WO2005103700A1

WO2005103700A1 - Ionic assay medium

Info

Publication number: WO2005103700A1
Application number: PCT/GB2005/001496
Authority: WO
Inventors: Neil Charles Bruce
Original assignee: The University Of York
Priority date: 2004-04-21
Filing date: 2005-04-20
Publication date: 2005-11-03
Also published as: GB2430266A; GB0408853D0; GB2430266A8; GB0622205D0; GB2430266B

Abstract

The present invention relates to a composition comprising an assay medium wherein the assay medium includes an ionic liquid and a specific binding member.

Description

IONIC ASSAY MEDIUM

The present invention relates to compositions comprising ionic liquids and to their use as solvents for carrying out assays, in particular immunoassays. rimunoassays are based on the formation of a thermodynamically stable antigen- antibody complex. These methods are used, especially in clinical chemistry, for the fast and safe detection of proteins, hormones and pharmaceutical agents.

Traditionally immunoassays are carried out in aqueous systems in which the target antigens are dissolved in solution before reaction with the antibody. A problem associated with performing immunoassays in aqueous solutions is that the target antigens are often insoluble or unstable in aqueous solution.

Attempts have been made to solubilise the analytes used in competitive immunoassays by the addition of surfactants to the aqueous solvent. Surfactants can, however, alter the tertiary structure of an antibody and possibly of any enzyme used in the assay.

There is a need for an alternative medium for carrying out assays, such as immunoassays, which does not suffer from the aforementioned problems.

According to a first aspect of the present invention there is provided a composition comprising an assay medium including an ionic liquid and a specific binding member.

The assay medium may comprise an ionic liquid and less than 10% water, for example less than 5% water or less than 1% water for example less than 0.1% water.

The assay medium of the present invention may be a single-phase reaction medium. As used herein, the term "ionic liquid" may relate to liquids which may have a melting point not higher than 100°C.

The ionic liquid used in all aspects of the invention may be made up of anions and cations or alternatively consist of zwitterions carrying both a positive and a negative charge on the same molecule. Most commonly the ionic liquid will comprise an anion and a cation.

The ionic liquid may be composed of catiomc and anionic portions according to the following general formula:

[A]„⁺[Y]„-

wherein n = 1 or 2 and wherein A represents the cationic portion and Y represents the anionic portion.

The cations utilised in the ionic liquids of the invention are typically composed of a quaternary nitrogen-based ion which may be aromatic, aliphatic, cyclic or acyclic. Preferably, the quaternary nitrogen-based ion is based on a nucleus selected from quaternary ammonium cations, pyrazolium cations, imidazolium cations, triazolium cations, pyridinium cations, pyridazinium cations, pyrimidinium cations, pyrazinium cations and triazinium cations. The heterocyclic nucleus may be substituted at any carbon or nitrogen atom by any alkyl, alkenyl, alkoxy, alkenedioxy, allyl, aryl, arylalkyl, aryloxy, amino, aminoalkyl, thio, thioalkyl, hydroxyl, hydroxyalkyl, oxoalkyl, carboxyl, carboxylalkyl, haloalkyl or halide function including all salts, ethers, esters, pentavalent nitrogen or phosphorus derivatives or stereoisomers thereof. Any of these functions may include a functional group selected from the group consisting of alkenyl, hydroxyl, amino, thio, carbonyl and carboxyl groups. By "alkenyl" is meant any alkenyl group, preferably an alkenyl group with a carbon chain length of between 2 and 20 carbon atoms. The alkenyl group may be a straight chain, branched or cyclic group.

Preferred cations are those based on an imidazolium heterocyclic nucleus. Particularly preferred are those cations based on 1.3-disubstituted imidazolium.

The anion may be selected from halogenated inorganic anions, nitrates, sulphates, carbonates, sulphonates and carboxylates. The alkyl groups of the sulphonates and carboxylates may be selected from Ci - C ₀ alkyl groups and may be substituted at any position with any alkyl, alkenyl, alkoxy, alkeneoxy, aryl, arylalkyl, aryloxy, amino, aminoalkyl, thio, thioalkyl, hydroxyl, hydroxyalkyl, carbonyl, oxoalkyl, carboxyl, carboxyalkyl or halide function, including all salts, ethers, esters, pentavalent nitrogen or phosphorus derivatives or stereoisomers thereof. For example, the anion may be selected from chloride, hexafluorophosphate, tetrafluoroborate, trifluoroacetate, benzoate, salicylate, ± -lactate, (-) lactate, (+) - pantothenate, (±) -tartrate, (+) -tartrate, (-) -tartrate, (±) -hydrogen tartrate, (+) - hydrogen tartrate, (-) -hydrogen tartrate, (±) -potassium tartrate, (+) -potassium tartrate, (-) -potassium tartrate, mesø-tartrate, meso-1 -hydrogen tartrate, meso-2- hydrogen tartrate, meso-l -potassium tartrate, masO-2-potassium tartrate. An especially preferred anion is an organic carboxylate. When the anion is required to include a labile proton then tartrate and lactate functional groups are preferred. Both tartrate and lactate comprise acid and hydroxyl functional groups.

In a further preferred embodiment, the ionic liquid includes an ion which comprises a functional group selected from the group consisting of alkenyl, hydroxyl, amino, thio, carbonyl and carboxyl groups.

Methods of synthesising ionic liquids are also disclosed in "Preparation and Characterization of New Room Temperature Ionic Liquids", Luis C. Branco et al., Chem. Eur. J., 2002, 8, 3671-3677 and "Ion conduction in zwitterionic-type molten salts and their polymers", Yoshizawa et al., J. Mater. Chem., 2001, 11, 1057-1062, and other suitable methods may be used.

As used herein, the "specific binding member" may be a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. Typically, one member of the pair of molecules has an area on its surface, which may be a protrusion or a cavity, which specifically binds to, and is therefore complementary to, a particular spatial and polar organisation of the other member of the pair of molecules. Thus, the members of the pair generally have the property of binding specifically to each other although those members which recognise, rather than specifically bind to, the other member of the pair are also included, for example, an antibody directed to a specific amino acid sequence may also recognise amino acids that are adjacent to the specific sequence. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate, protein-nucleic acid. The present invention is particularly concerned with antigen-antibody type reactions. The specific binding member e.g. antibody, may be in combination or association (e.g. bound to) with the specific binding member of its corresponding specific binding pair e.g. antigen.

An "antibody" is an immunoglobulin, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced.

As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.

A "ligand" may include, inter alia, a peptide or a nucleic acid molecule that binds to a target analyte. The peptide may bind to a target analyte which may be a protein or peptide fragment. The peptide may be a modified peptide.

h a preferred embodiment the specific binding member is an antibody, or an active binding fragment of an antibody. Preferably said antibody, or said binding fragment, is a monoclonal antibody. The antibody may be in combination or association with an antigen.

The specific binding member may comprise a detectable or functional label. A detectable label, for example a radiolabel such as ¹³¹I or ⁹⁹Tc, may be attached to specific binding members of the invention using conventional chemistry known in the art of antibody imaging. Labels also include horseradish peroxidase or alkaline phosphatase. Labels also include micro particles e.g. colloidal gold or fluorescent compounds such as fluorescein or rhodamine. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin. A label may include a labelled antibody (secondary antibody) which may be able to interact with a specific binding member, for example, an antibody (primary antibody).

The specific binding member may be immobilised on a solid support surface, for example a plastic, glass, paper, nylon, nitrocellulose, metal, silica or silicon surface.

In a further aspect, the present invention provides a kit of parts for determining the presence of a target compound in a sample, the kit comprising an assay medium including an ionic liquid and a specific binding member wherein the specific binding member carries a detectable or functional label. The composition or kit of the invention may further comprise an analyte (referred to herein as a "standard") which competes with a target analyte in the sample for binding to the specific binding member. This type of assay is typically referred to as a competitive assay. The standard will generally be provided in the composition in a known amount. The standard may be provided in a separate assay medium to the specific binding member; the assay medium optionally including an ionic liquid as described herein. The standard may be coupled to a detectable or functional label. The standard may be immobilised onto a solid support surface.

A further aspect provides an assay product comprising an assay medium including an ionic liquid and, optionally, including a detectable or functional label.

A further aspect provides an assay product comprising a composition according to the invention. The assay product may be a microarray, microtitre plate, lateral flow device, or part thereof, nylon/nitrocellulose membrane, beads e.g glass, latex or polystyrene.

hi a further aspect, the present invention provides an assay for determining the presence of a target analyte in a sample, the assay comprising: i) providing an assay medium comprising an ionic liquid and a specific binding member; ii) incubating a sample comprising a target analyte with the assay medium; and iii) determining binding of the specific binding member to the target compound.

The use of ionic liquids as a solvent for biological and/or chemical assays has several advantages over traditional aqueous solutions. Ionic liquids have an ability to dissolve a wide range of inorganic, organic, polymeric and biological materials, often to a very high concentration. They have a wide liquid range, allowing both high and low temperature processes to be carried out in the same solvent. They do not elicit solvolysis phenomena and most stabilise short-lived reactive intermediates. There are no pH effects in the solvents and there is practically zero vapour pressure over much of the liquid range. Ionic liquids also exhibit excellent electrical and thermal conductivity whilst being non-flammable, recyclable and generally of low toxicity. By selecting ionic liquids having particular functional groups, it is possible to tailor the ionic liquid to a particular reaction or assay, and thereby optimise the reaction/assay conditions.

Target analytes may include proteins e.g receptors, antigens, hormones, ligands, enzymes, antibodies, DNA binding proteins; nucleic acids e.g DNA or RNA; small molecules; pharamaceutical agents e.g. toxins, substances of abuse such as cocaine, nicotine, opoids (such as heroin), cannabinoids, barbiturates, benzodiazepines, amphetamines, hallucinogens, sedatives, hypnotics, inhalants and anxiolytics including ketamine, PCP (phencyclidine), dextromethorphan, LSD, Ecstasy, caffeine, alcohol, tobacco, cannabis; explosives; microorganisms e.g bacteria, fungi including yeast, viruses; biological or chemical agents e.g. biological warfare agents.

The sample to be assayed may be a water sample, body fluid, food or soil sample. Alternatively, the sample may be a drugs sample or an explosives sample. In a preferred aspect the sample is a body fluid, for example, serum, plasma or urine.

As used herein, "determining" encompasses, inter alia, a simple detection test whereby an indication of the interaction between the target analyte and specific binding member is provided. Alternatively, the determining step may be a quantitative test in which the concentration of the target analyte in the sample is measured.

Determination of the binding of the analyte to the specific binding member may involve techniques such as, for example, surface plasmon resonance (SPR), piezoelectric sensing to detect the mass or bulk of the bound analyte, or immunoprecipitation of the bound analyte-antibody. The assay may be a nonisotopic homogeneous immunoassay which allows the direct observation of the analyte bound to the antibody. This can be achieved, for example, by detecting a change of fluorescence of a fluorophore present in the antibody or antigen. Enzyme Multiplied Immunoassay Technique (EMIT) requires an antibody that can bind to both the target compound and an enzyme in a competitive manner, such that binding of the target compound prevents the enzyme from binding. The ability of the enzyme to perform catalysis is dependent on whether it is bound to the antibody or not. EMIT is particularly useful in the detection of drugs of abuse.

The assay may be a nonisotopically labelled heterogeneous test. Either the antibody or the analyte to be detected may be fixed to a solid interface via, for example, a covalent bond or via adsorption. Examples of this type of immunoassay are the Enzyme Linked Immunosorbent assay (ELISA) in which an antibody is immobilised on a solid carrier which may be, for example, a microtitre plate, polystyrene beads or a test tube and the Sandwich Immunoassay.

h a further aspect the invention provides the use of an assay medium comprising an ionic liquid in an assay, such as an immunoassay. The assay medium may further comprise a detectable marker system capable of detecting the binding of a specific binding member to a target analyte.

In a yet further aspect, the invention provides the use of a composition according to the invention in an assay, preferably an immunoassay. The assay may be useful in the detection of a target analyte as described herein.

The use of a composition according to the invention may allow assays, such as immunoassays, to be carried out in a single phase system in which both the analyte and the binding member are soluble and stable. Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis.

The present invention will now be described by way of example only:

EXAMPLE

Protocol for the detection of a test antigen using competitive ELISA in an ionic liquid

Unless otherwise specified, all solutions are prepared in the ionic liquid diethanolaminobutylammonium hexafluorophosphate (DEABA PF₆).

To a 96-well plate (with 8 rows labelled A to H and 12 columns numbered 1 to 12) 200 μl of standard antigen solution (2 μl/ml in DEABA PF₆) is added. The plate is incubated at 37 °C for 2 hours. Unbound antigen is washed from wells using DEABA PF₆

150 μl DEABA PF₆ (containing 0.1% BSA) is added to each well. To wells A2 to D2 50 μl of standard antigen solution (10 mM) are added with mixing. To wells E2 to H2 50 μl of test solution are added with mixing.

50 μl from each well in column 2 are removed and transferred to corresponding wells in lane 3, again with mixing, hi this manner serial dilutions across the plate up to lane 11 are performed.

Lane 1 is the negative control, and lane 12 is be the positive control.

To each well in columns 2 to 12 50 μl of antibody solution (1:1000 dilution of appropriate antibody that is linked to horseradish peroxidase) is added. The plate is covered and incubated at 37 °C for 2 hours, to allow antibody to bind to target antigen. Unbound antibody and antigen is washed from the wells using DEABA PF₆

The chromagemc substrate, 2,2'-azino-di-(3 ethylbenzthiazoline sulfonic acid (ABTS) solution (in water), is prepared as follows; o 274 mg of ABTS o 147 ml of 0.1 M citric acid o 103 ml of 0.2 M dibasic sodium hydrogen phosphate

To a 25 ml aliquot, 25 μl of 30% H₂O₂ is added and mixed. 200 μl of substrate solution is added to each well. Colour is allowed to develop for approximately 10 minutes. Absorbance of solutions in each well is measured at 416 nm.

Claims

1. A composition comprising an assay medium wherein the assay medium includes an ionic liquid and a specific binding member.

2. A composition as claimed in claim 1 wherein the assay medium comprises an ionic liquid and less than 10% water.

3. A composition as claimed in claim 2 wherein the assay medium comprises less than 1% water.

4. A composition as claimed in claim 1 wherein the ionic liquid is composed of cationic and anionic portions according to the following general formula:

[A]„⁺[Y]_n-

5. A composition as claimed in claim 4 wherein the cationic portion of the ionic liquid is composed of a quaternary nitrogen-based ion which is aromatic, aliphatic, cyclic or acyclic.

6. A composition as claimed in claim 5 wherein the quaternary nitrogen-based ion is based on a nucleus selected from quaternary ammonium cations, pyrazolium cations, imidazolium cations, triazolium cations, pyridinium cations, pyridazinium cations, pyrimidinium cations, pyrazinium cations and triazinium cations.

7. A composition as claimed in claim 1 wherein the ionic liquid includes an ion comprising a functional group selected from the group consisting of alkenyl, hydroxyl, amino, thio, carbonyl and carboxyl groups.

8. A composition as claimed in claim 4 wherein the anionic portion is selected from halogenated inorganic anions, nitrates, sulphates, carbonates, sulphonates and carboxylates.

9. A composition as claimed in claim 1 wherein the specific binding member is an antibody

10. A composition as claimed in claim 1 wherein the specific binding member is an active binding fragment of an antibody.

11. A composition as claimed in claim 9 or 10 wherein the antibody or binding fragment is a monoclonal antibody.

12. A composition as claimed in claim 9, 10 or 11 wherein the antibody or binding fragment is in association with an antigen.

13. A composition as claimed in claim 1 wherein the specific binding member carries a detectable or functional label.

14. A composition as claimed in claim 13 wherein the detectable label is a radiolabel

15. A composition as claimed in claim 13 wherein the detectable label is a labelled antibody.

16. A composition as claimed in claim 1 wherein the specific binding member is immobilised on a solid support surface.

17. A composition as claimed in claim 16 wherein the support surface is selected from the group consisting of plastic, glass, paper, nylon, nitrocellulose, metal, silica or silicon surfaces.

18. An assay product comprising an assay medium including an ionic liquid and, optionally, including a detectable or functional label.

19. An assay product comprising a composition as claimed in claim 1.

20. An assay product as claimed in claim 19 that is a microarray, microtitre plate, lateral flow device, or part thereof, nylon/nitrocellulose membrane or solid beads.

21. An assay for determining the presence of a target analyte in a sample, the assay comprising: i) providing an assay medium comprising an ionic liquid and a specific binding member; ii) incubating a sample comprising a target analyte with the assay medium; and iii) determining binding of the specific binding member to the target compound.

22. An assay as claimed in claim 21 wherein the target analyte is selected from the group consisting of receptors, antigens, hormones, ligands, enzymes, antibodies, peptides, DNA binding proteins, DNA, RNA, pharamaceutical agents, toxins, substances of abuse (narcotics) such as cocaine, nicotine, opoids (such as heroin), cannabinoids, barbiturates, benzodiazepines, amphetamines, hallucinogens, sedatives, hypnotics, inhalants and anxiolytics including ketamine, PCP (phencyclidine), dexfromethorphan, LSD, Ecstasy, caffeine, alcohol, tobacco, cannabis; explosives; microorganisms, biological warfare agents and chemical warfare agents.

23. An assay as claimed in claim 21 wherein the sample to be assayed is selected from water, body fluid, food, soil, drugs, sample or explosives.

24. An assay as claimed in claim 23 wherein the body fluid is serum, plasma or urine.

25. An assay as claimed in claim 21 wherein the assay is an immunoassay.

26. An assay as claimed in claim 25 that is an Enzyme Linked hnmunosorbent assay (ELISA).

27. An assay as claimed in claim 21 that is a competitive assay.

28. Use of an assay medium comprising an ionic liquid in an assay.

29. Use as claimed in claim 28 wherein the assay is an immunoassay.