HK1188826B - Use of signal enhancing compounds in electrochemiluminescence detection - Google Patents

Description

use of signal enhancing compounds in electrochemiluminescence detection

Technical Field

The present invention relates to methods for detecting an analyte in a sample by electrochemiluminescence using novel reagent compositions. Disclosed herein are novel reagent compositions, kits for measuring Electrochemiluminescence (ECL) and electrochemiluminescence detection methods using the novel reagent compositions. In particular, the present invention relates to the use of novel combinations of compounds that can be used in the measurements to provide improved assay performance.

Background

Methods for measuring electrochemiluminescence have been known for some years. The method exploits the ability of specific metal complexes to achieve an excited state by oxidation, from which they decay to the ground state, emitting electrochemiluminescence. For a review see Richter, m.m., chem.rev.104(2004) 3003-3036.

Currently, there are a wide variety of commercially available instruments that utilize Electrochemiluminescence (ECL) for analytical measurements. Substances that can cause ECL emission (ECL-active substances) have been used as ECL markers. Examples of ECL labels include organometallic compounds such as tris-bipyridyl-ruthenium (RuBpy) moieties, where the metals are, for example, group VII and VIII metals, including Re, Ru, Ir, and Os. In the ECL process, the substance that reacts with the ECL label is referred to herein as an ECL co-reactant. Common co-reactants for ECL include tertiary amines (e.g., Tripropylamine (TPA)), oxalates, and persulfates. Light is generated by ECL labels or ligands; the participation of the binding agent in the binding interaction can be monitored by measuring ECL emitted from ECL labels. Alternatively, ECL signals from ECL-active compounds may be indicative of the chemical environment (see, e.g., U.S. patents 5,641,623 and 5,643,713, which describe ECL assays that monitor for the presence or destruction of specific ECL co-reactants). For more background on ECL, ECL markers, ECL assays and instruments used to perform ECL assays, see U.S. patent 5,093,268; 5,147,806, respectively; 5,240,863, respectively; 5,308,754, respectively; 5,324,457, respectively; 5,591,581; 5,597,910, respectively; 5,679,519, respectively; 5,705,402; 5,731,147, respectively; 5,786,141, respectively; 5,846,485; 5,866,434, respectively; 6,066,448; 6,136,268 and 6,207,369 and EP0441875 and published PCT WO 97/36931; WO 98/12539; WO 99/14599; WO 99/32662; WO 99/58962; WO 99/63347; WO00/03233 and WO 98/57154.

Commercially available ECL instruments have shown excellent performance. They have been widely used for reasons including their superior sensitivity, dynamic range, accuracy and tolerance to complex sample matrices. Commercially available instruments use a flow cell based design with durable reusable flow cells.

The available sample volumes for determining analytes are often limited and more different analytes have to be determined from one sample. There is also a need to develop faster laboratory equipment for assay automation and more sensitive methods for detecting analytes. This leads to the need for highly sensitive and specific electrochemiluminescence assays and methods for performing them. In addition, improvements relating to hazardous safety or environmental issues should also be considered.

However, an even more sensitive detection of the analyte is highly advantageous. It is therefore an object of the present invention to improve the known methods and reagent compositions, in particular with respect to enhancing ECL signal and improving analyte detection in combination with electrochemiluminescence operation. It would be desirable to find new signal enhancing agents and/or compounds with improved electrochemiluminescence measurement properties.

Disclosure of Invention

The present invention relates in one embodiment to a method for measuring an analyte in a sample by electrochemiluminescence detection, comprising the steps of: a) incubating the sample with a detection reagent labeled with an electrochemiluminescent group, b) separating the labeled detection reagent bound to the analyte from the labeled detection reagent not containing the analyte, c) incubating the separated labeled detection reagent with a reagent composition comprising: i) at least one co-reactant, and II) at least one compound selected from carbonamides (carbonic acid amides) of formula I and formula II,

formula I

In the formula I, R₁=CH₃、CH₂F、CH₂Cl、CH₂CH₃、CHClCH₃、CH₂CH₂Cl、C(CH₃)₂CH₃、CH₂CH₂CH₃、CClHCH₂CH₃Or CH₂CH₂CH₂CH₃，R₂= H, and R₃=H，

Formula II

d) Electrochemically triggering the release of luminescence, and e) determining an Electrochemiluminescence (ECL) signal, thereby measuring the analyte.

The invention also relates to a reagent composition for determining ECL, comprising I) a compound selected from carboxamides of formula I and formula II, and II) at least one co-reactant.

The invention also relates to a reagent mixture comprising a reagent composition for determining ECL, comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, and II) at least one co-reactant, a sample to be investigated and at least one detection reagent labeled with an electrochemiluminescent group.

The invention also relates to a kit for measuring ECL, comprising a reagent composition for determining ECL, said reagent composition comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II) and II) at least one co-reactant.

The present invention together with additional objects, features and advantages thereof will be more fully understood from the following detailed description of certain preferred embodiments.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as "Molecular Cloning: A laboratory Manual", second edition (Sambrook et al, 1989); "Oligonucleotide Synthesis" (edited by m.j. gate, 1984); "Animal Cell Culture" (ed. r.l. freshney, 1987); "Methods in enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al, 1987 and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al eds., 1994).

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, Dictionary of microbiology and Molecular Biology, 2nd edition, J.Wiley & Sons, New York (1994); march, Advanced Organic Chemistry Reactions, mechanics and Structure, 4 th edition, John Wiley & Sons, New York (1992); lewis, B., Genes V, published by Oxford University Press (1994), ISBN 0-19-8542879); kendrew, J.et al (eds.), The Encyclopedia of Molecular Biology, published by Blackwell science Ltd (1994), ISBN 0-632-02182-9); and Meyers, R.A, (ed.), Molecular Biology and digital Biotechnology a Comprehensive Desk Reference, published by VCH Publishers, Inc. (1995), ISBN 1-56081-.

All references, including patent applications and publications, cited in this application are hereby incorporated by reference in their entirety.

Definition of

As used in this application, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used in this application to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an analyte" means one analyte or more than one analyte. The term "at least" is used to indicate that, optionally, one or more other objects may be present. For example, optionally, an array comprising at least two discrete regions may comprise two or more discrete test zones.

The expression "one or more" means 1 to 50, preferably 1 to 20, further preferably 2, 3, 4,5, 6, 7,8, 9, 10, 12 or 15.

Examples of "carboxamides" and their chemical structures are listed in table 1 below.

Table 1 carboxamides have the following general structure, unless otherwise indicated:

formula I as used herein represents:

in the formula I, R₁=CH₃、CH₂F、CH₂Cl、CH₂CH₃、CHClCH₃、CH₂CH₂Cl、C(CH₃)₂CH₃、CH₂CH₂CH₃、CClHCH₂CH₃Or CH₂CH₂CH₂CH₃，R₂= H, and R₃=H。

Formula II, as used herein, represents the following structure, referred to as 2-pyrrolidone, as shown in Table 1, number 24.

Embodiments of the present invention may be used to test a variety of samples that may contain an analyte or activity of interest. The sample may be in solid, emulsion, suspension, liquid or gaseous form. They may be, but are not limited to, samples containing or of human or animal origin: for example, cells (viable or dead) and cell derived products, immortalized cells, cell debris, cell fractions (cell fractions), lysates, organelles, cell membranes, hybridomas, cell culture supernatants (including supernatants from antibody producing organisms such as hybridomas), wastewater or drinking water, food, beverages, pharmaceutical compositions, blood, serum, plasma, hair, sweat, urine, fecal matter, feces, saliva, tissue, biopsies, effluents, separated and/or fractionated samples, separated and/or fractionated liquids, organs, plants, plant parts, plant by-products, soil, water, supplies, water sources, residues filtered from fluids (gas and liquid), bland low grade beer, absorbent materials, gels, cytoskeletons, unfractionated samples, unfractionated lysates, Nuclei, nuclear fractions, chemicals, chemical solutions, structural biological components, skeletal (ligaments, tendons) components, isolated and/or fractionated skeletal components, hair components and/or isolates, skin samples, skin components, dermis, endothelial layer, eukaryotic cells, prokaryotic cells, fungi, yeast, immune cells, drugs, therapeutic drugs, oils, extracts, mucus, sewage, environmental samples, organic solvents, or air. In one embodiment, the sample may further comprise, for example, water, alcohol, acetonitrile, dimethyl sulfoxide, dimethylformamide, N-methyl-pyrrolidone, methanol, or other organic solvent.

As used herein, a "sample" is obtained for the purpose of in vitro evaluation. As the skilled artisan will appreciate, any of the assessments are performed in vitro. If the sample is a patient sample, the patient sample is then discarded. Patient samples are only used for the in vitro diagnostic method of the present invention and no material of the patient sample is transferred back into the patient's body.

Analytes that can be measured include, but are not limited to, whole cells, cell surface antigens, protein complexes, cell signaling factors and/or components, second messengers, second messenger signaling factors and/or components, subcellular particles (e.g., organelles or membrane fragments), viruses, prions, dust mites or fragments thereof, viroids, immune factors, antibodies, antibody fragments, antigens, haptens, fatty acids, nucleic acids (and synthetic analogs), ribosomes, proteins (and synthetic analogs), lipoproteins, polysaccharides, inhibitors, cofactors, haptens, cell receptors, receptor ligands, lipopolysaccharides, glycoproteins, peptides, polypeptides, enzymes, enzyme substrates, enzyme products, nucleic acid processing enzymes (e.g., polymerases, nucleases, integrases, ligases, helicases, telomerase, etc.), protein processing enzymes (e.g., proteases, and the like, Kinases, protein phosphatases, ubiquitin-protein ligases, etc.), cellular metabolites, endocrine factors, paracrine factors, autocrine factors, cytokines, hormones, pharmaceutical agents, drugs, therapeutic drugs, synthetic organic molecules, organometallic molecules, sedatives, barbiturates, alkaloids, steroids, vitamins, amino acids, sugars, lectins, recombinant or derivative proteins, biotin, avidin, streptavidin, or inorganic molecules present in the sample.

Whole cells may be animal, plant or bacterial, and may be live or dead. Examples include plant pathogens such as fungi and nematodes. The term "subcellular particle" is intended to encompass, for example, subcellular organelles such as membrane particles from disrupted cells, cell wall fragments, ribosomes, multienzyme complexes, and other particles that can be derived from living organisms. Nucleic acids include, for example, chromosomal DNA, plasmid DNA, viral DNA, and recombinant DNA derived from multiple sources. Nucleic acids also include RNA, such as messenger RNA, ribosomal RNA, and transfer RNA. Polypeptides include, for example, enzymes, transport proteins, receptor proteins, and structural proteins such as viral coat proteins. Preferred polypeptides are enzymes and antibodies. Particularly preferred polypeptides are monoclonal antibodies. Hormones include, for example, insulin and T4 thyroid hormone. Drugs include, for example, cardiac glycosides. Of course, synthetic materials that are chemically similar to biological materials, such as synthetic polypeptides, synthetic nucleic acids, and synthetic membranes, vesicles, and liposomes are included within the scope of the invention. The foregoing is not intended to be a comprehensive list of biological materials suitable for use in the present invention, but is merely intended to illustrate the broad scope of the invention.

Also typically, the analyte of interest is present at 10^-3Present in a molar concentration of, e.g., at least as low as 10^-18The molar concentration of (c).

The term "analyte-specific agent" (ASR) according to the present invention must be understood as a molecule or a biomolecule (e.g. a protein or an antibody) having the ability to specifically bind to an analyte. An "analyte-specific reagent" (ASR) is a class of biomolecules that can be used to identify and measure the amount of an individual chemical in a biological sample. ASR are for example antibodies (polyclonal or monoclonal), specific receptor proteins, ligands, nucleic acid sequences and similar reagents intended for use in diagnostic applications by specific binding or chemical reaction with a substance in a sample to identify and quantify the individual chemical substance or ligand in a biological sample. Briefly, the analyte-specific reagent is the active ingredient of the assay. ASR will meet affinity criteria for binding to the analyte as well as specificity criteria.

The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. The term "antibody" encompasses various forms of antibody structures, including whole antibodies and antibody fragments. The antibodies of the invention are in one embodiment human antibodies, humanized antibodies, chimeric antibodies, antibodies derived from other animals such as mice, goats or sheep, monoclonal or polyclonal antibodies, or antibodies to a T-cell depleted antigen. Genetic engineering of antibodies is described, for example, in Morrison, S.L., et al, Proc.Natl.Acad Sci.USA81(1984) 6851-6855; US5,202,238 and US5,204,244; riechmann, L., et al., Nature332(1988) 323-327; neuberger, M.S., et al, Nature314(1985) 268-270; lonberg, N., nat. Biotechnol.23(2005) 1117-1125.

Any antibody fragment that retains the above-described criteria for the analyte-specific reagent may be used. Antibodies are produced by procedures known in the art, for example, as described in Tijssen (Tijssen, P., Practice and the order of enzymeimmunoassays,11, Elsevier Science Publishers B.V., Amsterdam, entire book, especially pages 43-78). Furthermore, the skilled artisan is familiar with immunoadsorption-based methods that can be used to specifically isolate antibodies. By these means, the quality of the antibody and thus its performance in an immunoassay can be improved (Tijssen, P., supra, page 108-115).

"detection reagents" according to the invention include analyte-specific reagents (ASR) labelled with an electrochemiluminescent group, or analyte homologues labelled with an electrochemiluminescent group. Depending on the test format, the skilled artisan knows which detection reagents have to be selected for the different assay formats (e.g., sandwich assay, dual antigen bridging assay (DAGS), competition assay, homogeneous assay, heterogeneous assay). The detection reagent in the heterogeneous immunoassay may be, for example, an antibody. It is known to those skilled in the art that the detection reagent may be immobilized on a solid phase. In one embodiment, the method of measuring an analyte in a sample by electrochemiluminescence detection may be performed as a homogeneous assay. In one embodiment, the method may be performed as a heterogeneous assay. In one embodiment, the method may be performed in a sandwich assay format. In one embodiment, the method may be performed as a competition assay. In yet another embodiment, the method may be performed as a dual antigen bridging assay (DAGS). Known Immunoassay formats are described in particular in the books Price, c.p. and Newman, d.j., Principles and practice of immunoassays, 2nd ed. (1997).

The term "label" as used herein refers to any substance capable of producing a detectable signal, whether visually or by use of a suitable instrument. Various labels suitable for use in the present invention include, but are not limited to, chromogens, fluorescent, chemiluminescent, or electrochemiluminescent compounds, catalysts, enzymes, enzyme substrates, dyes, colloidal metallic and non-metallic particles, and organic polymer latex particles.

The term "luminescence" refers to any emission of light that does not derive energy from the temperature of an energy source (e.g., electromagnetic radiation source, chemical reaction, mechanical energy). Generally, the source causes electrons of the atom to migrate from a lower energy state to an "excited" higher energy state; the electrons then release this energy in the form of emitted light as they fall back to a lower energy state. Such light emission typically occurs in the visible or near visible range of the electromagnetic spectrum. The term "luminescence" includes, but is not limited to, such light emission phenomena as phosphorescence, fluorescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, and thermoluminescence.

The term "luminescent label" refers to a label that generates a luminescent signal, e.g., light emission, that does not derive energy from the temperature of the emission source. The luminescent label may be, for example, a fluorescent molecule, a phosphorescent molecule, a radioluminescent molecule, a luminescent chelator, a phosphorus or phosphorus-containing compound, or a quantum dot.

An "electrochemiluminescence assay" or "ECLA" is an electrochemical assay that detects bound analyte molecules by labels attached to detection reagents (target molecules). The electrodes electrochemically initiate the luminescence of a chemical label attached to the detection reagent. The light emitted by the label is measured by a photodetector and indicates the presence or amount of bound analyte molecule/target molecule complexes. ECLA processes are described, for example, in us patent 5,543,112; 5,935,779, respectively; and 6,316,607. Signal modulation can be maximized for different analyte molecule concentrations for accurate and sensitive measurements.

In ECLA procedures, microparticles may be suspended in a sample to effectively bind an analyte. For example, the particles may have a diameter of 0.05 μm to 200 μm, 0.1 μm to 100 μm, or 0.5 μm to 10 μm, and a surface component capable of binding the analyte molecules. In a frequently used ECLA systemRocheDagnsotics, Germany), the microparticles have a diameter of about 3 μm. The microparticles may be formed from: crosslinked starch, dextran, cellulose, protein, organic polymer, styrene copolymer such as styrene/butadiene copolymer, acrylonitrile/butadiene/styrene copolymer, ethylene acetoacetate copolymer, vinyl chloride/acrylate copolymer, inert inorganic particles, chromium dioxide, iron oxide, silica mixtures, proteinaceous material or mixtures thereof, including but not limited to agarose beadsLatex beads, shell core particles, and the like. Preferably, the microparticles are monodisperse and may be magnetic, such as paramagnetic beads. See, e.g., U.S. Pat. nos. 4,628,037; 4,965,392, respectively; 4,695,393; 4,698,302, respectively; and 4,554,088. Microparticles may be used in amounts ranging from about 1 to 10,000 μ g/ml, preferably 5 to 1,000 μ g/ml.

The expression "of interest" refers to the possible associated analyte or substance that should be analyzed or determined.

"detecting" includes any means of detection, including direct and indirect detection. The term "detection" is used in the broadest sense and includes both qualitative and quantitative measurements of an analyte, which is measured herein. In one aspect, the detection method as described herein is used to identify the small presence of an analyte of interest in a sample. In another aspect, the method can be used to quantify the amount of analyte in a sample.

"reduce" or "inhibit" is a decrease or decrease in activity, function and/or amount as compared to a reference. By reduced or inhibited is meant the ability to cause an overall reduction of preferably 10% or greater, more preferably 25% or greater, and most preferably 50%, 75%, 90%, 95% or greater.

An "enhancement" such as "enhancing a specific signal" or "enhancing an ECL signal" is an increase or enhancement of activity, function and/or amount as compared to a reference. An increase or boost refers to the ability to cause an overall increase of preferably 10% or more, more preferably 25% or more, most preferably 50% or more.

The term "determining" is used in the present application for qualitative and quantitative detection of an analyte and may include determining the amount and/or concentration of the analyte.

The term "measuring" is scientifically a process of evaluating or determining the magnitude of a quantity (such as length or mass) relative to a unit of measure (such as meter or kilogram). The measurements use a reference point against which other things can be evaluated. The term measurement may also be used to refer to a specific result (determined value) obtained from the measurement process. It is the basis for comparison. The skilled person is aware of materials and methods for correlating a measured signal or a determined value with a concentration.

The "reagent composition" or "ECL-reagent composition" according to the present invention comprises reagents that support the generation of ECL-signal, such as co-reactants, buffers for pH control and optionally other components. The skilled artisan is aware of the components present in the reagent composition that are required for ECL signal generation in an electrochemiluminescence detection method.

As used herein, an "aqueous solution" is a homogeneous solution of particles, substances or liquid compounds dissolved in water. The aqueous solution may also comprise an organic solvent. Organic solvents are known to the person skilled in the art, for example methanol, ethanol or dimethyl sulfoxide. As used herein, it is also understood that the aqueous solution may contain up to 50% organic solvent.

The substances that participate in ECL labeling during ECL are referred to herein as ECL "co-reactants". Common co-reactants for ECL include tertiary amines (e.g., Tripropylamine (TPA)), oxalates, and persulfates. The skilled artisan is aware of useful co-reactants for use in electrochemiluminescence detection methods.

A "solid phase", also referred to as a "solid support", is an insoluble, functionalized polymeric material to which library members or reagents can be attached or covalently bound (often via a linker) to immobilize or allow them to be readily separated (by filtration, centrifugation, washing, etc.) by excess reagents, soluble reaction byproducts, or solvents. The solid phases used in the process of the invention are widely described in the prior art (see, for example, Butler, J.E., Methods22(2000) 4-23). The term "solid phase" refers to a non-fluid substance and includes particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glasses, and ceramics; gel substances such as silica gel, alumina gel and polymer gel; capillaries that can be made of polymers, metals, glass, and/or ceramics; zeolites and other porous materials; an electrode; a microtiter plate; a solid bar; and a cuvette, tube, chip or other spectrometer sample container. The solid phase components of the assay are distinguished from the inert solid surface with which the assay can be contacted in that the "solid phase" contains on its surface at least one moiety intended to interact with a capture antibody or capture molecule. The solid phase may be a fixed component such as a tube, strip, cuvette, chip or microtiter plate, or may be a non-fixed component such as beads and microparticles. Microparticles may also serve as a solid phase for a homogeneous assay format. A variety of microparticles that allow for non-covalent or covalent attachment of proteins and other substances may be used. Such particles include polymeric particles such as polystyrene and poly (methyl methacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass, and metal oxide particles. See, e.g., Martin, C.R. et al, analytical chemistry-News & Features70(1998)322A-327A, which is incorporated herein by reference.

The terms "chip", "biochip", "polymer chip" or "protein chip" are used interchangeably and refer to a collection of a large number of probes, markers or biochemical markers arranged on a shared substrate (e.g. a solid phase), which may be part of a silicon wafer, nylon strip, plastic strip or glass slide.

The method comprises the following steps:

in one embodiment, the present invention relates to a method of measuring an analyte in a sample by electrochemiluminescence detection, comprising the steps of: a) incubating the sample with a detection reagent labeled with an electrochemiluminescent group, b) separating the labeled detection reagent bound to the analyte from the labeled detection reagent not containing the analyte, c) incubating the separated labeled detection reagent with a reagent composition comprising: i) at least one co-reactant, and II) at least one compound selected from the group consisting of carboxamides of formula I and formula II,

formula I

In the formula I, R₁=CH₃、CH₂F、CH₂Cl、CH₂CH₃、CHClCH₃、CH₂CH₂Cl、C(CH₃)₂CH₃、CH₂CH₂CH₃、CClHCH₂CH₃Or CH₂CH₂CH₂CH₃，R₂= H, and R₃=H，

In the formula II, the compound is shown in the specification,

d) electrochemically triggering the release of luminescence, and e) determining an Electrochemiluminescence (ECL) signal, thereby measuring the analyte.

One aspect of the invention relates to improved ECL methods based on the reagent compositions of the invention, in particular ECL methods characterized by a low detection limit. The reagent composition unexpectedly enhances specific signal and reduces background signal. More specifically, the methods of the invention provide improved sensitivity at low detection levels by reducing background electrochemiluminescence in the absence of ECL labels.

The present inventors have unexpectedly found that the use of certain compounds from carboxamides provides a number of advantages, including improved signal generation in ECL detection methods, and thus improved ECL assay performance.

One feature of the present invention is a method for determining an analyte in a sample to be investigated using an electrochemiluminescent marker, wherein one of the following methods for measuring electrochemiluminescence phenomena is employed.

Unexpectedly, the method using compounds selected from carboxamides emitted less background luminescence than conventional test reagents without these compounds. This is particularly advantageous at low detection levels, where increasing the signal to background ratio (= signal to noise ratio) greatly improves sensitivity. Unexpectedly, the inventors of the present application found that electrochemiluminescence detection using the method of the present invention results in a 10% to 60% improvement in the signal-to-noise ratio of ECL detection.

The method for measuring an analyte in a sample by electrochemiluminescence detection according to the present invention may in one embodiment be performed in an aqueous solution.

In one embodiment, the carboxamide used in the method of the invention is selected from acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide and 2-chlorobutyramide.

In a preferred embodiment, the carbamide used in the method of the invention is selected from acetamide, 2-chloroacetamide, propionamide and butyramide.

In a preferred embodiment, the carboxamides used in the process of the invention are selected from the group consisting of acetamides, propionamides and butyramides.

In a preferred embodiment, the concentration of the carbamide used in the process of the invention is from 0.01M to 0.25M. In a further preferred embodiment, the concentration of carbamide used is 0.01M to 0.2M. In a further preferred embodiment, the concentration of carbamide used is 0.01M to 0.1M.

In one embodiment, the method of the invention is particularly suitable for detecting biomolecules, such as proteins, polypeptides, peptides, peptide fragments, hormones, peptide hormones, vitamins, provitamins, vitamin metabolites and amino acids, in a sample of interest.

The sample used in the method of the invention is in one embodiment a liquid sample, e.g. whole blood, serum or plasma. The sample, or more specifically the sample of interest, may, in one embodiment, comprise any bodily fluid and feces. In one embodiment, the sample will be a liquid sample, such as saliva, fecal extract, urine, whole blood, plasma, or serum. In one embodiment, the sample will be whole blood, plasma or serum.

The skilled person will appreciate that the steps "a) incubating the sample with a detection reagent labelled with an electrochemiluminescent group" and "b) separating the labelled detection reagent with bound analyte from the labelled detection reagent without analyte" in the method of the invention may be performed in the same location, e.g. in the same reactor. Said steps (a) and (b) may be carried out in an automated process controlled by a control means.

Non-specific sample components and labeled detection reagents that are free of analyte may be removed in a separate process in step (b) of the method of the invention. For example, labeled detection reagents that are bound to the analyte and free of the analyte can be separated using a washing step.

Other test components that support electrochemiluminescence detection of analytes can also be used in the methods of the invention.

One aspect of the present invention covers the need for effective preservation, for example for long term storage of reagent mixtures and reagent compositions. Suitable preservatives should have no effect or, in the ideal case, a positive effect on ECL signal development.

As suitable preservative compounds, boric acid and/or borates were identified as effective in controlling bacterial and fungal growth and unexpectedly enhancing specific ECL signaling. The ECL detection method using a reagent composition comprising boric acid and/or a borate salt as a preservative has a positive and unexpected effect of enhancing the specific ECL signal generated.

In one embodiment, the present invention relates to a method of measuring an analyte in a sample by electrochemiluminescence detection, comprising the steps of: a) incubating the sample with a detection reagent labeled with an electrochemiluminescent group, b) separating the labeled detection reagent bound to the analyte from the labeled detection reagent not containing the analyte, c) incubating the separated labeled detection reagent with a reagent composition comprising: i) at least one co-reactant, and ii) a preservative selected from boric acid and borate, d) electrochemically triggering the release of luminescence, and e) determining an Electrochemiluminescence (ECL) signal, thereby measuring the analyte.

In one embodiment, the method for measuring an analyte in a sample by electrochemiluminescence detection is characterized in that the reagent composition for ECL signal generation comprises a preservative selected from boric acid and borate at a concentration of 0.1% to 5%, preferably at a concentration of 0.5% to 4%, more preferably at a concentration of 0.5% to 2%.

In one embodiment, the method for measuring an analyte in a sample by electrochemiluminescence detection is characterized in that the reagent composition for ECL signal generation comprises boric acid as a preservative at a concentration of 0.1% to 5%, preferably at a concentration of 0.5% to 4%, more preferably at a concentration of 0.5% to 2%.

In one embodiment, the method for measuring an analyte in a sample by electrochemiluminescence detection is characterized in that the reagent composition for ECL signal generation comprises borate as a preservative at a concentration of 0.1% to 5%, preferably at a concentration of 0.5% to 4%, more preferably at a concentration of 0.5% to 2%.

The present inventors have unexpectedly discovered that a method of measuring an analyte in a sample by electrochemiluminescence detection that combines the effects of a carbamide and boric acid and/or borate in one reagent composition can result in further improvement of the signal-to-noise ratio in ECL detection. The accumulation of carbonamides with boric acid and/or borates in a reagent composition results in at least a 10%, 25% or 50% improvement in signal generation in ECL assays.

In one embodiment, the present invention relates to a method of measuring an analyte in a sample by electrochemiluminescence detection, comprising the steps of: a) incubating the sample with a detection reagent labeled with an electrochemiluminescent group, b) separating the labeled detection reagent bound to the analyte from the labeled detection reagent not containing the analyte, c) incubating the separated labeled detection reagent with a reagent composition comprising: i) at least one co-reactant, II) at least one compound selected from the group consisting of carboxamides of formula I and formula II, and iii) at least one preservative selected from the group consisting of boric acid and borates, d) electrochemically triggered release of luminescence, and e) determining an Electrochemiluminescence (ECL) signal, thereby measuring the analyte.

In one embodiment, the method for measuring an analyte in a sample by electrochemiluminescence detection is characterized in that the reagent composition further comprises a detergent and a buffer.

In one embodiment, the method for measuring an analyte in a sample by electrochemiluminescence detection is characterized in that the reagent composition further comprises a salt and/or an antifoaming agent.

In one embodiment, the invention relates to a method of performing an electrochemiluminescence assay, wherein electrochemiluminescence is caused in the presence of a reagent composition of the invention.

A typical ECL measurement process for an EDL immunoassay involves multiple exchanges of liquids and/or mixtures in ECL measurement cells (e.g., flow cells). A typical ECL measurement procedure consists of several steps as explained below.

The skilled artisan will recognize that ECL measurement cells must be conditioned or regenerated by rinsing the ECL measurement cells with the reagent composition of the invention and additionally applying an electrical potential prior to performing the ECL detection step. This step is part of the process of determining the analyte using ECL. It has been described in EP1051621 that during this conditioning step, during the measurement of an analyte in an ECL measurement cell, a layer forms on the surface of the measurement electrode supporting the generation of a signal.

For a typical ECL measurement procedure, the reagent mixture is introduced into clean and conditioned ECL measurement cells by entering the ECL measurement cell cavities through a fluid inlet channel. The mixture is an incubation of the sample, the reagent and the magnetic particles. The mixture introduced into the measurement cell may be surrounded by the reagent composition of the present invention which flows before and after the mixture.

In such ECL immunoassays, a detection reagent comprising a complex-molecule labeled with an electrochemical fluorophore and characterized for analysis is bound to these magnetic particles by a pair of specific biochemical binding partners (e.g. streptavidin and biotin). The magnetic particles are for example coated with streptavidin-polymer, whereas biotin is bound to the complex-molecules.

In ECL measurement cells, magnetic particles together with their bound labeled complex-molecules are trapped on the surface of an electrode in the magnetic field of a magnet arranged in the vicinity of the electrode. A magnetic field is applied during continuous flow of the mixture, whereby the incubation and/or reagent composition is expelled from the ECL measurement cell cavity through the fluid outlet channel.

After the magnetic particles are captured, the reagent composition of the invention containing the ECL co-reactant is introduced into the ECL measurement cells in the next step, whereby the magnetic particles are washed with the reagent composition. This washing step is to remove unbound components of the incubation from the electrode, which potentially interfere with the electrochemical reaction.

The release of the Electrochemiluminescence (ECL) signal is then electrochemically initiated by applying an electrical potential, whereby the luminescence intensity is detected by means of a photosensor and can be evaluated as a measure for the concentration of the labeled complex-molecules on the magnetic particles at the surface of the electrodes, whereby this concentration is again used as a measure for the concentration of the analyte in the sample.

After electrochemiluminescence detection, ECL measurement cells are typically rinsed with a clean fluid.

Devices for carrying out detection methods by means of electrochemiluminescence are mentioned in the examples section (examples 1, 2 or 3) or are described in EP1892524(a 1). Furthermore, the device may comprise means for controlling the temperature of the measurement unit and/or the liquid container. A measuring cell is understood to be a cell in which electrochemiluminescence is measured. The liquid container can be a storage container or a feeding device; for example, a tube for a reagent solution is contained in the measurement unit during measurement.

Composition (A):

one aspect of the invention relates to improved reagent compositions for ECL-signal generation, which result in improved signal-to-noise ratios. More specifically, the reagent compositions of the invention provide improved sensitivity at low detection levels by reducing background electrochemiluminescence in the absence of ECL labels. Unexpectedly, reagent compositions comprising compounds such as carboxamides emit less background luminescence than conventional test reagents that do not contain these compounds. This is particularly advantageous at low detection levels, where increasing the signal to background ratio (= signal to noise ratio) greatly improves sensitivity. The improved reagent composition comprises a compound from the group of carboxamides as well as other compounds that support the process of producing ECL. Unexpectedly, the inventors of the present application found that electrochemiluminescence detection using the reagent composition of the present invention results in an improvement of the signal-to-noise ratio of ECL detection of 10% to 60%.

One aspect of the invention relates to reagent compositions that give high signal to background ratios in electrochemiluminescence assays. The signal difference between the specific signal and the background signal is increased. The improved properties have been achieved by the advantageous combination of identified ECL co-reactants, pH buffers, detergents and pH, in particular by using a compound selected from carboxamides.

The reagent composition provides an environment suitable for effectively causing ECL markers to emit ECL and for sensitively measuring ECL markers by measuring ECL. The reagent compositions of the present invention may optionally comprise other components including preservatives, detergents, antifoams, ECL actives, salts, acidic and basic compounds for pH control (buffers), metal ions and/or metal chelators. Reagent compositions of the invention may also include components of a bioassay (which may be labeled with ECL labels in some cases), including binding agents, enzymes, enzyme substrates, cofactors, and/or enzyme inhibitors. The invention also includes assay reagents, compositions, kits, systems and system components comprising the reagent compositions of the invention, and optionally other assay components. The invention also includes methods of using the reagent compositions of the invention for ECL assays.

In one embodiment, the present invention relates to a reagent composition for determining ECL comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, and II) at least one co-reactant.

In one embodiment, the carboxamide in the reagent composition is selected from the group consisting of acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide and 2-chlorobutyramide.

In a preferred embodiment, the carbamide is selected from acetamide, 2-chloroacetamide, propionamide and butyramide. In a further embodiment, the carboxamide is selected from acetamide, propionamide and butyramide. The carboxamide has the individual concentration which is optimal for ECL enhancement. As shown in the experiments (in particular, tables 2, 3 and 4), the skilled person knows to select an appropriate concentration for the selected carbamide in the reagent composition. The skilled person is aware of methods for determining the optimum concentration for the carbamide in the reagent composition.

In one embodiment, the reagent composition comprises carbamide in a concentration of 0.01M to 0.25M. In a further embodiment, the reagent composition comprises carbamide in a concentration of 0.01M to 0.2M. In a further embodiment, the reagent composition comprises carbamide in a concentration of 0.01M to 0.1M.

The co-reactant of the reagent composition is selected from tertiary amines (e.g., Tripropylamine (TPA)), oxalates, and persulfates in one embodiment. In a preferred embodiment, the co-reactant is TPA.

It may be advantageous to include a preservative to prevent microbial growth when the reagent composition is stored. In addition, suitable preservatives have been identified to control bacterial and fungal growth so that the reagent compositions can be stored and used for long periods of time. The reagent composition of the present invention may additionally comprise one or more preservatives. In one embodiment of the invention, the reagent composition comprises a preservative (preservative agent).

In one embodiment, the present invention relates to a reagent composition for determining ECL comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, and iii) at least one preservative.

Preferably, the preservative has no or a positive effect on ECL signal production. Oxazolidines (e.g., Oxaban a or 4, 4-dimethyloxazolidine), azides, and related preservatives are known to those skilled in the art to be compatible with ECL. Oxazolidine concentrations of 0.01% to 1% are typically used in the test reagents. In one embodiment, the reagent composition comprises a preservative selected from oxazolidines, preferably Oxaban a. In one embodiment, the reagent composition comprises a preservative at a concentration of 0.01% to 1%, in another embodiment the reagent composition comprises a preservative at a concentration of 0.1% to 1%. It may also be advantageous to use a mixture of two or more preservatives.

The carboxamide 2-Chloroacetamide (CAA) already mentioned above has a preservative function in addition to its ECL signal enhancing effect.

As noted above, one aspect of the present invention encompasses the need to add effective preservatives that have no or a positive effect on ECL signaling. Boric acid and/or borates have been identified as effective control of bacterial and fungal growth as suitable inorganic compounds. Unexpectedly, the present inventors have found that the presence of boric acid and/or borate in the reagent composition used to determine ECL does not negatively affect ECL signaling. It has been unexpectedly found that reagent compositions comprising boric acid or borate as a preservative have a positive effect on the ECL signalling process, i.e. an increase in specific signalling. Furthermore, their high activity and low level of problems associated with hazardous safety or environmental concerns are advantageous. In contrast to some other commonly used preservatives in reagent compositions, boric acid or borates are halogen-free and do not release formaldehyde. The results for the boronic acid present in the ECL signal generation are shown in the examples section, e.g., example 2.

In one embodiment, the present invention relates to a reagent composition for determining ECL comprising i) at least one co-reactant, and iii) at least one preservative selected from boric acid and borate.

In one embodiment, the present invention relates to a reagent composition for determining ECL comprising i) at least one co-reactant, and iii) the preservative boric acid.

In one embodiment, the present invention relates to a reagent composition for determining ECL comprising i) at least one co-reactant, and iii) a preservative borate.

In one embodiment, the present invention relates to a reagent composition for determining ECL comprising I) a compound selected from carboxamides of formula I and formula II, II) at least one co-reactant, iii) at least one preservative selected from boric acid and borates.

In one embodiment, the reagent composition of the invention comprises boric acid or a borate as preservative in a concentration of 0.1% to 5%, preferably in a concentration of 0.5% to 4%, particularly preferably in a concentration of 0.5% to 2%.

In a preferred embodiment, the reagent composition according to the invention comprises boric acid as preservative in a concentration of 0.1% to 5%, preferably in a concentration of 0.5% to 4%, particularly preferably in a concentration of 0.5% to 2%.

In a preferred embodiment, the reagent composition according to the invention comprises borate as preservative in a concentration of 0.1% to 5%, preferably in a concentration of 0.5% to 4%, particularly preferably in a concentration of 0.5% to 2%.

The reagent compositions of the present invention optionally further comprise other test components. Other test components are selected from at least one detergent, at least one signal enhancing compound, a buffer comprising acidic and basic reagents for pH control, and water.

In one embodiment, the present invention relates to a reagent composition for determining ECL comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, iii) at least one preservative, iv) a buffer, v) at least one detergent, vi) a salt and/or an antifoaming agent and vii) optionally other test components.

Detergents suitable for use in the reagent compositions of the invention are those selected from the group consisting of: fatty acid alcohol ethoxylates, including poly (ethylene glycol) ethers, e.g., polidocanol or others having formula C_XEO_YPoly (ethylene glycol) ethers of (X = 8-18 and Y =2-9), Genapol (isotridecyl poly ((ethylene glycol ether)_n)，(alkylpolyglycoside), octylglycoside (octyl- β -D-glucopyranoside), and zwitterionic detergents such as Zwittergent3-12 or mixtures thereof. The detergent is used at a concentration of 0.01% to 2%. The optimum concentration of each detergent can be easily determined. The most suitable concentrations are those of 0.05% to 1%.

In one embodiment, the reagent composition of the invention comprises a detergent selected from polidocanol or other compounds having formula C_XEO_Y(X = 8-18 and Y =2-9), octyl glycoside (octyl- β -D-glucopyranoside) or zwitterionic detergents such as Zwittergent3-12 or mixtures thereof. In a preferred embodiment, the reagent composition comprises a detergent selected from polidocanol, octyl glycoside (octyl- β -D-glucopyranoside) and Zwittergent3-12, or mixtures thereof.

Further, the electrochemiluminescence signal can also be improved by adjusting the pH to 6.0-8.0, preferably 6.0-7.5, and particularly preferably 6.2-6.9. This can be conveniently done by using a pH buffer suitable for this range known to those skilled in the art. In one embodiment, suitable buffers for the reagent composition include KOH and phosphoric acid (H)₃PO₄)。

Still further, the signal may be enhanced by the addition of salts, including inorganic salts such as NaBr, NaCl, NaJ. The salt (specifically NaCl) is added at a concentration of 1mM to 1M, preferably 10mM to 100mM, and most preferably 10mM to 50 mM.

Avoiding the generation of bubbles or foam may be advantageous, especially in HTS applications. Thus, it may be desirable to add an anti-foaming agent to the reagent composition. A variety of commercial antifoams, including Antifoam o-30, Antifoam204, Antifoam A, Antifoam SE-15, Antifoam SO-25 and Antifoam289, may be added to the reagent compositions of the present invention.

Reagent compositions of the invention may include ECL labels. The ECL label may be a conventional ECL label. Examples of ECL markers include tris-bipyridyl-ruthenium (RuBpy) and other organometallic compounds in which the metal is, for example, a metal from groups VII and VIII, including Re, Ru, Ir, and Os. Those skilled in the art use these ECL labels to label analyte-specific reagents with an electrochemiluminescent group, or to label the analyte itself with an electrochemiluminescent group. In one embodiment, the reagent composition of the invention comprises a labelled analyte and/or a labelled analyte-specific reagent, wherein the ECL label is selected from ECL labels disclosed in US5,310,687(a) (BPRu = Ru (bpy)2-bpyCO-OSu) US2003/0124572(a1) (Sulfo-BPRu NHS ester), EP720614(a1) (Bpy2-Ru-bpy-CO-UEEK-korks. -OSu) and WO2002/027317(a2) (BPRu- (UE) -25-K and BPRu2-SK4), respectively.

The reagents and mixtures thereof used in the reagent compositions may be provided in liquid, frozen, deep-frozen, evaporatively-frozen, lyophilized, gaseous, solid or dry form prior to use. The reagent is dissolved in the solvent at least prior to use of the reagent composition. The reagent composition of the invention will be an aqueous solution. In a preferred embodiment, the reagent is dissolved in water.

These improved formulations are particularly valuable in high sensitivity assays. In some embodiments of the invention, the performance of ECL assays is improved even further by optimal combination of reagent composition and electrode composition. Suitable ECL electrode compositions include Ir, Pt or carbon electrodes.

These advantageous combinations include the aforementioned ECL-enhancing carboxamides and a suitable preservative selected from boric acid and borates, both of which have improved properties. These include higher dynamic range and improved ratio of ECL signal from bound label to ECL background signal when using the reagent compositions disclosed herein. This increased sensitivity is important, for example, in assays that benefit from lower detection limits (e.g., TroponnT assays (TNThs; Order-No.:05092744), hepatitis-B envelope antigen assays (HBeAg; Order-No.:11820583), anti-thyroid stimulating hormone receptor assays (anti-TSHR; Order-No.:04388780) -see, in particular, the examples section).

These improved reagent composition formulations may give better accuracy, which may result in lower detection limits in ECL assays.

Another aspect of the invention relates to reduced cost due to the reduced volume required for the sample, test-specific reagents and/or test reagents. The loss of signal for lower reagent volumes can be compensated for by using advantageous reagent compositions of the invention.

Yet another aspect of the invention relates to improved systems and apparatus comprising the reagent compositions of the invention and/or improved systems and apparatus suitable for performing the methods of the invention.

ECL signal generation may also be improved when the above findings are used alone or in combination with each other.

Reagent mixture:

for determining ECL, the reagent compositions of the present invention may be mixed with additional compounds to form a reagent mixture. In one embodiment, the present invention relates to a reagent mixture for determining ECL comprising a reagent composition for determining ECL, said reagent composition comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, iii) a sample to be investigated and iv) at least one detection reagent labeled with an electrochemiluminescent group.

In one embodiment, the present invention relates to a reagent mixture for determining ECL comprising a reagent composition for determining ECL comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, iii) at least one preservative, iv) a sample to be investigated and v) at least one detection reagent labeled with an electrochemiluminescent group.

In one embodiment, the present invention relates to a reagent mixture for determining ECL comprising a reagent composition for determining ECL comprising I) a compound selected from carboxamides of formula I and formula II, II) at least one co-reactant, iii) a preservative selected from boric acid and borates, iv) a sample to be investigated and v) at least one detection reagent labeled with an electrochemiluminescent group.

In a further embodiment, the present invention relates to a reagent mixture for determining ECL comprising a reagent composition for determining ECL, said reagent composition comprising i) a preservative selected from boric acid and borates, ii) at least one co-reactant, iii) a sample to be investigated and iv) at least one detection reagent labeled with an electrochemiluminescent group. In a preferred embodiment, the reagent mixture comprises the preservative boric acid. In a preferred embodiment, the reagent mixture comprises a preservative borate.

The present invention also relates in one embodiment to a reagent mixture for determining ECL comprising a reagent composition for determining ECL, said reagent composition comprising i) a preservative selected from boric acid and borate, ii) at least one co-reactant, iii) a sample to be investigated, iv) a detergent, v) a buffer, vi) at least one detection reagent labeled with an electrochemiluminescent group, and vii) a salt and/or an antifoaming agent.

In a further preferred embodiment, the present invention relates to a reagent mixture for determining ECL, comprising a reagent composition for determining ECL, said reagent composition comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, iii) a preservative selected from the group consisting of boric acid and borates, iv) a sample to be investigated, v) a detergent, vi) a buffer and vii) at least one detection reagent labeled with an electrochemiluminescent group.

The reagent mixture may also comprise at least one detergent and a buffer for controlling the pH. Optionally, the reagent mixture may comprise a salt and/or an antifoaming agent.

Other test components in the reagent mixture are selected from the group consisting of unlabeled analyte-specific reagents, analyte homologs, solid phase coatings, and interference-reducing agents.

The application is as follows:

one aspect of the invention relates to the use of the improved reagent composition and/or improved reagent mixture of the invention in performing an electrochemiluminescence detection method.

In one embodiment, the invention relates to the use of a carboxamide selected from formula I and formula II for performing an electrochemiluminescence assay. In one embodiment, the invention relates to the use of a carboxamide selected from formula I and formula II for performing an electrochemiluminescence detection method operation.

In a preferred embodiment, the invention relates to the use of a carboxamide selected from the group consisting of acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide and 2-chlorobutyramide for performing electrochemiluminescence assays.

In another preferred embodiment, the invention relates to the use of a carboxamide selected from acetamide, 2-chloroacetamide, propionamide and butyramide for performing electrochemiluminescence detection. In another embodiment, the invention relates to the use of a carboxamide selected from acetamide, propionamide and butyramide for performing electrochemiluminescence detection.

In one embodiment, the present invention relates to the use of a reagent composition comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, and iii) at least one preservative in the determination of ECL.

In one embodiment, the present invention relates to the use of a reagent composition comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, and iii) a preservative selected from the group consisting of boric acid and borates in the determination of ECL.

In one embodiment, the present invention relates to the use of a reagent composition comprising i) a compound selected from the group consisting of carboxamides selected from the group consisting of acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide and 2-chlorobutyramide, ii) at least one co-reactant, and iii) a preservative, in the determination of ECL.

In one embodiment, the reagent composition of the invention is suitable for conditioning or regenerating ECL measurement cells and determining ECL signals. In one embodiment, the reagent composition is used as a conditioning solution. In one embodiment, the reagent composition of the invention is used to condition or regenerate ECL measurement cells. In one embodiment, the reagent composition is for conditioning or regenerating ECL measurement cells comprising a compound selected from the group consisting of a carboxamide selected from the group consisting of acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide, and 2-chlorobutyramide. In another embodiment, the reagent composition for conditioning or regenerating ECL measurement cells comprises a compound selected from the group consisting of carboxamides selected from the group consisting of acetamides, propionamides, and butyramides.

For use in performing an electrochemiluminescence detection method, the reagent composition may be mixed with additional compounds to form a reagent mixture, such as a sample to be investigated, at least one detection reagent having an electrochemiluminescence group, and other components described below that support the method.

In one embodiment, the present invention relates to the use of a reagent mixture comprising a reagent composition a) comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, and iii) a preservative, b) a sample to be investigated, and c) at least one detection reagent labeled with an electrochemiluminescent group for determining ECL.

The invention relates to the use of a reagent mixture comprising a reagent composition for determining ECL, a) comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant, and iii) a preservative selected from the group consisting of boric acid and borates, b) a sample to be investigated, and c) at least one detection reagent labeled with an electrochemiluminescent group, for determining ECL.

In a further embodiment, the invention relates to the use of boric acid or a borate salt for performing electrochemiluminescence detection. In yet another embodiment, the present invention relates to the use of a preservative selected from boric acid and borates in performing an electrochemiluminescence detection method operation.

In one embodiment, the present invention relates to the use of a reagent mixture comprising a) a reagent composition for determining ECL comprising i) a preservative selected from boric acid and borates and ii) at least one co-reactant, b) a sample to be investigated, and c) at least one detection reagent labeled with an electrochemiluminescent group for determining ECL.

In addition, the reagent mixture for determining ECL may comprise a component selected from the group consisting of detergents and buffers for pH control. Optionally, the reagent mixture used may comprise a salt and/or an antifoaming agent. Other test components in the reagent mixture are selected from the group consisting of unlabeled analyte-specific reagents, analyte homologs, solid phase coatings, and interference-reducing agents.

The kit comprises:

one aspect of the invention relates to kits comprising one or more components of the reagent compositions of the invention in one or more containers. These components may optionally be combined with additional agents to form the reagent compositions of the present invention. The kit may also comprise additional assay-related components in one embodiment such as ECL labels, ECL-labeled assay reagents, diluents, wash solutions, protein denaturing reagents, enzymes, binding reagents, assay plates, disposables, and the like.

In one embodiment, the reagent composition is contained in one or more glass or plastic containers, with appropriate labeling of information about the contents of the reagent composition and instructions for proper storage and use. Information regarding reagent composition content, lot number, date of manufacture, shelf life, and instructions regarding proper storage and use can also be stored on RFID chips placed within glass or plastic containers. The information stored on the RFID chip can be read out via an antenna connected to an RFID reader and further processed in a control device.

In one embodiment, some or all of the components of the reagent composition may be stored in a liquid or dry state in one embodiment.

In one embodiment, the present invention relates to a kit for measuring ECL comprising a reagent composition for determining ECL, said reagent composition comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II and II) at least one co-reactant.

In a preferred embodiment, the invention relates to a kit for measuring ECL comprising a reagent composition for determining ECL, said reagent composition comprising i) a carboxamide selected from the group consisting of acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide and 2-chlorobutyramide and ii) at least one co-reactant.

In another preferred embodiment, the present invention relates to a kit for measuring ECL comprising a reagent composition for determining ECL, said reagent composition comprising i) a carboxamide selected from acetamide, 2-chloroacetamide, propionamide and butyramide and ii) at least one co-reactant.

In one embodiment, the present invention relates to a kit for measuring ECL comprising a reagent composition for determining ECL, said reagent composition comprising I) a compound selected from the group consisting of carboxamides of formula I and formula II, II) at least one co-reactant and iii) a preservative.

In a preferred embodiment, the present invention relates to a kit for measuring ECL, comprising a reagent composition for determining ECL, said reagent composition comprising I) a compound selected from carboxamides of formula I and formula II, II) at least one co-reactant and iii) a preservative selected from boric acid and borates.

In another preferred embodiment, the present invention relates to a kit for measuring ECL comprising a reagent composition for determining ECL comprising i) a carboxamide selected from the group consisting of acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide and 2-chlorobutyramide, ii) at least one co-reactant and iii) a preservative selected from the group consisting of boric acid and borate.

In another preferred embodiment, the present invention relates to a kit for measuring ECL comprising a reagent composition for determining ECL, said reagent composition comprising i) a carboxamide selected from acetamide, 2-chloroacetamide, propionamide and butyramide, ii) at least one co-reactant and iii) a preservative selected from boric acid and borates.

In one embodiment, the present invention relates to a kit for measuring ECL comprising a reagent composition for determining ECL, said reagent composition comprising at least i) a preservative selected from boric acid and a borate salt and ii) at least one co-reactant.

The aforementioned measurements themselves have significantly improved the known operation. Moreover, by combining these measurements it is possible to further significantly improve the sensitivity and/or dynamic measurement range of the analyte detection assay.

The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made to the operations set forth without departing from the spirit of the invention.

Drawings

FIG. 1: measurement of propionamide (X-axis) at a concentration of 0.001M to 0.25M; measurement of Δ manual assay (manual assay-assay buffer background), assay buffer background and relative recovery (percentage relative to reference) of the free label assay are shown on the Y-axis. See example 1 for details.

FIG. 2: measurement of 2-chloroacetamide (X-axis) at a concentration of 0.001M to 1M; measurement of Δ manual assay (manual assay-assay buffer background), assay buffer background and relative recovery (percentage relative to reference) of the free label assay are shown on the Y-axis. See example 1 for details.

FIG. 3: measurement of butyramide at a concentration of 0.001M to 1M (X-axis); measurement of Δ manual assay (manual assay-assay buffer background), assay buffer background and relative recovery (percentage relative to reference) of the free label assay are shown on the Y-axis. See example 1 for details.

FIG. 4: measurement of acetamide at a concentration of 0.001M to 1M (X-axis); measurement of Δ manual assay (manual assay-assay buffer background), assay buffer background and relative recovery (percentage relative to reference) of the free label assay are shown on the Y-axis. See example 1 for details.

FIG. 5: measurement of boric acid at a concentration of 0 to 5% (X-axis); manual assays are used as examples of high specificity signals; TSH graduator 1 gives a background signal as a low level graduator (TSH Cal 1); the TSH graduator 2 gives a signal at a high detection level (TSH Cal 2). Results are plotted as a percentage of the reference reagent composition without added boric acid. See example 2 for details.

Example 1

ECL measurement Using assay buffer (reagent composition) containing Carbonamide

ECL measurement Using Roche2010, using the protocol described below that can be used for this assay.

Compounds selected from carboxamides at the concentrations shown in tables 2, 3 and 4 were added to the following assay buffers:

180mM Tripropylamine (TPA)

0.1% polidocanol

300mM phosphate buffer

The final pH was determined using KOH/H₃PO₄Adjusted to pH 6.8. Assay buffer can also be used as a blank value.

A compound selected from the group of carboxamides (the formula of the carboxamides is shown in table 1) was added to the assay buffer (reagent composition) at the indicated concentration. Results are reported as signal recovery relative to measurements using assay buffers lacking these compounds.

Assay buffer background measurements of assay buffers containing the concentrations of carbamide shown in table 2 were performed. Values below 100% indicate that addition of the selected carbamide at the indicated concentration reduced ECL background signal. Reducing background electrochemiluminescence in the absence of ECL label is particularly advantageous at low detection levels, where increasing the signal to background ratio (= signal to noise ratio) greatly improves the sensitivity of the assay.

Table 2:

n.d. = undetermined

In a similar experiment, the signal of free label was determined. The free label value represents the signal generated by a solution containing free ECL label in the absence of microparticles (10 nM RuBpy in assay buffer). The values are listed in table 3 in percentages relative to the assay buffer without any other compound. This assay format is also referred to as homogeneous measurement or homogeneous assay format. Values above 100% indicate that addition of the selected carbamide at the selected concentration enhanced ECL signal. The results are shown in Table 3.

Table 3:

n.d. = undetermined

In addition, values were determined using a simplified assay including beads. The artificial assay is an assay for high specificity signals comprising RuBpy-labeled microparticles. This assay format is also referred to as a heterogeneous measurement or heterogeneous assay format. The difference between specific artificial assay-signal and background signal (assay buffer background) using the above assay buffer and other compounds is expressed as a delta artificial assay in% relative to the assay buffer without any other compound. Values above 100% indicate that addition of the selected carbamide at the selected concentration enhanced ECL signal. The results are shown in Table 4.

Table 4:

n.d. = undetermined

The results for propionamide as shown in tables 2, 3 and 4 are illustrated in figure 1. The results for 2-chloroacetamide as shown in tables 2, 3 and 4 are illustrated in figure 2. The results of butyramide as shown in tables 2, 3 and 4 are illustrated in fig. 3. The results of the acetamides as shown in tables 2, 3 and 4 are illustrated in figure 4.

Example 2

Boric acid as signal enhancing preservative

Use of ECL measurementsThe 2010 apparatus was run using the assay recommendation protocol described below.

The following ECL assay buffer was used to determine blank values:

180mM Tripropylamine (TPA)

0.1% polidocanol

0.1%Oxaban A

300mM phosphate buffer

Boric acid was added to the assay buffer in increments as indicated. Using KOH/H₃PO₄The final pH was adjusted to pH 6.8.

Assay buffer background measurements were performed with assay buffers containing boric acid at the concentrations shown in table 5. The free label value represents the signal generated by a solution containing free ECL label in the absence of microparticles (10 nM RuBpy in assay buffer, measured homogeneously) in% relative to assay buffer without any other compound. The manual assay is an assay for high specificity signals including RuBpy labeled microparticles. As a commercial in vitro diagnostic assay,TSH assay (forThe thyroid stimulating hormone assay of (1); Order-No. 11731459) was used to determine Δ TSH. TSH Scale 1(TSH Cal set; Order-No.:04738551) as a low level scale (no analyte present) was used in the TSH assay to give a background signal (TSH Cal 1); the TSH graduator 2 used in the TSH assay gave high signal values (TSH Cal 2).

The results of the manual assay, TSH Cal1 and TSH Cal2 are depicted in fig. 5, and the relative recovery (in%) of the buffer was determined as a reference without added boric acid.

Specifically, the following measurements were performed.

Table 5:

the addition of boric acid as a preservative to the assay buffer improved the specific heterogeneous signal, especially in the manual assay and in the determination of TSH Cal 2.

Example 3

Assay buffer pair containing propionamide and boric acidInfluence of lower detection Limit of the assay

The lower detection limits of several commercial in vitro assays (HBeAg: Roche Order-No.:11820583, anti-TSHR: Roche Order-No.:04388780, TNThs: Roche Order-No.:05092744) were determined to compare the two assay buffer formulations.

Assay buffer a:

180mM TPA, 0.1% polidocanol, 300mM phosphate buffer, 0.1% Oxaban A

Assay buffer B:

180mM TPA, 0,1% polidocanol, 50mM propionamide, 300mM phosphate buffer and 1% boric acid

Determination of the final pH of buffers A and B KOH/H was used₃PO₄Adjusted to pH 6.8.

The three commercially available assays described above have been analyzed to show the effect of carbamide, i.e., propionamide, and a preservative, i.e., boric acid, on the performance of the assay to detect very low analyte concentrations.

Is measured inThe analyzer was measured on the analyzer and calibrated as described in its packaging insert. To calculate the lower detection limit, the signal of samples without analyte (HBeAg, anti-TSHR) or with very low analyte concentration (TNThs) was determined. The standard deviation determined for the 21 samples (21-fold) was calculated, multiplied by 2(2SD) or 3(3SD) and added (HBeAg, TNThs) or subtracted (anti-TSHR, competition assay) to the mean of the signals. The corresponding concentration of the calculated signal is then determined using the calibration curve for each assay. For samples with low analyte concentrations (TNThs), the analyte concentration of the sample is subtracted from these calculated concentrations.

These 3 assays benefit from the improved reagent compositions of the present invention containing a carbamide as well as containing boric acid (boric acid also has a preservative function). The results of the HBeAg, anti-TSHR and TNThs assays are shown in tables 6, 7 and 8, respectively.

Table 6:

table 7:

table 8:

Claims (16)

1. A method of measuring an analyte in a sample by electrochemiluminescence detection, comprising the steps of:

a) incubating the sample with a detection reagent labeled with an electrochemiluminescent group,

b) separating the labeled detection reagent with the bound analyte from the labeled detection reagent without the analyte,

c) incubating the separated labeled detection reagent with a reagent composition comprising:

i) at least one co-reactant, and

II) at least one compound selected from the group consisting of carboxamides of the formula I and of the formula II,

in the formula I, R₁＝CH₃、CH₂F、CH₂Cl、CH₂CH₃、CHClCH₃、CH₂CH₂Cl、C(CH₃)₂CH₃、CH₂CH₂CH₃、CClHCH₂CH₃Or CH₂CH₂CH₂CH₃，R₂Is H, and R₃＝H，

d) Electrochemically triggered release of luminescence, and

e) determining an Electrochemiluminescence (ECL) signal, thereby measuring the analyte.

2. The method of claim 1, characterized in that the measurement of the analyte in the sample using ECL is performed in aqueous solution.

3. Process according to claim 1 or 2, characterized in that the carbonic acid amide is selected from the group consisting of acetamide, 2-fluoroacetamide, 2-chloroacetamide, propionamide, 2-chloropropionamide, 3-chloropropionamide, butyramide and 2-chlorobutyramide.

4. Method according to any one of claims 1 to 2, characterized in that the reagent composition comprises carbamide in a concentration of 0.01M to 0.25M.

5. The method according to any one of claims 1 to 2, characterized in that the reagent composition of step c) comprises a preservative.

6. The method according to claim 5, characterized in that the reagent composition of step c) comprises a preservative in a concentration of 0.1% to 5%.

7. The method according to claim 1 or 2, characterized in that the reagent composition of step c) comprises a preservative selected from boric acid and borates.

8. The method according to any one of claims 1 to 2, characterized in that the reagent composition of step c) comprises a detergent and a buffer.

9. The method according to claim 8, characterized in that the reagent composition of step c) further comprises a salt and/or an antifoaming agent.

10. A reagent composition for determining electrochemiluminescence, comprising:

i) a compound selected from the group consisting of carboxamides of formula I and formula II, and

ii) at least one co-reactant,

wherein the co-reactant is selected from the group consisting of tertiary amines, oxalates, and persulfates,

the compound of the carboxamide of formula I is:

in the formula I, R₁＝CH₃、CH₂F、CH₂Cl、CH₂CH₃、CHClCH₃、CH₂CH₂Cl、C(CH₃)₂CH₃、CH₂CH₂CH₃、CClHCH₂CH₃Or CH₂CH₂CH₂CH₃，R₂Is H, and R₃＝H，

The compound of the carboxamide of formula II is:

11. reagent composition according to claim 10, characterized in that the tertiary amine is tripropylamine.

12. Reagent composition according to claim 10, characterized in that it further comprises a preservative selected from boric acid and borates.

13. A reagent mixture for determining electrochemiluminescence comprising a reagent composition according to claim 10 or 12, a sample to be investigated and at least one detection reagent labeled with an electrochemiluminescent group.

14. Use of a reagent composition according to claim 10 or 12 for determining electrochemiluminescence.

15. Use of a carboxamide selected from formula I and formula II for performing an electrochemiluminescence detection operation,

wherein the compound of the carboxamide of formula I is:

in the formula I, R₁＝CH₃、CH₂F、CH₂Cl、CH₂CH₃、CHClCH₃、CH₂CH₂Cl、C(CH₃)₂CH₃、CH₂CH₂CH₃、CClHCH₂CH₃Or CH₂CH₂CH₂CH₃，R₂Is H, and R₃＝H，

The compound of the carboxamide of formula II is:

16. a kit for measuring electrochemiluminescence comprising the reagent composition of claim 10 or 12.