the second binding material, the analyte (if present in the original sample) will form a bridge between the analyte-binding material on the microparticles and the analyte-binding material affixed to the strip, resulting in the immobilization of the microparticles at that point on the strip where the analyte-binding material is affixed. This immobilization of the microparticles results in a visible signal (e.g., a colored band or dot) at this point on the strip. If the analyte is not present in the sample, the microparticles will continue past this location on the chromatographic strip and a visible signal will not be produced.
It will be appreciated that other labels (e.g., fluorescent labels) besides microparticles can be utilized. In addition, a single chromatography strip can contain reagents to detect or quantify a number of different analytes.
It will also be appreciated that the lateral flow strip can use an analyte detection that does not involve an antibody-antigen recognition system. Thus, for example, the strip can be impregnated in a detection zone with a chemical that reacts with the analyte itself to produce a signal.
Existing lateral flow chromatographic devices often fail to provide sufficient stability to the components of the test device, such as peptides, proteins (e.g. antibodies and fragments thereof), ligands, and nucleic acids. In addition, currently existing devices do not provide adequate control over the time in which the analyte is allowed to bind to detectable reagents of the assay. By providing a novel bulking material, the present invention solves these and other needs.
BRIEF SUMMARY OF THE INVENTION
It has been discovered that, surprisingly, bulking materials having non-sugar sweeteners effectively enhance chromatographic detection methods by providing component stability and controlled release. Thus, the novel components and methods disclosed herein provide a completely new modality of chromatographic analyte detection.
In a first aspect, the present invention provides a test device for detecting the presence of an analyte in a sample. The device includes a chromatographic medium having a sample release zone. The sample release zone includes a bulking material having a non-sugar sweetener component.
In an exemplary embodiment, the test device is a lateral flow test device used to detect the presence of an analyte in a sample. The device includes a chromatographic medium that, upon wetting, allows the sample to migrate through various zones that are in fluid communication. The chromatographic medium includes an analyte binding zone, an assay zone and a sample release zone. The analyte binding zone includes a mobile detectable analyte binding reagent capable of binding to the analyte to form a mobile reagent-analyte complex. The assay zone includes an immobilized assay reagent capable of immobilizing the mobile detectable analyte binding reagent. The sample release zone includes a bulking material having a non-sugar sweetener compound. The non-sugar sweetener compound is typically selected from polyalcohol sweetener, peptidyl sweetener, halo-substituted sugar derivative, hydroxypyrone sweetener, and sulfimide sweetener. The assay zone is in fluid communication with the analyte binding zone upon wetting of the chromatographic medium. The sample release zone is upstream of the assay zone and in fluid communication with the assay zone upon wetting of the chromatographic medium.
In another aspect, the present invention provides a method of stabilizing an antibody in a dry state. The method includes contacting the antibody with a bulking material. Bulking materials are described above and are equally applicable to the present methods. The bulking material includes a non-sugar sweetener compound. In an exemplary embodiment, the non-sugar sweetener compound selected from polyalcohol sweetener, peptidyl sweetener, hydroxypyrone sweetener, halo-substituted sugar derivative, and sulfimide sweetener.
In another aspect, the present invention provides methods of determining the presence or absence of an analyte in a sample using the test device of the present invention. The method includes contacting the analyte binding zone of the test device with a sample and allowing the sample to flow from the sample release zone to the assay zone.
In another aspect, the present invention provides kits for use in detecting the presence of an analyte in a sample. The kit includes a test device of the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the results of anti-mouse antibody visualization studies using bulking materials with various non-sugar sweeteners or trehalose (a sugar).
FIG. 2 illustrates the results of THC-BSA conjugate visualization studies using bulking materials with various non-sugar sweeteners or trehalose (a sugar).



DETAILED DESCRIPTION OF THE INVENTION
Introduction
The present invention provides methods, compositions, and kits for rapid detection of analytes in a sample. Surprisingly, it has been found that bulking materials having non-sugar sweeteners effectively enhance chromatographic detection methods by providing component stability and controlled release. The methods and devices are useful in a variety of testing formats, including lateral flow fertility tests, drug of abuse tests, infectious disease tests, environmental tests, and food inspection tests.
Definitions
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.
As used herein, “nucleic acid” means DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g., phosphorothioates, methylphosphonates), 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole. Modifications can also include 3′ and 5′ modifications such as capping with a fluorophore or another moiety.
“Peptide” refers to a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a polypeptide or protein. Unnatural amino acids, for example, β-alanine, phenylglycine and homoarginine may also be included. Amino acids that are not gene-encoded may also be used in the present invention. Furthermore, amino acids that have been modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules and the like may also be used in the invention. All of the amino acids used in the present invention may be either the D- or L-isomer. The L-isomer is generally preferred. In addition, other peptidomimetics are also useful in the present invention. As used herein, “peptide” refers to both glycosylated and unglycosylated peptides. Also included are peptides that are incompletely glycosylated by a system that expresses the peptide. For a general review, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).
The term “amino acid,” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
A “natural peptide,” as used herein, is a peptide that includes only naturally occurring amino acids and not amino acid analogs.
The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. Alkyl groups which are limited to hydrocarbon groups are termed “homoalkyl”.
The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkyl, as exemplified, but not limited, by —CH2CH2CH2CH2—. Likewise, the term “heteroalkylene” means a divalent radical derived from an heteroalkyl. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2, —S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, —CH═CH—N(CH3)—CH3, O—CH3, —O—CH2—CH3, and —CN. Up to two heteroatoms may be consecutive, such as, for example, —CH2—NH—OCH3 and —CH2—O—Si(CH3)3. Similarly, the term “heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, —CH2—CH2—S—CH2—CH2— and —CH2—S—CH2—CH2—NH—CH2—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O)2R′— represents both —C(O)2R′— and —R′C(O)2—. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R′′, —OR′, —SR′, and/or —SO2R′. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —NR′R′′ or the like, it will be understood that the terms heteroalkyl and —NR′R′′ are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —NR′R′′ or the like.
The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene” and “heterocycloalkylene,” by themselves or as part of another substituent, means a divalent radical derived from a cycloalkyl or heterocycloalkyl, respectively.
The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (preferably from 1 to 3 rings) which are fused together or linked covalently. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazol 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms “arylene” and “heteroarylene,” by themselves or as part of another substituent, means a divalent radical derived from a aryl or heteroaryl, respectively.
For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).
The term “oxo” as used herein means an oxygen that is double bonded to a carbon atom.
Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and “heteroaryl”) are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R′′, —SR′, -halogen, —SiR′R′′R′′′, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R′′, —OC(O)NR′R′′, —NR′′C(O)R′, —NR′—C(O)NR′′R′′′, —NR′′C(O)2R′, —NR—C(NR′R′′R′′′)═NR′′′′, —NR—C(NR′R′′)═NR′′′, —S(O)R′, —S(O)2R′, —S(O)2NR′R′′, —NRSO2R′, —CN and —NO2 in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R′, R′′, R′′′ and R′′′′ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R′′, R′′′ and R′′′′ groups when more than one of these groups is present. When R′ and R′′ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R′′ is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., —CF3 and —CH2CF3) and acyl (e.g., —C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like).
Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R′′, —SR′, -halogen, —SiR′R′′R′′′, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R′′, —OC(O)NR′R′′, —NR′′C(O)R′, —NR′—C(O)NR′′R′′′, —NR′′C(O)2R′, —NR—C(NR′R′′R′′′)═NR′′′′, —NR-C(NR′R′′)═NR′′′, —S(O)R′′, —S(O)2R′, —S(O)2NR′R′′, —NRSO2R′, —CN and —NO2, —R′, —N3, —CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R′′, R′′′ and R′′′′ are preferably independently selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R′′, R′′′ and R′′′′ groups when more than one of these groups is present.
Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —T—C(O)—(CRR′)q-U- , wherein T and U are independently —NR—, —O—, —CRR′— or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r—B—, wherein A and B are independently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)2—, —S(O)2NR′— or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —(CRR′)s—X′—(C′′R′′′)d—, where s and d are independently integers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)2—, or —S(O)2NR′—. The substituents R, R′, R′′ and R′′′ are preferably independently selected from hydrogen or substituted or unsubstituted (C1-C6)alkyl.
As used herein, the term “heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention.
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
A “sample” as used herein, refers to a representative part or a single item from a larger group chosen for analysis using the methods and/or test device of the present invention. A variety of samples may be analyzed using methods and devices of the present invention. Samples include environmental or biological materials derived from a bodily, cellular, viral and/or prion source. Some samples are derived from biological fluids such as saliva, blood and urine. In some embodiments, the biological fluids include whole cells, cellular organelles or cellular molecules such as a protein, protein fragment, peptide, carbohydrate or nucleic acid. The biological material can be endogenous or non-endogenous to the source. For example, in one embodiment, the biological material is a recombinant protein harvested from a bacteria and engineered using molecular cloning techniques (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference). In another embodiment, the sample comprises a chemically synthesized biological material such as a synthetic protein, protein fragment, peptide, carbohydrate or nucleic acid.
The term “wetting,” as used herein, means contacting with a liquid. The liquid may include, for example, organic solvents and/or aqueous solvents. For example, the chromatographic media may be wetted by contacting the chromatographic media with a liquid sample or with a liquid solvent, such as an organic solvent, water, or an aqueous solvent.
A “sugar,” as used herein refers to a naturally occurring simple sugar in linear or cyclic form. When in linear form, the simple sugar is an aldehyde or ketone derivative of a straight chain polyhydroxy alcohol. Typically, the sugar includes from three to six carbon atoms (e.g., aldotrioses, tetroses, pentoses, and hexoses). When in cyclic form, the sugar ring contains an oxygen heteroatom as in, for example, a pyranose or furanose sugar structure. A “sugar,” as used herein, does not include non-natural sugar derivatives.
“A” or “an,” as used in a claim of the present application, means at least one.
Test Devices
In a first aspect, the present invention provides a test device for detecting the presence of an analyte in a sample. The device includes a chromatographic medium having a sample release zone. The sample release zone includes a bulking material having a non-sugar sweetener component. The chromatographic device may further include additional test zone(s) (e.g. reaction zones, detection zones, and the like), which are well known in the art of analyte testing.
The chromatographic medium used herein may include a porous, solid phase material that allows liquid samples to migrate into various reaction and detection zones. The migration may proceed through any appropriate means of chromatographic transport, such as capillary action, pressure, electrophoresis, gravity, and the like.
In an exemplary embodiment, the test device is a lateral flow chromatographic device. The use of a chromatographic medium for the detection of analytes is well known, especially in the art of lateral flow chromatographic assays (e.g. home pregnancy testing and rapid drug analysis testing). Lateral flow chromatographic test formats useful in the present invention are discussed in detail in the background section and, for example, in U.S. Pat. Nos. 5,120,643; 4,168,146, 4,517,288; 4,452,901; 4,094,647; 4,361,537; 6,187,598, and 6,534,320, which are herein incorporated by reference in their entirety for all purposes.
In an exemplary embodiment, the lateral flow chromatographic device is a “lateral flow chromatography strip.” A lateral flow chromatography strip refers to a test strip utilized for lateral flow chromatography. Lateral flow (chromatography) assays typically involve the application of a liquid test sample suspected of containing an analyte to be detected. The sample may be applied to a lateral flow chromatographic strip. The strip includes a chromatographic media (e.g., paper, nitrocellulose, etc., see, e.g., U.S. Pat. No. 5,569,608) through which a liquid sample can flow by capillary action. Where the detection of the analyte utilizes an antibody or antibody fragment, the assay may be referred to as a lateral flow immunochromatography assay and the strip a lateral flow immunochromatography strip.
Although lateral flow chromatographic test devices are well known in the art, no currently known lateral flow chromatographic devices include non-sugar sweetener components for sample release and/or component stability. Therefore, the present invention is not limited by any particular chromatographic medium or test device format. Rather, the current invention encompasses all known lateral flow test device formats with the improvement of a sample release zone that includes a bulking material having at least one non-sugar sweetener component. The devices of this invention can be readily assembled with any of a variety of commercially available lateral flow chromatography assays (e.g., Syntron QuickScan 6, Syntron Bioresearch, Carlsbad, Calif.; Avitar Visualine II, Avitar Technologies, Canton, Mass.; Determine HIV-1/2, Abbott Laboratories, Abbott Park, Ill.; Clear Blue and E.P.T., Inverness Medical, Waltham, Mass.; Tirage BNP Test, Biosite, Inc., San Diego, Calif.; QuickVue tests, Quidel Corporation, San Diego, Calif.; OSOM tests, Genzyme, Waltham, Mass.; Directigen, BD Diagnostic Systems, Sparks, Md.).
A variety of analytes may be detected using the methods of the present invention, including hormones, drugs of abuse, impurities, infectious agents, nucleic acids, and disease markers. Thus, the devices, methods and kits of the present invention are useful, for example, in fertility tests, (e.g., urine and serum pregnancy tests, and LH ovulation predicator tests; tumor marker tests); drug of abuse (DOA) tests (e.g., methamphetamine, amphetamine, morphine, cocaine, marijuana (THC), PCP, benzodiazepine, barbiturates, methadone, tricyclic, MDMA and multi-drug test formats); infectious disease tests (e.g. HIV, Hepatitis-B, Hepatitis-C, Syphilis, Chlamydia, HPV, Herpes); H. Pylori fecal test (e.g. legionella pneumonia urine test); biowarfare agents (anthrax, ricin toxin, smallpox); and food inspection (e.g. rotavirus test, aflatoxin, genetically modified organisms-GMOs).
Exemplary Lateral Flow Assay Formats
In an exemplary embodiment, the lateral flow test device used to detect the presence of an analyte in a sample includes a chromatographic medium that, upon wetting, allows the sample to migrate through various zones that are in fluid communication. The chromatographic medium typically includes an analyte binding zone, an assay zone and a sample release zone. The analyte binding zone includes a mobile detectable analyte binding reagent capable of binding to the analyte to form a mobile reagent-analyte complex. The assay zone includes an immobilized assay reagent capable of immobilizing the mobile detectable analyte binding reagent. The sample release zone includes a bulking material having a non-sugar sweetener compound. The non-sugar sweetener compound is typically selected from polyalcohol sweetener, peptidyl sweetener, halo-substituted sugar derivative, hydroxypyrone sweetener, and sulfimide sweetener.
The assay zone is in fluid communication with the analyte binding zone upon wetting of the chromatographic medium. The sample release zone is upstream of the assay zone and in fluid communication with the assay zone upon wetting of the chromatographic medium. Therefore, a liquid sample may migrate from the sample release zone to the assay zone.
In an exemplary embodiment, the sample is in liquid form and contains at least one analyte. The analyte is applied to the chromatographic medium thereby wetting the chromatographic medium. The mobile detectable analyte binding reagent binds to the analyte after the analyte enters the analyte binding zone, thereby forming a mobile reagent-analyte complex. The mobile reagent-analyte complex migrates to the assay zone. By detecting the presence or absence of the mobile detectable analyte binding reagent in the assay zone, the presence of an analyte in a sample may be determined.
The lateral flow test device may optionally include a control zone and/or a labeling zone. The control zone includes an immobilized capture reagent that binds to any labeled reporter agent, including a mobile detectable analyte binding reagent. The immobilized capture reagent binds to the mobile detectable analyte binding reagent and/or the mobile detectable analyte binding reagent portion of the mobile reagent-analyte complex. The control zone is downstream of the assay zone and in fluid communication with the assay zone upon wetting of the chromatographic medium.
The labeling zone includes a detectable labeling reagent capable of binding to the mobile detectable analyte binding reagent. The labeling zone is located downstream of the analyte binding zone and upstream of the assay zone. The labeling zone is in fluid communication with the sample release zone and the assay zone upon wetting of the chromatographic medium.
For reasons of clarity, the lateral flow test device may be discussed in terms of a single chromatographic medium having zones with individual components. However, it should be understood that test devices having more than one chromatographic medium are also within the scope of the invention. In addition, each chromatographic medium may include more than one type of zone, region and/or components thereof. For example, each chromatographic medium may include more than one analyte binding zone, assay zone, and/or sample release zone. Each zone may include more than one reagent within the zone. Thus, the methods of the present invention may be used to detect the presence of multiple analytes in parallel.
In some embodiments, the sample release zone partially or fully overlaps with the analyte binding zone. Thus, the sample release zone and the analyte binding zone may form a single zone, sometimes referred to herein as an release-binding zone. The release-binding zone includes one or more non-sugar sweetener compounds and a mobile detectable analyte. The release-binding zone is in fluid communication with the assay zone upon wetting of the chromatographic medium.
The sample release zone may also form a zone that is separate and distinct from the analyte binding zone. Where the sample release zone is non-overlapping with the analyte binding zone, the sample release zone is in capillary contact with the analyte binding zone. The sample release zone may be upstream or downstream from the analyte binding zone. In an exemplary embodiment, the sample release zone is downstream from the analyte binding zone.
The sample release zone may be in capillary contact with the assay zone in addition to the analyte binding zone, upon wetting of the medium. The sample release zone is typically downstream of the analyte binding zone and upstream of the assay zone.
Analyte Binding Zone
The analyte binding zone includes a mobile detectable analyte binding reagent that is capable of binding to the analyte in a sample. A wide variety of mobile detectable analyte binding reagents are useful in the present invention.
Mobile detectable analyte binding reagents useful in conjunction with the present invention include those materials which are members of a specific binding pair, also referred to herein as a ligand and receptor binding pair. The ligand and receptor are related in that the receptor specifically binds to the ligand, being capable of distinguishing the ligand from other materials having similar characteristics. Exemplary binding pairs include any known specific ligand and receptor pair found in nature or constructed using recombinant techniques (e.g. fusion proteins), chemical synthesis, or immunonological methods including antibody or peptide expressing phage display libraries. Therefore, mobile detectable analyte binding reagents include any appropriate biomolecule, such as peptides, proteins, carbohydrates, steroids, nucleic acids, sugars (including simple and complex sugars), and natural or unnatural derivatives thereof.
For example, analytes in a sample may be recombinantly engineered to include a protein tag able to specifically bind a protein tag binder. The protein tag binder is, or forms a portion of, the mobile detectable analyte binding reagent. A wide variety of protein tag and tag binder pairs are known in the art and are useful in the present invention, including, for example: glutathione-S-transferase (GST) and Glutathione (GSH), maltose-binding protein and maltose, chitin-binding protein and cellulose, thioredoxin and OligoGlutamic acid, HA peptide and HA; His and NTA (Nitrilotriacetic acid, with a metal such as Ni, Co, Fe, Cu); S and S-peptide; PKA peptide and PKA; polyArg (6-10 Arg) and polyGlutamic acid (10-15 amino acids); GBD and galactose; streptavidin and biotin; thioredoxin and polyglutamic acid (10-15 amino acids); polyAsp and polyarginine (10-15 amino acids); and KSI and polyphenylalanine or polyleucine (10-30 amino acids).
Methods of constructing and expressing genes that encode fusion proteins are well known to those of skill in the art. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (2000 Supplement) (Ausubel); Cashion et al., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864.
The mobile detectable analyte binding reagent may be, or form a portion of, an antibody or antibody fragment that binds specifically to the analyte. Antibodies useful with the present invention include, for example, intact antibodies, Fab, F(ab′)2, Fv, Fd, Fd′ and scFv structures. In an exemplary embodiment, the antibodies are, or are derived from, IgG antibodies, IgM antibodies, or mixtures thereof. The antibodies are typically essentially free of association with antibodies capable of binding with non-analyte molecules. The antibodies may be polyclonal or monoclonal. Antibodies may be commercially available or may be obtained by mouse ascites, tissue culture or other techniques known to the art. A typical description of hybridoma procedure for the production of monoclonal antibodies may be found in Wands, J. R., and V. R. Zurawski, Gastroenterology 80:225 (1981); Marshak-Rothstein, A., et al.; J. Immunol. 122:2491 (1979); Oi, V. Y. and L. A. Herzenberg, “Immunoglobulin Producing Hybrid”, Mishell, B. B. and S. M. Shiigi (eds.) Selected Methods in Cellular Immunology, San Francisco: W. H. Freeman Publishing, 1979; and U.S. Pat. No. 4,515,893 issued to Kung, et al. The use of mixtures of monoclonal antibodies of differing antigenic specificities or of monoclonal antibodies and polyclonal antibodies may be employed. Regardless of the particular source or type of antibodies, however, it is preferred that they be generally free of impurities. The antibodies may be purified by column chromatographic or other conventional means but are preferably purified according to known affinity purification techniques.
Useful antigens and haptens include those materials, whether natural or synthesized, which present antigenic determinants for which the analyte antibodies are specifically reactive when presented on the chromatographic strip materials of the invention. Synthesized antigens include those which are constructed according to conventional chemical syntheses as well as those constructed according to recombinant DNA techniques. Antigen materials may also be labeled with enzymes and colloidal particles according to the invention and used in sandwich type assays for the detection of antibody analytes or in competition assays for the detection of antigen analytes.
In some embodiments, the mobile detectable analyte binding reagent includes a nucleic acid. Thus, the devices of the present invention are useful in the practice of a wide range of nucleic acid hybridization assays. DNA and RNA hybridization materials useful in practicing the present invention include DNA and RNA polynucleotide probes having base sequences generally complementary to those of analyte gene materials. The probes of the invention will generally have between about 17 and about 10,000 bases and preferably between about 20 and about 5,000 bases. The probes need not be perfectly complementary to the base sequences of analyte gene materials and will generally hybridize provided about 70% or greater homology exists between the base sequences. Conditions relating to DNA and RNA hybridization are disclosed generally in Crosa, et al., J. Bact. 115(3), 904-911 (1973). Polynucleotide probe materials may be obtained according to techniques well known in the art. See, e.g., Kornberg, DNA Replication, W. H. Freeman and Co., San Francisco, 670-679 (1978); Dallas, et al., J. Bacteriol. 139, 850-858 (1979) and So, et al., Nature, 277, 453-456 (1979).
Detectable Labels
The mobile detectable analyte binding reagents of the present invention optionally include a detectable label. Any appropriate label is useful in the current invention, including, for example, luminescent labels, radioactive isotopic labels, enzymatic labels, and other labels well known in the art. Useful labels may be detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, magnetic, electromagnetic, optical or chemical means. Exemplary labels include magnetic bead labels (e.g., DynabeadsTM); fluorescent dye labels (e.g., fluorescein isothiocyanate, texas red, rhodamine, green fluorescent protein, and the like); radiolabels (e.g., H3, I125, S35, C14, or P32); enzyme labels (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA); calorimetric labels such as colloidal gold, silver, selenium, or other metals and metal sol labels (see U.S. Pat. No. 5,120,643, which is herein incorporated by reference in its entirety for all purposes), or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) bead labels; up-converting reporter labels; carbon black labels; and semiconducting particle labels (e.g. quantum dot conjugates made from nanometer-scale crystal of semiconductor material (CdSe) coated with an additional semiconductor shell (ZnS)). Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; 4,366,241; 6,312,914; 5,990,479; 6,207,392; 6,423,551; 6,251,303; 6,306,610; 6,322,901; 6,319,426; 6,326,144; and 6,444,143, which are herein incorporated by reference in their entirety for all purposes.
A variety of fluorescent labels can be incorporated into the compositions of the invention. Many such labels are commercially available from, for example, the SIGMA chemical company (Saint Louis, Mo.), Molecular Probes (Eugene, Oreg.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill. Furthermore, those of skill in the art will recognize how to select an appropriate fluorophore for a particular application and, if it not readily available commercially, will be able to synthesize the necessary fluorophore de novo or synthetically modify commercially available fluorescent compounds to arrive at the desired fluorescent label.
Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, and fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.
Detectable labels may be associated with the mobile detectable analyte binding reagents by any appropriate means, including, for example, covalent bonding, hydrogen bonding, van der Waal forces, π bond stacking, hydrophobic interactions, and ionic bonding.
The detectable labels may be covalently attached to the mobile detectable analyte binding reagents of the present invention using a reactive functional group, which can be located at any appropriate position. When the reactive group is attached to an alkyl, or substituted alkyl chain tethered to an aryl nucleus, the reactive group may be located at a terminal position of an alkyl chain. Reactive groups and classes of reactions useful in practicing the present invention are generally those that are well known in the art of bioconjugate chemistry. Currently favored classes of reactions available with reactive known reactive groups are those which proceed under relatively mild conditions. These include, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982.
Useful reactive functional groups include, for example:
(a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;
(b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.
(c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
(d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups;
(e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
(f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;
(g) thiol groups, which can be converted to disulfides or reacted with acyl halides;
(h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated or oxidized;
(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc;
(j) epoxides, which can react with, for example, amines and hydroxyl compounds; and
(k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis.
The reactive functional groups can be chosen such that they do not participate in, or interfere with, the reactions necessary to assemble the labeled mobile detectable analyte binding reagents. Alternatively, a reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those of skill in the art will understand how to protect a particular functional group from interfering with a chosen set of reaction conditions. For examples of useful protecting groups, See Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.
Linkers may also be employed to attach the detectable labels to the mobile detectable analyte binding reagents. Linkers may include reactive groups at the point of attachment to the detectable label and/or the mobile detectable analyte binding reagents. Any appropriate linker may be used in the present invention, including substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycoalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and substituted or unsubstituted heteroarylene. Other useful linkers include those having a polyester backbone (e.g. polyethylene glycol), nucleic acid backbones, amino acid backbones, and derivatives thereof. A wide variety of useful linkers are commercially available (e.g. polyethylene glycol based linkers such as those available from Nektar, Inc. of Huntsville, Ala.).
The detectable label may also be non-covalently attached to the mobile detectable analyte binding reagents using any appropriate binding pair. Binding pairs are discussed above in the context of analyte—mobile detectable analyte binding reagent interactions and are equally applicable to attaching a detectable label to a mobile detectable analyte binding reagent.
Assay Zone
The assay zone includes an immobilized assay reagent capable of immobilizing the mobile detectable analyte binding reagent. Typically, the immobilized assay reagent specifically binds to the mobile detectable analyte binding reagent, analyte, or mobile reagent-analyte complex.
A wide variety of immobilized assay reagents may be used to implement an array of lateral flow device formats. For example, the immobilized assay reagent may form half of a binding pair. Binding pairs are discussed above in the context of mobile reagent-analyte complexes and are equally applicable to the binding of immobilized assay reagents to the mobile detectable analyte binding reagent, analyte, or mobile reagent-analyte complex. Exemplary binding pairs include any known specific ligand and receptor pair found in nature or constructed using recombinant techniques (e.g. fusion proteins) or immunonological methods. Therefore, immobilized assay reagents include any appropriate biomolecule, such as peptides, proteins, carbohydrates, steroids, nucleic acids, sugars (including simple and complex sugars), and natural or unnatural derivatives thereof.
In an exemplary embodiment, the lateral flow chromatographic device provides a format conducive to a sandwich-type assay. In this embodiment, the immobilized assay reagent is specifically reactive with the analyte and not specifically reactive with the mobile detectable analyte binding reagent. Thus, the immobilized assay reagent is capable of binding the analyte. Typically, the mobile detectable analyte binding reagent includes a detectable label. Therefore, where the mobile detectable analyte binding reagent binds to the analyte to form a mobile reagent-analyte complex, the immobilized assay reagent will bind to the analyte portion of the mobile reagent-analyte complex to form an immobilized mobile reagent-analyte complex. Detection of the detectable label within the assay zone indicates the presence of the analyte in the sample.
In another exemplary embodiment, the lateral flow chromatographic device provides a format conducive to a competition-type assay. In this embodiment, the immobilized assay reagent is specifically reactive with the mobile detectable analyte binding reagent and not specifically reactive with the analyte. For example, the immobilized assay reagent may bind to substantially the same site on or within the mobile detectable analyte binding reagent as the analyte. Thus, when the analyte is bound to the mobile detectable analyte binding reagent, binding of the immobilized assay reagent the mobile detectable analyte binding reagent is substantially reduced. Typically, the mobile detectable analyte binding reagent includes a detectable label. Detection of the detectable label within the assay zone indicates the absence or low concentration of analyte in the sample. In a related embodiment, the immobilized assay reagent is an immobilized analyte or derivative thereof. In another related embodiment, the lateral flow chromatographic device further comprises a control zone, which is discussed below.
A variety of methods may be employed to immobilize the assay reagents of the present invention to the chromatographic medium. Useful techniques include covalent and non-covalent binding, including the use of binding pairs and reactive functional groups discussed above in the context of attaching detectable labels to mobile detectable analyte binding reagents.
Control Zone
The lateral flow chromatographic devices of the present invention may optionally include a control zone. The control zone includes an immobilized capture reagent that immobilizes a reagent having a detectable label, such as the mobile detectable analyte binding reagent. The control zone is downstream of the assay zone and in fluid communication with the assay zone upon wetting of the chromatographic medium.
The mobile detectable analyte binding reagent may be immobilized by any appropriate means, including covalent and non-covalent binding (e.g. covalent bonding, hydrogen bonding, van der Waal forces, π bond stacking, hydrophobic interactions, and ionic bonding). Thus, binding pairs and reactive functional groups discussed above are equally applicable to the immobilized capture reagents.
In some embodiments, the immobilized capture reagent binds directly to the mobile detectable analyte binding reagent. In other embodiments, the immobilized capture reagent immobilizes the mobile detectable analyte binding reagent by binding to a reagent that is bound to the mobile detectable analyte binding reagent. The reagent that is bound to the mobile detectable analyte binding reagent is also referred to herein as an immobilization tag.
A wide variety of immobilized capture reagents are useful in binding to the immobilized capture reagent or the immobilization tag, including those reagents making up one half of a binding pair, as discussed above, as well as non-specific binders, such as protein A, protein G, and poly-L-lysine. For example, where the mobile detectable analyte binding reagent or immobilization tag includes an antibody or antibody fragment having an Fc region, then the immobilized capture reagent may be an Fc binder such as protein G, protein A, or recombinant protein A/G (a gene fusion product secreted from a non-pathogenic form of Bacillus which contains four Fc binding domains from protein A and two from protein G). Where the mobile detectable analyte binding reagent or immobilization tag is a nucleic acid, the immobilized capture reagent may include a nucleic acid that is capable of hybridizing to the mobile detectable analyte nucleic acid or immobilization tag nucleic acid. The immobilized capture reagent may also include a general binding agent, such as poly-L-lysine, that binds to the immobilization tag or the mobile detectable analyte.
In some embodiments, the immobilization reagent is a blocking reagent or a biomolecule, that is bound to some portion of the mobile detectable analyte binding reagent (e.g. the colloidal gold label). In related embodiments, the immobilized capture agent is an anti-BSA antibody, an anti-biomolecule antibody, or a general binder, such as poly-L-lysine.
As described above, the immobilized capture reagent may immobilize the mobile detectable analyte binding reagent, either alone or in combination with other reagents attached thereto (such as an analyte or immobilization tag). Thus, the immobilized capture reagent may bind to the immobilization reagent, and/or the mobile detectable analyte binding reagent portion of the mobile reagent-analyte complex. Thus, the immobilized capture reagent may immobilize the mobile detectable analyte binding reagent in the presence, and/or in the absence, of the analyte. Typically, the mobile detectable analyte binding reagent includes a detectable label. Therefore, the detection of the detectable label confirms the presence of the mobile detectable analyte thereby indicating that the lateral flow chromatographic device is functioning properly.
In some embodiments, the reagent having a detectable label is not the mobile detectable analyte binding reagent. Rather the reagent is simply a mobile control reagent having a detectable label. The mobile control reagent may be, or include, an immobilization tag as described above. Immobilization and subsequent detection of the mobile control reagent indicates that the lateral flow chromatographic device is functioning properly.
Labeling Zone
The lateral flow chromatographic device optionally includes a labeling zone. The labeling zone contains a detectable labeling reagent capable of binding to the mobile detectable binding reagent. The labeling zone is downstream of the analyte binding zone and upstream of the assay zone. The labeling zone is in fluid communication with the sample release zone and the assay zone upon wetting of the chromatographic medium. Therefore, as the mobile detectable binding reagent passes through the labeling zone, the detectable labeling reagent attaches to form a labeled mobile detectable binding reagent.
Useful detectable labels and methods for their attachment to the mobile detectable binding reagent are discussed above and are equally applicable to the detectable labeling reagents.
Sink Zone
The lateral flow chromatographic device optionally includes a sink zone downstream of, and in fluid communication with the assay zone upon wetting of the chromatographic medium. The sink zone includes a sorbent material. The function of the sink zone is to aid in the capillary movement of a sample through the lateral flow chromatographic device. Therefore, any appropriate sorbent material (i.e. adsorbent and/or absorbent) is useful as a component of the sink zone. Adsorbent materials are insoluble materials that coat liquid on the material surface, including pores and capillaries, without substantial swelling (e.g., less than about 50%). Absorbent materials are insoluble materials that draw in and retain liquid causing substantial swelling (e.g. more than about 50%).
A wide variety of sorbent materials are known in the art and are applicable to the current invention, including, for example, inorganic microporous sorbent materials (e.g., zeolites, clays, silica gels, activated alumina, and the like), polyesters, coated plastics, coated rubbers, sponges (both natural and synthetic), and paper products (e.g. cellulose and nitrocellulose based materials).
Chromatographic Media
Chromatographic media useful with the present invention include those chromatographic substrate materials having capillarity and the capacity for chromatographic solvent transport of non-immobilized reagents and reactive sample components by means of a selected chromatographic solvent. The chromatographic substrate materials used with the invention may be in the form of strips or other forms including, but not limited to, particles or gel materials in a chromatographic column. While a wide variety of chromatographic strip materials such as woven and non-woven fibrous materials used for paper chromatography are suitable for use with the invention, microporous or microgranular thin layer chromatography substrates are also useful and may improve the speed and resolution of the assays. When used in the final test format, the materials should preferably be inert and generally not reactive physically or chemically with any of the sample components, reagents, colloidal particle labels, buffers or reaction products. The addition of materials, such as polyvinyl alcohol or any suitable blocking agent can be used to block a reactive material while the test runs or prior to running the test.
Thin layer chromatographic substrate materials particularly suitable for use with the present invention include granular thin layer chromatographic materials such as silica or microgranular cellulose. Useful non-granular microporous materials include microporous cellulose esters, for example, esters of cellulose with an aliphatic carboxylic acid, such as an alkane carboxylic acid, having from 1 to 7 carbon atoms, e.g., acetic acid, propionic acid, or any of the butyric acids or valeric acids. Also useful are microporous materials made from nitrocellulose, by which term any nitric acid ester of cellulose is intended. Suitable materials include nitrocellulose in combination with any of the carboxylic acid cellulose esters. Thus, pure nitrocellulose esters can be used as consisting of an ester of cellulose having approximately 3 nitric groups per 6 carbon atoms (e.g. a Type SMWP material (Millipore Corp., Bedford, Mass.)).
The various chromatographic substrate materials may be used as such in suitable shapes such as films, strips or sheets. They may also be coated onto or bonded or laminated to appropriate inert support materials such as paper, glass, plastic, metal or fabrics (e.g. Mylar). The support material may provide structural support to the chromatographic substrate material as well as prevent evaporation of reagent and solvent materials during the assay procedure. Cover plates may also be fashioned of such inert materials. Cover plates, although not required for practice of the invention, lend additional structural support and further prevent evaporation of reagent and solvent materials during the assay procedure. Such cover plates may be transparent for viewing the progression of the assay and may comprise ports for addition of sample materials, chromatographic solvent or reagents.
The chromatographic medium upon which the assays are conducted may be any shape or size. In an exemplary embodiment, the medium is a strip of thickness in the range of from about 0.01 mm to about 0.5 mm. In a related embodiment, the thickness is about 0.1 mm. The strips may vary widely in their other dimensions and may be kept fairly small in order to shorten the assay development time and minimize material usage. When the strips are extremely small in size they may be attached to a suitable handle or holder in order to aid in handling and observation of results. Strips approximately 2-10 mm wide and up to 100 mm long have been found to be particularly suitable in the fabrication of single pathway devices.
The pore size may vary within wide limits. The pore size is between about 0.05 μm and 20 μm. In a related embodiment, the pore size is about 5 μm. Pore size is limited on the lower end by the size of the transported analytes, reagents and colloidal particle labels. If the pore size is too small, assay materials will be transported slowly or not at all. On the higher end, pore size is limited by binding capacity.
The chromatographic transport may be rapid with the transport and assay being completed within less than five minutes, and possible less than or about two minutes. Chromatographic transport should not be so rapid that specific binding capacity is lost as reagents do not have time to specifically bind. The combination of pore size and substrate thickness may thus be varied according to the characteristics of the chromatographic solvents, specific reagents, sample materials and detectable labels used in order to obtain the desired speed and resolution.
In forming the strip materials of the present invention, it may be desirable to minimize irregularities in the materials or in the edges of the materials which might cause uneven flow through the material. Means of fashioning the strip materials include the use of a paper cutter with a tungsten carbide rotary blade. Alternatively, a laser cutting device may be used that is suitable for use in mass production techniques.
As discussed above, various reagents may be immobilized (e.g. immobilized capture reagent and immobilized and immobilized assay reagent). Where the media is nitrocellulose or a mixed nitrocellulose ester, immobilization of reagents may not require any specific linkage. As discussed above, various reactive groups may be used for other materials and reagents, including functionalization with materials such as carbonyldiimidazole, glutaraldehyde or succinic acid, or treatment with materials such as cyanogen bromide. Other suitable reactions include treatment with Schiff bases and borohydride for reduction of aldehydic, carbonyl and amino groups. DNA, RNA and certain antigens may be immobilized against solvent transport by baking onto the chromatographic material. For nucleic acids, baking may be carried out at temperatures ranging from about 60° C. to about 120° C. for times varying from about five minutes to about 12 hours, but preferably at about 80° C. for about two hours.
In some embodiments, components of the present invention (e.g. binding reagents, bulking material, assay reagents, labeling reagents, and/or capture reagents) are impregnated and dried onto the chromatographic media of the devices. The components may be resolubilized upon wetting and subsequently transported by means of conventional chromatographic solvent systems. The ability to impregnate chromatographic media with the device components, which may then be resolubilized, makes possible the practice of a variety of assay procedures.
Release Zone
As discussed above, the purpose of the sample release zone of the present invention is to provide reagent stability and/or controlled release of the reagents. Controlled release of the reagents may provide additional time for the components (e.g. the analyte and the mobile detectable analyte binding reagent) of the test device to interact when wetted, thereby providing improved detectability.
The release zone is upstream of the assay zone and in fluid communication with the assay zone upon wetting of the chromatographic medium. The release zone may completely or partially overlap with the analyte binding zone. Thus, the release zone may be combined with the analyte binding zone to form a single zone, also referred to herein as a release-binding zone. The release zone may also partially or completely overlap with the labeling zone. In an exemplary embodiment, a single zone is formed by combining the release zone and the labeling zone, also referred to herein as a labeling release zone.
The sample release zone includes a bulking material. The bulking material includes a non-sugar sweetener (as described below) and optionally additional reagents, such as salts, buffers, surfactants, blocking agents, preservatives, flow reagents and biomolecules (such as starch, nucleic acid, protein, and amino acids). Desirable bulking materials provide stability and flow release of the reagents of the present invention.
A variety of starches are useful in the present invention, including hydrolyzed and non-hydrolyzed starches. Useful hydrolyzed starches include maltodextrin of various lengths. In an exemplary embodiment, the maltodextrin had a dextrin equivalence (DE) of less than or equal to 20. In a related embodiment, maltodextrin has a dextrose equivalence from 2 to 20. In another related embodiment, the maltodextrin has a DE from 11 to 18. In another related embodiment, the maltodextrin has a DE of 18. Typically, the concentration of starch used in the bulking material is between 0.01% and 60% (w/v). In an exemplary embodiment, the concentration is between 1% and 20%. In another exemplary embodiment, the concentration is about 10%.
Surfactants are useful as additional reagents in the bulking material. Exemplary surfactants include, for example, non-ionic surfactants and ionic surfactants. Non-ionic surfactants do not ionize in aqueous solutions. Exemplary non-ionic surfactants include sodium deoxycholate, octylglucoside, digitonin, octaethyleneglycol mono n-dodecyl ether (C12E8), lubrol, polyoxyethylated octyl phenol (Triton X-100), Ethylphenolpoly-(ethyleneglycolether) (Nonidet P-40), [Octylphenoxy]polyethoxyethanol (Nonidet P-40 substitute), polyoxyethylene sorbitan monooleate (polyoxyethylenesorbitanmonooleate) (Tween 80), polyoxyethylene sorbitan monolaureate (Tween-20), BRIG 35, dodecyl maltopyranoside, heptyl thioglucopyranoside, block-copolymer surfactants such as Pluronic and Tetronic surfactants available from BASF (e.g. Pluronic F127, Pluronic F98, Tetronic 904 and Tetronic T1307), Isotridecyl(PEG-ether)8 (also referred to as Genapol X-080), N-alkanoyl-N-methylglucamide surfactants (e.g. the MEGA series surfactant including MEGA 9 AND MEGA 10), and the like. Ionic surfactants include anionic surfactants, cationic surfactants, and amphoteric surfactants. Useful anionic surfactants include, for example, sodium dodecyl sulfate, cholate and deoxycholate, and the like. Exemplary cation surfactants include cetyltrimethyl-ammonium bromide (CTAB) and the like. Amphoteric surfactants useful in the present invention include, for example, LysoPC, CHAPS, Zwittergent 3-14, and the like.
In some embodiments, the bulking material includes a Tetronic and/or a Pluronic detergent. Tetronic and Pluronic are registered trademarks of BASF Corporation. Tetronic surfactants are tetrafunctional block copolymers derived from the addition of ethylene oxide and propylene oxide to ethylenediamine. Pluronic surfactants are block copolymers of propylene oxide and ethylene oxide.
In an exemplary embodiment, the surfactant is selected from octylglucoside, digitonin, C12E8, lubrol, Triton X-100, Nonidet P-40, Tween-80, Tween-20, BRIG 35, dodecyl maltopyranoside, heptyl thioglucopyranoside, Pluronic F127, Pluronic F98, Genapol X-080, Tetronic T1307, Tetronic 904, Pluronic F98 and MEGA 10. In another exemplary embodiment, the surfactant is selected from Tetronic T1307, Tetronic 904 and Pluronic F98. The concentration of surfactant in the bulking material may be from about 0.01% to about 10%. In an exemplary embodiment, the concentration of the surfactant is between 1% and 5%. In another exemplary embodiment, the concentration is from about 1.0% to about 2.0%.
In an exemplary embodiment, the surfactant is selected from octylglucoside, digitonin, C12E8, lubrol, Triton X-100, Nonidet P-40, Tween 80, Tween-20, BRIG 35, dodecyl maltopyranoside, heptyl thioglucopyranoside, Pluronic F-127, Genapol X-080, Tetronic T1307, and MEGA 10. In another exemplary embodiment, the surfactant is Tetronic T1307. The concentration of surfactant in the bulking material may be from about 0.01% to about 10%. In an exemplary embodiment, the concentration of the surfactant is between 1% and 5%. In another exemplary embodiment, the concentration is about 1.5%.
Preservatives may also be included in the bulking material. Useful preservative include, for example, azides (e.g. sodium azide), aromatic alcohols (e.g. xylenols, benzyl alcohols), e-amino-n-caproic acid (EACA), thimerosal (sodium ethylmercurithiosalicylate), mercury containing preservatives (e.g. phenyl mercury acetate), and antibiotics (e.g. gentamicin).
Flow reagents may also be included in the bulking material to adjust the smooth and even release of the assay reagents. Useful flow agents include polymers and polymeric particles such as polyvinylpyrrolidone (e.g. PVP-10). In an exemplary embodiment, the concentration of the flow agent is between about 0.01% to about 5.0%. In another exemplary embodiment, the concentration of the flow agent is between about 0.01% to about 1.0%. In another exemplary embodiment, the concentration of the flow agent is between about 0.01% to about 0.1%. In another exemplary embodiment, the concentration of the flow reagent is about 0.05%.
Buffers are also useful as additional reagents in the bulking material. One skilled in the art will recognize that the choice of buffer will depend upon the solvent used in the assay and the desired pH. For biological samples, the desired pH is typically between 5.0 and 9.0. In an exemplary embodiment, the desired pH is between 6.0 and 8.0. In another exemplary embodiment, the desired pH is between 7.0 and 8.0. In yet another exemplary embodiment, the desired pH is about 8.0. Exemplary buffers include N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine, and tris(hydroxymethyl)aminomethane (Tricine), acetate, borate, citrate, HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), BES (N,N-bis[2-hydroxyethyl]-2-amino-ethanesulfonic acid), TES (N-tris[Hydroxymethyl]methyl-2-aminoethanesulfonic acid), MOPS (morpholine propanesulphonic acid), PIPES (piperazine-N,N′-bis[2-ethane-sulfonic acid]), and MES (2-morpholino ethanesulphonic acid). In an exemplary embodiment, the buffer is Tricine.
The bulking material may also include a blocking reagent to prevent binding of the assay components to the chromatographic media. The choice of blocking reagent will depend on the solvent used in the assay, the analyte to be detected, the pH of the solution, and the reactivity of the blocking agents. Useful blocking agents include meta-soluble proteins (e.g., casein, zein as disclosed in U.S. Pat. No. 6,534,320), alcohol blocking reagents (e.g. polyvinyl alcohol (PVA-10)), and soluble protein blocking agents. Soluble protein blocking agents include, for example, serum albumin (including bovine, horse, sheep, and goat); hydrolyzed proteins (including hydrolyzed casein); and commercially available polypeptides, proteolytic digest, and synthetic peptide (e.g. those commercially available, for example, from Sigma Chemicals or Bachem Inc.). The concentration of the blocking reagent may range from about 0.01% to about 20%. In an exemplary embodiment, the concentration is between about 0.05% to about 10%. In another exemplary embodiment, the concentration is between about 0.05% to about 5%. In another exemplary embodiment, the concentration is between about 0.05% to about 1.0%. In another exemplary embodiment, the concentration is between about 0.05% to about 0.5%. In another exemplary embodiment, the concentration is about 0.1%.
Flow agents may also be included in the bulking material to adjust the smooth and even release of the assay reagents. Useful flow agents include polymers and polymeric particles such as polyvinylpyrrolidone (e.g. PVP-10). In an exemplary embodiment, the concentration of the flow agent is between about 0.01% to about 0.1%. In another exemplary embodiment, the concentration of the flow reagent is about 0.05%.
A wide variety of salts are useful as additional reagents in the bulking material of the present invention, including, for example, potassium gluconate, potassium benzoate, sodium benzoate, potassium tartrate, sodium tartrate, potassium sodium tartrate, sodium borate, potassium borate, potassium sulfate, sodium acetate, potassium acetate, ammonium acetate, potassium oxalate, sodium oxalate, sodium citrate, sodium ascorbate, sodium sorbate, potassium sorbate, potassium sorbitol, sodium sorbitol, potassium glutamate, sodium glutamate, ammonium glutamate, ammonium citrate lactic acid, succinic acid, sodium propionate, potassium propionate, and malonic acid.
Nucleic acids useful as additional reagents in the bulking material include DNA, RNA, and derivatives thereof.
Other useful additional reagents include those materials found in commercial artificial sweetener mixture, such as dextrose. In an exemplary embodiment, the bulking material is a commercial artificial sweetener mixture, such as Equal®, Sweet Measure®, Sweet'n Low®, and the like. Thus, the bulking material may include dextrose (e.g., at a concentration between about 0.1% and 30%), maltodextrin (e.g., at a concentration between about 0.1% and 50%), and aspartame (e.g., at a concentration between about 0.01%-5.0%). In another exemplary embodiment, the bulking material may include dextrose (e.g., at a concentration between about 0.1% and 5%), maltodextrin (e.g., at a concentration between about 0.1% and 20%), and sucralose (e.g., at a concentration between about 0.1%-25%) or aspartame (e.g., at a concentration between about 0.1%-1.5%). In a related embodiment, the concentration of sucralose is about 8%. In a related embodiment, the concentration of aspartame is about 1%-1.5%.
In another exemplary embodiment, the bulking material includes maltitol (e.g., at a concentration between about 0.01% and 25%) and saccharine (e.g., at a concentration between about 0.01% and 25%). In a related embodiment, the bulking material includes from 1% to 20% maltitol and from 1% to 10% saccharine. In another related embodiment, the bulking material includes from 5% to 15% maltitol and from 2% to 6% saccharine. In another related embodiment, the bulking material includes from 8% to 12% maltitol and from 3% to 5% saccharine. In another related embodiment, the bulking material includes about 10% maltitol and about 4% saccharine. In another related embodiment, the bulking material includes 4% maltitol and 10% saccharine.
In some embodiments, the bulking material includes maltitol, saccharine, a preservative, a surfactant, a buffer, a blocking agent, and a flow agent. In a related embodiment, the surfactant is a block-copolymer surfactants such as a Pluronic or Tetronic surfactant available from BASF (e.g. Pluronic F127, Pluronic F98, Tetronic 904 and Tetronic T1307). The buffer may maintain the pH of the solution between 6.0 and 8.0, inclusive (e.g. tricine). In some embodiments, the preservative is an azide (e.g. sodium azide). The blocking agent may be polyvinyl alcohol. The flow agent may be polyvinylpyrrolidone.
Non-Sugar Sweetener Components
The bulking material of the present invention includes a non-sugar sweetener or combination of non-sugar sweeteners. Non-sugar sweeteners are compounds that lack a sugar moiety while retaining the molecular properties known to impart sweetness. For sweetness to be perceived by an individual, a molecule must: (1) be soluble in the chemical environment of the receptor site on the tongue, and (2) have the proper electronic distribution, commonly referred to as the “AH-B-X system.”
The AH-B-X system requires three basic components to a sweetener: AH, B, and X. These components are typically represented as shown in the formula below:




In Formula (I), the numbers represent distances between components in angstroms (Å). A and B are electronegative components of a molecule. H is a hydrogen atom bonded to an atom within the A component. Together, -A-H from an electron acceptor and B an electron donor. X represents a hydrophobic component of the molecular sweetener. The dashed lines signify that the components AH, B, and X form part of a single molecule. AH, B, and/or X may be directly or indirectly conjugated together.
Using a computational approach, possible AH, B, and X regions of potential non-sugar sweeteners may be identified. For example, a computer algorithm may be employed to map the electrostatic potential (elpot) properties and the Highest Occupied Molecular Orbitals (HOMO) of potential non-sugar sweeteners to identify electronic distributions conforming to the AH, B, X system. See Guley et al., “Comparison of Relative Sweetness to Molecular Properties of Artificial and Natural Sweeteners,” http://www.shodor.org/succeed/compchem/projects/fall00/sweeteners/; Immel et al., XV th International Carbohydrate Symposium, July 5-10, Abstract C163 (1992).
Potential compounds may also be assayed by determining whether the non-sugar sweetener binds to the appropriate receptors on the tongue to impart a sweet flavor. Appropriate assays may be performed in vitro, for example, using recombinant or purified receptors and known methods of detecting ligand-receptor binding. Alternatively, potential compounds may be given to animal test subjects to determine the level of relative sweetness.
In an exemplary embodiment, the non-sugar sweetener is not a natural peptide. In another exemplary embodiment, the bulking material does not include sugar. In a related embodiment, the bulking material does not include a sugar or a complex sugar.
Thus, the non-sugar sweeteners of the present invention are not sugars, as defined herein. See definitions section above. However, the non-sugar sweeteners of the present invention are soluble in the chemical environment of the receptor site on the tongue, and have the proper AH-B-X electronic distribution system to impart sweetness.
Exemplary classes of non-sugar sweeteners of the present invention include non-natural peptidyl sweeteners, polyalcohol sweeteners, halo-substituted sugar derivatives, sulfimide sweeteners, and hydroxypyrone sweeteners. In an exemplary embodiment, the non-sugar sweetener of the present invention is selected from aspartame, neotame, acesulfame, dihydrochalcone, cyclamate, saccharine, sucralose, stevia, maltitol, erythritol, zylitol, glycyrrhizin, maltol (including ethyl maltol), and alitame, various salts and acids thereof, and derivatives thereof. In particular, the sodium, calcium and potassium salts of the relevant acids have been proven to have suitable sweetening properties.
Non-Natural Peptidyl Sweeteners
In an exemplary embodiment, the non-sugar sweetener of the present invention is a non-natural peptidyl sweetener. A wide variety of non-natural peptidyl sweeteners useful in the present invention are known in the art.
Examples of non-natural peptidyl sweeteners include L-aspartic acid derived sweeteners, such as L-aspartyl-L-phenylalanine methyl ester (aspartame); L-α-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide hydrate; methyl esters of L-aspartyl-L-phenylglycerine and L-aspartyl-L-2,5,dehydrophenylglycine; L-aspartyl-2,5-dihydro-L-phenylalamine; L-aspartyl-L-(1-cyclohexyen)-alanine; esters of L-aspartyl-D-serine and L-aspartyl-D-threonine (Aryoshi et al., Bull. Chem. Soc. Jap., 47:326 (1974)); esters of L-aspartyl-D-alaninol and L-aspartyl-D-serinol (U.S. Pat. No. 3,971,822), 3-L-α-aspartyl-D-alanylamido-2,2,4,4-tetramethylthietanylamine(alitame; EP Pat. App. 0034876); N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine 1-methyl ester (neotame); N-[N-[3-(4-hydroxy-3-menthoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester and N-[N-(3-phenylpropyl)-L-α-aspartyl]-L-phenylalanine 1-methyl ester; and many others. Neotame (CAS Number 165450-17-9; Formula: C20H30N2O5) is a dipeptide methyl ester derivative, and is prepared by the reductive alkylation of N-L-α-aspartyl-L-phenylalanine 1-methyl ester (aspartame).
Non-natural peptidyl sweeteners are described in detail in a variety of publications, including U.S. Pat. Nos. 3,642,491; 3,492,131; 3,475,403; 3,280,099; 4,028,319; 4,025,551; 5,456,932; 6,329,343; 5,503,862; 4,810,817; 6,652,901; 6,692,778; 5,480,668; 5,510,508; and 5,728,862. Each of the forgoing applications are incorporated by reference in their entirety for all purposes.
Polyalcohol Sweeteners
A variety of polyalcohol sweeteners are useful in the present invention. Polyalcohol sweeteners may also be referred to as polyhydroxy alcohols, sugar alcohols or polyols. The polyalcohol sweeteners of the present invention may be derived from fruits or commercially produced from dextrose. Like sugars, polyalcohol sweeteners are sweet to the taste but are absorbed more slowly, are only partially utilized, and metabolized differently than sugars. They are not readily utilized by ordinary mouth bacteria, and may actually help prevent tooth decay.
Typically, polyalcohol sweeteners contain four to ten fully saturated carbons in a linear chain. Polyalcohol sweeteners may be substituted with three or more hydroxyl groups. In some embodiments, each carbon is substituted with a hydroxyl moiety. Unlike sugars, polyalcohol sweeteners do not contain aldehyde or ketone moieties at a chain terminus. Exemplary polyalcohol sweeteners include sorbitol, lactitol, mannitol, maltitol, rythritol, arabitol, ribitol, galactitol and rhamnitol. In an exemplary embodiment, the polyalcohol sweetener is not xylitol.
Halo-Substituted Sugar Derivatives
In another exemplary embodiment, the non-sugar sweetener of the present invention is a halo-substituted sugar derivative. The halo substituted sugar derivatives of the present invention include sugars derivatized with one or more halogen substituents. The halogen substituent may be conjugated to any appropriate portion of the sugar molecule, including a sugar backbone carbon. In an exemplary embodiment, the halogen is chlorine. In another exemplary embodiment, the sugar that is derivatized is sucrose. In a related embodiment, the halo-substituted sugar derivative is sucralose or derivative thereof.
Sucralose and its derivatives are discussed in detail in U.S. Pat. application No. 4,405,654, which is herein incorporated by reference in its entirety for all purposes.
Hydroxypyrone Sweeteners
In another exemplary embodiment, the non-sugar sweetener is a hydroxypyrone sweetener. Hydroxypyrone sweeteners typically have the formula




In Formula (II), R1, R2, and R3 are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In an exemplary embodiment, R1, R2, and R3 are independently selected from hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In a related embodiment, R1, R2, and R3 are independently selected from hydrogen, substituted or unsubstituted (C1-C5)alkyl, and substituted or unsubstituted 2 to 5 membered heteroalkyl.
In another exemplary embodiment, R1 and R2 are hydrogen and R3 is selected from hydrogen, substituted or unsubstituted (C1-C5)alkyl, and substituted or unsubstituted 2 to 5 membered heteroalkyl. In a related embodiment, R3 is substituted or unsubstituted (C1-C5) alkyl. In a further related embodiment, R3 is methyl or ethyl.
Exemplary hydroxypyrone sweeteners include maltol (3-hydroxy-2-methyl-4-pyrone) and ethyl maltol (3-hydroxy-2-ethyl-4-pyrone).
Sulfimide Sweeteners
In another exemplary embodiment, the non-sugar sweetener is a sulfimide sweetener. A variety of sulfimide sweeteners useful in the present invention are well known in the art, including cyclamate and its derivatives (see U.S. Pat. Nos. 2,275,125 and 2,288,976), acelsulfame and its various salt forms including acelsulfame-K (see U.S. Pat. No. 3,689,486), and saccharin. The above patents are herein incorporated by reference in their entirety for all purposes.
In an exemplary embodiment, the sulfimide sweetener has the formula:

R1—NH—S(O)2—R2  (III).

In Formula (III), R1 is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. R2 is selected from —OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. R2A is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
R1 and R2 are optionally joined to form a substituted or unsubstituted ring. R1 and R2A may also be joined to form a substituted or unsubstituted ring.
In another exemplary embodiment, R1 and R2 are joined to form a substituted or unsubstituted 5 to 6 membered ring. In a related embodiment, the ring is a substituted 5 membered ring. In another related embodiment, the ring is a 5-membered ring fused to a phenyl group. In another related embodiment, the sulfimide is substituted or unsubstituted saccharin. In still another related embodiment, the sulfimide is saccharin.
In another exemplary embodiment, R1 and R2A are joined to form a substituted or unsubstituted 5 to 6 membered ring. In a related embodiment, the ring is a substituted 6 membered ring. In another related embodiment, the sulfimide has the formula




In Formula (IV), R3 and R4 are members independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In a related embodiment, R3 and R4 are independently selected from hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl. In another related embodiment, R3 and R4 are independently selected from hydrogen and substituted or unsubstituted (C1-C5)alkyl. In still another related embodiment, R3 and R4 are independently selected from hydrogen, methyl, and ethyl. In another related embodiment, R4 is hydrogen and R3 is methyl.
In another exemplary embodiment of the compound of Formula (III), R2 is —OH and R1 is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In a related embodiment, R1 is selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In another related embodiment, R1 is substituted or unsubstituted cycloalkyl. In another related embodiment, R1 is substituted or unsubstituted cyclohexyl. In still another related embodiment, R1 is cyclohexyl.
Methods
In another aspect, the present invention provides a method of stabilizing a protein (e.g. an antibody) in a dry state. The method includes contacting the antibody with a bulking material. Bulking materials are described above and are equally applicable to the present methods. The bulking material includes a non-sugar sweetener compound. In an exemplary embodiment, the non-sugar sweetener compound selected from polyalcohol sweetener, peptidyl sweetener, hydroxypyrone sweetener, halo-substituted sugar derivative, and sulfimide sweetener.
In another aspect, the present invention provides methods of determining the presence of an analyte in a sample using the test device described above. The method includes contacting the analyte binding zone of the test device of the present invention with a sample and allowing the sample to flow from the sample release zone to the assay zone. The presence of the analyte is determined by detecting the presence or absence of the detectable label associated with the mobile detectable analyte binding reagent as described above.
Kits
The present invention further includes kits for use in detecting the presence of an analyte in a sample. The kit includes a test device of the present invention. Test devices are discussed in detail above and are equally applicable to the kits of the present invention.
The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described, or portions thereof, it being recognized that various modifications are possible within the scope of the invention claimed. Moreover, any one or more features of any embodiment of the invention may be combined with any one or more other features of any other embodiment of the invention, without departing from the scope of the invention. For example, the features of test devices of the present invention are equally applicable to the kits and methods of the present invention described herein. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
EXAMPLES
Example 1
Test of Various Reagents for use in Bulking Materials by Determining the Stability of the Antibodies when Incubated at 57° C., and the Flow of the Antibodies on a Lateral Flow Chromatographic Strip
Monoclonal antibodies against THC, cocaine, opiates, benzodiazapene, amphetamine, PCP, methamphetamine, and methodone were conjugated to colloidal gold (40-60 nanometer) by absorbing each antibody (from 0.5 μm/OD/mL to 4 μgm/OD/mL) to the gold colloid for 5 minutes at room temperature then blocking with 30 μm of BSA/OD gold colloid for 10 minutes at room temperature. The antibody coated gold was then diluted in a solution of 50-150 mM Tricine pH 8.0 with Tween-20 ranging from (0.5%-0.9%) along with various concentrations of salts, proteins, and sugar substitutes (see table below). Fifteen microliters of the mixture was applied to a glass fiber sample pad (Millipore) and baked overnight at 57° C. to test for stability. These gold release pads were then assembled into lateral flow strips using drug conjugates corresponding to each antibody striped onto Prima-40 nitrocellulose membrane (Schleicher and Schuell) at 1 mg/mL in PBS. The mylar-backed nitrocellulose membrane was affixed to an adhesive backing (G&L Precision Die Cutting) along with a sorbent pad (Whatman 222 or equivalent), assembled with the gold coated sample pad, then tested for the ability of the gold labeled monoclonal to bind to the corresponding drug conjugate using urine standards spiked at varying levels of drug concentration.
Results were read visually after 7 minutes. Table 1 represents the summation of hundreds of permutations. The table indicates whether the reagent provided stability at 57° C., which normally denatures the antibodies. The table also indicates whether the gold released to flow readily and whether the tests worked.







TABLE 1







Flow Release vs. Stability for Various Materials















Antibody






Stability



Reagent
% (w/v)
Flows
at 57° C.







Hydrolyzed soy
4
yes
slight



Hydrolyzed gelatin
4
yes
some



Hydrolyzed casein
4
yes
modest



Polypep- porcine
4
yes
yes



Sucrose
 2-10
yes
yes



Sodium Benzoate
2-8
yes
some



Aspartame
 5-20
yes
yes



(nutrasweat)



Sucralose (Splenda)
10 
yes
yes



Potassium gluconate
2-6
yes
modest



Sodium borate
2-6
yes
modest



Potassium sulfate
2-6
yes
some



Sodium acetate
2-6
yes
slight



Potassium sodium
2-6
yes
slight



tartrate



Sorbitol
2-6
yes
slight



Ascorbic acid, sodium
2-6
yes
modest



Potassium oxalate
2-6
yes
slight



BSA
2-8
yes
modest



Potassium acetate
2-6
yes
modest



Ammonium chloride
4
yes
some



Calcium chloride
2-6
yes
slight



Lithium chloride
2-6
yes
slight



Ammonium citrate
2-6
yes
modest



Potassium citrate
2-6
yes
modest



d,I-Lactic acid
2-6
yes
some



Gelatin
2-6
yes
modest



Ammonium glutamate
4
yes
modest



Potassium glutamate
2-6
yes
modest



Sodium glutamate
2-6
yes
modest



Malonic acid
2-6
yes
modest



Sodium propionate
2-6
yes
modest



Potassium sorbate
2-6
yes
modest



Sorbitol
2-6
yes
modest



Succinic acid
2-6
yes
modest



Ammonium tartrate
2-6
yes
modest



Dipotassium tartrate
2-6
yes
modest







Notes:



“Slight” indicates little stability and was marginally above buffer only.



“Some” indicates that about 25% of activity remained.



“Modest” indicated about 25-50% of activity remains.



“Yes” indicated virtually no reduction in antibody reactivity and is highlighted.






As shown in Table 1, a variety of salts allowed the quick release of the antibody coated gold colloid, but offered little stability for antibody reactivity. Aspartame (in the form of a Nutrasweet® packet) and sucralose (in the form of a Splenda®) provided good stability and release.
Example 2
Test of Various Non-sugar Sweeteners for use in the Bulking Materials
A stock solution of THC gold was made by coating gold colloid at 0.8 μm/OD/mL with monoclonal anti-THC (MedTox clone) for 5 minutes at room temperature (RT) followed by blocking for NSB with 30 μgm/OD of BSA (Sigma Chemical) for 10 minutes at room temperature. The THC gold was diluted in 1% Tetronic T1307 (BASF), 100 mM Tricine (Sigma Chemical), 0.1% polyvinyl alcohol (10 kd, Sigma Chemical), 0.05% PVA-10 (10 kd, Sigma Chemical) to 3 OD/mL and 15 microliters of this mixture spotted onto 5 mm wide by 22 mm long glass fiber membranes (Millipore GF), dried at 1 hour at 57° C., tested using a THC negative urine, then subjected to overnight baking at 57° C. and retested using a negative urine sample. Readings were by visual examination at 7 minutes.







TABLE 2







Comparison of Non-sugar Sweeteners












THC(−) 1
THC(−) 3-5


Designator
Reagent
hr 57° C.
day@57° C.





A
None
++ to +++
+−


B
1% Na Saccharine
++ to +++
+ to ++


C
2% Na Saccharine
++ to +++
+ to ++


D
4% Na Saccharine
++
++


E
8% Na Saccharine
+
+−


F
5% Tagatose
++
+−


G
10% Tagatose
++
+−


H
20% Tagatose
++
+−


I
20% Sweet Measure
++ to +++
++ to +++



(Aspartame)


J
5% Maltitol
++
+−


K
10% Maltitol
++ to +++
++


L
20% Maltitol
++
+ to ++


M
5% Xylitol
+
+−


N
10% Xylitol
+− to +
+−


O
20% Xylitol
 + to ++
+−


P
1% pure aspartame
++
++


Q
.5% pure aspartame
++
+ to ++


R
1.25% Maltodextrin
++
+−


S
2.5% Maltodextrin
++
+− to + 


T
5% Maltodextrin
++
+


U
10% Maltodextrin
++
+


V
15% Maltodextrin
++
++









In Tables 2-9, a − means not visible, a −+ signal indicates the line is barely visible, a +− indicates a light line, a + signal is a line that is easily seen. These are graded up to a ++++ line, which is as dark as a control line (nearly black).
As shown in Table 2, sodium saccharine helps stabilize the antibody, but begins to inhibit the reactivity at 8% w/v. Tagatose (i.e. Nutralose) and Xylitol did not provide optimal stabilizing effects, although they did release nicely. Sweet Measure (commercial product from Food Lion) was comprised of aspartame and maltodextrin, both of which showed stabilizing effects on antibody reactivity, although increasing concentrations of maltodextrin (a partially hydrolyzed starch) released the colloidal gold more slowly. Maltitol demonstrated good stabilizing effects at 10% and gave excellent gold release. Tests using Sweet Measure were repeated with other DOA antibodies with similar results.
Example 3
Determination of Concentrations and Formulations of Sucralose useful in Bulking Materials
Pure sucralose solution was obtained from McNeil (makers of Splenda). Buffer (1.5% T1307, 0.1% PVA-10, 0.05% PVP-10, 100 mM Tricine pH 8) with various amounts of sucralose were tested against THC.8 and PCP4 gold. Conjugates were baked overnight at 57° C. with negative urine.







TABLE 3







Sucralose Formulations












Reagent-1
Reagent-2
PCP-4
THC.8







None
None
+− to +
+− to +



1% sucralose
None
+
+− to +



2% sucralose
None
+
+− to +



4% sucralose
None
+ to ++
+ to ++



8% sucralose
None
+ to ++
++



5% sucralose
10% maltitol
+ to ++
++



5% sucralose
1% NaSaccharine
+ to ++
++










As shown in Table 3, pure sucralose provides stability to heat with THC and PCP antibodies. Sucralose in combination with maltitol or with sodium saccharine provides excellent stability.
Example 4
Test of Size Ranges of Maltodextrin for use in a Bulking Materials
Prepared PCP colloidal gold using a MedTox monoclonal anti-PCP antibody conjugated to colloidal gold at 4 μgm/OD/mL (pH 8) and THC colloidal gold using a MedTox monoclonal anti-THC antibody at 0.8 μgm/OD/mL (pH 8) as previously described. Tested colloidal golds at 3 OD/mL using maltodextrin (Cargill) as a non-sugar bulking agent for its ability to stabilize these labile monoclonals with a dextrose equivalence rating of 11 and 18 in a solution of 1.5% Tetronic T1307, 0.1% PVA-10, 0.05% PVP-10, and 100 mM Tricine pH 8.0. Tested 15 microliters spotted onto 5 mm by 22 mm Millipore GF membrane baked at 1 hr vs. ON at 57° C.







TABLE 4







Maltodextrin Formulations










Reagent
PCP-4
THC.8
Comments










Baked 1 hour at 57° C. (initial look)










15% DE11
ND
++
Slow gold release


15% DE18
ND
++
Slow gold release


20% Sweet Measure
++
ND
Combo of maltodextrin and





aspartame


10% maltitol
++
ND







Baked 2 days at 57° C.










15% DE11
ND
+ to ++



15% DE18
ND
++





ND = not done






As shown in Table 4, DE 11 and DE18 provided stabilization.
Example 5
Test of Sodium Saccharine Formulations for use in Bulking Materials
Tested 4% saccharine formulation (1.5% T1307, 0.1% PVA-10, 0.05% PVP-10, 100 mM Tricine pH 8.0) with all eight DOA to see if it caused any problems. Baked for 4 hours at 57° C., tested, then baked ON at 57° C. Ran using negative urine only and analyzed line intensity at 7 min.







TABLE 5.0







4% Saccharine Formulation











Test
4 hr at 57° C.
ON at 57° C.







PCP-4
++
+ to ++



THC.8
+ to ++
+



OPI-2
++++
+++



AMP-2
++
+



MAMP-4
++
+



BZO-4
++
+



MTD-2
+++++ (black)
+++++ (black)



BE-2 (coc)
++++
+++










Example 5.1
Test of Maltitol Formulations for use in Bulking Materials
Tested all eight DOA in 10% maltitol in 1.5% T1307/0.1PVA/0.05PVP/100 mM Tricine, baked ON at 57° C. with negative urine.







TABLE 5.1







Stabilizing Effect of 10% Maltitol Formulation










Test
ON at 57° C.







PCP-4
+ to ++



THC.8
+ to ++



OPI-2
+++ to ++++



AMP-2
++



MAMP-4
+



BZO-4
+



MTD-2
+++++



BE-2 (coc)
+++










Example 6
Test of Combinations of Maltitol and Saccharine, Maltitol and Aspartame, and Aspartame and Sodium Saccharine for use in Bulking Materials
Tested PCP4 vs. THC.8 baked ON at 57° C. with (−) urine.







TABLE 6







Comparison of Combinations of Non-sugar Sweeteners











Material
PCP-4
THC.8







1% saccharine/10% maltitol (std buffer)
++
+



2% saccharine/10% maltitol (std buffer)
+ to ++
+



4% saccharine/10% maltitol (std buffer)
+ to ++
+ to ++



.8% aspartame/10% maltitol (std buffer)
+
+



1% aspartame/1% NaSaccharine
+ to ++
+










Example 7
Test of Additional Reagents for use with Maltitol and Saccharine
The following formulations was tested for use as bulking materials.
Formulation (I): 1.5% Tetronic T1307 (BASF, tetrafunctional block copolymer surfactant terminating in primary hydroxyl groups, aka poloxamine 1307); 10% Maltitol; 4% sodium saccharine; 150 mM Tricine, pH 8.0; 0.02% sodium azide; 0.1% polyvinyl alcohol (PVA-10); and 0.05% polyvinylpyrrolidone (PVP-10)
Formulation (II): 2% Tetronic T1307 (BASF); 2% Tetronic T904 (BASF, same as T1307 but altered polyethylene to polypropylene lengths); 10% Maltitol; 4% sodium saccharine; 150 mM Tricine, pH 8.0; 0.02% sodium azide; 0.1% polyvinyl alcohol (PVA-10); and 0.05% polyvinylpyrrolidone (PVP-10).
Formulation (III): same as Formulation (II) at pH 6.0.
Formulations (I), (II), and (III) shown below (Tables 7-9) demonstrated good stabilizing effects at 57° C., which completely denatured the reagents after 1-2 days without the formulations. Formulation (I) provided excellent stability at 130 days at 57° C. (Table 7). Formulation (II) provided slightly better signals while maintaining good stability (Table 8). The barbiturate assay (phenobarbital) was extremely labile and previously had to be stored at 4° C. with a very limited shelf life. However, Formula (III) (pH modification of Formula (II)) provided stability of at least 16 days at 57° C., which was not possible without the stabilizing effects of the non-sugar stabilizers employed.







TABLE 7







Stability Assay using Formulation (I) at 57° C.









Material
DAY 1
DAY 130





THC Assay
+++
++


OPI2000 Assay
+++
++


AMP Assay
+++
++


COC Assay
+++
++


PCP Assay
++
+ to ++


*Without the non-sugar stabilizers, the
++ to +++
−


tests above denatured after 2 days
















TABLE 8







Stability Assay using Formulation (II) at 57° C.











Material
DAY 1
DAY 40







THC Assay
+++
++



OPI2000 Assay
+++
++



AMP Assay
+++
+++



COC Assay
+++
++



PCP Assay
+++
++



*Without the non-sugar stabilizers, the
+++
−



tests above denatured after 2 days

















TABLE 9







Stability of Barbiturate Assay using Formulation (III) at 57° C.











Material
Day 1
Day 16







Formulation (III)
++++
+++



No stabilizers
+++
−










Example 8
Stabilization of Antibodies using Non-sugar Sweeteners or a Sugar in Bulking Materials
Rabbit anti-mouse antibodies were striped onto Millipore nitrocellulose at 1 mg/mL in a 1×PBS with 2% of the following reagents: trehalose, sodium saccharine, maltodextrin (DE18), sucralose, or maltitol at 0.03 ul/mm using an striper (IVEK Cororation). The membranes were then baked at 57° C. to simulate aging and to stress the stability of the reagents. The baked membranes where laminated onto polystyrene cards (G&L Precision Die Cutting), affixed with a sink pad and a sample pad, which contained colloidal gold labeled anti-THC monoclonal. The cards were cut into 4.8 mm strips, placed into a diagnostic cassette, and negative urine sample was applied to the pad. After seven minutes, the intensity of the bands was measured using a digitizing scanner (Bio-Dot) and the grey scale readings determined as shown in FIG. 1.
As shown in FIG. 1, the stability of the rabbit anti-mouse antibody rapidly decayed to a baseline after 42 days whereas those using non-sugar sweeteners remained more stable over time as compared to the non-stabilized antibody. This was especially evident when comparing maltitol and sucralose to the non-stabilized antibody.
Example 9
Test of Additional Reagents for use with Maltitol and Saccharine
THC-BSA conjugate (protein and chemically coupled drug) was striped onto Prima-40 nitrocellulose (Schleicher & Schuell) at 1 mg/mL in a 1×PBS with 2% one of the following reagents: trehalose, sodium saccharine, maltodextrin (DE18), or sucralose (maltitol was inadvertently omitted) at 0.03 ul/mm using a striper (IVEK Corporation). The membranes were then baked at 57° C. to simulate aging and to stress the stability of the stabilizing agents. The baked membranes where laminated onto polystyrene cards (G&L), affixed with a sink pad and a sample pad, which contained colloidal gold labeled anti-THC monoclonal. The cards were cut into 4.8 mm strips, placed into a diagnostic cassette, and negative urine sample was applied to the pad. After seven minutes, the intensity of the bands was measured using a digitizing scanner (Bio-Dot) and the grey scale readings determined as shown in FIG. 2
As shown in FIG. 2, the stability of the THC-BSA drug conjugate, which represents a protein and drug, degraded within 10 days when baked at 57° C. as compared to conjugate baked in the presence of a sugar (trehalose) or non-sugar sweetener (sodium saccharine, sucralose, and maltodextrin).
GENERAL CONCLUSIONS
The use of maltodextrin, sodium saccharine, sucralose, aspartame, and maltitol demonstrated added thermal stability of the antibodies tested. The combination of 4% saccharine and 10% maltitol demonstrated an excellent stabilizing combination for PCP. Additional testing was performed with twelve DOA monoclonals using this combination of saccharine and maltitol with excellent gold release and thermal stability even after 21 days at 57° C.

## Claims

1. A test device for detecting the presence of an analyte in a sample, said device comprising a chromatographic medium comprising:
(a) an analyte binding zone comprising a mobile detectable analyte binding reagent capable of binding to said analyte, and
(b) an assay zone comprising an immobilized assay reagent capable of immobilizing said mobile detectable analyte binding reagent, wherein said assay zone is in fluid communication with said analyte binding zone upon wetting said chromatographic medium; and
(c) a sample release zone comprising a bulking material comprising a non-sugar sweetener compound selected from the group consisting of polyalcohol sweetener, hydroxypyrone sweetener, peptidyl sweetener, halo-substituted sugar derivative, and sulfimide sweetener, wherein said sample release zone is upstream of said assay zone and in fluid communication with said assay zone upon wetting said at least one chromatographic medium.

2. The device of claim 1, wherein said immobilized assay reagent is capable of binding to an analyte.

3. The device of claim 1, wherein said immobilized assay reagent is capable of binding to said a mobile detectable analyte binding reagent, wherein the binding of said a mobile detectable analyte binding reagent to said an immobilized assay reagent is reduced when said an analyte is bound to said a mobile detectable analyte binding reagent.

4. The device of claim 1, further comprising, downstream of, and in fluid communication with said assay zone, a control zone comprising an immobilized capture reagent that immobilizes said a mobile detectable analyte binding reagent in the presence, and in the absence, of said at least one analyte.

5. The device of claim 1, further comprising, downstream of, and in fluid communication with said assay zone, a control zone comprising an immobilized capture reagent that immobilizes a mobile control reagent, said mobile control reagent comprising a detectable label.

6. The device of claim 1, wherein said sample release zone completely or partially overlaps with said analyte binding zone.

7. The device of claim 6, further comprising, downstream of said analyte binding zone and upstream of said assay zone, a labeling zone in fluid communication with said sample release zone and said assay zone upon wetting of said at least one medium, said labeling zone comprising at least one detectable labeling reagent capable of binding to said mobile detectable analyte binding reagent.

8. The device of claim 7, wherein said at least one detectable labeling reagent comprises a detectable label selected from the group consisting of radioisotope label, luminescent label, magnetic bead label, fluorescent dye label, enzyme label, colloidal metal label, colored glass bead label, colored latex bead label, up-converting reporter label, carbon black label, and semiconducting particle label.

9. The device of claim 1, wherein said sample release zone is downstream of said analyte binding zone and is in fluid communication with said analyte binding zone upon wetting of said at least one medium.

10. The device of claim 9, wherein said sample release zone further comprises at least one detectable labeling reagent capable of binding to said mobile detectable analyte binding reagent.

11. The device of claim 1, wherein said a mobile detectable analyte binding reagent comprises at least one detectable label.

12. The device of claim 11, wherein said at least one detectable label is selected from the group consisting of radioisotope label, luminescent label, magnetic bead label, fluorescent dye label, enzyme label, colloidal metal label, colored glass bead label, colored latex bead label, up-converting reporter label, carbon black label, and semiconducting particle label.

13. The device of claim 1, further comprising a sink zone downstream of, and in fluid communication with said assay zone upon wetting of said medium, wherein said sink zone comprises a sorbent material.

14. The device of claim 1, wherein said bulking material further comprises one or more additional reagents selected from the group consisting of an amino acids, salts, surfactants, soluble proteins, hydrolyzed starches, buffers and nucleic acids.

15. The device of claim 14, wherein said hydrolyzed starch is maltodextrin.

16. The device of claim 14, wherein said hydrolyzed starch is maliodextrin having a dextrose equivalence less than or equal to 20.

17. The device of claim 14, wherein said hydrolyzed starch is maltodextrmn having a dextrose equivalence from 2 to 20.

18. The device of claim 14, wherein said salt contains one or more salt reagents selected from the group consisting of potassium gluconate, potassium benzoate, sodium beuzoate, potassium tartrate, sodium tartrate, potassium sodium tartrate, sodium borate, potassium borate, potassium sulfate, sodium acetate, potassium acetate, ammonium acetate, potassium oxalate, sodium oxalate, sodium citrate, sodium ascorbute, sodium sorbate, potassium sorbate, potassium sorbitol, sodium sorbitol, potassium glutamate, sodium glutamate, ammonium glutamate, arnmonium citrate lactic acid, succinic acid, sodium propionate, potassium propionate, and malonic acid.

19. The device of claim 14, wherein said buffer is a member selected from the group consisting of sodium borate, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine, and tris(hydroxymethyl)aminomethane.

20. The device of claim 14, wherein said soluble protein is a member selected from the group consisting of serum albumin, hydrolyzed proteins, polypeptide, proteolytic digest, and synthetic peptide.

21. The device of claim 14, wherein said nucleic acid is DNA.

22. The device of claim 1, wherein said polyalcohol sweetener is a member selected from the group consisting of maltitol, mannitol, lactitol, polyglycitol, erythritol, glycerol, and sorbitol.

23. The device of claim 1, wherein said peptidyl sweetener is a member selected from the group consisting of aspartame and neotame.

24. The device of claim 1, wherein said halo-substituted sugar derivative is sucralose.

25. The device of claim 1, wherein said sulfimide sweetener is a compound having the formula:


R1—NH—S(O)2—R2  (III)

wherein
R1 is a member selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;
R2 is a member selected from the group consisting of —OR2A, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, wherein
R2A is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl, and
R1 and R2A are optionally joined to form a substituted or unsubstituted ring; and


R1 and R2 are optionally joined to form a substituted or unsubstituted ring.

26. The device of claim 1, wherein said sulfimide sweetener is a member selected from the group consisting of acesulfame, saccharine, and cyclamate.

27. The device of claim 14, wherein said bulking material comprises dextrose, maltodextrin, and aspartame.

28. The device of claim 14, wherein said bulking material comprises dextrose, maltodextrin, and sucralose.

29. The device of claim 1, wherein said bulking material comprises maltitol and saccharine.

30. The device of claim 1, wherein said bulking material comprises maltitol, saccharine, a preservative, a surfactant, a buffer, a blocking agent, and a flow agent.