Patent Application
20220137034
The present invention relates to an in vitro method for determining the presence, absence and/or concentration of an analyte in a sample. In particular, the present invention relates to a method using a fluorescently based competition assay comprising a fluorescently labelled analyte binding protein and a fluorescently labelled analyte analogue.
The quantification of small molecule analytes in particular in complex biological samples such as blood is of importance e.g. for determining the concentration of a specific drug molecule in the blood of patients. Traditionally this is done by pre-treatment of the blood sample and analysis by HPLC-MS in clinical biochemical departments. This is however time consuming, require specialists and require sophisticated equipment.
Few point-of-care (POC) technologies are available for the detection of small molecules, where the glucose meter is an outstanding exception, which relies on the enzymatic conversion of glucose.
For almost all small molecule drugs, such enzymatic conversion is not available and other techniques must be applied.
Surface plasmon resonance (SPR) is an assay principle that has been employed in a variety of setups for small molecule detection. Surface plasmon resonance is a heterogeneous assay type that utilizes mass changes at a surface caused by binding, usually as competitive immunoassays. The setup allows for detection of a wide range of compounds at the relevant ranges, however it is an expensive technology. (Sensors 2010, 10, 7323-7346).
The proximity hybridisation technique, usually used for detection of compounds exhibiting multiple epitopes, was recently adapted for small molecule detection. Employing the bivalent binding interaction of antibodies allowed for detection of digoxin in the nanomolar range. The assay exhibited detection times of 10-30 mins. (Anal. Chem. 2018, 90, 9667-9672)
Another type of detection system which is used for small molecule detection is the so-called quench body system (Anal. Methods, 2016, 8, 7774-7779), where a fluorophore close to the binding site is in part quenched in the absence of the analyte. This is not a ratiometric system and furthermore it must be optimized significantly for each analyte.
Ricci and co-workers displayed a sensor in which structural change of DNA induced by proximity hybridisation allowed for detection of antibodies in 10% plasma in a homogenous fashion (J. Am. Chem. Soc. 2018, 140, 947-953).
Plaxco and coworkers showed measurement of small molecule targets in the μM range directly in undiluted whole blood with overall good precision employing a heterogenous aptamer based sensor system. The setup has likewise been implemented directly in live animals for real time measurement of small molecule targets. The real time measurement allowed for precise control of drug in the animal model based on feedback-controlled dosing (Proc. Natl. Acad. Sci. USA 2017, 114, 645-650).
In recent work a homogenous for detection of cortisol showed drastic improvement relative to the conventional heterogeneous cortisol assay types. The work was implemented in a Point of care pchip for single molecule array measurements with vastly increased sensitivity compared to conventional methods (J. Am. Chem. Soc. 2018, 140, 18132-18139).
In a series of papers and a patent Kai Johnsson has described semisynthetic protein peptide systems that provides a FRET readout upon small molecule binding in solution (J. Am. Chem. Soc. 2009, 131, 5873; J. Am. Chem. Soc., 2009, 131, pp 5873-5884; Angew. Chem. Int. Ed. 2014, 53, 1302, WO2015067302A1).
A so-called strand displacement assay for detection of small molecules has also been developed. (J. Am. Chem. Soc. 2014, 136, 11115-11120; EP2895617A1). A platform for digoxin detection within the relevant range, employing a highly selective assay and a recyclable and passively driven G-chip has also been developed. (Adv. Sci. 2019, doi/10.1002/advs.201802051). Furthermore, a rapid amplification based homogenous assay for detection of Methotrexate in human plasma within 4 minutes within the relevant ranges has been developed (ACS Sens. 2018, 3, 9, 1706-1711).
Most of the assays described above have, however, not been developed for detection of analytes in blood. In the few examples where this is the case the assay is not generic, too slow or not suitable for a POCT device.
Hence, an improved method for fast determination of an analyte would be advantageous, and in particular a more efficient and/or reliable method for fast determination of the concentration of an analyte in a blood sample would be advantageous.
The present invention relates to an in vitro method for determining the presence, absence and/or concentration of an analyte (1) in a sample. The method uses an optically based competition assay comprising a optically labelled analyte binding protein (3) and a optically labelled analyte analogue (6). The concentration/presence of the analyte (1) is determined by inhibitory binding of the analyte (1) to the analyte binding protein (3) thereby impeding binding of the analyte analogue (6) to the analyte binding protein (3). The invention further relates to kits, solid supports (7), cartridges (8), detection chips (10) and uses thereof. The method is schematically outlined in
Thus, an object of the present invention relates to the provision of a sensitive method for determining the presence, and in particular the concentration, of an analyte in a sample. In particular, it is an object of the present invention to provide a method that solves the problems of the prior art with fast determination of the concentration of an analyte in a sample.
Thus, one aspect of the invention relates to an (in vitro) method for determining the presence and/or concentration of an analyte (1) in a sample (2), said method comprising
In a preferred embodiment, the first member (5A) of the optical signal pair (5) is covalently coupled to the analyte binding protein (3) through a first oligonucleotide linker (preferably a DNA linker) and the analyte analogue (6) is covalently coupled to the second member (5B) of the optical signal pair (5) through a second oligonucleotide linker (preferably a DNA linker). This setup is tested in the example section.
In another preferred embodiment, said sample (2) is brought in contact with the analyte binding protein (3) before being brought in contact with said analyte analogue (6). Example 11 documents that the order of mixing the assay species influences on the efficiency of the assay.
Another aspect of the present invention relates to relates to a kit comprising
Yet another aspect of the present invention is to provide a porous solid support comprising
In a preferred embodiment, the solid support is configured for receiving a sample, and bringing said sample in contact with the analyte binding protein (3) before bringing said sample in contact with said analyte analogue (6).
A further aspect of the invention relates to a cartridge (8):
Yet a further aspect relates to a detection chip (10) comprising the solid support (8) according to the invention mounted in a cartridge (8) according to the invention.
Still another aspect of the present invention is to provide the use of the kit according to the invention and/or the porous solid support (7) according to the invention and/or the cartridge (8) according to the invention and/or the detection chip (10) for determining the presence, absence and/or concentration of an analyte (1) in a sample (2).
The present invention will now be described in more detail in the following.
Prior to discussing the present invention in further details, the following terms and conventions will first be defined:
Small Molecules
In the Present Context, the Term “Small Molecule” Relates to a Low Molecular weight (<900 daltons) organic compound that may regulate a biological process, with a size on the order of 1 nm. Most drugs are small molecules. Larger structures such as nucleic acids and proteins, and many polysaccharides are not small molecules.
Antibody
The term “antibody” as used herein refers to a protein of the immunoglobulin (Ig) superfamily that binds non-covalently to certain substances (antigens/analytes) to form an antibody-antigen/analyte complex. Antibodies can be endogenous, or polyclonal wherein an animal is immunized to elicit a polyclonal antibody response or by recombinant methods resulting in monoclonal antibodies produced from hybridoma cells or other cell lines. It is understood that the term “antibody” as used herein includes within its scope any of the various classes or sub-classes of immunoglobulin derived from any of the animals conventionally used.
Antibody Fragments
The term “antibody fragments” as used herein refers to fragments of antibodies that retain the principal selective binding characteristics of the whole antibody. Particular fragments are well-known in the art, for example, Fab, Fab′, and F(ab′)2 which are obtained by digestion with various proteases, pepsin or papain, and which lack the Fc fragment of an intact antibody or the so-called “half-molecule” fragments obtained by reductive cleavage of the disulfide bonds connecting the heavy chain components in the intact antibody. Such fragments also include isolated fragments consisting of the light-chain-variable region, “Fv” fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker. Other examples of binding fragments include (i) the Fd fragment, consisting of the VH and CH1 domains; (ii) the dAb fragment, which consists of a VH domain; (iii) isolated CDR regions; and (iv) single-chain Fv molecules (scFv) described above. In addition, arbitrary fragments can be made using recombinant technology that retains antigen-recognition characteristics.
Analyte
The term “analyte” as used herein refers to any entity that an analyte binding protein has affinity for.
Analyte Analogue
The term “analyte analogue” as used herein refers to an analyte that has been modified to contain a reporter molecule and optionally to alter the affinity of the analyte analogue for the analyte binding protein, compared to an unmodified analyte.
Analyte Binding Protein
The term “analyte binding protein” as used herein refers to a protein that has affinity for a discrete epitope, antigen or analyte that can be used with the methods of the present invention. Preferably, the analyte binding protein is an antibody or fragment thereof.
Affinity
The term “affinity” as used herein refers to the strength of the binding interaction of two molecules, such as an antibody and its antigen (or the analyte binding protein and the analyte/analyte analogue according to the invention). For bivalent molecules such as antibodies, affinity is typically defined as the binding strength of one binding domain for the antigen, e.g. one Fab fragment for the antigen. The binding strength of both binding domains together for the antigen is referred to as “avidity”. As used herein “High affinity” refers to a ligand that binds to an antibody having an affinity constant (Ka) greater than 104 M−1, typically 105-1011 M−1; as determined by inhibition ELISA or an equivalent affinity determined by comparable techniques such as, for example, Scatchard plots or using Kd/dissociation constant, which is the reciprocal of the Ka, etc.
Energy Transfer
The term “energy transfer” as used herein refers to the process by which the excited state energy of an excited group, e.g. fluorescent reporter dye, is conveyed through space or through bonds to another group, e.g. a quencher moiety or fluorophor, which may attenuate (quench) or otherwise dissipate or transfer the energy to another reporter molecule or emit the energy at a longer wavelength. Energy transfer typically occurs through fluorescence resonance energy transfer (FRET).
Optical Pair
The term “optical pair” as used herein refers to any two moieties that can form a pair allowing for optical determination of such a pair. A pair could be to parts of a protein pair, which are only functional when they are in proximity. An example of such a pair is Cy3 and Cy5.
Fluorescent Pair
The term “fluorescent pair” as used herein refers to any two moieties that participate in energy transfer. Typically, one of the moieties acts as a fluorescent reporter, i.e. donor, and the other acts as an acceptor, which may be a quenching compound or a compound that absorbs and re-emits energy in the form of a fluorescent signal.
Quencher
The term “quencher” or “quenching moiety” as used herein refers to a compound that is capable of absorbing energy from an energy donor that is not re-emitted (non-fluorescent) or re-emitted at a detectably different wavelength from the energy emitted by the donor molecule. In this respect, quenchers may be essentially non-fluorescent or fluorescent.
Sample
The term “sample” as used herein refers to any material that may contain an analyte of interest. Typically, the sample comprises a population of cells, cellular extract, subcellular components, tissue culture, a bodily fluid, tissue, and reaction mixtures. The sample may be in an aqueous solution, a viable cell culture or immobilized on a solid or semi-solid surface such as a gel, a membrane, a glass surface, a microparticle or on a microarray. Preferably, the sample is a blood sample, such as a blood plasma sample.
Kit
The term “kit” as used herein refers to a packaged set of related components, typically one or more compounds or compositions.
Method for Determining the Presence and/or Concentration of an Analyte in a Sample
A described above, the present invention relates to a method where the concentration of an analyte can be determined in a sample, preferably a blood plasma sample. As outlined in
Preferably, the method is based on FRET technology. The method further takes advantage of site-directed labelling of the analyte binding protein making the use of FRET very efficient. Thus, an aspect of the invention relates to an (in vitro) method for determining the presence and/or concentration of an analyte (1) in a sample (2), said method comprising
A special feature of the method of the invention is that it can determine the concentration of the analyte in the sample. Thus, in a preferred embodiment, the concentration of the analyte in the sample is determined. Concentration determination of different analytes are further described in examples 3-8.
In a preferred embodiment, the first member (5A) of the optical signal pair (5) is covalently coupled to the analyte binding protein (3) through a first oligonucleotide linker (preferably a DNA linker) and the analyte analogue (6) is covalently coupled to the second member (58) of the optical signal pair (5) through a second oligonucleotide linker (preferably a DNA linker). This setup is used in the example section. The use of the oligonucleotide linkers also makes it possible to make specific labelling instead of random labelling. This is shown in e.g. examples 9 and 10.
In another preferred embodiment, said sample (2) is brought in contact with the analyte binding protein (3) before being brought in contact with said analyte analogue (6). Example 11 documents that the order of mixing the assay species influences on the efficiency of the assay.
To determine the presence or absence of an analyte, it may be advantageous to compare the fluorescent to one or more reference levels. Thus, in another embodiment, in said determination step V.,
The method of the invention is based on a change in signal when an analyte (1) binds to the labelled analyte binding protein (3), thereby releasing the labelled analyte analogue (6). Thus, in a further embodiment, in said determination step V. and/or VI., the determination is performed by illuminating the sample (2) with an appropriate wavelength and observing the sample (2) at relevant wavelengths, wherein the sample (2) generates a change in detectable signal in the presence of the analyte (1) in the sample (2). It is noted that such effect can preferably be obtained e.g. using a FRET pair or a fluorophore-quencher pair. In the example section, FRET is used.
When determining the concentration in a sample other reference levels may be used. Thus, in an embodiment, in said determination step VI., a reference level is a subset of known concentrations, such as obtained from a titration curve of the analyte. Examples 3-8+
The sample (2) to be analyzed for the presence of the analyte in question may be from different sources. Thus, in an embodiment said sample (2) is a biological sample, a water sample, environmental sample, a food sample, a beverage, a surface swap, a medical formulation, a drug formulation, an addictive substance or formulation. Medical formulations may be dissolved or diluted. A medical formulation could also be an illegal drug.
In yet an embodiment, said biological sample (2) has been obtained from a human or animal, such as a mammal. The sample may have been previously obtained from a subject, meaning that the method is an in vitro method. In yet a further embodiment said biological sample (2) is selected from the group consisting of a blood sample, such as whole blood, such as blood plasma or blood serum, saliva, urine, CSF and a tissue sample.
The analyte to be determined may be of different types. Thus, in an embodiment, said analyte (1) is selected from the group consisting of a small molecule, a peptide and a protein, preferably a small molecule. In yet an embodiment said small molecule has a molecular weight of less than 900 Dalton, such as in the range 100-900, 200-900, 300-900, 400-900 or 500-900 Daltons. In a more specific embodiment, said small molecule is selected from the group consisting of an anticoagulant, such as Dabigatran and Apixaban, an antibiotic, such as such as Linezolid, a drug, such as an anti-cancer drug. Data for these compounds are presented in examples 3-8.
In a further embodiment, said analyte (1) is selected from the group consisting of anticoagulants such as Warfarin, Edoxaban, Rivaroxaban and Betrixaban, and immusupressants such as Methotrexate, Cyclosporine, Tacrolimus, Sirolimus, and Everolimus, and illegal drugs such as Cannaboids, Cocaine, Opiates (Heroin), Methamphetamine, Amphetamine, and Phencyclidine.
In yet a further embodiment, said analyte (1) is selected from the groups consisting of:
1)
Drugs such as selected from the group consisting of Atorvastatin, Levothyroxine, Lisinopril, Omeprazole, Metformin, Amlodipine, Simvastatin, Metoprolol, Losartan, Azithromycin, Zolpidem, Hydrochlorothiazide, Furosemide, Metoprolol, Pantoprazole, Gabapentin, Amoxicillin, Prednisone, Sertraline, Tamsulosin, Fluticasone, Pravastatin, Tramadol, Montelukast, Escitalopram, Carvedilol, Alprazolam, Warfarin, Meloxicam, Clopidogrel, Amoxicillin, Allopurinol, Bupropion, Lisinopril, Citalopram, Losartan, Atenolol, Cialis, Duloxetine, Fluoxetine, Fenofibrate, Crestor, Venlafaxine, Ventolin, Cyclobenzaprine, Trazodone, and Methylprednisolone.
2)
(Illegal) food additives, such as selected from the group consisting of cinnamyl anthranilate, cobalt salts, coumarin, cyclamate, diethyl pyrocarbonate (DEPC), dulcin (p-ethoxy-phenylurea), monochloroacetic acid, nordihydroguaiaretic acid (NDGA), oil of calamus, polyoxyethylene-8-stearate (Myrj 45), safrole, thiourea, and melamine.
3)
Vitamins such as selected from the group consisting of all-trans-Retinol, Retinals, and alternative provitamin A-functioning Carotenoids including all-trans-beta-carotene, Thiamine, Riboflavin, Niacin, Niacinamide, Nicotinamide riboside, Pantothenic acid, Pyridoxine, Pyridoxamine, Pyridoxal, Biotin, Folates, Folic acid, Cyanocobalamin, Hydroxocobalamin, Methylcobalamin, Adenosylcobalamin, Ascorbic acid, Cholecalciferol (D3), Ergocalciferol (D2), Tocopherols, Tocotrienols, Phylloquinone, and Menaquinones.
4)
Exogenous Anabolic Androgenic Steroids (AAS), such as selected from the group consisting of 1-androstendiol, 1-androstendione, bolandiol, bolasterone, boldenone, boldione, calusterone, clostebol, danazol, dehydrochlormethyltestosterone, desoxymethyltestosterone, drostanolone, ethylestrenol, fluoxymesterone, formebolone, furazabol, gestrinone, 4-hydroxytestosterone, mestanolone, mesterolone, metenolone, methandienone, methandriol, methasterone, methyldienolone, methyl-1-testosterone, methylnortestosterone, methyltrienolone, methyltestosterone, mibolerone, nandrolone, 19-norandrostenedione, norboletone, norclostebol, norethandrolone, oxabolone, oxandrolone, oxymesterone, oxymetholone, prostanozol, quinbolone, stanozolol, stenbolone, 1-testosterone, tetrahydrogestrinone and trenbolone. Endogenous Anabolic Androgenic Steroids (AAS), such as selected from the group consisting of androstenediol, androstenedione, dihydrotestosterone, prasterone and testosterone.
5)
Other Anabolic Agents, such as selected from the group consisting of clenbuterol, selective androgen receptor modulators (SARMs), tibolone, zeranol and zilpaterol.
6)
Short-acting 82 agonists (SABAs), such as selected from the group consisting of bitolterol—Tornalate, fenoterol—Berotec, isoprenaline (INN) or isoproterenol (USAN)—Isuprel, levosalbutamol (INN) or levalbuterol (USAN)—Xopenex, orciprenaline (INN) or metaproterenol (USAN)—Alupent, pirbuterol—Maxair, procaterol, ritodrine—Yutopar, salbutamol (INN) or albuterol (USAN)—Ventolin, terbutaline—Bricanyl and albuterol—Ventolin/Proventil.
7)
Aromatase inhibitors such as including, but not limited to aminoglutethimide, anastrozole, exemestane, formestane, letrozole and testolactone.
8)
Selective estrogen receptor modulators (SERMs) including, but not limited to raloxifene, tamoxifen and toremifene.
9)
Other anti-estrogenic substances, including but not limited to clomiphene, cyclofenil and fulvestrant.
10)
Diuretics, such as selected from the group consisting of acetazolamide, amiloride, bumetanide, canrenone, chlorthalidone, etacrynic acid, furosemide, indapamide, metolazone, spironolactone, thiazides, triamterene, epitestosterone and probenecid.
11)
Stimulants, such as selected from the group consisting of adrafinil, adrenaline, amfepramone, amiphenazole, amphetamine, amphetaminil, benzphetamine, benzylpiperazine, bromantan, cathine, clobenzorex, cocaine, cropropamide, crotetamide, cyclazodone, dimethylamphetamine, ephedrine, etamivan, etilamphetamine, etilefrine, famprofazone, fenbutrazate, fencamfamin, fencamine, fenetylline, fenfluramine, fenproporex, furfenorex, heptaminol, isometheptene, levmethamfetamine, meclofenoxate, mefenorex, mephentermine, mesocarb, methamphetamine (D−), methylenedioxyamphetamine, methylenedioxymethamphetamine, methylamphetamine, methylephedrine, methylphenidate, modafinil, nikethamide, norfenefrine, norfenfluramine, octopamine, ortetamine, oxilofrine, parahydroxyamphetamine, pemoline, pentetrazol, phendimetrazine, phenmetrazine, phenpromethamine, phentermine, 4-phenylpiracetam (carphedon), prolintane, propylhexedrine, selegiline, sibutramine, strychnine and tuaminoheptane.
12)
Antibiotics such as selected from the group consisting of Vancomycin, Teicoplanin, Linezolid, Daptomycin, Trimethoprim/sulfamethoxazole, Doxycycline, Ceftobiprole,
Ceftaroline, Clindamycin, Dalbavancin, Fusidic acid, Mupirocin (topical), Omadacycline, Oritavancin, Tedizolid, Telavancin, Tigecycline, Pseudomonas aeruginosa, Carbapenems, Ceftazidime, Cefepime, Ceftobiprole, Ceftolozane, Fluoroquinolones, Piperacillin, Ticarcillin, Streptogramins, Tigecycline, Daptomycin, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin,
Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Ansamycins, Geldanamycin, Herbimycin, Rifaximin, Carbacephem, Loracarbef, Carbapenems, Ertapenem, Doripenem, Imipenem, Meropenem, Cefadroxil, Cefazolin, Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan, Cefamandole, Cefmetazole, Cefonicid, Loracarbef, Cefprozil, Cefuroxime, Cephalosporins, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cephalosporins, Cefepime, Cephalosporins, Ceftaroline fosamil, Ceftobiprole, Glycopeptides, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Lincosamides, Clindamycin, Lincomycin, Lipopeptide, Daptomycin, Macrolides(Bs), Azithromycin, Clarithromycin, Erythromycin, Roxithromycin, Telithromycin, Spiramycin, Fidaxomicin, Monobactams, Aztreonam, Nitrofurans, Furazolidone, Nitrofurantoin(Bs), Oxazolidinones(Bs), Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfonamidochrysoidine (archaic), Tetracyclines, Demeclocycline, Doxycycline, Metacycline, Minocycline, Oxytetracycline, Tetracycline, Drugs against mycobacteria, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol(Bs), Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin.
13)
Compounds selected from the group consisting of Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole and Trimethoprim.
Thus the invention can find use in the detection/quanitification of many different groups of compounds/small molecules.
The binding affinity to the analyte binding protein (3) may be different for the analyte and the analyte analogue. Thus, in an embodiment said analyte analogue (6) has a higher, a lower or an equal affinity for the binding site (4) in the analyte binding protein (3), compared to the analyte (2), preferably the binding affinity is lower.
In a further embodiment, said analyte binding protein (3) is selected from the group consisting of an antibody or fragment thereof, such as a Fab fragment, a
F(ab′)2, a Fv, a Fd, a dAb, a scFv fragment or a single-domain antibody (sdAb) such as a Nanobody, and an affibody.
In yet an embodiment, the analyte binding protein is selected from the group consisting of idarucizumab, an anti-apixaban antibody and an anti-linezolid antibody.
Preferably, the optical signal pairs are fluorescent pairs. Different types of fluorescent pairs may find use as optical signal pairs in the method of the invention. Thus in an embodiment the fluorescent pair (5) is selected from the group consisting of fluorescent pairs.
In an embodiment, the fluorescent pairs are Quantum dots.
In another embodiment, the first member (5A) of the optical signal pair (5) is (covalently) coupled to the analyte binding protein (3) through a first linker, preferably an oligonucleotide linker, even more preferably a DNA linker. In yet another embodiment, the second member (5B) of the optical signal pair (5) is (covalently) coupled to the analyte analogue (6) through a second linker, preferably an oligonucleotide linker, even more preferably a DNA linker. Preferably, the members of the optical pairs are covalently coupled, even more preferably through DNA linkers.
Advantages of using oligonucleotide linkers are:
In the event oligonucleotides are used as linkers, it may be an advantage that the oligonucleotides do not hybridize to each other. Thus, in an embodiment, the first oligonucleotide linker and the second oligonucleotide linker are non-complementary. In a specific embodiment, the first oligonucleotide linker and the second oligonucleotide linker are identical in sequence.
In an embodiment, the first linker and the second linkers consist only of an oligonucleotide, preferably a DNA linker. In another embodiment, the first linker and the second linker does not comprise any peptides, proteins, alkyls or polyethylene glycol (PEG).
In an embodiment, the first and/or second oligonucleotide linker comprises one or more modified/artificial nucleotides, such as LNA, PNA, L or D acyclic threoniniol nucleic acid (aTNA), 2-fluoro-DNA or 2-MeO-DNA, and morpholino-DNA.
In yet an embodiment the DNA strands used in the invention are SEQ ID NO's 1 and 4. With reference to
In a further embodiment, the first oligonucleotide and the second oligonucleotide are partly complementary, such as by having maximum 10 complementary bases such as maximum 9 or such as maximum 8 complementary bases. By having a weak complementary between the first oligonucleotide and the second oligonucleotide, binding efficiency can be increased for weak bindings between analyte analogue (6) and the analyte binding protein (3) or for increasing the FRET signal. The skilled person knows of methods for determining complementary (sequence identify), such as by using the software NUPACK (http://www.nupack.org/), or other oligonucleotide tools available online.
In an embodiment, the first oligonucleotide is at most 90% complementary to the second oligonucleotide, such as at most 80%, such as at most 70%, such as at most 60%, such as at most 50%, such as at most 40%, preferably at most 30%, more preferably at most 20%, most preferably at most 10% complementary to the second oligonucleotide. Preferably, the first and second oligonucleotides are DNA.
In the present context, when referring to “complementary”, G pairs to C, A pairs to T and U and vice versa. In some embodiments, G may also pair to U and vice versa to form a so-called wobble base pair. A wobble base pair is a non-Watson-Crick base pairing between two nucleotides in RNA molecules. The four main wobble base pairs are guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine (G-U, I-U, I-A and I-C).
Thus, in the present context “complementarity” is a measure of complementarity between nucleic acids at nucleotide level. The nucleic acid complementarity may be determined by comparing the nucleotide sequence in a given position in each sequence of the first and second linkers when the sequences are aligned.
To determine the percent identity of two nucleic acids sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first nucleic acid sequence for optimal alignment with a second nucleic acid sequence). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are the same length.
In another embodiment, the two sequences are of different length and gaps are seen as different positions. One may manually align the sequences and count the number of identical nucleotides. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST program of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention.
To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilising the NBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. “scoring matrix” and “gap penalty” may be used for alignment. In the context of the present invention, the BLASTN default settings may be advantageous.
The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted. An embodiment of the present invention thus relates to sequences of the present invention that has some degree of sequence variation.
In yet another embodiment, the first member (5A) of the fluorescent pair (5) is covalently coupled to the analyte binding protein (3) through a first linker. In yet another embodiment the second member (5B) of the fluorescent pair (5) is covalently coupled to the analyte analogue (3) through a second linker. In yet an embodiment, the first linker is not a peptide or a protein-based molecule having affinity for the analyte binding protein (3), such as an antibody or fragment thereof.
The magnitude of the FRET signal depends on the proximity between the first member (5A) and second member (5B) of the optical signal pair (5). Thus, through adjustment of the length of the linkers, the FRET signal can be fine-tuned by altering the proximity of the first member (5A) and second member (5B) of the optical signal pair (5). Therefore, in an embodiment, the first linker is an oligonucleotide consisting of between 2 and 100 nucleotides, such as between 2 and 75 nucleotides, preferably between 5 and 40 nucleotides. In another embodiment, the second linker is an oligonucleotide consisting of between 2 and 100 nucleotides, such as between 2 and 75 nucleotides, preferably between 5 and 40 nucleotides. In a further embodiment, the first linker and second linker are oligonucleotides linkers, each comprising at least 5 nucleotides, preferably at least 10 nucleotides. In yet another embodiment, the first linker and second linker are oligonucleotides linkers, each comprising at most 100 nucleotides, such as at most 75 nucleotides, preferably at most 50 nucleotides. Preferably, the oligonucleotides are DNA.
In an embodiment, the first member (5A) of the optical signal pair (5) is a donor fluorophore of a FRET pair and the second member (5B) of the optical signal pair (5) is an acceptor fluorophore of a FRET pair. In another embodiment, the first member (5A) of the optical signal pair (5) is an acceptor fluorophore of a FRET pair and the second member (5B) of the optical signal pair (5) is a donor fluorophore of a FRET pair. Preferably, the donor and acceptor fluorophores are coupled to the first and second oligonucleotide linkers, preferably DNA linkers.
Different types of references signals may be used in a method according to the present invention. Thus, in an embodiment the reference fluorescent signal is a negative control (such as a corresponding sample known not to comprise the analyte in question).
When the concentration of the analyte is determined, it may be determined within a certain range. Thus, in an embodiment, the concentration determination is in the range 0 nM to 2000 nM, such as in the range 0 to 1500 nM, such as 0.1 to 1200 nM, such as in the range 0.1-200 nM, such as in the range 2-20 nM OR such as in the range 10 to 1000 nM, such as 10 to 400 nM, or such as 10 to 200 nM. In examples 3-8 concentrations in the range 0-100 nM (
The method of the invention may take place in solution and/or using a solid support format. Thus, in an embodiment, said analyte binding protein (3) and said analyte analogue (6) are provided on a (porous) solid or (porous) semi-solid support.
In yet an embodiment, said analyte binding protein (3) and said analyte analogue (6) are provided on independently distinct regions on (porous) the semi-solid or (porous) solid support. Examples 4, 6 and 8 show data for the method in a solid support format. In a further embodiment, the semi-solid or solid support is selected from the group consisting of a paper, a membrane, a polymeric gel, a fiber, a polymer, a polymeric fiber, a polymeric particle, a polymeric microparticle or an array.
The order of which the components are brought in contact with each other can also vary. Thus, in an embodiment said sample (2) is brought in contact with the analyte binding protein (3) before being brought in contact with said analyte analogue (6). In yet another embodiment, said analyte analogue (6) is brought in contact with a mixture of the sample (2) and the analyte binding protein (3). Example 11 documents that the order of mixing the assay species influences on the efficiency of the assay.
In yet another embodiment, said sample is provided in a dissolved state to the first region and is subsequently transported to the second region and subsequently detected, e.g. in a dedicated detection chamber (9). In a related embodiment, said transport is by capillary forces.
It is important for FRET analysis (or for fluorophore-quencher pairs) that the molecules are in close proximity. Thus, in an embodiment, the distance between the first member 5A and the second member 5B, when the analyte analogue is bound to the analyte binding protein, is in the range 1-100 Angstrom, such as 10-100 Ångstrom, such as 10-50 Ångstrom.
In another embodiment, the distance between the first member (5A) and the second member (5B), when the analyte analogue is bound to the analyte binding protein, is in the range 10-30 Ångstrom, such as 10-20 Ångstrom, such as 10-15 Ångstrom.
The proximity between the first member (5A) and the second member (5B) is amongst others influenced by the coupling technique of the first member (5A) to the analyte binding protein (3). Thus, coupling techniques that ensure coupling of the first member (5A) near the binding site (4) for the analyte (1) is preferred. In an embodiment, the first member (5A) is coupled to the analyte binding protein (3) using a first guiding oligonucleotide (DNA) strand (11) conjugated to an analyte (1), and hybridized to a reactive oligonucleotide (DNA) strand (13) conjugated to the first member (5A), and a reactive group. In another embodiment, the reactive group binds in the vicinity of the binding site (4) for the analyte (1), such as within 5-30 Å of the binding site (4). In a further embodiment, the guiding strand (11) binds non-covalently to the binding site (4). In yet another embodiment, a releasing strand (12) is applied to remove the guiding strand (11) by strand displacement. The analyte binding protein (3) may be e.g. natural and recombinant proteins, such as antibodies Fab domains and other proteins with affinity for a small molecule ligand.
Alternative coupling techniques can be employed as well with the method described herein. Thus, in an embodiment, the analyte binding protein (3) is an antibody and the first member (5A) is coupled to the Fab region of said antibody. For coupling to the Fab region, the first member (5A) may be conjugated to a linker, such as an oligonucleotide linker, preferably a DNA linker. In another embodiment, the first member (5A) is coupled to the analyte binding protein (3) by thiol coupling, such as coupling to cysteine residues of the analyte binding protein (3). For thiol coupling to the analyte binding protein (3), the first member (5A) may be conjugated to a linker, such as an oligonucleotide linker, preferably a DNA linker.
Optical detection signals can be determined using different setups. Thus, in an embodiment, the optical signal is determined using spectroscopy, such as FRET as shown in
It is to be understood that the method of the invention may be performed in a detection chip (10) according to the invention using a solid support (7) and a cartridge (8) according to the invention as further described below. See also
Kit
The present invention also relates to a kit enabling performing the method of the invention. Thus, in an aspect the invention relates to a kit comprising
As shown in examples 3, 5 and 7 the method of the invention may be performed in a setup using the kit components of the invention.
Solid Support
In yet an aspect the invention relates to a (porous) solid support which may be used in the method of the invention or form part of a kit according to the invention, where component I. and component II. of the kit are deposited on the solid support. Thus, an aspect of the invention relates to a porous solid support (7) comprising
In a variant of the solid support, the solid support is configured for receiving a sample, and bringing said sample in contact with the analyte binding protein (3) before bringing said sample in contact with said analyte analogue (6). Thus, in an embodiment, the first region and the second region are arranged in serial connection on the solid support. In another embodiment, the solid support comprises an anterior end and a posterior end, wherein the anterior end comprises the first region and the posterior end comprises the second region. In a further embodiment, the solid support comprises an inlet for receiving a sample. In another embodiment, the inlet is positioned in the anterior region of the solid support. In an embodiment, the posterior end of the solid support is placed in fluid connection with a detection chamber (9). In a further embodiment, the posterior end of the solid support or the detection chamber (9) comprises an outlet. Preferably, the sample moves through the solid support by capillary forces. As shown in example 11, the order of mixing may improve the efficiency of the assay. Thus, in a preferred embodiment, the solid support is configured for receiving a sample, and bringing said sample in contact with the analyte binding protein (3) before bringing said sample in contact with said analyte analogue (6).
In a preferred embodiment, said first region and said second region are at independent regions at the solid support (7). Such as setup is depicted in
Cartridge
The porous solid support (7) of the invention may be mounted in a cartridge (8). Thus, in yet an embodiment, the solid support is mounted in a cartridge (8). In another embodiment, the cartridge (8) further comprises a detection chamber (9) or is in fluid connection to a detection chamber (9). In an additional embodiment, said detection chamber (9), is adapted to receive fluid (to be investigated for the presence, absence and/or concentration of the analyte) when said fluid has passed through said first region and said second region of the porous solid support (mounted in the cartridge (8)). In yet an embodiment, the porous solid support is adapted to transport a fluid by the use of capillary forces from, such as through the first region, to the second region and to the detection chamber (9). In a further embodiment, the cartridge (8) comprises an outlet. In another embodiment, the outlet is positioned in the detection chamber (9). Again, preferably, the sample moves through the solid support by capillary forces.
In an embodiment the cartridge (8) comprises or consists of a thermoplastic, preferably poly(methyl methacrylate) (PMMA). PMMA also known as acrylic or acrylic glass as well as by the trade names Crylux, Plexiglas, Acrylite, Lucite, and Perspex among several others, is a thermoplastic (which may be transparent) often used in sheet form as a lightweight or shatter-resistant alternative to glass.
In an embodiment, the cartridge (8) is designed to enable the fluid (sample (2)) to migrate by capillary forces from/through the solid support (7) to the detection chamber (9) and stop when the detection chamber (9) is filled.
In another embodiment, the part of the cartridge (8) comprising the detection chamber (9) comprises an optically transparent material, such as optically transparent PMMA, allowing for optical determination of the analyte in the sample. In yet an embodiment the remaining part of the cartridge (8) is optically non-transparent, such as optically non-transparent PMMA.
In a further embodiment, the cartridge (8) is adapted to be mounted with several solid supports (7) each solid support being in connection with individual detection chambers (9). Such a setup is shown in
Thus, an aspect of the invention relates to a cartridge (8) mounted with the porous solid support (7) according to the invention.
In yet an embodiment, the invention relates to a cartridge (8) comprising a groove (14) for mounting of the solid support (7) and a detection chamber (9), wherein the detection chamber (9) is made of an optically transparent material allowing for optical determination of the analyte (1) in the sample (2). In
Detection Chip
The invention relates to a detection chip (10). Thus, in an aspect the invention relates to a detection chip (10) comprising the solid support (7) according to the invention mounted in a cartridge (8) according to the invention. Phrased in another way, the detection chip (10) is adapted for the fluid (such as a blood sample (2)) to be applied to one end of the solid support (7) loaded in the cartridge (8) (in sum the detection chip (10)). The fluid may move by capillary forces through the first region and afterwards through the second region of the solid support (7) and finally end up in the detection chamber (9), where the presence, absence or concentration of an analyte (1) in the sample (2) may be determined. See also
Uses
In yet another aspect, the invention relates to the use of the kit according to the invention and/or the porous solid support (7) according to the invention and/or the cartridge (8) according to the invention and/or the detection chip (10) for determining the presence, absence and/or concentration of an analyte (1) in a sample (2).
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
The invention will now be described in further details in the following non-limiting examples.
The analyte binding protein (3) being covalently linked to the first member (5A) of a fluorescent pair (5), applied for the assay may be prepared in the following way. As shown in
The assay in action using the above described produced analyte binding protein (3) is shown in
In the presence of the analyte (1), the paratope (4) for the analyte (1) of the analyte binding protein (3) is blocked and when the analyte binding protein (3) is exposed to the analyte analogue (6), the analyte analogue (6) cannot bind to the protein (3) (Shown in the top sequence for presence of analyte). This results in emission only from the FRET donor (5A or 5B) when the donor is excited and serves as a clear zero point signal.
In the absence of the analyte (1) the analyte analogue (6) can bind to the analyte binding protein (3). Hence, the proximity of the first member (5A) and the second member (5B) of the fluorescent pair (5) efficiently provide a FRET signal, i.e. when the donor of the fluorescent pair is excited, energy is transferred to the acceptor and emission from the acceptor is observed. This serves as the maximal signal. If the max signal is used as a reference, any sub-stoichiometric amount of the analyte (relative to antibody) can be quantified based on a calibration curve. The readout using a fluorometer for dabigatran at different stoichiometry relative to antibody is shown in
Explanatory example of one embodiment of the method of the invention, wherein the method is performed on a solid support with reference to the figures.
The solid support assay is conducted on a conjugate pad technology in conjunction with liquid phase fluorescence spectroscopy using a transparent detection chamber (9). The conjugate pad (e.g. pieces of porous paper, microstructured polymer or sintered polymer) is prepared as shown in
The analyte binding protein (3) (being covalently linked to the first member (5A) of a fluorescent pair (5)) is spotted on one position of a solid support (7) while the analyte analogue (6) (being covalently coupled to a second member (58) of the fluorescent pair (5)) is spotted on another point (further to the left relative to the first spotting on
Examples 4, 6 and 8 show detection of analytes in such a solid support format.
Aim
The aim of these experiments was to detect the presence/concentration of the anticoagulant dabigatran in both buffer and plasma by measuring FRET using a spectrofluorometer.
Materials and Methods
An analyte binding protein (3) and an analyte analogue (6) is used for this experiment. The DNA-strands are modified with internal Cy3 or Cy5 fluorophores and contain 3′-amino modifier for the functionalization with dabigatran.
Strand Sequences:
Cy5-ACC-
Y 3′
The strands were purchased from Integrated DNA technologies (IDT DNA). In general, the chemicals were purchased from Sigma-Aldrich. Dabigatran was purchased at Cayman Chemical Company. Idarucizumab (Praxbind®) was supplied by Aarhus University Hospital.
Methods:
Modification of DNA Strands:
Preparation of the Reactive Strand (13):
The protocol was adapted from Rosen et al. (Nature Chemistry, 6, 2014, 804-809).
To a solution of DNA (10 nmol) in water (50 μL), MeCN (50 μL) and Et3N (1 μL) Bis(2,5-dioxopyrrolidin-1-yl) octanedioate in DMF (50 μM, 50 μL) was added. The solution was shaken at rt for 30 min. The DNA was ethanol precipitated by addition of aq. NaOAc (3 M, 18 μL, pH=5.2), cold ethanol (500 μL) and glycogen (1 μL, 20 mg/mL). The solution was incubated in liq. N2 for 1 min followed by cold centrifugation for 45 min (4° C., 20000 g). The supernatant was removed, and the pellet dissolved in 200 μL TEAA buffer (0.1 M) and subjected to purification by RP-HPLC 10-20% MeCN in 0.1 TEAA over 15 min. T=25° C., flow rate=1 mL/min. The fractions containing product was pooled and an equivalent volume of 2% aq. TFA solution was added. The mixture was aliquoted, lyophilized and stored at −20° C. Rt=9.8 min (24%).
Guiding Strand (Dabigatran) (11) and Analyte Analogue (Dabigatran) (6):
DNA (10 nmol) in water (5 μL) was added to a solution of carbonate buffer (200 μL, 20 mM, pH 8.5), water (5 μL), dabigatran (50 μL, 7.6 mM in DMSO with 10% 0.1 M HCl) and freshly prepared DMTMMCI (40 μL, 0.5 M in H2O). The mixture was shaken for 2 h at 25° C. Ethanol precipitation was performed by addition of aq. NaOAc (3 M, 32 μL, pH=5.2), cold EtOH (990 μL) and glycogen (1 μL, 20 mg/mL). The mixture was cooled in liq. N2 followed by cold centrifugation for 45 min (4° C., 20000 g). The supernatant was removed, and the pellet dissolved in 0.1 M TEAA buffer, which was subjected to purification by RP-HPLC 5-20% MeCN in 0.1 TEAA over 30 min. T=25° C., flow rate=1 mL/min.
Dabigatran guiding strand: Rt=19.9 min, LCMS: [M]; Calc.: 8026, found: 8026. Dabigatran acceptor strand: HPLC 10-35% MeCN in TEAA over 15 min. Rt=10.1 min, LCMS: [M]; Calc.: 11426, found: 11426.
Generic Protein Labelling Protocol (Analyte Binding Protein (3))
The protein (0.25 μM, 1 eq.) was added to a solution of the reactive strand (13) (0.3 μM, 1.2 eq.) and guiding strand (11) (0.3 μM, 1.2 eq.) in HEPES buffer (50 mM, pH 8.0) and NaCl (400 mM). The reaction mixture was incubated overnight at rt. The releasing strand (12) (0.6 μM, 2 eq.) was added, and the reaction mixture incubated for 30 min at rt. The mixture was concentrated by Amicon Ultra® centrifugal filters (MWCO 30K, 14100 g for 10 min) and purified by anion exchange HPLC. Anion exchange was performed with a Thermo Scientific™ Dionex™ DNAPac™ PA-100 4×250 mm column on a Hewlett Packard Agilent 1100 Series HPLC system. Purification was performed with Buffer A (25 mM Tris) and Buffer B (25 mM Tris, 1 M NaCl) using an increasing gradient of buffer B (0-75%) over 10 min (flow rate: 1 mL/min, T=25° C.). Fractions containing the analyte binding protein (3) were collected and washed twice with HEPES buffer (10 mM, pH 7.0, 0.02 v/v % Tween®-20) in Amicon Ultra® centrifugal filters (MWCO 30K, 14100 g for 30 min). The concentration of product was determined by the Cy3 absorbance at 550 nm. For the dabigatran assay, idarucizumab was used as antibody.
Rt (analyte binding protein (3) for dabigatran)=9.5 min
Assay Setup
In a typical assay, the analyte binding protein (3) conjugate and analyte analogue (6) are kept at a 1:1 stoichiometry. The conjugate (50 nM) was mixed with the analyte (1) dabigatran (500 nM) in either HEPES (50 mM, pH 7.0) or human plasma and incubated at room temperature for 5 min. The analyte analogue (6) (50 nM) was added and the FRET measurements were initiated.
FRET Measurements
To a quartz cuvette, that is washed 3 times with MilliQ water between samples, was added 70 μL of the assay solution. Excitation of the Cy3 dye was performed at 530 nm, and the spectrum was measured from 540-750 nm. A background measurement without analyte binding protein (3) conjugate and analyte analogue (6) was subtracted from the measured spectra. FRET was calculated as E=IA/(IA+ID), where the IA was measured at 650 nm for Cy5 and ID was measured at 550 nm for Cy3.
For kinetic measurements, the analyte analogue (6) was added directly to the cuvette containing the remaining assay solution, and the FRET measurements were initiated with timepoints every 45 sec for 10 min. For temperature measurements, the cuvette containing the assay solution without the acceptor strand was incubated for 10 min at the investigated temperature.
Results
The binding of the analyte binding protein (3) to the analyte analogue (6) brings the Cy3 dye on the conjugate in close proximity to the Cy5 dye on the analyte analogue (6), which is measured as a high FRET signal. However, when analyte (1) dabigatran is present in the assay it will block the binding of the acceptor strands, which results in a decrease in the FRET signal compared to a sample without dabigatran present. Since the decrease in the FRET signal is dependent on the concentration of dabigatran, a titration curve of known concentration of dabigatran was constructed (
In a conventional fluorometer and cuvette setup the assay is able to rapidly measure the analyte up to 500 nM in both buffer and plasma. The important threshold for clinical decision making is 74 nM, which is within the dynamic range of the reported assay.
The aim of these experiments was to detect the presence/concentration of the anticoagulant dabigatran at 74 nM in human plasma (and other complex matrices) and whole blood by measuring FRET using a LED as excitation source in the lateral flow chip (
Materials:
The strands and conjugate from example 3 were used for these experiments.
Methods:
Solid support (7) preparation.
Fusion 5 filter papers (23×6 mm for plasma, 40×6 mm for whole blood) were spotted with 1 pmol of analyte binding protein (3) at an 8 mm distance from the end and 1 pmol analyte analogue (6) was spotted at a 2 mm distance from the same end. Before use, the solid support (7) were stored under reduced pressure overnight after addition of the biomolecules.
Chip Setup
A small strip of prepared filter paper containing the biomolecules is placed in the cartridge shown in
Results
The chips loaded with 1 pmol analyte binding protein (3) and 1 pmol analyte analogue (6) were titrated with varying concentrations of dabigatran (0-100 ng/mL) in human plasma/whole blood (
The detection chip (10) fitted with a solid support (7) containing the reagents for the dabigatran assay. The system readily detects the analyte in various complex matrices and is performing similar to the conventional standard for dabigatran measurements.
Aim of Study
The aim of these experiments was to detect the presence/concentration of the anticoagulant apixaban at 54.4 nM in human plasma by measuring FRET using a spectrofluorometer.
Materials:
An analyte binding protein (3) and an analyte analogue (6) is used for this experiment. The DNA-strands are modified with internal Cy3 or Cy5 fluorophores and contain 3′-amino modifier for the functionalization with a carboxylic acid containing analogue of apixaban.
Strand Sequences:
The starting material strands were the same as used in example 3 and were further modified according to the protocols in the methods section.
The strands were purchased from Integrated DNA technologies. In general, the chemicals were purchased from Sigma-Aldrich. An apixaban analogue containing a carboxylic acid in place of the primary amide was purchased at Acesys Pharmatech. The anti-apixaban antibody 79-2 was developed and provided by BioPorto A/S.
Methods:
Modification of DNA Strands:
The reactive strand (13) for protein conjugate formation was the same as used in example 3.
Preparation of Apixaban Modified Analyte Analogue (6) and Guiding Strand (11):
To a solution of DNA (20 nmol) in water (10 μL) was added carbonate buffer (200 μL, 20 mM, pH 8.5), water (30 μL), acid modified apixaban derivative (20 μL, 10 mg/mL in DMSO) and freshly prepared DMTMMCI (40 μL, 0.5 M in H2O). The mixture was shaken at 25° C. for 2 h. The DNA was precipitated by addition of aq. NaOAc (3 M, 32 μL, pH=5.2), cold EtOH (990 μL) and glycogen (1 μL, 20 mg/mL). The mixture was cooled in liq. N2 followed by centrifugation for 45 min (4° C., 20000 g). The supernatant was removed, and the pellet dissolved in 0.1 TEAA buffer, which was subjected to purification by RP-HPLC 10-20% MeCN in 0.1 TEAA over 15 min. T=25° C., flow rate=1 mL/min.
Apixaban guiding strand (11): Rt=10.8 min, LCMS: [M]; Calc.: 8015, found: 8016.
Apixaban analyte analogue (6): Rt=12.6 min, LCMS: [M]; Calc.: 11414, found: 11416.
Antibody Conjugate
The antibody conjugate of the anti-apixaban antibody, 79-2, was prepared by the generic protein conjugation protocol from example 3.
Rt (analyte binding protein (3) for apixaban)=9.7 min
Assay Setup
In a typical assay, the analyte binding protein (DNA(Cy3)-anti apixaban antibody conjugate) (3), and the analyte analogue (apixaban) (6) is kept at a 1:2.5 stoichiometry. The conjugate (25 nM) was mixed with apixaban (0-1000 nM) in human plasma and incubated at room 5-45° C. for 10 min. The analyte analogue (6) (63 nM) was added and the FRET measurements were initiated.
FRET Measurements
To a quartz cuvette, that is washed 3 times with MilliQ water between samples, was added 70 μL of the assay solution. Excitation of the Cy3 dye was performed at 530 nm, and the spectrum was measured from 540-750 nm. A background measurement without DNA-protein conjugate and acceptor strand was subtracted from the measured spectra. FRET was calculated as E=IA/(IA+ID), where the IA was measured at 650 nm for Cy5 and ID was measured at 550 nm for Cy3. For kinetic measurements, the acceptor strand was added directly to the cuvette containing the remaining assay solution, and the FRET measurements were initiated with timepoints every 45 sec for 10 min. For temperature measurements, the cuvette containing the assay solution without the acceptor strand was incubated for 10 min at the investigated temperature.
Results
The binding of the antibody binding protein (3) (DNA-anti apixaban antibody) to the analyte analogue (apixaban) (6) was evaluated by FRET measurements (
The assay is able to rapidly distinguish the presence and absence of the small molecule apixaban.
The aim of these experiments was to detect the presence/concentration of the anticoagulant apixaban at 54.4 nM (threshold concentration used at hospitals) in human plasma by measuring FRET using an LED as excitation source in the chip (
Materials:
The strands and conjugate from example 5 was used for these experiments.
Methods:
Solid Support (7) Preparation.
Fusion 5 filter papers (23×6 mm) were spotted with 0.5 pmol of analyte binding protein (3) at an 8 mm distance from the detection chamber and 1.25 pmol analyte analogue was spotted at a 2 mm distance from the same end. The analyte binding protein (3) was spotted in a 5/5% sucrose/trehalose buffer whereas the analyte analogue (6) was spotted in water. The solid support (7) was stored under reduced pressure for 1 hour.
Chip Setup
A small strip of prepared solid support (7) (23×6 mm) containing the assay reagents is placed in the cartridge according to
Results
The chips loaded with 0.5 pmol analyte binding protein (3) and 1.25 pmol analyte analogue (6) were titrated with varying concentrations of apixaban (0-500 nM) in human plasma (
The apixaban assay in the detection chip (10) allows for detection of apixaban in the range 0-500 nM which gives rise to a linear response.
The aim of these experiments was to detect the presence/concentration of the antibiotic linezolid at the nanomolar range in human plasma by measuring FRET using a spectrofluorometer and testing the stability of the assay at different temperatures.
Materials:
An analyte binding protein (3) and an analyte analogue (6) are used for this experiment. The DNA-strands are modified with internal Cy3 or Cy5 fluorophores and contain 3′-amino modifiers for the functionalization with an analogue of linezolid containing an amine in place of the amide. The functionalization is performed in a sequential manner.
Strand Sequences:
The starting material strands were the same as in example 3. These strands were modified according to the protocols in the methods section below.
The strands were purchased from Integrated DNA technologies. In general, the chemicals were purchased from Sigma-Aldrich. A linezolid analogue containing an amine in place of the amide was purchased at Matrix Scientific. The anti-linezolid antibody 74-6 was developed and provided by BioPorto A/S.
Methods:
Modification of DNA Strands:
The reactive strand (13) for protein conjugate formation was the same as used in example 3.
The Analyte Analogue (6) and Guiding Strand (11) for Protein Conjugate Formation:
To a solution of DNA (10 μL, 200 μM) in sodium carbonate buffer (60 μL, 20 mM, pH 8.5) was added acetonitrile (40 μL) and bis-NHS-ester linker (disuccinimidyl glutarate) (20 μL, 10 mg/mL in DMF). The reaction mixture was incubated at 25° C. for 30 minutes followed by ethanol precipitation. The pellet was redissolved in sodium carbonate buffer (50 μL, 20 mM, pH 8.5) and a solution of Linezolid amine analogue (25 μL, 5 mg/mL in DMF) was added and the mixture was incubated at 25° C. for 2 h followed by ethanol precipitation and purification by RP-HPLC 10-35% MeCN in 0.1 TEAA over 15 min. T=25° C., flow rate=1 mL/min. Linezolid guiding strand: Rt=7.4 min, LCMS [M]; Calc.: 7963.1, found: 7963.4 Linezolid acceptor strand: Rt=11.3 min, LCMS [M]; Calc.: 11362.9, found: 11363.8
Antibody Conjugate
The analyte binding protein (3) (anti-linezolid antibody, 74-6), was prepared by the generic protein conjugation protocol from example 3. Two different analyte binding proteins (3) were prepared; one protein containing a dye (5A) close to one of the two paratopes (4) (termed single-modified protein) and one protein containing dyes (5A) close to both of the two paratopes (4) (termed double-modified protein).
Rt (linezolid single-modified binding protein)=9.6 min
Rt (linezolid double-modified binding protein)=10.5 min
Assay Setup
In a typical assay, the analyte binding protein (3) and analyte analogue (6) is kept at a 1:2.5 stoichiometry. The conjugate (25 nM) was mixed with linezolid (0-1000 nM) in human plasma and incubated at room 5-45° C. for 10 min. The acceptor strand (63 nM) was added and the FRET measurements were initiated.
FRET Measurements
To a quartz cuvette that is washed 3 times with MilliQ water between samples, was added 80 μL of the assay solution. Excitation of the Cy3 dye was performed at 530 nm, and the spectrum was measured from 540-750 nm. A background measurement without DNA-protein conjugate and acceptor strand was subtracted from the measured spectra. FRET was calculated as E=IA/(IA+ID), where the IA was measured at 650 nm for Cy5 and ID was measured at 550 nm for Cy3. For kinetic measurements, the acceptor strand was added directly to the cuvette containing the remaining assay solution, and the FRET measurements were initiated with timepoints every 45 sec for 9 min. For temperature measurements, the cuvette containing the assay solution without the acceptor strand was incubated for 10 min at the investigated temperature prior to addition of the acceptor strand.
Results
FRET measurements were performed to evaluate if the DNA-anti-linezolid antibody could bind the linezolid modified acceptor strand. The measurements showed high FRET signals, when the components were mixed together. Upon binding addition of small molecule linezolid prior to addition of the acceptor strand, a concentration dependent decrease in the FRET signal was observed. Thereby, it was possible to measure the linezolid.
The assay was evaluated by kinetic measurements at 5-45° C. The measurements at 25° C. demonstrated, the assay reached a maximum signal after only 2-3 min (
The linezolid assay (both single-modified protein and double-modified protein) performs well in 87/79% human plasma (for single-modified protein/double-modified protein, respectively), and allows for rapid detection of linezolid.
The aim of these experiments was to detect the presence/concentration of the antibiotic linezolid at 50 nM in human plasma by measuring FRET using a LED as excitation source in the lateral flow chip (
Materials:
The strands and conjugate (only double-modified protein) from example 7 were used for these experiments.
Methods:
Solid Support (7) Preparation.
Fusion 5 filter papers (23×6 mm for plasma, 40×6 mm for whole blood) were spotted with 0.5 pmol of analyte binding protein (3) at an 8 mm distance from the end and 1.25 pmol analyte analogue (6) was spotted at a 2 mm distance from the same end. Before use, the solid support (7) were stored under reduced pressure overnight after addition of the biomolecules.
Chip Setup
A small strip of prepared filter paper containing the biomolecules (23×6 mm) is placed in the cartridge shown in
Results
The chips loaded with 0.5 pmol analyte binding protein (3) and 1.25 pmol analyte analogue (6) were titrated with varying concentrations of linezolid (0-1000 nM) in human plasma (
The detection chip (10) fitted with a solid support (7) containing the reagents for the linezolid assay allowed for detection of linezolid in the nanomolar range both in 93% plasma and 93% whole blood.
The aim of these experiments was to quantify the performance of unspecific labelled protein conjugates in buffer in a fluorometer setup.
Materials:
The starting material strands were the same as used in Example 3 and were further modified according to the protocols in the methods section Methods:
An analyte binding protein (3), a protein covalently linked to the first member (5A) of the optical pair (5) in a unspecific manner (Global Cy3 Conjugate (Glo-Cy3), a protein covalently linked to a DNA strand containing first member (5A) of the optical pair (5) in a unspecific manner (Glo-DNA), and an analyte analogue (6) is used for this experiment. The DNA-strands are modified with internal Cy3 or Cy5 fluorophores and contain 3′-amino-modifier for the functionalization with dabigatran.
Modification of DNA Strands:
Preparation of the DBCO Strand
Dibenzocyclooctyne-N-hydroxysuccinimidyl (DBCO) ester in DMF (100 μL, 20 mM) was added to a solution of DNA (20 nmol) in water (100 μL) and Et3N (1 μL). The solution was shaken overnight at rt. Ethanol precipitation of the DNA was performed by addition of aq. NaOAc (3 M, 28 μL, pH=5.2), cold ethanol (502) and glycogen (1 μL, 20 mg/mL). The solution was incubated in liq. N2 for 1 min followed by centrifugation for 45 min (4° C., 20000 g). The supernatant was removed immediately after, and the pellet was dissolved in 200 μL MilliQ and subjected to purification by RP-HPLC 10-20% MeCN in 0.1 M TEAA over 15 min. T=25° C., flow rate=1 mL/min. The fractions containing product was pooled. DBCO-donor strand: Rt=12.4 min (24%), LCMS MS [M]; Calc.: 7655.4, found: 7652.2
Antibody Conjugate
Preparation of Global DNA Conjugate (Glo-DNA)
To a solution of the NHS azide (2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate) (25 μM, 5 eq.) in HEPES buffer (50 mM, pH 8.0) was added the antibody (5 μM, 1 eq.). The reaction mixture was incubated at rt overnight. The reaction mixture concentrated by Amicon Ultra® centrifugal filters (MWCO 3K, 14100 g for 30 min) and washed thrice with HEPES buffer (50 mM, pH 8.0). The azide modified antibody was used without further purification.
To a solution of the DBCO strand (5 μM, 1 eq.) in HEPES buffer (50 mM, pH 8.0) was added the azide modified antibody (25 μM, 5 eq.). The reaction mixture was incubated at rt overnight. The reaction was purified by anion exchange HPLC. Anion exchange was performed with a Thermo Scientific™ Dionex™ DNAPac™ PA-100 4×250 mm column on a Hewlett Packard Agilent 1100 Series HPLC system. The purification was performed with Buffer A (25 mM Tris) and Buffer B (25 mM Tris, 1 M NaCl) with an increasing gradient of buffer B (0-75%) over 10 min (flow rate: 1 mL/min, T=25° C.). Fractions containing the conjugates were collected and washed twice with HEPES buffer (10 mM, pH 7.0, 0.02 v/v % Tween®-20) in Amicon Ultra® centrifugal filters (MWCO 3K, 14100 g for 30 min) to remove the majority of the salt from the samples. DNA-protein conjugate Rt=10.4 min. The concentration of the conjugates was determined using the Cy3 absorbance at 550 nm.
Preparation of Global Cy3 Conjugate (Glo-Cy3)
To a solution of the NHS-Cy3 (3H-Indolium, 2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-1-propen-1-yl]-1-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-3,3-dimethyl-, tetrafluoroborate) (100 μM, 5 eq.) in DMSO was added the antibody (20 μM, 1 eq.) in HEPES buffer (50 mM, pH 8.0). The final DMSO concentration was 5%. The reaction mixture was incubated at rt overnight. The reaction mixture concentrated by Amicon Ultra® centrifugal filters (MWCO 3K, 14100 g for 30 min) and washed thrice with HEPES buffer (50 mM, pH 8.0). The protein was used without further purification. The concentration of the protein was determined using the absorbance at 280 nm and the concentration of Cy3 using the absorbance at 550 nm. This yielded an antibody to Cy3 ratio of 1.6.
Assay Setup
In a typical assay, the analyte binding protein (3) conjugate and analyte analogue (6) are kept at a 1:1 stoichiometry. The conjugate (25 nM) was mixed with either the analyte (1) dabigatran (250 nM) or the analyte analogue (6) in HEPES (50 mM, pH 7.0) incubated at room temperature for 10 min. The analyte analogue (6) (25 nM) or the analyte (250 nM) was added and the FRET measurements were initiated.
FRET Measurements
To a quartz cuvette, that is washed 3 times with MilliQ water between samples, was added 70 μL of the assay solution. Excitation of the Cy3 dye was performed at 530 nm, and the spectrum was measured from 540-750 nm. A background measurement without analyte binding protein (3) conjugate and analyte analogue (6) was subtracted from the measured spectra. FRET was calculated as E=IA/(IA+ID), where the IA was measured at 650 nm for Cy5 and ID was measured at 550 nm for Cy3.
For kinetic measurements, the analyte analogue (6)/dabigatran was added directly to the cuvette containing the remaining assay solution, and the FRET measurements were initiated with timepoints every 2 min for 10 min.
Results
The binding of the analyte binding protein (3) to the analyte analogue (6) brings the Cy3 dye on the conjugate in close proximity to the Cy5 dye on the analyte analogue (6), which is measured as a high FRET signal. However, when analyte (1) dabigatran is present in the assay it will block the binding of the acceptor strands, which results in a decrease in the FRET signal compared to a sample without dabigatran present. The results are shown in
In a conventional fluorometer and cuvette setup, the global labelled controls performed significant worse than the assay described in Example 3.
The aim of these experiments was to quantify the performance of the unspecific conjugates (Glo-Cy3 and Glo-DNA) in human plasma by measuring FRET using a LED as excitation source in the lateral flow chip (
Materials:
The strands and conjugate from Example 9 were used for these experiments.
Methods:
Solid Support (7) Preparation
Fusion 5 filter papers (40×6 mm for plasma) were spotted with 1 pmol of analyte binding protein (3)/Glo-Cy3/Glo-DNA at an 8 mm distance from the end and 1 pmol analyte analogue (6) was spotted at a 2 mm distance from the same end. Before use, the solid support (7) were stored under reduced pressure overnight after addition of the biomolecules.
Chip Setup
A small strip of prepared filter paper containing the biomolecules is placed in the cartridge shown in
Results
The FRET in presence and absence of 1 μM dabigatran was measured and the data is shown in
The unspecific labelled proteins, Glo-Cy3 and Glo-DNA, do not perform well in the lateral flow chip. The FRET signal is either low or no signal from the protein conjugate is present in the detection chamber (10).
The aim of these experiments was to investigate to what extend outcompetition between analyte binding protein (3), analyte analogue (6) (dabigatran) and dabigatran is occurring. The aim is likewise to investigate whether or not the order of addition of the assay species is crucial. Phrased in another way the aim is likewise to investigate the effect of the order of addition of the assay species.
Materials:
An analyte binding protein (3) (dabigatran) and an analyte analogue (6) (dabigatran) is used for this experiment. The strands and conjugate from Example 3 were used for these experiments.
Methods:
Assay Setup
In a typical assay, the analyte binding protein (3), and the analyte analogue (dabigatran) (6) is kept at a 1:1 stoichiometry. The conjugate (25 nM) was mixed with either dabigatran (250 nM, 25 nM, 0 nM) or analyte analogue (6) (25 nM) in HEPES pH 7.5 and incubated at rt for 10 min. To the sample lacking analyte analogue (6) was added analyte analogue (6), and to the sample lacking dabigatran was added dabigatran. Hereafter the FRET measurements were initiated.
FRET Measurements
To a quartz cuvette, that is washed 3 times with MilliQ water between samples, was added 70 μL of the assay solution. Excitation of the Cy3 dye was performed at 530 nm, and the spectrum was measured from 540-750 nm. A background measurement without DNA-protein conjugate and acceptor strand was subtracted from the measured spectra. FRET was calculated as E=IA/(IA+ID), where the IA was measured at 650 nm for Cy5 and ID was measured at 550 nm for Cy3. For kinetic measurements, the FRET measurements were initiated with timepoints every 2 min for 10 min.
Results
The binding of the antibody binding protein (3) to the analyte analogue (6) was evaluated by FRET measurements (
Outcompeting the binding of analyte binding protein (3) and analyte analogue (6) is only limited in presence of both 1 and 10 eq. of dabigatran. This concludes that the order of addition of the assay species is important. Thus, the order of addition of the assay species influences the efficiency of the assay.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19159127.0 | Feb 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/054587 | 2/21/2020 | WO | 00 |
