Reducing interference in immunoassays

Abstract

The present invention relates to a method for determining an analyte in a sample, comprising the steps of: a) contacting said sample with at least first and second detection compounds; b) determining the amount of complexes comprising at least one detection compound; and c) determining said analyte in the sample based on the results of step b), wherein said first detection compound comprises a first binding moiety and a first label, and said second detection compound comprises a second binding moiety and a second label, and wherein the first label and the second label are not identical. The present invention further relates to a kit for detecting an analyte in a sample, comprising at least a first and a second detection compound for the analyte, wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label, and the first and second labels are not identical; and a device for determining an analyte in a sample, comprising at least a first and a second detection compound for the analyte, wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label, and the first and second labels are not identical, and the device comprises means for determining at least one signal obtained from the first and second labels; and the use of a composition comprising at least a first and a second detection compound for detecting an analyte, wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label, and the first and second labels are not identical.

Description

[Technical field]

[0001] The present invention relates to a method for determining an analyte in a sample, comprising the steps of: a) contacting said sample with at least a first and a second detection compound; b) determining the amount of a complex comprising at least one detection compound; and c) determining said analyte in the sample based on the result of step b), wherein said first detection compound comprises a first binding moiety and a first label, and said second detection compound comprises a second binding moiety and a second label, and wherein the first label and the second label are not identical. The present invention further relates to a kit for detecting an analyte in a sample, comprising at least a first and a second detection compound for said analyte, said first detection compound comprising a first binding moiety and a first label, said second detection compound comprising a second binding moiety and a second label, and said first and second labels being different; and to a device for determining an analyte in a sample, comprising at least a first and a second detection compound for said analyte, said first detection compound comprising a first binding moiety and a first label, said second detection compound comprising a second binding moiety and a second label, and said first and second labels being different; and to a means for determining at least one signal obtained from said first and said second labels; and to a use of a composition comprising at least a first and a second detection compound for detecting an analyte, wherein said first detection compound comprises a first binding moiety and a first label, said second detection compound comprises a second binding moiety and a second label, and said first and second labels being different. Summary of the Invention

[0002] Clinical testing, and in particular immunological testing, has become an invaluable tool in the diagnosis of diseases. In particular, immunoassays have offered the possibility to specifically detect single analytes or groups of analytes in complex mixtures, such as body fluids, such as blood, serum or plasma. However, several sources of interference in immunoassays have been identified, such as (to name a few) cross-reactivity of interfering substances with the capture compound, non-specific binding of the detection compound to the solid phase, "cross-linking" of the capture compound and the detection compound by heterophilic antibodies or human anti-mouse antibodies (HAMA) (see e.g. WO2016097116 A1; Park & ​​Kricka (2013), Chapter 5.3 - Interferences in Immunoassay in The Immunoassay Handbook (4th ed.) edited by David Wild (Elsevier, Oxford: 403); Schiettecatte (2012), Interferences in Immunoassays, Advances in Immunoassay Technology, Dr. Norman HL Chiu (editor)). Further targets of interference can be labels, i.e., signal-generating units in the assay (e.g., Buijs et al. (2011), Ann Clin Biochem. 48(Pt 3):276; Heijboer et al. (2009), Ann Clin Biochem.46(Pt 3):263; Ando et al. (2007), Intern Med. 46(15):1225).

[0003] In competitive immunoassays, the analyte contained in the sample competes with a labeled analyte (specifier) ​​for binding to a capture compound, which is often an antibody or, for example, an antigen of a pathogen, if the presence of antibodies against said pathogen is to be tested. If the concentration of analyte in the sample is high, there is a strong competition resulting in a reduced binding of the specifier to the capture compound, causing a reduction in the signal, which, depending on the test format, results in a quantitative or qualitative test result. If there is a reduction in the signal caused by anti-label interference, this can cause a false positive result.

[0004] In non-competitive immunoassay, analyte is detected by contacting the analyte with a compound that specifically binds to the analyte and either carries a label itself or is the target of a second molecule that carries a label.Thus, in non-competitive immunoassay, the amount of analyte is determined by determining the amount of complex formed between the analyte and the detection compound that carries a label.Thus, similar specificity problems can be faced as above.Anti-label interference tends to reduce the signal by binding to the label, resulting in a reduction in signal yield, thereby resulting in false negative results.In some cases, anti-label interference can increase the signal even in non-competitive assay format, thereby resulting in false positive results.

[0005] Currently, two different strategies are known on how to reduce anti-labeling interference: the most commonly used method is to add the target of the interfering substance (a label or a slightly modified target (labeled analogue) that cannot generate a signal) to the assay, usually at a concentration that exceeds the original label concentration. The addition of the label or labeled analogue serves as an alternative target for the interfering substance, which leads to a reduction in the binding of the interfering substance to the label, eliminating or reducing the interference. However, it may not be possible to sufficiently reduce the interference by adding more active labels, for example, because the addition of active labels would interfere with the assay. In this case, the method of choice is to add inactive labeled analogues that do not generate a signal but are targeted by the interfering substance. However, because there is a structural difference between reactive labels and labeled analogues, the efficiency of reducing anti-labeling interference of labeled analogues may be limited.

[0006] The second possibility to reduce label-specific interference is to specifically target the affected label by a binding partner that is added to the assay, which is non-reactive in the assay but blocks the binding of interfering substances to the label, thus also leading to reduced interference.(See, for example, DE19519973A1, WO2017093271A1; Sapin et al. (2007), Clin Chern Lab Med. 45(3):416; DeForge (2010), J Immunol Methods. 362(1-2):70).However, such a procedure inevitably involves a significant reduction in signal yield, which may cause a reduction or impairment of detection limit.

[0007] Problem to be solved It is therefore an object of the present invention to provide an improved immunoassay which avoids the problems mentioned above.

[0008] Means for solving the problem These problems are solved by methods, kits, devices and compositions having the features of the independent claims. Exemplary aspects, which may be realized in isolation or in any arbitrary combination, are recited in the dependent claims.

[0009] Thus, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: a) contacting the sample with at least a first and a second detection compound; b) determining the amount of complex containing at least one detection compound; and c) determining said analyte in the sample based on the result of step b); wherein said first detection compound comprises a binding moiety and a first label, and said second detection compound comprises a binding moiety and a second label, and wherein the first label and the second label are not identical. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] When used below, the terms "has", "comprise" or "include" or any optional grammatical variation thereof are used in a non-exclusive manner. These terms can therefore refer both to the situation in which no further features are present in the entity described in this context besides the features introduced by these terms, and to the situation in which one or more further features are present. As an example, the expressions "A has B", "A comprises B" and "A includes B" can refer both to the situation in which no other elements are present in A besides B (i.e. the situation in which A consists solely of B and the situation in which A consists exclusively of B) and the situation in which one or more further elements are present in entity A besides B, such as element C, elements C and D, or even further elements.

[0011] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "particularly", "more particularly" or similar terms are used in conjunction with any feature without restricting further possibilities. Features introduced by these terms are therefore optional features and are not intended to limit the scope of the claims in any way. As a person skilled in the art will understand, the invention can be implemented by using alternative features. Similarly, features introduced by "in one embodiment of the invention" or similar expressions are intended to be optional features, without any limitations on further aspects of the invention, without any limitations on the scope of the invention, and without any limitations on the possibility of combining the feature so introduced with other optional or non-optional features of the invention.

[0012] Unless otherwise indicated, the term "about" refers to the indicated value with the generally accepted technical precision in the relevant field, preferably ±20%, more preferably ±10%, and most preferably ±5% of the indicated value. Furthermore, the term "essentially" indicates that there is no deviation having an effect on the indicated result or use, i.e., possible deviations do not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Thus, "consisting essentially of" means including the specified components, but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process used to provide the component, and components added for purposes other than achieving the technical action of the invention. For example, a composition defined using the phrase "consisting essentially of" includes any known acceptable additives, excipients, diluents, carriers, and the like. Preferably, a composition consisting essentially of a set of components will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of the unspecified component(s).

[0013] The method of the present invention is, in one embodiment, an in vitro method. Moreover, it can include steps in addition to the specifically mentioned steps. In particular, step a) can be preceded by a step of providing a sample, or step a) and / or b) can include the addition of additional compounds to facilitate binding and detection. Moreover, some or all steps can be assisted by automated equipment. In one embodiment, the method is an immunological method, i.e., in one embodiment, at least one of the analyte and the detection compound is or includes an antibody.

[0014] The term "biological molecule" is known to those skilled in the art and typically relates to a molecule produced by the metabolism of at least one living organism. Thus, the term "biological macromolecule" in one embodiment relates to a polymer produced by an organism from monomeric precursors. Exemplary biological macromolecules are polypeptides, DNA, RNA or polysaccharides.

[0015] The term "polypeptide" as used herein, in one embodiment, includes variants and fragments of the specifically indicated polypeptide. Variants include polypeptides that contain an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the specifically indicated amino acid sequence. Percent identity values ​​are preferably calculated over the entire amino acid sequence region. A range of programs based on various algorithms are available to those skilled in the art for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the alignment of sequences, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))], which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991)], should be used. The sequence identity values ​​quoted above in percentage (%) should be determined using the program GAP, preferably with the following settings over the entire sequence region: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which shall always be used as the standard settings for sequence alignments unless otherwise stated. Polypeptides comprising fragments of any of the aforementioned polypeptide sequences are also encompassed in one embodiment as polypeptides of the present invention.A fragment is a polypeptide that still has activity suitable for use as an analyte or in a detection compound as specified elsewhere herein, e.g., a binding moiety as specified elsewhere herein. Thus, a polypeptide can comprise or consist of a domain of the polypeptide of the invention that confers said biological activity. A fragment as contemplated herein preferably comprises at least 50, at least 100, at least 250 or at least 500 consecutive amino acid residues of any one of the amino acid sequences, including at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 consecutive amino acids of any one of the amino acid sequences. A polypeptide of the invention can also contain further polypeptide sequences. In particular, a polypeptide of the invention can be a fusion protein, where one partner of the fusion protein is a polypeptide as specified herein. Such fusion proteins may contain as additional moieties polypeptides for monitoring expression (e.g. green, yellow, blue or red fluorescent proteins, alkaline phosphatase, etc.) or so-called "tags" which may serve as detectable markers or purification purposes or as an aid for binding said polypeptides to solid surfaces. Tags for different purposes are well known in the art and include the FLAG tag, the 6-histidine tag, the MYC tag, biotin, etc.

[0016] The term "antibody" as used herein includes monoclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, so long as they exhibit the desired binding activity specified elsewhere herein. In one embodiment, the antibody is not an antibody contained in an antiserum, and typically is not a polyclonal antibody or a polyclonal serum. Thus, in one embodiment, the antibody is an antibody contained in a mixture, wherein at least 80%, in one embodiment at least 90%, and in a further embodiment at least 95% of the antibody molecules contained in said mixture are detection compounds of the present invention. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a full-length antibody or an antibody fragment.

[0017] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five main immunoglobulin classes: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunit structures and three-dimensional organization of the different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed., WB Saunders, Co. (2000). An antibody can be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0018] The terms "full-length antibody", "complete antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, and not to an antibody fragment as defined below. The terms specifically refer to an antibody having a heavy chain containing the Fc region. An "antibody fragment" includes, in one embodiment, a portion of an intact antibody, including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, the name reflecting its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. "Fv" is the smallest antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure similar to that in the two-chain Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site. The six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or even half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site. The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment contains a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).

[0019] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to an analyte, where the polypeptide sequence that binds to the analyte was obtained by a process that includes the selection of a single analyte-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a pool of multiple clones, e.g., hybridoma clones, phage clones, or recombinant DNA clones. In contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0020] The term "analyte" as used herein refers to a chemical molecule, in one embodiment an organic molecule, that binds to a detection compound of the present invention with sufficient affinity to allow detection of the analyte / detection compound complex. In one embodiment, the dissociation constant (K d ) can be up to 10 -7 mol / L, in a further embodiment up to 10 -8 mol / l, in a further embodiment up to 10 -9 mol / L. In one embodiment, the analyte is a biological molecule, and in a further embodiment, the analyte is a biological macromolecule. In a further embodiment, the analyte is a polypeptide.

[0021] In one embodiment, when the analyte is a polypeptide, the polypeptide is an antigen produced by an infectious agent, e.g., a virus, bacterium, or protozoan; or the analyte is an antibody produced by the subject against an antigen produced by the infectious agent, e.g., a virus, bacterium, or protozoan.

[0022] In one embodiment, the analyte is a bacterial antigen, such as a Treponema pallidum antigen, a Borrelia antigen, a Brucella antigen, a Chlamydia antigen, a Mycoplasma antigen, a Listeria antigen or an antigen from a single-celled organism such as a protozoan, in one embodiment a Trypanosoma cruzi antigen. In one embodiment, the analyte is an antibody against a bacterial antigen, in a further embodiment an antibody against a bacterial polypeptide. In one embodiment, the antibody is an anti-Treponema pallidum antibody, an anti-Borrelia antibody, an anti-Brucella antibody, an anti-Chlamydia antibody, an anti-Mycoplasma antibody or an anti-Listeria antibody. In yet another embodiment, the analyte is an antibody against a single-celled organism, in one embodiment an anti-Trypanosoma cruzi antibody. In a further embodiment, the analyte is an anti-autoantibody, i.e. an antibody that recognizes an antigen produced by the subject itself, which in one embodiment is diagnostic and / or prognostic of a disease, in one embodiment an autoimmune disease. Diagnostic and prognostic anti-autoantibodies are known in the art and include, for example, antinuclear antibodies, which are indicative of systemic lupus erythematosus or polymyositis, antithyroid antibodies, which are indicative of Hashimoto's thyroiditis or Grave's disease, anti-intrinsic factor antibodies, which are indicative of anemia, and anti-tissue transglutaminase antibodies, which are indicative of celiac disease or gluten sensitivity.

[0023] In one embodiment, the analyte is a viral antigen, such as an antigen of a virus, in one embodiment selected from the list consisting of hepatitis virus, arbovirus, adenovirus, coxsackie virus, echovirus, influenza virus, parainfluenza virus, cytomegalovirus, herpes virus, Epstein-Barr virus, mumps virus, norovirus, rotavirus, rubella virus, measles virus, human immunodeficiency virus, varicella zoster virus, poliovirus and enterovirus. In one embodiment, the analyte is an antibody against a viral antigen, in a further embodiment against a viral polypeptide, or in a further embodiment against a viral capsid polypeptide, in one embodiment of a medically relevant virus, in one embodiment selected from the list specified herein above. In one embodiment, the virus is hepatitis A virus, hepatitis B virus, hepatitis C virus. In one embodiment, the viral capsid polypeptide is a hepatitis virus capsid polypeptide, in one embodiment a hepatitis A (HA) virus capsid polypeptide. Thus, the analyte, in one embodiment, is an antibody against a Hepatitis virus capsid polypeptide, for example, against a Hepatitis A virus capsid polypeptide.

[0024] In one embodiment, the analyte is a protozoan antigen, for example from Toxoplasma, in one embodiment from T. gondii. In one embodiment, the analyte is an antibody against a protozoan antigen, in a further embodiment against a protozoan polypeptide. In one embodiment, the analyte is an antibody against T. gondii p30 polypeptide (Genbank Accession Number: S85174.1).

[0025] However, it is also contemplated that the analyte is a low molecular weight compound determinable by complex formation with a detection compound. In one embodiment, the analyte has a molecular mass of at least 100 (100 atomic mass units corresponds to 100 Da; 1 Da is 1.66×10 -27In one embodiment, the analyte has a molecular mass of at least thyroxine (T4), triiodothyronine (T3), folate, folate binding protein, vitamin B 12 or intrinsic factors.

[0026] As used herein, the term "detection compound" refers to a compound comprising a binding moiety and a label, both as specified elsewhere herein. In one embodiment, the binding moiety and the label form an affinity complex in said detection compound, in one embodiment, wherein the dissociation constant (K d ) can be up to 10 -8 mol / L, in a further embodiment up to 10 -9 mol / L, in a further embodiment up to 10 -10 mol / L. In one embodiment, the binding moiety and the label are covalently linked. In one embodiment, the method for determining an analyte is a non-competitive immunoassay. In a further embodiment, the method for determining an analyte is a competitive immunoassay; as will be understood, in such a case, the detection compound is a compound of the assay, which is also called a "specific agent" in the art, i.e., a compound linked to a label, which competes with the analyte for binding to the capture compound.

[0027] The term "binding moiety" as used herein refers to the structure of the detection compound, not the label, as specified elsewhere herein. Thus, for example, if the detection compound is a dye-labeled antibody, the antibody would be considered the binding moiety; if the detection compound is a dye-labeled F(ab) fragment, the F(ab) fragment would be the binding moiety. In contrast, the term "affinity domain" is used to refer to the substructure of the detection compound that mediates the affinity of the detection compound for the analyte. Thus, the affinity domain is the substructure of the binding moiety that includes the atoms that contact the analyte in the detection compound / analyte complex. Thus, the binding moiety can include more than one affinity domain, for example, two identical affinity domains in one embodiment when the binding moiety is an IgG, ten identical affinity domains in a further embodiment when the binding moiety is an IgM, and two different affinity domains in a further embodiment when the binding moiety is a diabody. In a further embodiment, the binding moiety can include multiple, i.e., any number of identical or non-identical affinity domains, in the case of a binding moiety being a polyepitopic antigen, greater than two.

[0028] In one embodiment, the binding moiety of the first detection compound is not identical to the binding moiety of the second detection compound. Thus, in one embodiment, the first and second detection compounds can be different monoclonal antibodies that in one embodiment recognize the same epitope of the antibody. In one embodiment, in such a case, the dissociation constant of the complex formed between the first detection compound and the analyte and the dissociation constant of the complex formed between the second detection compound and the analyte differ by no more than 5-fold; in one embodiment, no more than 2-fold; in a further embodiment, no more than 1.5-fold. In one embodiment, the dissociation constant of the complex formed between the first detection compound and the analyte has a value between 70% and 130% of the dissociation constant of the complex formed between the second detection compound and the analyte. In a further embodiment, the dissociation constant of the complex formed between the first detection compound and the analyte has a value between 80% and 120% of the dissociation constant of the complex formed between the second detection compound and the analyte. In a further embodiment, the dissociation constant of the complex formed between the first detection compound and the analyte has a value of 90% to 110% of the dissociation constant of the complex formed between the second detection compound and the analyte. In a further embodiment, the dissociation constant of the complex formed between the first detection compound and the analyte and the dissociation constant of the complex formed between the second detection compound and the analyte are essentially equal or equal.

[0029] In a further embodiment, the binding moiety of the first detection compound and the binding moiety of the second detection compound are essentially identical, i.e., they differ only in structural determinants that are not related to the binding of the analyte.In a further embodiment, the binding moiety of the first detection compound and the binding moiety of the second detection compound are identical; thus, in one embodiment, the first detection compound and the second detection compound differ only in the label.As will be understood by those skilled in the art, this difference can include the difference defined in one embodiment by the different coupling chemistry of the first label and the second label.

[0030] The term "label", as used herein, relates to a compound adapted to make the presence of a molecule or complex containing said label detectable. Typically, a label has a detectable property, typically an optical property or / and an enzymatic property. However, it is also envisaged that said detectable property is a property that emits radioactivity. As will be understood, the parameter determined to detect said detectable property will also be called "signal" or "detectable signal". For the avoidance of doubt, it is noted that the signal may be detected to be zero or below the detection limit.

[0031] The term "enzymatic property" as used herein relates to the property of a label to generate a detectable product from a substrate by biological catalysis. Thus, the enzymatic property is typically conferred by the presence of a polypeptide having said enzymatic property in said label. Typically, the enzymatic property is at least one enzymatic activity selected from the group consisting of a phosphatase activity (e.g. in alkaline phosphatase), a peroxidase activity (e.g. in horseradish peroxidase) and a glycosidase activity (e.g. in beta-galactosidase). Exemplary substrates for enzymatic activities are well known in the art. Typically, said enzymatic activity generates a product having a detectable optical property as specified herein above or / and said enzymatic activity generates a product that is detectable by an electronic device.

[0032] The term "optical property" as used herein relates to any property that can be detected by an optical instrument. In particular, the optically determinable property can be or include at least one property selected from the group consisting of reflection, transmission, emission, scattering, fluorescence, phosphorescence, diffraction and polarization properties. Further optical properties envisaged by the present invention are color, fluorescence, luminescence or refraction. In one embodiment, the optically determinable property as referred to herein refers to a property of a chemical compound that can be optically detected, such as light absorption, emission, light remission or properties related thereto. It will be understood that the detection of an optically determinable property as used herein encompasses the detection of the presence of a property that was not previously detectable, the detection of the absence of a property that has been previously detected, and the detection of a quantitative change in the property, i.e. a change in signal intensity that correlates to the degree of change in at least one optical property. It is understood that the term "optical property" also relates in one embodiment to luminescence, in one embodiment to chemiluminescence, and in a further embodiment to electrochemiluminescence, also known as electrogenerated chemiluminescence. Thus, in one embodiment the detectable signal can be a luminescent signal, in one embodiment a chemiluminescent signal, in a further embodiment an electrochemiluminescent signal. In accordance with the above, in one embodiment the label is a dye, and in one embodiment a chemiluminescent compound.

[0033] As mentioned herein, the first detection compound comprises a first binding moiety and a first label, the second detection compound comprises a second binding moiety and a second label, and the first label and the second label are not identical. The first and second labels of the present invention are not identical, i.e., the two labels can be distinguished in at least one physical, biological and / or chemical property. For example, the first and second labels can be distinguished by their interaction with specific antisera or antibodies, or by chromatography and / or mass spectrometry. In one embodiment, the distinguishing property is a structural property. Thus, in one embodiment, the first and second labels differ in at least one feature of their chemical structure, for example, by at least one atom, chemical bond and / or charge. In one embodiment, the distinguishing property of the first and second labels is unrelated to the detectable property of the first and second labels; thus, in one embodiment, the detectable property of the first and second labels is the same, but the first and second labels are structurally different. In a further embodiment, the characteristic that distinguishes the first and second labels relates to a detectable property of the first and second labels; thus, in one embodiment, the detectable properties of the first and second labels are not identical and the first and second labels are structurally distinct.

[0034] In one embodiment, the first and second (non-identical) labels provide the same quality of detectable property. As used herein, the term "quality of detectable property" relates to the physical properties of the detectable property that define its mode of detection; thus, two labels having the same quality of detectable property are two labels that allow the detection of their detectable property by the same measurement principle, i.e., measurement of emission, transmission, luminescence, including chemiluminescence, absorbance, etc. In a further embodiment, the first and second labels provide essentially the same detectable property, i.e., allow the detection of their detectable property by essentially the same method, and the two methods are "essentially the same" when at most one measurement parameter, e.g., the detection wavelength, is readjusted by a factor of at most 2, in one embodiment at most 1.5, in a further embodiment at most 1.2, in a further embodiment at most 1.1. In a further embodiment, the first and second labels provide the same detectable property, i.e., allow the detection of their detectable property by the same method. As will be understood, in the latter case, the detectable properties of the first and second labels can be detected simultaneously. As will be further appreciated, detection of the detectable property of the first and / or second label can be performed under non-optimal conditions for one or both labels; for example, if two labels have overlapping absorption spectra, absorption can be measured at a wavelength of non-maximum absorption where both labels have the same molar extinction coefficient. Thus, in one embodiment, determining the amount of the complex in step b) comprises detecting a property of the first detection compound and a property of the second detection compound, and in one embodiment, simultaneously detecting a property of the first detection compound and a property of the second detection compound.

[0035] In one embodiment, the first label is Ru(bpy)2-bpyCO-OSu (also referred to as "BP-Ru"; CAS Registry Number 137323-76-3, = ruthenium(2+), bis(2,2'-bipyridine-κN 1 ,κN 1’ )[1-[4-(4'-methyl[2,2'-bipyridine]-4-yl-κN 1 ,κN 1’)-1-oxobutoxy]-2,5-pyrrolidinedione]-, (OC-6-33) Ru(bpy)2-bpyCO2H (=BPRu or Ru-bpy) reactive ester, CAS Registry Number 115239-59-3), and the second label is sulfo-BPRu NHS ester (also called "sulfo-Ru"; CAS Registry Number 482618-42-8, known in the art as ruthenate(2-), bis[[2,2'-bipyridine]-4,4'-dimethanesulfonato(2-)-κN 1 ,κN 1’ ][1-[4-(4'-methyl[2,2'-bipyridin]-4-yl-κN 1 ,κN 1’ )-1-oxobutoxy]-2,5-pyrrolidinedione]-, sodium (1:2), also known as (OC-6-31).

[0036] As used herein, the term "determining" refers to determining at least one detectable characteristic of the analyte to be determined by the method of the present invention in a sample. In one embodiment, the method of the present invention is applicable to any determination involving the use and detection of at least one detection compound as specified herein. Thus, in one embodiment, determining includes determining the direct or indirect interaction of the analyte characteristic with the detection compound. In one embodiment, the analyte characteristic is a binding domain that binds, in one embodiment specifically binds, to the detection compound. Thus, in one embodiment, the analyte detectable characteristic is a receptor or a receptor domain thereof, a lectin, an aptamer (which can be a peptide aptamer or a polynucleotide aptamer), anticalin, a Designed Ankyrin Repeat Protein, or an immunoglobulin or a binding subdomain thereof, and the detection compound comprises a chemical structure that is specifically bound by said detectable characteristic. Conversely, in one embodiment, the detection compound comprises a receptor or a receptor domain thereof, a lectin, an aptamer (which may be a peptide aptamer or a polynucleotide aptamer), anticalin, a designed ankyrin repeat protein or an immunoglobulin or a binding subdomain thereof, and the detectable feature of the analyte comprises a chemical structure that is specifically bound by said detection compound.

[0037] In the context of the present invention, an "aptamer" is a macromolecule that specifically binds to its interaction partner. Aptamers can be peptide or polynucleotide aptamers and are in principle known to the skilled person. The term "peptide aptamer", as used herein, relates to a peptide that specifically binds to its interaction partner and comprises 8 to 80 amino acids, in one embodiment 10 to 50 amino acids, in a further embodiment 15 to 30 amino acids. They can be isolated from randomized peptide expression libraries in suitable host systems such as, for example, baker's yeast (see, for example, Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-1998).

[0038] As used herein, the term "anticalin" refers to an artificial polypeptide derived from lipocalin that specifically binds to its interaction partner. Similarly, "designed ankyrin repeat proteins" or "DARPins" as used herein are artificial polypeptides that contain several ankyrin repeat motifs and specifically bind to their interaction partners.

[0039] In one embodiment, the detectable feature of the analyte and / or the detection compound comprises an immunoglobulin or a binding subdomain thereof. Thus, in one embodiment, determining comprises determining a direct or indirect interaction of the immunological feature of the analyte with the detection compound and / or a direct or indirect interaction of the feature of the analyte with the detection compound. Thus, in one embodiment, the method is an immunoassay. An immunological feature according to the invention is, in one embodiment, a structural feature of the analyte that facilitates the detection of the analyte in the sample by immunological means. In one embodiment, said immunological feature facilitates the identification, in a further embodiment, the quantification, of the analyte by immunological means. Thus, a typical immunological feature is a feature that facilitates the differentiation of said analyte from other chemical compounds in the sample. In one embodiment, determining the analyte is to establish whether the analyte is present or absent in the sample at a concentration above the detection limit of the method. Methods for determining the detection limit are known to those skilled in the art. In a further embodiment, determining is to semi-quantitatively or quantitatively determine the amount or concentration of the analyte in the sample. For a quantitative determination, the absolute or exact amount of the analyte will be determined, or the relative amount of the analyte will be determined. A relative amount can be determined in cases where the exact amount of the analyte cannot or should not be determined. In such cases, it can be determined whether the amount of the analyte present is increased or decreased with respect to a second sample containing said analyte in a second amount.

[0040] As will be understood by those skilled in the art, typically, it will not be necessary to determine the first detection compound / analyte and the second detection compound / analyte complexes separately. Thus, in one embodiment, the complex of the first detection compound and the analyte and the complex of the second detection compound and the analyte are determined together, i.e. indiscriminately between said first detection compound and said second detection compound. Thus, in one embodiment, the total amount of analyte present in the complex with either the first detection compound or the second detection compound or both is determined. This can be accomplished in one embodiment, as specified herein above, by simultaneously determining the detectable properties of the first and second labels in step b); or by determining the detectable properties of the first and second labels separately in step b) but determining the sum of the properties of the first detection compound and the sum of the properties of the second detection compound in step c). As will be understood from the above, the first and second labels can be bound to the same type of binding moiety; thus, the first and second binding moieties can be different, but can also be the same. Thus, the determination can be achieved by contacting the sample with a monoclonal antibody, a first fraction of which is bound to a first label and a second fraction of which is bound to a second label. The same applies mutatis mutandis to polyclonal antibody preparations.

[0041] As will be further appreciated by those skilled in the art, the determination of the analyte / capture compound complex can include further steps and / or the use of further compounds. For example, one or more further detection compounds that are not identical to the first and second detection compounds at least in terms of label can be used. In one embodiment, the first and second detection compounds are used in a ratio of 1:5 to 5:1, typically 1:2 to 2:1, about 1:1, or 1:1. In a further embodiment, three detection compounds are used in a ratio of about 2:1:1, 1:2:1 or 1:1:2, about 1:1:1 or 1:1:1. Thus, in one embodiment, the first and second detection compounds and the potentially further detection compounds are present in the reaction mixture in approximately the same amount, in a further embodiment in the same amount. In one embodiment, 2 to 10 detection compounds, in one embodiment, 2 to 5 detection compounds, in one embodiment, 2 to 4 detection compounds, in one embodiment, 2 to 3 detection compounds are used, and the labels of all detection compounds used are not identical to each other. In one embodiment, the detection compounds are used essentially equal, in one embodiment in equal amounts.

[0042] Furthermore, depending on the assay format selected, a capture antibody can be used to bind the analyte and the analyte / detection compound(s) complex to a solid surface. In one embodiment, the assay is a competitive immunoassay, typically a competitive heterogeneous immunoassay, i.e., an immunoassay, in which the analyte competes with a labeled derivative of said analyte for binding to a capture compound bound to a solid surface, and in which the amount of the labeled derivative of said analyte bound to said capture compound is determined. Thus, in such a case, the method further comprises mixing a specific agent with the sample. In one embodiment, the method of the present invention is a heterogeneous competitive immunoassay, and comprises indirectly determining the amount of the complex formed between the analyte and two non-identical capture compounds by determining the amount of the complex formed between the specific agent and the non-identical capture compounds. In one embodiment, the competitive assay is an assay for anti-HAV (anti-hepatitis A virus), anti-HBc (anti-hepatitis B core antigen), anti-HBe (anti-hepatitis B e-antigen), folate, folate RBC (red blood cells), anti-TSH-R, total vitamin D, vitamin B12, thyroxine T4, FT4 (free thyroxine), thyroxine T3, FT3 (free triiodothyronine), testosterone, progesterone, digitoxin, anti-TG, anti-TPO (anti-thyroid peroxidase), DGEA or estradiol.

[0043] In a further embodiment, the immunoassay is a double antigen sandwich assay ("DAGS"), in which a bivalent analyte, e.g., an antibody, binds to a capture compound bound to a solid surface, and in which the amount of analyte / capture compound complex is determined by binding of a detection compound, as specified herein below, to said analyte / capture compound complex. In one embodiment, the DAGS assay is an assay for anti-Toxoplasma IgG, anti-Rubella IgG, anti-HBs (Hepatitis B Virus Surface Antigen) antibodies, HCV (Hepatitis C Virus) core antigen or anti-HCV antibodies, CMV antibodies (Cytomegalovirus) IgG, Treponema pallidum antibodies, HTLV (Human T-cell Lymphotropic Virus) antibodies, or Chagas (American Trypanosomiasis) antibodies.

[0044] The term "capture compound" as used herein relates to a chemical molecule that directly or indirectly binds to the analyte as specified herein above. In one embodiment, the capture compound is bound to or adapted to bind to a solid surface. In one embodiment, the capture compound is an organic molecule, and in a further embodiment, a biological macromolecule as specified herein above, such as a polypeptide as specified herein above. In one embodiment, the capture compound indirectly binds to the analyte of the present invention with sufficient affinity to allow detection of a complex comprising the analyte and the capture compound; i.e., in such a case, the capture compound is an indirect ligand. The term "indirect binding" as used herein relates to a binding in which the ligand does not directly contact the analyte but binds to the analyte, in one embodiment, contacting a chemical molecule that specifically binds to the analyte, where in one embodiment, said molecule that binds to the analyte is a molecule that directly binds to the analyte, i.e., a direct ligand. Thus, in one embodiment, the analyte, the chemical molecule that binds to the analyte and the indirect ligand form a complex with the properties indicated above, in particular with the dissociation constant indicated. In a further embodiment, the capture compound directly binds to the analyte of the invention with sufficient affinity to allow detection of the analyte / capture compound complex, as specified herein above. Thus, in one embodiment, the capture compound is a direct ligand.

[0045] As used herein, the term "solid surface" relates to any suitable solid surface adapted to bind the capture compounds of the invention and to be separated from the sample, for example by physical means. In one embodiment, said solid surface is the surface of beads, in one embodiment microbeads, such as magnetic microbeads or paramagnetic microbeads. In one embodiment, said surface is adapted to improve the binding of the capture compounds, for example by covalently or non-covalently attaching molecules that bind to the substructures of the capture compounds. Typical molecules that bind to the substructures of the capture compounds are, for example, antibodies, streptavidin, complexed nickel ions, any component of the X-anti-X system, where compound "X" specifically binds to "anti-X", such as sugars and sugar-binding proteins / lectins or hormones and their receptors, etc. In a further embodiment, the solid surface binds to said capture compounds by covalent or non-covalent bonds, for example by hydrophobic interactions. Thus, in one embodiment, said solid surface is the surface of a multicluster plate. In one embodiment, the surface of a multicluster plate is pretreated to increase the affinity and / or capacity for binding of the capture compounds. Suitable pretreatments are known in the art.

[0046] Methods for binding biological molecules, typically polypeptides, to solid surfaces are well known in the art and include, for example, binding by hydrophobic interactions, binding via biotinylated and immobilized streptavidin, covalent binding, antibody-antigen interactions, etc., or combinations of these interactions, such as antibody-antigen interactions between an antibody and a pathogen polypeptide, where said antibody is biotinylated and bound to a solid surface via immobilized streptavidin. Thus, the capture antibody can also preferably be a capture complex. In one embodiment, the capture compound is a compound of the capture complex that directly binds to the analyte. Those skilled in the art know how to bind the capture compound or complex to a solid surface depending on the solid surface selected. In one embodiment, the capture compound is a viral polypeptide, such as a viral capsid polypeptide. In one embodiment, said capture compound is a hepatitis virus capsid polypeptide, and in one embodiment, a hepatitis A virus (HAV) capsid polypeptide. However, it is also envisaged that the capture compound is an antibody.

[0047] The term "sample" as used herein relates to a sample of bodily fluid, a sample from a tissue or organ, or a sample of washing / rinsing fluid or a swab or smear obtained from an external or internal body surface. In one embodiment, the sample is suspected to contain an analyte as specified herein. The sample, in one embodiment, contains at least one analyte as specified elsewhere herein. In one embodiment, the sample is a blood, plasma, serum, urine, saliva or tear sample. Samples can be obtained by using a brush, (cotton) swab, spatula, rinse / rinsing fluid, punch biopsy device, puncture of a cavity with a needle or lancet, or by surgical instrumentation. However, samples obtained by well-known techniques including scrapes, swabs or biopsies from the genitourinary tract, perianal region, anal canal, oral cavity, upper aerodigestive tract and epidermis in one embodiment are also included as samples of the present invention. The cell-free fluids can be obtained from body fluids or tissues or organs by lysis techniques such as homogenization and / or by separation techniques such as filtration or centrifugation. In one embodiment, the sample is obtained from a body fluid known to contain HA viral polypeptides or / and antibodies against at least one HA viral polypeptide, i.e. in one embodiment blood, plasma, serum, saliva, etc., in one embodiment plasma or serum. It should be understood that the sample can be further processed to carry out the method of the invention. In particular, cells can be removed from the sample by methods and means known in the art. Furthermore, at least one analyte can be extracted and / or purified from the sample by methods and means known in the art. Thus, the term sample can also relate to a preparation that contains or is suspected to contain at least one analyte that has been diluted, enriched, purified and / or extracted from the sample.

[0048] The term "contacting" as used in the context of the methods of the invention is understood by the skilled artisan. In one embodiment, the term relates to physically contacting a compound of the invention with a sample or with a further compound, thereby, for example, allowing the sample and the compound to interact.

[0049] The term "subject" as used herein relates to a vertebrate, in one embodiment a mammalian subject. In one embodiment, the subject is a livestock, companion animal or laboratory animal, such as a cow, sheep, goat, horse, cat, dog, guinea pig, mouse or rat. In one embodiment, the subject is a human. In a further embodiment, the subject is a human suspected of containing an analyte as specified herein.

[0050] Advantageously, it has been found in the research underlying the present invention that the interference caused by anti-labeling compounds from samples can be reduced by using different labels that can provide essentially the same quantity and / or quality of signal in immunoassay.For example, the signal reduction caused by anti-labeling interference, which causes false positive results in competitive immunoassay, can be reduced.Similarly, in sandwich / double antigen sandwich immunoassay, anti-labeling interference tends to reduce signal by binding to label, which causes a reduction in signal yield, thereby resulting in false negative results.In some cases, signal increase has also been observed, thereby causing false negative results.At least in all of these cases, the use of at least two different labels avoids interference causing false results.

[0051] The definitions made above apply mutatis mutandis below. The additional definitions and explanations further made below also apply mutatis mutandis to all aspects described herein.

[0052] The invention also relates to a method for improving the specificity of detection of an analyte in an assay, the method comprising replacing 10% to 90% of a first detection compound by a second detection compound having a non-identical label.

[0053] As used herein, the term "replace a percentage of a first detection compound with a second detection compound" refers to omitting a indicated percentage of a first detection compound from an assay and including a new amount of a second detection compound corresponding to the amount of the first detection compound omitted. In one embodiment, the percentages and amounts of the first and second detection compounds are calculated on a molar basis; thus, if the concentration of the first detection compound in the assay is initially 10 pM and 50% is replaced, the second detection compound will be included at a concentration of 5 pM. In a further embodiment, the percentages and amounts of the first and second detection compounds are calculated based on signal yield. Thus, for example, the concentration of the first detection compound in the original assay can be set to an arbitrary signal yield of 100%; thus, if 50% of the first detection compound is to be replaced based on signal yield, an amount of the first detection compound is omitted so that the signal yield is reduced to 50%, and a second detection compound is added so that the signal yield is raised to 100% again. As will be appreciated, the above applies mutatis mutandis when a first detection compound is replaced by more than one further detection compound and when more than one detection compound is replaced by one or more further detection compound(s).

[0054] Typically, the proportion of capture or detection compounds to be displaced will be selected such that a measurable effect of displacement can theoretically be expected. As will be understood by those skilled in the art, the expected value of the measurable effect will depend on the number of non-identical labels used for displacement. For example, if three detection compounds are used in the improved assay instead of one, it is preferred that, for example, 66% of the initial detection compounds are displaced. In principle, it is assumed that the proportion of a given detection compound is (100% / n)±50%, where n=(the number of non-identical detection compounds used in the assay). In another embodiment, a proportion of (100% / n)±20% is used. As will be understood by those skilled in the art, the sum of the proportions used will add up to 100%. Thus, in one embodiment, the proportion of detection compounds to be displaced is a proportion of 10% to 90%, in one embodiment a proportion of 25% to 75%, in a further embodiment a proportion of 40% to 60%, and in a further embodiment a proportion of about 50%.

[0055] The present invention further relates to a kit for detecting an analyte in a sample, comprising at least a first and a second detection compound for said analyte, said first detection compound comprising a first binding moiety and a first label, said second detection compound comprising a second binding moiety and a second label, and wherein the first and second labels are not identical.

[0056] The term "kit" as used herein refers to a collection of said compounds, means or reagents of the present invention, which may or may not be packaged together. The components of the kit may be constituted by separate vials (i.e. as a kit of separate parts) or may be provided in a single vial. Moreover, in one embodiment, the kit of the present invention is for use in carrying out the method mentioned herein above. In one embodiment, it is envisaged that all components are provided in a ready-to-use manner for carrying out the method mentioned above. Moreover, the kit, in one embodiment, comprises instructions for carrying out said method. The instructions may be provided by an instruction manual in paper or electronic form. For example, the instructions may comprise instructions for interpreting the results obtained in carrying out said method using the kit of the present invention. In one embodiment, the kit for detecting an analyte in a sample comprising at least two non-identical detection compounds further comprises at least one capture compound. In a further embodiment, the kit further comprises a solid support for immobilizing said capture compound or for immobilizing the analyte. In one embodiment, the kit contains 2 to 10 types of detection compounds for the analyte, in one embodiment, 2 to 5 types of detection compounds for the analyte, in one embodiment, 2 to 4 types of detection compounds, and in one embodiment, 2 to 3 types of detection compounds for the analyte.

[0057] Furthermore, the present invention relates to a device for determining an analyte in a sample, comprising at least a first and a second detection compound for said analyte, wherein said first detection compound comprises a first binding moiety and a first label, and said second detection compound comprises a second binding moiety and a second label, and wherein the first label and the second label are not identical, and means for determining at least one signal resulting from said first label and said second label.

[0058] The term "device" as used herein refers to a system of means, including said means operatively linked to each other to at least allow a determination. Exemplary means for determining the amount of an analyte and means for performing a determination, such as means for determining at least one signal, are disclosed above in connection with the method of the present invention. As will be understood by those skilled in the art, a means for determining at least one signal includes a means capable of determining one signal; as will also be understood by those skilled in the art, a means for determining a signal is a means capable of detecting said signal, even when the signal is zero or below the detection limit for a particular sample.

[0059] How to link the means in the operating method will depend on the type of means included in the device. In one embodiment, the means are included in a single device. Thus, said device may comprise (i) an analysis unit for the measurement of the amount of analyte in the applied sample and (ii) a computer unit for processing the resulting data for evaluation. Exemplary means for detection are disclosed in connection with the embodiment relating to the method of the invention above. In such a case, the means are operatively linked in that a user of the system compiles the result of the determination of the detectable property of the label (in one embodiment the optically or / and electrochemically determinable property of the label) based on the instructions and interpretations given in the manual; or said instructions and interpretations are included in an executable program code included in the device, such that the amount or concentration of the analyte in the applied sample is output to the user as a result of the determination. The skilled person will understand how to link the means without further ado. Exemplary devices are devices that can be applied without the special knowledge of a specialist technician, such as test strips or electronic devices that only require loading the sample. The results can be given as a raw data output that requires interpretation by a technician. However, in one embodiment, the output of the device is processed, i.e. evaluated, raw data, the interpretation of which does not require a technician. Further exemplary devices comprise an analysis unit / device (e.g. biosensor, array, solid support bound to a ligand that specifically recognizes a peptide, surface plasmon resonance device, NMR spectrometer, mass spectrometer, etc.) or an evaluation unit / device as mentioned above according to the method of the invention. In one embodiment, 2-10 detection compounds for said analyte, in one embodiment 2-5 detection compounds for said analyte, in one embodiment 2-4 detection compounds, in one embodiment 2-3 detection compounds for said analyte are comprised in said device.

[0060] Furthermore, the present invention relates to the use of a composition comprising at least a first and a second detection compound for detecting an analyte, wherein said first detection compound comprises a first binding moiety and a first label, said second detection compound comprises a second binding moiety and a second label, and wherein the first and second labels are not identical; the present invention relates to the use of at least a first and a second detection compound for an analyte for the manufacture of a diagnostic composition or device, wherein said first detection compound comprises a first binding moiety and a first label, said second detection compound comprises a second binding moiety and a second label, and wherein the first and second labels are not identical; and the present invention relates to the use of a composition comprising at least a first and a second detection compound for an analyte for determining said analyte in a sample, wherein said first detection compound comprises a first binding moiety and a first label, said second detection compound comprises a second binding moiety and a second label, and wherein the first and second labels are not identical.

[0061] In view of the above, the following aspects are particularly envisaged. 1. A method for determining an analyte in a sample, comprising the steps of: a) contacting said sample with at least first and second detection compounds; b) determining the amount of complex containing at least one detection compound; and c) determining said analyte in the sample based on the result of step b); wherein the first detection compound comprises a first binding moiety and a first label, the second detection compound comprises a second binding moiety and a second label, and the first label and the second label are not identical.

[0062] 2. The method of embodiment 1, wherein the first and second labels provide the same quality of detectable property. 3. The method of embodiment 1 or 2, wherein the first and second labels provide the same detectable property.

[0063] 4. The method of any one of embodiments 1 to 3, wherein the first and second labels provide essentially the same quality of the detectable property. 5. The method of any one of embodiments 1 to 4, wherein the detectable property provided by the first and / or second detection compound is an emissive property, in one embodiment a luminescent property, in a further embodiment a chemiluminescent property.

[0064] 6. The method of any one of aspects 1 to 5, wherein the label in the first detection compound comprises Ru(bpy)2-bpyCO-OSu (CAS Registry No. 137323-76-3).

[0065] 7. The method of any one of embodiments 1 to 6, wherein the label in the second detection compound is sulfo-BPRu NHS ester (CAS Registry No. 482618-42-8). 8. The method of any one of embodiments 1 to 7, wherein determining the amount of the complex in step b) comprises detecting a property of the first detection compound and a property of the second detection compound, and in one embodiment comprises simultaneously detecting the property of the first detection compound and the property of the second detection compound.

[0066] 9. The method of any one of aspects 1 to 8, wherein determining the analyte in step c) comprises determining a sum value of a property of a first detection compound and a sum value of a property of a second detection compound.

[0067] 10. The method of any one of embodiments 1-9, wherein the binding moiety specifically binds to said analyte or specifically binds to a capture compound that specifically binds to the analyte. 11. The method of any one of embodiments 1-9, wherein the binding moiety competes with the analyte for binding to the capture compound.

[0068] 12. The method of any one of embodiments 1 to 11, wherein the binding moiety is a biological molecule or a fragment thereof, in one embodiment a polypeptide or a fragment thereof.

[0069] 13. The method of any one of embodiments 1 to 12, wherein the binding moiety is an antibody or a fragment thereof. 14. The method of any one of aspects 1 to 13, wherein the method is an immunoassay.

[0070] 15. The method of any one of embodiments 1 to 14, wherein the method is a competitive assay. 16. The method of any one of aspects 1 to 14, wherein the method is a sandwich assay, and in one aspect a double antigen sandwich assay.

[0071] 17. The method of any one of embodiments 1 to 16, wherein said method is a qualitative or semi-quantitative assay. 18. The method of any one of embodiments 1 to 17, wherein the method is a quantitative assay.

[0072] 19. The method of any one of embodiments 1 to 18, wherein the analyte is a polypeptide. 20. The method of any one of aspects 1 to 19, wherein the analyte is an antibody, in one aspect an antibody against an antigen of a pathogenic organism, in one aspect an antibody against a viral antigen, in one aspect an antibody against a protozoan antigen.

[0073] 21. The method of any one of aspects 1 to 20, wherein the analyte is an anti-Hepatitis A antibody or an anti-Toxoplasma antibody. 22. The method of any one of embodiments 1 to 19, wherein the analyte is an antigen from a pathogenic organism, and in one embodiment a bacterial antigen.

[0074] 23. The method of any one of aspects 1 to 22, wherein 2 to 10 types of detection compounds, in one aspect 2 to 5 types of detection compounds, in one aspect 2 to 4 types of detection compounds, and in one aspect 2 to 3 types of detection compounds are used, and the labels of all of the detection compounds are not identical to one another.

[0075] 24. A method for improving the specificity of detection of an analyte in an assay, comprising replacing 10% to 90% of a first detection compound with a second detection compound having a non-identical label.

[0076] 25. The method of embodiment 24, wherein 25% to 75%, in a further embodiment 40% to 60%, and in a further embodiment about 50% of the first detection compound is replaced. 26. A method for identifying a sample containing an interfering substance that confounds the determination of an analyte by a detection compound having a first label, comprising the steps of: a) contacting an aliquot of said sample with a first detection compound having said first label; b) contacting an aliquot of said sample with a second detection compound having a second label; c) determining a first signal produced by the first label; d) determining a second signal produced by the second label; e) comparing the first signal of step c) with the second signal of step d) to identify samples containing interfering substances that confound the determination of the analyte by the detection compound having the first label; The method includes:

[0077] 27. The method of embodiment 26, further comprising determining a plurality of analytes. 28. A kit for detecting an analyte in a sample, comprising at least a first and a second detection compound for the analyte, wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label, and wherein the first and second labels are not identical.

[0078] 29. The kit of embodiment 28, wherein said kit further comprises at least one capture compound for said analyte. 30. The kit of embodiment 28 or 29, wherein said kit further comprises a solid support for immobilizing said capture compound or at least a component of said sample, including said analyte.

[0079] 31. A device for determining an analyte in a sample, comprising at least first and second detection compounds for said analyte, wherein said first detection compound comprises a first binding moiety and a first label, and said second detection compound comprises a second binding moiety and a second label, and wherein the first label and the second label are not identical, and means for determining at least one signal resulting from said first label and said second label.

[0080] 32. Use of a composition comprising at least a first and a second detection compound for detecting an analyte, wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label, and the first and second labels are not identical.

[0081] 33. Use of at least a first and a second detection compound for an analyte for the manufacture of a diagnostic composition or device, wherein said first detection compound comprises a first binding moiety and a first label, and said second detection compound comprises a second binding moiety and a second label, and wherein the first and second labels are not identical.

[0082] 34. The use of embodiment 32 or 33, wherein the analyte is an analyte as specified in any one of embodiments 19 to 22. 35. Use of a composition comprising at least a first and a second detection compound for an analyte for determining said analyte in a sample, wherein said first detection compound comprises a first binding moiety and a first label, said second detection compound comprises a second binding moiety and a second label, and said first label and said second label are not identical.

[0083] 36. The method of any one of embodiments 1 to 27, the kit of any one of embodiments 28 to 30, the device of embodiment 31 and / or the use of any one of embodiments 32 to 35, wherein the affinity domains and / or binding moieties of the first and second detection compounds are identical.

[0084] 37. The method of any one of embodiments 1 to 27, the kit of any one of embodiments 28 to 30, the device of embodiment 31 and / or the use of any one of embodiments 32 to 35, wherein the affinity domains and / or binding moieties of the first and second detection compounds are not identical.

[0085] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein. The following examples are merely illustrative of the present invention and should not be construed in any way as limiting the scope of the present invention. EXAMPLES

[0086] Example 1: Increased specificity in qualitative and competitive Elecsys anti-Hepatitis A virus (anti-HAV) assays Immunoassays for the in vitro determination of anti-HAV antibodies were performed on an automated Elecsys® cobas analyzer (Roche Diagnostics GmbH) according to the manufacturer's instructions. Elecsys® is a registered trademark of the Roche group.

[0087] The assay was carried out according to the competitive principle. In the first incubation, 50 μl of the sample was incubated with the added HAV antigen so that the anti-HAV of the sample binds to the HAV antigen. In the second (subsequent) incubation step, a biotinylated antibody specific for the HAV antigen and a ruthenium-labeled antibody were added to the sample-HAV antigen mixture together with streptavidin-coated microparticles so that the still free binding sites on the HAV antigen were occupied. The entire complex was bound to the solid phase (microparticles) by the interaction of biotin and streptavidin. The reaction mixture was then aspirated into the measuring cell, where the microparticles were magnetically captured on the surface of the electrode. Unbound substances were then removed by ProCell / ProCell M (a buffer containing tripropylamin, necessary for signal generation). The application of a voltage to the electrode then induced chemiluminescence, which was measured by a photomultiplier tube. The results were determined by a calibration curve generated instrument-specifically by a two-point calibration (Cal1 = negative calibrator containing human anti-HAV negative serum; Cal2 = positive calibrator containing human anti-HAV in human serum). <hav>The M-2.157-F(ab')2-antibody fragment was used, one aliquot of which was labeled with BP-Ru and another aliquot of which was labeled with Sulfo-Ru; these were used to generate three different versions of the anti-HAV assay: Assay 1: MAK used in R2 <hav>100% of M-2.157-F(ab')2 is labeled with sulfo-Ru.

[0088] Assay 2: MAK used in R2<HA V> 100% of M-2.157-F(ab')2 is labeled with BP-Ru. Assay 3: A 1+1 mixture of Assay 1 R2 and Assay 2 R2 (meaning a mixture of BP-Ru and Sulfo-Ru) was used. The contributions of the BP-Ru and Sulfo-Ru signals to the overall signal were similar.

[0089] The first label was Ru(bpy)2-bpyCO-OSu, also known as "BP-Ru" (CAS Registry Number 137323-76-3, = ruthenium(2+), bis(2,2'-bipyridine-κN 1 ,κN 1’ )[1-[4-(4'-methyl[2,2'-bipyridine]-4-yl-κN 1 ,κN 1’ )-1-oxobutoxy]-2,5-pyrrolidinedione]-, (OC-6-33) Ru(bpy)2-bpyCO2H (=BPRu or Ru-bpy) reactive ester, CAS Registry Number 115239-59-3), and the second label is "sulfo-Ru" also known as sulfo-BPRu NHS ester (CAS Registry Number 482618-42-8, known in the art as ruthenate(2-),bis[[2,2'-bipyridine]-4,4'-dimethanesulfonato(2-)-κN 1 ,κN 1’ ][1-[4-(4'-methyl[2,2'-bipyridin]-4-yl-κN 1 ,κN 1’ )-1-oxobutoxy]-2,5-pyrrolidinedione]-, sodium (1:2), also known as (OC-6-31).

[0090] Results with calibrators and standards are shown in Table 1. In addition, 27 known anti-labeled interference samples, such as the anti-Sulfo-Ru interference sample, as well as one known anti-BP-Ru interference sample, were tested by all three assays (Table 2). These samples are known to give false positive results when used in the respective anti-HAV assay variants. As expected, the anti-Sulfo-Ru interference sample is a false positive in Assay 1 but a true negative in Assay 2, and the anti-BP-Ru interference sample is a true negative in Assay 1 but a false positive in Assay 2. Interestingly, all samples except one anti-Sulfo Ru interference sample (PN0206_0925) are true negative in Assay 3, which contains a labeled mixture of BP-Ru and Sulfo Ru, implying a significant improvement in specificity.

[0091] The cut-off index (COI) is calculated as follows: The cutoff was determined using a two-point calibration using negative (Cal1) and positive (Cal2) calibrators. The cutoff index (COI) was determined by dividing the counts (sample) by the cutoff. Results are interpreted as reactive if COI ≤ 1 and non-reactive if COI > 1.

[0092] Example 2: Increasing the sensitivity of the quantitative DAGS Elecsys Toxo IgG assay Immunoassays for in vitro determination of Toxo-IgG antibodies were performed on an automated Elecsys® cobas analyzer (Roche Diagnostics GmbH) according to the manufacturer's instructions. Elecsys® is a registered trademark of the Roche Group.

[0093] The assay was performed according to the sandwich principle (IgG antibodies are sandwiched between two Toxo-p30 antigens). In the first incubation, 10 μl of sample (biotinylated recombinant T. gondii specific antigen and T. gondii specific recombinant antigen labeled with ruthenium complex) form a sandwich complex. In the second step, streptavidin-coated microparticles were added so that the immune complex of sample antibody and Toxo antigen binds to the solid phase via biotin and streptavidin interaction. The reaction mixture was then aspirated into the measuring cell, where the microparticles were magnetically captured on the surface of the electrode. Unbound material was then removed by ProCell / ProCell M (a buffer containing tripropylamin, necessary for signal generation). Application of a voltage to the electrodes then induced chemiluminescence, which was measured by a photomultiplier tube. Results were determined by instrument-specifically generated calibration curves with a two-point calibration (Cal1 = negative calibrator containing human serum that is anti-Toxoplasma negative; Cal2 = positive calibrator containing human serum reactive to anti-Toxoplasma IgG).

[0094] In detail, two differently labeled recombinant Toxo-p30 antigen quantities were used, one labeled with BP-Ru and the other with Sulfo-Ru (see Example 1 for chemical names of labels). Using the two recombinant Toxo-p30 antigen quantities, three different versions of the Toxo IgG assay were generated: 100% of the recombinant Toxo-p30 antigen used in Assay 1:R2 is labeled with Sulfo-Ru.

[0095] Assay 2: 100% of the recombinant Toxo-p30 antigen used in R2 is labeled with BP-Ru. Assay 3: A 1+1 mixture of Assay 1 R2 and Assay 2 R2 (representing a mixture of BP-Ru and Sulfo-Ru) was used. The contributions of the BP-Ru and Sulfo-Ru signals to the overall signal were similar.

[0096] Results were interpreted as non-reactive below 1 IU / mL, indeterminate between 1 and 3 IU / mL, and reactive above 3 IU / mL, according to the manufacturer's instructions. Results with calibrators and standards are shown in Table 3. Furthermore, several anti-Sulfo-Ru and anti-BP-Ru interfering samples that give false negative or false indeterminate results in the respective assay variants were tested in all three assays (Table 4). As expected, the anti-Sulfu-Ru interfering samples are false negative or false indeterminate in assay 1, but true indeterminate or true positive in assay 2. Vice versa, the same is true for the anti-BP-Ru interfering samples that interfere with assay 2. Interestingly, all samples were found to be true indeterminate or true negative in assay 3, which contains a labeling mixture of BP-Ru and Sulfo-Ru, implying a significant increase in sensitivity.

[0097] References cited: Ando et al. (2007), Intern Med. 46(15):1225 Buijs et al. (2011), Ann Clin Biochem. 48(Pt 3):276 DE 19519973 A1 DeForge (2010), J Immunol Methods. 362(1-2):70) Heijboer et al. (2009), Ann Clin Biochem.46(Pt 3):263 Klevenz et al., Cell Mol Life Sci. 2002, 59: 1993-1998 Park & ​​Kricka (2013), Chapter 5.3 - Interferences in Immunoassay in The Immunoassay Handbook (4th ed.), edited by David Wild, Elsevier, Oxford: 403 Sapin et al. (2007), Clin Chern Lab Med. 45(3):416 Schiettecatte (2012), Interferences in Immunoassays, Advances in Immunoassay Technology, Dr. Norman H.L. Chiu (Editor) International Publication No. 2016 / 097116 A1 International Publication No. 2017 / 093271 A1

[0098] [Table 1]

[0099] [Table 2-1]

[0100] [Table 2-2]

[0101] [Table 3-1]

[0102] [Table 3-2]

[0103] [Table 4-1]

[0104] [Table 4-2] < / hav> < / hav>

Claims

1. 1. A method for determining an analyte in a sample, comprising the steps of: a) contacting the sample with at least a first and a second detection compound; b) determining the amount of complexes containing at least one detection compound; and c) determining the analyte in the sample based on the result of step b); wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label; the first label and the second label are not identical and provide the same detectable property; the detectable property provided by the first and / or second detection compound is an emissive, luminescent, or chemiluminescent property; and The method, wherein the method is an immunoassay.

2. The label in the first detection compound is Ru(bpy). ２ 2. The method of claim 1, wherein the label in the second detection compound comprises -bpyCO-OSu (CAS Registry No. 137323-76-3) and / or the label in the second detection compound comprises sulfo-BPRu NHS ester (CAS Registry No. 482618-42-8).

3. The method of claim 1 or 2, wherein determining the amount of the complex in step b) comprises detecting a detectable property of the first detection compound and a detectable property of the second detection compound, or comprises simultaneously detecting a detectable property of the first detection compound and a detectable property of the second detection compound.

4. The method according to any one of claims 1 to 3, wherein the sample is a body fluid, blood, serum or plasma sample.

5. The method of any one of claims 1 to 4, wherein the analyte is a polypeptide.

6. The method of any one of claims 1 to 5, wherein the analyte is an antibody, an anti-Hepatitis A antibody or an anti-Toxoplasma antibody.

7. The method of any one of claims 1 to 5, wherein the analyte is an antigen from a pathogenic organism, a viral antigen or a bacterial antigen.

8. 1. A kit for detecting an analyte in a sample by immunoassay, comprising at least a first and a second detection compound for the analyte, the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label; the first label and the second label are non-identical and provide the same detectable property; and the detectable property provided by said first and / or said second detection compound is an emissive, luminescent, or chemiluminescent property; The kit.

9. 9. The kit of claim 8, further comprising at least one capture compound for said analyte and / or a solid support for immobilizing said capture compound or at least a component of a sample containing said analyte.

10. 1. A device for determining an analyte in a sample by immunoassay, comprising at least first and second detection compounds for the analyte, wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label; wherein the first label and the second label are not identical and provide the same detectable property; and the detectable property provided by said first and / or said second detection compound is an emissive, luminescent, or chemiluminescent property; and means for determining at least one signal resulting from said first label and said second label.

11. 1. Use of a composition comprising at least a first and a second detection compound for detecting an analyte by immunoassay, wherein the first detection compound comprises a first binding moiety and a first label, and the second detection compound comprises a second binding moiety and a second label; the first label and the second label are non-identical and provide the same detectable property; and the detectable property provided by said first and / or said second detection compound is an emissive, luminescent, or chemiluminescent property; The above uses.

12. The method according to any one of claims 1 to 7, the kit according to claim 8 or 9, the device according to claim 10 and / or the use according to claim 11, wherein the binding moieties of the first and the second detection compounds are identical.

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