Reducing interference in immunoassays

Abstract

The present invention relates to a method for determining an analyte in a sample comprising a) contacting the sample with at least a first and a second detection agent compound; b) determining the amount of a complex comprising at least one detector compound; and c) determining the analyte in the sample based on the result of step b) wherein the first detector compound comprises a first binding moiety and a first marker and the second detector compound comprises a second binding moiety and a second marker, and wherein the first marker and the second marker are different. The invention further relates to a kit for detecting an analyte in a sample; and means for determining at least one signal obtained from the first marker and the second marker; and the use of a composition comprising at least the first and second detection agent compounds for detecting an analyte.

Description

[0001] This application is a divisional application of the invention patent application filed on March 13, 2019, with application number 201980018844.X and invention title "Reducing Interference in Immunoassays". Technical Field

[0002] This application relates to the field of immunological assays. Specifically, this application relates to methods, kits, and apparatus for determining analytes in samples, wherein interference in immunoassays is reduced. Background Technology

[0003] Laboratory tests, particularly immunological tests, have become invaluable tools in disease diagnosis. Specifically, immunoassays have provided the possibility of specifically detecting a single analyte or a group of analytes in complex mixtures, such as bodily fluids like blood, serum, or plasma. However, several causes of interference in immunoassays have been identified, such as cross-reactivity of interfering substances with capture compounds, nonspecific binding of detection compounds to the solid phase, and “bridging” binding of capture and detection compounds by heterophilic antibodies or human anti-mouse antibodies (HAMA), to name just a few (see, for example, WO2016097116 A1; Park & ​​Kricka (2013), Ch. 5.3- Interferences in Immunoassay, The Immunoassay Handbook (4th edition), edited by David Wild, Elsevier, Oxford: 403; Schiettecatte (2012), Interferences in Immunoassays, Advances in Immunoassay Technology, Dr. Norman HL Chiu (ed.)). Another target of interference could be a marker, i.e. a signal-generating unit in the measurement (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).

[0004] In competitive immunoassays, the analyte contained in the sample competes with a labeled analyte (the marker) for binding to a capture compound, which is often an antibody, or, in the case of an antigen of a pathogen, an antibody against the pathogen. High concentrations of the analyte in the sample result in strong competition, leading to reduced binding of the marker to the capture compound and causing a signal reduction, which, depending on the assay, results in either a quantitative or qualitative test. If this signal reduction is due to interference from the labeled analyte, it can cause false positives.

[0005] In non-competitive immunoassays, the analyte is detected by contacting a compound that specifically binds to the analyte and carries the label itself, or acts as a target molecule carrying the label. Therefore, in non-competitive immunoassays, the amount of analyte is determined by measuring the amount of the complex formed between the analyte and the label-carrying detection compound. Consequently, similar specificity issues may arise, as described above. Anti-label interference tends to reduce the signal by binding to the label, causing a decrease in signal yield and thus false negative results. In some cases, anti-label interference can also increase the signal in a non-competitive assay, leading to false positive results.

[0006] Currently, two different strategies exist for reducing label interference: the most common approach is to add the target of the interfering substance (the label or a slightly modified target that does not generate a signal (a label-analyte)) to the assay at a concentration typically exceeding that of the original label. The addition of the label or label-analyte acts as a substitute target for the interfering substance, leading to a reduction in the binding of the interfering substance to the label, thus eliminating or reducing the interference. However, it is not possible to sufficiently reduce interference simply by adding more active label, for example, because the addition of an active label can interfere with the assay. In such cases, the preferred approach is to add an inactive label-analyte that does not generate a signal but is targeted by the interfering substance. However, the efficiency of label-analytes in reducing label interference can be limited due to the structural differences between reactive labels and label-analytes.

[0007] A second possibility for reducing marker-specific interference is to specifically target the affected marker with a binding partner added to the assay. This binding partner is non-reactive in the assay but shields the interfering substance from binding to the marker, thus also reducing interference (see, for example, DE 19519973 A1, WO 2017093271 A1; Sapin et al. (2007), Clin Chern Lab Med. 45(3):416; DeForge (2010), J ImmunolMethods. 362(1-2):70). However, this process may require a significant reduction in signal yield, which in turn may lead to a decrease or impairment of the detection limit. Summary of the Invention

[0008] Therefore, the object of the present invention is to provide an improved immunoassay method that avoids the aforementioned problems. These problems are addressed by methods, kits, apparatus, and compositions having the features of the independent claims. Typical embodiments that may be implemented separately or in any arbitrary combination are set forth in the dependent claims.

[0009] The present invention relates to a method for determining an analyte in a sample, comprising a) contacting the sample with at least first and second detection reagent compounds; b) determining an amount of a complex comprising at least one detection reagent compound; and c) determining the analyte in the sample based on the result of step b), wherein the first detection reagent compound comprises a first binding moiety and a first label, and the second detection reagent compound comprises a second binding moiety and a second label, wherein the first label and the second label are different. The present invention further relates to a kit for detecting an analyte in a sample, comprising at least first and second detection compounds of the analyte, wherein the first detection compound comprises a first binding portion and a first label, and the second detection compound comprises a second binding portion and a second label, wherein the first label and the second label are different; and an apparatus for determining an analyte in a sample, comprising at least first and second detection compounds of the analyte, wherein the first detection compound comprises a first binding portion and a first label, and the second detection compound comprises a second binding portion and a second label, wherein the first label and the second label are different; and an apparatus for determining at least one signal obtained from the first label and the second label; and use of a composition comprising at least first and second detection compounds for detecting an analyte, wherein the first detection compound comprises a first binding portion and a first label, and the second detection compound comprises a second binding portion and a second label, wherein the first label and the second label are different.

[0010] Therefore, the present invention relates to a method for determining an analyte in a sample, comprising: a) Contact the sample with at least the first and second detection reagent compounds; b) Determining the amount of a complex containing at least one detection reagent compound; and, c) Determine the analyte in the sample based on the results of step b). The first detection agent compound comprises a binding portion and a first marker, and the second detection agent compound comprises a binding portion and a second marker, wherein the first marker and the second marker are different.

[0011] As used below, the terms “have,” “contain,” or “include,” or any of their grammatical variations, are used in a non-exclusive manner. Thus, these terms can refer either to a situation where no other features exist in the entity described in this context besides those introduced by these terms, or to a situation where one or more other features exist. For example, the expressions “A has B,” “A contains B,” and “A includes B” can refer either to a situation where no other element exists in A besides B (i.e., where A consists solely of B), or to a situation where one or more other elements, such as element C, elements C and D, or even other elements, exist in entity A besides B.

[0012] Furthermore, as used below, the terms “preferred,” “more preferred,” “most preferred,” “specifically,” “more specifically,” “particularly,” “more particularly,” or similar terms are used in combination with optional features without limiting further possibilities. Therefore, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As those skilled in the art will recognize, the invention can be practiced by using alternative features. Similarly, features introduced by phrases such as “in an embodiment of the invention” or similar expressions are intended to be optional features, without any limitation on further embodiments of the invention, without any limitation on the scope of the invention, and without any limitation on the possibility of combining features introduced in this manner with other optional or non-optional features of the invention.

[0013] Unless otherwise specified, the term "about" refers to an indication value with technical precision generally accepted in the relevant field, preferably involving an indication value ±20%, more preferably ±10%, and most preferably ±5%. Further, the term "substantially" indicates that there is no deviation affecting the result or use of the indication, i.e., potential deviations do not cause the result of the indication to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, "substantially composed of..." means including the specified components but excluding other components, except for materials present as impurities, unavoidable materials present due to the process used to provide said components, and components added for purposes other than achieving the technical effects of the invention. For example, compositions defined using the phrase "substantially composed of..." encompass any known and acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition substantially composed of one 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 unspecified components.

[0014] In one embodiment, the method of the present invention is an in vitro method. Furthermore, it may include steps other than those specifically mentioned. Specifically, step a) may precede a step of providing a sample, or steps a) and / or b) may include the addition of other compounds to promote binding and detection. Additionally, some or all of the steps may 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 detection compound is an antibody or contains an antibody.

[0015] The term "biomolecule" is known to those skilled in the art and generally refers to a molecule produced by the metabolism of at least one organism. Therefore, in one embodiment, the term "biomacromolecule" refers to a polymer produced by an organism from a monomeric precursor. Typical biomacromolecules are polypeptides, DNA, RNA, or polysaccharides.

[0016] In one embodiment, the term "peptide" as used herein includes variants and fragments of a specifically designated peptide. Variants include peptides comprising an amino acid sequence having 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% identity with a specifically designated amino acid sequence. The percentage identity value is 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 yield particularly reliable results. For sequence alignment, 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))] can be used, which are part of the GCG software package [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)]. Preferably, the sequence identity values ​​expressed as percentages (%) of the above sequences are determined using a procedure GAP across the entire sequence region with the following settings: vacancy weight: 50, length weight: 3, average match: 10.000, and average mismatch: 0.000, which, unless otherwise specified, should always be used as the standard settings for sequence alignment. In one embodiment, peptides comprising fragments of any of the aforementioned peptide sequences are also included as peptides of the present invention. Such fragments are peptides that are still suitable as analytes or for use in detection reagent compounds specified elsewhere herein, for example, having activity as a binding moiety as specified elsewhere herein. Thus, peptides may comprise or be composed of the domains of the present invention peptides that confer said biological activity. Fragments as indicated herein preferably comprise at least 50, at least 100, at least 250, or at least 500 consecutive amino acid residues of any of the aforementioned amino acid sequences, comprising at least 20, at least 30, at least 50, at least 80, at least 100, or at least 150 consecutive amino acids of any of the aforementioned amino acid sequences. The peptides of the present invention may also contain additional peptide sequences. Specifically, the polypeptide of the present invention may be a fusion protein, wherein one of the spouse bodies of the fusion protein is a polypeptide as specified herein.Such fusion proteins may contain, as other parts, peptides for monitoring expression (e.g., green, yellow, blue, or red fluorescent proteins, alkaline phosphatase, etc.) or so-called "tags," which can be used as detectable markers or as aids for purification purposes or for binding the peptides to solid surfaces. Tags for various purposes are well known in the art and include FLAG-tags, 6-histidine-tags, MYC-tags, biotin, etc.

[0017] As used herein, the term "antibody" includes monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, and antibody fragments, provided they exhibit the desired binding activity as specified elsewhere herein. In one embodiment, the antibody is not an antibody contained in antiserum, and is generally not a polyclonal antibody or polyclonal serum. Thus, in one embodiment, the antibody is an antibody contained in a mixture, comprising at least 80% in said mixture, at least 90% in another embodiment, and at least 95% in a further embodiment, of the antibody molecules being the detection compound 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.

[0018] Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of their constant domains in their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known and are often described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed., WB Saunders, Co. (2000). Antibodies can be part of a larger fusion molecule formed by the covalent or non-covalent association of an antibody with one or more other proteins or peptides.

[0019] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein to refer to an antibody in its substantially complete form, rather than an antibody fragment as defined below. The terms specifically refer to antibodies having a heavy chain containing an Fc region. In one embodiment, an “antibody fragment” comprises a portion of an intact antibody containing its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; dimeric antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody yields two identical antigen-binding fragments (referred to as “Fab” fragments, each having a single antigen-binding site) and a remaining “Fc” fragment (whose name reflects its tendency to crystallize). Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of cross-linking the antigen. An “Fv” is the smallest antibody fragment containing an intact antigen-binding site. In one embodiment, the dichain Fv species consists of a dimer of a tightly, non-covalently associated heavy chain variable domain and a light chain variable domain. In single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a “dimeric” structure similar to that of double-chain Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define the antigen-binding site. A total of six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than the entire binding site. The term “dimer” refers to an antibody fragment having two antigen-binding sites, the fragment containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).

[0020] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possibility of a small number of mutations (e.g., naturally occurring mutations). Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies typically comprise an antibody containing a polypeptide sequence that binds to an analyte, wherein the analyte-binding polypeptide sequence is obtained by a method comprising selecting a single analyte-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a combination of many clones such as hybridoma clones, phage clones, or recombinant DNA clones. In contrast to polyclonal antibody preparations, which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally not contaminated by other immunoglobulins.

[0021] As used herein, the term "analyte" refers to a chemical molecule, in one embodiment an organic molecule, that binds to the detection reagent compound of the present invention with sufficient affinity to allow detection of the analyte / detector compound complex. In one embodiment, the dissociation constant (Ki) of the analyte / detector compound complex is... d (At most 10) -7 mol / L, and in further embodiments, up to 10 mol / L. -8 mol / L, and in further embodiments, up to 10 mol / L. -9 mol / L. In one embodiment, the analyte is a biomolecule; in a further embodiment, the analyte is a biomacromolecule. In a further embodiment, the analyte is a polypeptide.

[0022] In one embodiment, if the analyte is a polypeptide, the polypeptide is an antigen produced by a pathogen, such as a virus, bacteria, or protozoan organism; or the analyte is an antibody produced by the subject against an antigen produced by a pathogen, such as a virus, bacteria, or protozoan organism.

[0023] In one embodiment, the analyte is a bacterial antigen, such as Treponema pallidum antigen, Treponema pallidum antigen, Brucella antigen, Chlamydia antigen, Mycoplasma antigen, Listeria antigen, or an antigen derived from a single-celled organism, such as a protozoan, or, in one embodiment, 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-Treponema pallidum 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, or, in one embodiment, an anti-Trypanosoma cruzi antibody. In a further embodiment, the analyte is an anti-autoantibody, i.e., an antibody that recognizes antigens produced by the subject themselves; in one embodiment, it is a diagnostic and / or prognostic anti-autoantibody for a disease; in another embodiment, it is a diagnostic and / or prognostic anti-autoantibody for an autoimmune disease. Diagnostic and prognostic anti-autoantibodies are known in the art and include, for example, antinuclear antibodies indicating systemic lupus erythematosus or polymyositis, antithyroid antibodies indicating Hashimoto's thyroiditis or Graves' disease, anti-intrinsic factor antibodies indicating anemia, and anti-tissue transglutaminase antibodies indicating celiac disease or gluten sensitivity.

[0024] In one embodiment, the analyte is a viral antigen, such as a viral antigen selected from the list of: hepatitis viruses, arboviruses, adenoviruses, Coxsackieviruses, echoviruses, influenza viruses, parainfluenza viruses, cytomegaloviruses, herpesviruses, Epstein-Barr virus, mumps virus, norovirus, rotavirus, rubella virus, measles virus, human immunodeficiency virus, varicella-zoster virus, poliovirus, and enteroviruses. In one embodiment, the analyte is an antibody against a viral antigen; in a further embodiment, an antibody against a viral polypeptide; or in a further embodiment, an antibody against a viral capsid polypeptide; in one embodiment, an antibody against a medically relevant virus; and in one embodiment, an antibody against a virus selected from the list specified above. In one embodiment, the virus is hepatitis A virus, hepatitis B virus, or hepatitis C virus. In one embodiment, the viral capsid polypeptide is a hepatitis virus capsid polypeptide; and in one embodiment, a hepatitis A (HA) virus capsid polypeptide. Therefore, in one embodiment, the analyte is an antibody against a hepatitis virus capsid polypeptide, such as an antibody against a hepatitis A virus capsid polypeptide.

[0025] In one embodiment, the analyte is a protozoan antigen, for example, from Toxoplasma gondii, specifically Toxoplasma glabrata. In one embodiment, the analyte is an antibody against a protozoan antigen; in a further embodiment, an antibody against a protozoan polypeptide. In one embodiment, the analyte is an antibody against the Toxoplasma glabrata p30 polypeptide (Genbank accession number: S85174.1).

[0026] However, it is also envisioned that the analyte is a low molecular weight compound, which can be determined by forming a complex with the detection reagent compound. In one embodiment, the analyte has a molecular mass of at least 100 (corresponding to 100 atomic mass units and 100 Da; 1 Da corresponds to 1.66 × 10⁻⁶). −27 In a further embodiment, the molecular weight is at least 250 kg, at least 500 kg, or at least 1000 kg. In one embodiment, the analyte is thyroxine (T4), triiodothyronine (T3), folic acid, folic acid-binding protein, or vitamin B12. 12 Or intrinsic factors.

[0027] As used herein, the term "detector 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 the detector compound, and in one embodiment, the dissociation constant (K0) of the binding moiety / label complex is... d (At most 10) -8 mol / L, and in further embodiments, up to 10 mol / L. -9 mol / L, in a further embodiment, up to 10 mol / L. -10 mol / L. In one embodiment, the binding moiety and the label are covalently linked. In one embodiment, the method for determining the analyte is a non-competitive immunoassay. In a further embodiment, the method for determining the analyte is a competitive immunoassay; as will be understood, in this case, the detection compound is the compound to be measured, also referred to in the art as a "targeting agent," i.e., a compound that is bonded to the label and competes with the analyte for binding to the capture compound.

[0028] As used herein, the term "binding moiety" refers to the structure of the detection compound, not a marker as specified elsewhere herein. Thus, for example, in the case where the detection compound is a dye-labeled antibody, the antibody would be the binding moiety; and in the case where the detection compound is a dye-labeled F(ab) fragment, the F(ab) fragment would be the binding moiety. Conversely, the term "affinity domain" is used to refer to a substructure of the detection compound that mediates the affinity of the detection compound for the analyte. Thus, the affinity domain is a substructure of the binding moiety that contains atoms of the analyte in the detection compound / analyte complex that contact the analyte. Therefore, the binding moiety may contain more than one affinity domain; for example, in one embodiment, two identical affinity domains (in the case where the binding moiety is IgG), in a further embodiment, ten identical affinity domains (in the case where the binding moiety is IgM), and in a further embodiment, two different affinity domains (in the case where the binding moiety is a dimer). In a further embodiment, in the case where the binding moiety is a multi-epitope antigen, the binding moiety may contain a plurality, i.e., any number greater than two, of identical or different affinity domains.

[0029] In one embodiment, the binding portion of the first detection compound is different from the binding portion of the second detection compound. Therefore, in one embodiment, the first and second detection compounds may be different monoclonal antibodies, recognizing the same epitope of the antibody in one embodiment. In one embodiment, in this case, the dissociation constant of the complex formed between the first detection compound and the analyte differs from the dissociation constant of the complex formed between the second detection compound and the analyte by no more than 5 times; in another embodiment, by no more than 2 times; and in a further embodiment, by no more than 1.5 times. In one embodiment, the dissociation constant of the complex formed between the first detection compound and the analyte has a value of 70% to 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 of 80% to 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 substantially equal or equal.

[0030] In a further embodiment, the binding portions of the first and second detection compounds are substantially identical, i.e., differing only in structural determinant clusters unrelated to analyte binding. In a further embodiment, the binding portions of the first and second detection compounds are identical; therefore, in one embodiment, the first and second detection compounds differ only in the label. As those skilled in the art will understand, in one embodiment, this difference may include differences determined by different coupling chemistry of the first and second labels.

[0031] As used herein, the term "label" refers to a compound suitable for making the presence of a molecule or complex containing the label detectable. Typically, the label has a detectable property, usually optical and / or enzymatic. However, it is also contemplated that the detectable property is a radioactive emission property. As will be understood, the parameter determined for detecting the detectable property will also be referred to as a "signal" or "detectable signal." For the avoidance of doubt, note that the signal can also be detected as zero or below a detection limit.

[0032] As used herein, the term "enzyme properties" refers to the characteristics of a label that produces a detectable product from a substrate through biocatalysis. Therefore, enzyme properties are typically conferred by the presence of a polypeptide in the label that possesses the enzyme properties. Typically, enzyme properties are selected from at least one enzyme activity: phosphatase activity (e.g., in alkaline phosphatase), peroxidase activity (e.g., in horseradish peroxidase), and glycosidase activity (e.g., in β-galactosidase). Typical substrates for enzyme activities are well known in the art. Typically, the enzyme activity produces a product having detectable optical properties as described above, and / or a product detectable by electronic instruments.

[0033] As used herein, the term "optical property" refers to any property that can be detected by an optical instrument. Specifically, an optically measurable property can be or may include at least one property selected from the group consisting of: reflectivity, transmissivity, emissivity, scattering, fluorescence, phosphorescence, diffraction, and polarization. Other optical properties contemplated by the present invention are color, fluorescence, luminescence, or refraction. In one embodiment, the optically measurable property referred to herein refers to the property of a chemical compound that can be optically detected, such as light absorption, luminescence, diffuse reflection, or related properties. It should be understood that detecting an optically measurable property as used herein includes detecting the presence of a previously undetectable property, detecting the absence of a previously detected property, and detecting a quantitative change in the property, i.e., detecting a change in signal intensity related to the degree of change in at least one optical property. It should be understood that in one embodiment, the term "optical property" also relates to luminescence, in one embodiment chemiluminescence, and in a further embodiment electrochemiluminescence, also referred to as electrochemiluminescence. Thus, in one embodiment, the detectable signal can be a luminescent signal, in one embodiment a chemiluminescent signal, and in a further embodiment an electrochemiluminescent signal. Based on the above, in one embodiment, the marker is a dye, and in another embodiment, it is a chemiluminescent compound.

[0034] As mentioned herein, 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 different. The first and second labels of the present invention are different, i.e., the two labels may differ in at least one physical, biological, and / or chemical property. For example, the first and second labels may be distinguished by their interaction with certain antisera or antibodies or by chromatographic and / or mass spectrometric analysis. 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, differing in at least one atom, chemical bond, and / or charge. In one embodiment, the distinguishing feature of the first and second labels is independent of the detectability of the first and second labels; therefore, in one embodiment, the detectability of the first and second labels is the same, although the first and second labels are structurally different. In a further embodiment, the distinguishing feature of the first and second labels is related to the detectability of the first and second labels; therefore, in one embodiment, the detectability of the first and second labels is different, and the first and second labels are structurally different.

[0035] In one embodiment, the (different) first and second markers provide the same quality of detectability. As used herein, the term "quality of detectability" refers to the physical properties of the detectability that determine its detection mode; therefore, two markers having the same quality of detectability are two markers that allow their detectability to be detected by the same measurement principles, i.e., measurements of radiation, transmission, luminescence (including chemiluminescence), absorbance, etc. In a further embodiment, the first and second markers provide substantially the same detectability, i.e., allow their detectability to be detected by substantially the same method, whereby the two methods are "substantially identical" if at most one measurement parameter, such as the detection wavelength, is readjusted by at most 2 times, at most 1.5 times in one embodiment, at most 1.2 times in a further embodiment, and at most 1.1 times in a further embodiment. In a further embodiment, the first and second markers provide the same detectability, i.e., allow their detectability to be detected by the same method. As will be understood, in the latter case, the detectability of the first and second markers can be detected simultaneously. As will be further understood, the detection of the detectable properties of the first and / or second markers can be performed under suboptimal conditions for one or both markers; for example, in the case where the two markers have overlapping absorption spectra, absorption can be measured at a non-maximum absorption wavelength where the two markers have the same molar extinction coefficient. Therefore, in one embodiment, determining the amount of the complex in step b) includes detecting the properties of the first and second detector compounds; in another embodiment, it includes simultaneously detecting the properties of the first and second detector compounds.

[0036] In one implementation, the first marker is Ru(bpy)2-bpyCO-OSu (also known as "BP-Ru"; CAS Registry No. 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-pyrrolidone]-, (OC-6-33) Ru(bpy)2-bpyCO2H reactive ester (= BPRu, or Ru-bpy, CAS Registry No. 115239-59-3), and the second label is sulfonyl-BPRuNHS ester (also known as "sulfonyl-Ru"; CAS Registry No. 482618-42-8, also known in the art as ruthenate (2-), bis[[2,2'-bipyridine]-4,4'-dimethylsulfonic acid alkyl(2-)-κN1 ,κN 1' ][1-[4-(4'-methyl[2,2'-bipyridine]-4-yl-κN] 1 ,κN 1' [1-Oxobutoxy]-2,5-pyrrolidone]-,sodium(1:2),(OC-6-31).

[0037] As used herein, the term "assay" refers to the determination of at least one detectable feature of an analyte in a sample to be determined by the method of the present invention. In one embodiment, the method is applicable to any assay, including the use and detection of at least one detection compound as specified herein. Thus, in one embodiment, the assay includes determining the direct or indirect interaction between a feature of the analyte and the detection compound. In one embodiment, the feature of the analyte is a domain that binds to the detection compound; in another embodiment, it is a domain that specifically binds to the detection compound. Thus, in one embodiment, the detectable feature of the analyte is a receptor or its receptor domain, a lectin, an aptamer (which may be a peptide aptamer or a polynucleotide aptamer), an anticalin, a designed ankylosing repeat protein, or an immunoglobulin or its binding subdomain, and the detection compound comprises a chemical structure specifically bound by said detectable feature. Conversely, in one embodiment, the detection compound comprises a receptor or its receptor domain, a lectin, an aptamer (which may be a peptide aptamer or a polynucleotide aptamer), an anticarrier protein, a designed ankylosing repeat protein, or an immunoglobulin or its binding subdomain, and the detectable feature of the analyte comprises a chemical structure specifically bound by the detection compound.

[0038] In the context of this invention, an "aptamer" is a macromolecule that specifically binds to its interacting partner. An aptamer can be a peptide or a polynucleotide aptamer, and is known in principle to those skilled in the art. As used herein, the term "peptide aptamer" refers to a peptide that specifically binds to its interacting partner and comprises 8-80 amino acids, 10-50 amino acids in one embodiment, and 15-30 amino acids in a further embodiment. They can, for example, be isolated from randomized peptide expression libraries in a suitable host system (such as baker's yeast) (see, for example, Klevenz et al., Cell Mol Life Sci. 2002, 59:1993–1998).

[0039] As used herein, the term "anticarrier protein" refers to an artificial polypeptide derived from a lipid carrier protein that specifically binds to its interacting partner. Similarly, "designed ankyrin repeat protein" or "DARPin" as used herein is an artificial polypeptide containing several ankyrin repeat motifs and specifically binding to its interacting partner.

[0040] In one embodiment, the detectable characteristics of the analyte and / or the detection agent compound comprise an immunoglobulin or its binding subdomain. Therefore, in one embodiment, the determination includes measuring the direct or indirect interaction between the immunological characteristics of the analyte and the detection agent compound and / or the direct or indirect interaction between the characteristics of the analyte and the immunological characteristics of the detection agent compound. Therefore, in one embodiment, the method is an immunoassay. In one embodiment, the immunological characteristic according to the invention is a structural characteristic of the analyte that facilitates the immunological detection of the analyte in the sample. In one embodiment, the immunological characteristic facilitates identification, and in a further embodiment, quantification of the analyte by immunological means. Therefore, a typical immunological characteristic is one that facilitates the differentiation of the analyte from other compounds in the sample. In one embodiment, determining the analyte is determining whether the analyte is present in the sample at a concentration higher than the method detection limit. Methods for determining the detection limit are known to those skilled in the art. In a further embodiment, the determination is a semi-quantitative or quantitative determination of the amount or concentration of the analyte in the sample. For quantitative determination, the absolute or precise amount of the analyte is determined, or the relative amount of the analyte is determined. The relative amount can be determined when the precise amount of the analyte cannot be determined or should not be determined. In this case, it can be determined whether the amount of analyte present increases or decreases relative to a second sample containing the second amount of the analyte.

[0041] As those skilled in the art will understand, it is generally not necessary to separately determine the first and second detective compound / analyte complexes. Therefore, in one embodiment, the complexes of the first and second detective compounds and the analyte are determined together, i.e., without distinction between the first and second detective compounds. Therefore, in one embodiment, the total amount of analyte present in the complex of the first or second detective compound or both is determined. In one embodiment, this can be accomplished by simultaneously determining the detectability of the first and second markers in step b), as specified above; or by separately determining the detectability of the first and second markers in step b), but determining the sum of the properties of the first and second detective compounds in step c). As understood above, the first and second markers can bind to the same type of binding moiety; therefore, the first and second binding moieties can be different, but they can also be the same. Therefore, the determination can be achieved by contacting the sample with a monoclonal antibody (whose first fraction binds to the first marker and its second fraction binds to the second marker). This, plus necessary modifications, also applies to polyclonal antibody preparations.

[0042] As those skilled in the art will further understand, the determination of the analyte / capture compound complex may include additional steps and / or the use of additional compounds. For example, one or more additional detection compounds that differ from the first and second detection compounds at least in relation to the label may 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, the 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 potentially additional 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 are used; in another embodiment, 2 to 5 detection compounds; in yet another embodiment, 2 to 4 detection compounds; in yet another embodiment, 2 to 3 detection compounds, and the labels of all the detection compounds used are different from each other. In one embodiment, the detection compounds are used in substantially equal amounts; in yet another embodiment, they are used in equal amounts.

[0043] Furthermore, depending on the chosen assay method, capture antibodies can be used to bind analytes and analyte / detector compound complexes 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 the analyte for binding to a capture compound bound to the solid surface, and in which the amount of the labeled derivative of the analyte bound to the capture compound is measured. Therefore, in this case, the method further includes mixing a specifying agent into the sample. In one embodiment, the method of the present invention is a heterogeneous competitive immunoassay and includes indirectly determining the amount of the complex formed between the analyte and the two different capture compounds by measuring the amount of the complex formed between the specifying agent and the different capture compounds. In one implementation, 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), folic acid, folic acid RBC (red blood cells), anti-TSH-R, total vitamin D, vitamin B12, thyroxine (T4), FT4 (free thyroxine), thyroxine (T3), FT3 (free triiodothyronine), testosterone, progesterone, digitalisin, anti-TG, anti-TPO (anti-thyroid peroxidase), DGEA, or estradiol.

[0044] In another embodiment, the immunoassay is a double-antigen sandwich assay (“DAGS”), wherein a bivalent analyte, such as an antibody, binds to a capture compound that binds to a solid surface, and wherein the amount of the analyte / capture compound complex is determined by the binding of a detection reagent compound to the analyte / capture compound complex as described below. In one embodiment, the DAGS assay is an anti-Toxoplasma gondii IgG, anti-rubella IgG, anti-HBs (hepatitis B virus surface antigen) antibody, HCV (hepatitis C virus) core antigen or anti-HCV antibody, CMV (cytomegalovirus) antibody, syphilis (Treponema pallidum) antibody, HTLV (human T-cell lymphoblastic virus) antibody, or Chagas disease (American trypanosomiasis) antibody.

[0045] As used herein, the term "capture compound" refers to a chemical molecule that binds directly or indirectly to an analyte as specified above. In one embodiment, the capture compound binds to or is adapted to bind to a solid surface. In one embodiment, the capture compound is an organic molecule; in a further embodiment, it is a biomacromolecule as described above, such as a polypeptide. In one embodiment, the capture compound binds indirectly to the analyte of the present invention with sufficient affinity to allow the detection of a complex comprising the analyte and the capture compound; i.e., in this case, the capture compound is an indirect ligand. As used herein, the term "indirect binding" means binding in which the ligand does not directly contact the analyte but contacts the chemical molecule that binds the analyte; in one embodiment, the chemical molecule that specifically binds the analyte is specifically bound to the analyte; in one embodiment, the molecule that binds the analyte is the molecule that directly binds the analyte, i.e., the direct ligand. Thus, in one embodiment, the analyte, the chemical molecule that binds the analyte, and the indirect ligand form a complex having the properties described above, particularly having the dissociation constant as shown. In a further embodiment, the capture compound binds directly to the analyte of the present invention with sufficient affinity to allow the detection of the analyte / capture compound complex as described above. Therefore, in one embodiment, the capturing compound is a direct ligand.

[0046] As used herein, the term "solid surface" refers to any suitable solid surface suitable for binding the capture compounds of the present invention and suitable for separation from a sample, for example, by physical means. In one embodiment, the solid surface is the surface of beads, and in another embodiment, it is microbeads, such as magnetic or paramagnetic microbeads. In one embodiment, the surface is adapted to improve the binding of the capture compounds, for example, by covalently or nonvalently attaching molecules that bind the substructures of the capture compounds. Typical molecules that bind the substructures of the capture compounds are, for example, antibodies, streptavidin, complex nickel ions, components of any X-anti-X system (such as, for example, sugars and sugar-binding proteins / lectins or hormones and their receptors) in which compound "X" specifically binds "anti-X". In a further embodiment, the solid surface binds the capture compounds by covalent or nonvalent bonds, for example, by hydrophobic interactions. Thus, in one embodiment, the solid surface is the surface of a multi-cluster plate. In one embodiment, the surface of the multi-cluster plate is pretreated to increase the affinity and / or ability to bind the capture compounds. Suitable pretreatments are known in the art.

[0047] Methods for binding biomolecules (typically peptides) to solid surfaces are well known in the art and include, for example, binding via hydrophobic interactions, biotinylation and binding via immobilized streptavidin, covalent binding, antibody-antigen interactions, or combinations of these interactions, such as antibody-antigen interactions between an antibody and a pathogen peptide, wherein the antibody is biotinylated and bound to the solid surface via immobilized streptavidin. Therefore, the capture compound can also preferably be a capture complex. In one embodiment, the capture compound is a capture complex that directly binds to the analyte. Those skilled in the art know how to bind capture compounds or complexes to solid surfaces, depending on the solid surface chosen. In one embodiment, the capture compound is a viral peptide, such as a viral capsid peptide. In one embodiment, the capture compound is a hepatitis virus capsid peptide, and in another embodiment, a hepatitis A virus (HAV) capsid peptide. However, it is also contemplated that the capture compound is an antibody.

[0048] As used herein, the term "sample" refers to a sample of bodily fluids, a sample from a tissue or organ, or a sample of a washing / rinsing solution, or a swab or smear obtained from an external or internal body surface. In one embodiment, the sample is suspected of containing an analyte as specified herein. In one embodiment, the sample 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, rinsing / washing solution, drill biopsy equipment, puncture a cavity with a needle or lancet, or by surgical instrumentation. However, samples obtained by well-known techniques, including, in one embodiment, scrapes, swabs, or biopsies from the urogenital tract, perianal region, anal canal, oral cavity, upper respiratory and digestive tract, and epidermis, are also included as samples of the present invention. Cell-free liquids can be obtained from bodily 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 bodily fluid known to contain HA virus polypeptides and / or antibodies against at least one HA virus polypeptide, i.e., blood, plasma, serum, saliva, etc. in one embodiment, and plasma or serum in another embodiment. It should be understood that the sample can be further processed to implement the method of the invention. Specifically, cells can be removed from the sample by methods and approaches known in the art. Furthermore, at least one analyte can be extracted and / or purified from the sample by methods and approaches known in the art. Therefore, the term "sample" can also refer to a preparation containing or suspected of containing at least one analyte diluted, enriched, purified, and / or extracted from the sample.

[0049] The term "contact" as used in the context of the methods of this invention is understood by those skilled in the art. In one embodiment, the term refers to bringing the compound of this invention into physical contact with a sample or with another compound, thereby allowing, for example, the sample and the compound to interact.

[0050] As used herein, the term "subject" refers to a vertebrate, and in one embodiment, a mammalian subject. In one embodiment, the subject is a farm animal, 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 person suspected of containing an analyte as specified herein.

[0051] Advantageously, in the work upon which this invention is based, it has been found that interference from anti-labeled compounds from the sample can be reduced by using different labels (which can provide substantially the same amount and / or characteristics of signal) in the immunoassay. For example, in competitive immunoassays, signal reduction caused by anti-labeled interference that leads to false positive results can be reduced. Similarly, in sandwich / double-antigen sandwich immunoassays, anti-labeled interference tends to reduce the signal by binding to the label, causing a decrease in signal yield and thus false negative results. In some cases, signal increase has also been observed, thereby causing false negative results. At least in all these cases, using at least two different labels avoids interference that causes erroneous results.

[0052] The definitions made above, with necessary modifications, apply to the following. Further definitions and explanations below, with necessary modifications, also apply to all embodiments described in this specification.

[0053] The present invention also relates to a method for improving the detection specificity of an analyte in an assay, comprising replacing 10% to 90% of a first detection reagent compound with a second detection reagent compound having a different label.

[0054] As used herein, the term "replacing a fraction of the first detection compound with the second detection compound" means omitting a fraction of the first detection compound from the assay and newly including the second detection compound in an amount corresponding to the omitted first detection compound. In one embodiment, the fractions 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 fractions 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 any signal yield of 100%; thus, if 50% of the first detection compound should be replaced based on signal yield, a certain amount of the first detection compound is omitted, reducing the signal yield to 50%, and the second detection compound is added, increasing the signal yield back to 100%. It will be understood that if the first detection compound is replaced by more than one additional detection compound, and if more than one detection compound is replaced by one or more additional detection compounds, the above, plus necessary modifications, apply.

[0055] Typically, the fraction of the alternative capture or detection compound is selected such that a theoretically predictable measurable effect of the substitution can be expected. As those skilled in the art will understand, the expected measurable effect will depend on the number of different markers used for substitution. For example, if three are used in an improved assay to replace one detection compound, it is preferable to replace, for example, 66% of the initial detection compound. Typically, a fraction of (100% / n) ± 50% is envisioned for a given detection compound, where n = (the number of different detection compounds used in the assay). In another embodiment, a fraction of (100% / n) ± 20% is used. As those skilled in the art will understand, the sum of the fractions used will be 100%. Thus, in one embodiment, the fraction of the alternative detection compound is 10% to 90%, in another embodiment it is 25% to 75%, in a further embodiment it is 40% to 60%, and in yet another embodiment it is about 50%.

[0056] The present invention further relates to a kit for detecting an analyte in a sample, comprising at least first and second detection compounds of 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 different.

[0057] As used herein, the term "kit" refers to a collection of the compounds, devices, or reagents of the present invention that may or may not be packaged together. The components of the kit may be contained in or provided in a single vial (i.e., as a separate component of the kit). Furthermore, in one embodiment, the kit of the present invention is used to perform the methods described above. In one embodiment, it is envisioned that all components are provided ready to use for performing the methods described above. Furthermore, in one embodiment, the kit contains instructions for performing the methods. These instructions may be provided in paper or electronic form by a user manual. For example, the manual may contain instructions for explaining the results obtained when performing the methods described above using the kit of the present invention. In one embodiment, a kit for detecting an analyte in a sample (containing at least two different detection compound compounds) further contains at least one capture compound. In a further embodiment, the kit further contains a solid support for immobilizing the capture compound or for immobilizing the analyte. In one embodiment, the kit contains 2 to 10 detection compound compounds for the analyte; in another embodiment, 2 to 5 detection compound compounds for the analyte; in another embodiment, 2 to 4 detection compound compounds; and in yet another embodiment, 2 to 3 detection compound compounds for the analyte.

[0058] Furthermore, the present invention relates to an apparatus for determining an analyte in a sample, comprising at least first and second detection compounds of the analyte, wherein the first detection compound comprises a first binding portion and a first marker, and the second detection compound comprises a second binding portion and a second marker, wherein the first marker and the second marker are different; and an apparatus for determining at least one signal obtained from the first marker and the second marker.

[0059] As used herein, the term "device" refers to a system of devices comprising at least the aforementioned operable interconnected means to allow measurement. The foregoing description, in conjunction with the method of the present invention, discloses typical devices for determining the amount of an analyte and devices for performing such determinations, such as devices for determining at least one signal. As will be understood by those skilled in the art, a device for determining at least one signal includes a means capable of determining a signal; and as will also be understood by those skilled in the art, a device 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.

[0060] How the device is connected operatively will depend on the type of device included in the apparatus. In one embodiment, the apparatus is comprised of a single device. Thus, the device may include (i) an analytical unit for measuring the amount of analyte in the applied sample and (ii) a computer unit for processing the resulting data for evaluation. Typical apparatuses for detection are disclosed, along with embodiments relating to the methods of the present invention described above. In this case, the apparatus is operatively connected in such a way that the user of the system combines the results of measurements of the detectable properties of the marker, the optical and / or electrochemically measurable properties of the marker in one embodiment, based on the instructions and explanations given in the manual; or the instructions and explanations are included in executable program code contained in the apparatus, such that the amount or concentration of the analyte in the applied sample is output to the user as a result of the measurement. Those skilled in the art will appreciate how the apparatus can be connected without further complication. Typical apparatuses are those that can be used without the specific knowledge of a skilled technician, such as test strips or electronic devices that only require sample loading. The results may be given as the output of raw data, which requires interpretation by a skilled technician. However, in one embodiment, the output of the apparatus is processed, i.e., raw data for evaluation, the interpretation of which does not require a skilled technician. Other typical devices include analytical units / devices (e.g., biosensors, arrays, solid supports coupled to ligands of specifically recognizing peptides, plasma surface resonance devices, NMR spectrometers, mass spectrometers, etc.) or the evaluation units / devices mentioned above according to the method of the present invention. In one embodiment, the device contains 2 to 10 detection compounds for the analyte; in another embodiment, 2 to 5 detection compounds for the analyte; in another embodiment, 2 to 4 detection compounds; and in yet another embodiment, 2 to 3 detection compounds for the analyte.

[0061] Furthermore, the present invention relates to the use of compositions comprising at least first and second detection compounds 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, wherein the first label and the second label are different; and the present invention relates to the use of first and second detection compounds comprising at least an analyte for preparing a diagnostic composition or diagnostic device, 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 different; and the present invention relates to the use of compositions comprising at least first and second detection compounds comprising an analyte for determining the analyte in a sample, 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 different.

[0062] In view of the above, the following implementation plan is specifically proposed: 1. A method for determining an analyte in a sample, comprising: a) Contact the sample with at least the first and second detection reagent compounds; b) Determining the amount of a complex containing at least one detection reagent compound; and, c) Determine the analyte in the sample based on the results of step b). The first detection agent compound comprises a first binding portion and a first marker, and the second detection agent compound comprises a second binding portion and a second marker, wherein the first marker and the second marker are different.

[0063] 2. The method of embodiment 1, wherein the first and second markers provide the same detectable property.

[0064] 3. The method of implementation 1 or 2, wherein the first and second markers provide the same detectable properties.

[0065] 4. The method of any one of embodiments 1 to 3, wherein the first and second markers provide substantially the same amount of detectable properties.

[0066] 5. The method of any one of embodiments 1 to 4, wherein the detectable property provided by the first and / or second detection agent compound is a radiation property, in one embodiment a luminescence property, and in a further embodiment a chemiluminescence property.

[0067] 6. The method of any one of embodiments 1 to 5, wherein the marker in the first detection reagent compound includes Ru(bpy)2-bpyCO-OSu (CAS Registry No. 137323-76-3).

[0068] 7. The method of any one of embodiments 1 to 6, wherein the marker in the second detection reagent compound is sulfonyl-BPRu NHS ester (CAS Registry No. 482618-42-8).

[0069] 8. The method of any one of embodiments 1 to 7, wherein determining the amount of the complex in step b) includes detecting the properties of the first detection compound and the second detection compound, and in one embodiment includes simultaneously detecting the properties of the first detection compound and the second detection compound.

[0070] 9. The method of any one of embodiments 1 to 8, wherein determining the analyte in step c) comprises determining the sum of the characteristics of the first detection compound and the characteristics of the second detection compound.

[0071] 10. The method of any one of embodiments 1 to 9, wherein the binding portion specifically binds the analyte or a capture compound that specifically binds the analyte.

[0072] 11. The method of any one of embodiments 1 to 9, wherein the binding portion competes with the analyte for binding to the capture compound.

[0073] 12. The method of any one of embodiments 1 to 11, wherein the binding portion is a biomolecule or a fragment thereof, and in one embodiment a polypeptide or a fragment thereof.

[0074] 13. The method of any one of embodiments 1 to 12, wherein the binding portion is an antibody or a fragment thereof.

[0075] 14. The method of any one of embodiments 1 to 13, wherein the method is an immunoassay.

[0076] 15. The method of any one of embodiments 1 to 14, wherein the method is a competitive assay.

[0077] 16. The method of any one of embodiments 1 to 14, wherein the method is a sandwich assay, and in one embodiment is a double-antigen sandwich assay.

[0078] 17. The method of any one of embodiments 1 to 16, wherein the method is a qualitative or semi-quantitative determination.

[0079] 18. The method of any one of embodiments 1 to 17, wherein the method is a quantitative determination method.

[0080] 19. The method of any one of embodiments 1 to 18, wherein the analyte is a polypeptide.

[0081] 20. The method of any one of embodiments 1 to 19, wherein the analyte is an antibody, in one embodiment an antibody against an antigen from a pathogenic organism, in one embodiment an antibody against a viral antigen, and in one embodiment an antibody against a protozoan antigen.

[0082] 21. The method of any one of embodiments 1 to 20, wherein the analyte is an anti-hepatitis A antibody or an anti-toxoplasmosis antibody.

[0083] 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.

[0084] 23. The method of any one of embodiments 1 to 22, wherein 2 to 10 detection compounds are used, in one embodiment 2 to 5 detection compounds, in one embodiment 2 to 4 detection compounds, in one embodiment 2 to 3 detection compounds, and wherein the markers of all detection compounds are different from each other.

[0085] 24. A method for improving the detection specificity of an analyte in a assay, comprising replacing 10% to 90% of a first detection reagent compound with a second detection reagent compound having a different label.

[0086] 25. The method of embodiment 24, wherein 25% to 75%, 40% to 60% in a further embodiment, and approximately 50% in a further embodiment, the first detection compound is replaced.

[0087] 26. A method for identifying a sample containing an interfering substance, said interfering substance disrupting the determination of an analyte using a detection reagent compound having a first label, said method comprising: a) Contacting equal aliquots of the sample with a first detection reagent compound having the first marker; b) Contacting an equal aliquot of the sample with a second detection reagent compound having a second marker; c) Measure the first signal generated by the first marker; d) Measure the second signal generated by the second marker; e) Identify a sample containing interfering substances that disrupt the determination of an analyte using a detection reagent compound with a first marker by comparing the first signal from step c) with the second signal from step d).

[0088] 27. The method of implementation scheme 26, further comprising the determination of multiple analytes.

[0089] 28. A kit for detecting an analyte in a sample, comprising at least first and second detection compounds of 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 different.

[0090] 29. The kit of embodiment 28, wherein the kit further comprises at least one capture compound for the analyte.

[0091] 30. The kit of embodiment 28 or 29, wherein the kit further comprises a solid support for immobilizing the captured compound or components of the sample containing at least the analyte.

[0092] 31. An apparatus for determining an analyte in a sample, comprising at least first and second detection compounds of the analyte, wherein the first detection compound comprises a first binding portion and a first label, and the second detection compound comprises a second binding portion and a second label, wherein the first label and the second label are different; and an apparatus for determining at least one signal obtained from the first label and the second label.

[0093] 32. Use of a composition comprising at least first and second detection reagent compounds for detecting an analyte, wherein the first detection reagent compound comprises a first binding moiety and a first marker, and the second detection reagent compound comprises a second binding moiety and a second marker, wherein the first marker and the second marker are different.

[0094] 33. Use of at least the first and second detection compounds of an analyte for the preparation of a diagnostic composition or diagnostic device, wherein the first detection compound comprises a first binding moiety and a first marker, and the second detection compound comprises a second binding moiety and a second marker, wherein the first marker and the second marker are different.

[0095] 34. Use of embodiment 32 or 33, wherein the analyte is the analyte specified in any one of embodiments 19 to 22.

[0096] 35. Use of a composition comprising at least first and second detection reagent compounds of an analyte for determining the analyte in a sample, wherein the first detection reagent compound comprises a first binding moiety and a first marker, and the second detection reagent compound comprises a second binding moiety and a second marker, wherein the first marker and the second marker are different.

[0097] 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 portions of the first and second detection reagent compounds are identical.

[0098] 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 portions of the first and second detection reagent compounds are different.

[0099] All references cited in this specification are incorporated herein by reference for the purposes of their entire disclosure and for the specific disclosures mentioned herein. Detailed Implementation

[0100] The following examples are intended to illustrate the invention only. They should not in any way be construed as limiting the scope of the invention.

[0101] Example 1: Increased specificity of qualitative and competitive Elecsys anti-hepatitis A virus (anti-HAV) assay According to the manufacturer's instructions, in the automated Elecsys ® An immunoassay for the in vitro determination of anti-HAV antibodies was performed on a cobas analyzer (Roche Diagnostics GmbH). Elecsys ® It is a registered trademark of the Roche Group.

[0102] The assay is performed based on a competitive principle. In the first incubation, 50 µl of sample is incubated with added HAV antigen, allowing the sample to bind to the HAV antigen. In the second (subsequent) incubation step, biotinylated and ruthenium-labeled antibodies specific to the HAV antigen are added to the sample-HAV antigen mixture along with streptavidin-coated microparticles, causing any remaining free binding sites on the HAV antigen to become occupied. The entire complex binds to the solid phase (microparticles) via the interaction of biotin and streptavidin. Next, the reaction mixture is drawn into the measurement chamber, where the microparticles are magnetically trapped onto the electrode surface. Unbound material is then removed with ProCell / ProCell M (a buffer solution containing tripropylamine necessary for signal generation). A voltage is then applied to the electrode to induce chemiluminescence emission, which is measured by a photomultiplier tube. The results, obtained via calibration curves, are from instruments specifically generated through 2-point calibration (Cal1 = negative calibrator containing human anti-HAV negative serum; Cal2 = positive calibrator containing human anti-HAV in human serum). Specifically, monoclonal MAK was used. <hav>M-2.157-F(ab')2-antibody fragment, one aliquot labeled with BP-Ru, and the other aliquot labeled with sulfonyl-Ru; these were used to generate three different versions of the anti-HAV assay: Determination 1: 100% MAK used in R2 <hav>M-2.157-F(ab')2 labeled with sulfido-Ru Assay 2: 100% MAK<HA V> M-2.157-F(ab')2 labeled with BP-Ru used in R2 Assay 3: 1 + 1 mixture (meaning mixture of BP-Ru and sulfido-Ru) of Assay 1 R2 and Assay 2 R2 has been used. The signal contribution of BP-Ru and sulfido-Ru to the total signal is similar.

[0103] The first label is "BP-Ru", also known as Ru(bpy)2-bpyCO-OSu (CAS Registry Number 137323-76-3, = Ruthenium(2+), bis(2,2'-bipyridine-kN 1 ,κN 1' )[1-[4-(4'-methyl[2,2'-bipyridine]-4-yl-kN 1 ,κN 1' )-1-oxobutoxy]-2,5-pyrrolidinedione]-, (OC-6-33) Ru(bpy)2-bpyCO2H, reactive ester (= BPRu, or Ru-bpy), CAS Registry Number 115239-59-3) and the second label is "sulfido-Ru", also known as sulfido-BPRu NHS ester (CAS Registry Number 482618-42-8, also known in the art as Ruthenium acid (2-), bis[[2,2'-bipyridine]-4,4'-dimethylsulfonato(2-)-kN 1 ,κN 1' ][1-[4-(4'-methyl[2,2'-bipyridine]-4-yl-kN 1 ,κN 1' )-1-oxobutoxy]-2,5-pyrrolidinedione]-, sodium (1 :2), (OC-6-31).

[0104] The results with calibrators and standard samples are shown in Table 1. In addition, 27 known anti-label interference samples, such as anti-sulfido-Ru interference samples and one known anti-BP-Ru interference sample were tested with all 3 assay formats (Table 2). These samples are known to cause false positive results when used in the respective anti-HAV assay variants. As expected, the anti-sulfido-Ru interference sample was false positive in Assay 1, but correctly negative in Assay 2, and the anti-BP-Ru interference sample was correctly negative in Assay 1, but false positive in Assay 2. Interestingly, all samples except one anti-sulfido Ru interference sample (PN0206_0925) were correctly negative in Assay 3 containing a mixture of labels BP-Ru and sulfido Ru, meaning a significant increase in specificity.

[0105] The cut-off index (COI) was calculated as follows: The cut-off value was determined using a 2-point calibration with a negative (Cal1) and a positive (Cal2) calibrator. The cut-off index (COI) was determined by dividing the counts (sample) by the cut-off value. If the COI < 1, the result is interpreted as reactive, and if the COI > 1, it is non-reactive.

[0106] Example 2: Increase in sensitivity of the quantitative DAGS Elecsys Toxo IgG assay The immunoassay for the in vitro determination of Toxo-IgG antibodies was performed on an automated Elecsys cobas analyzer (Roche Diagnostics GmbH) according to the manufacturer's instructions. The Elecsys Toxo IgG assay is a sandwich immunoassay for the quantitative determination of IgG antibodies to Toxoplasma gondii in human serum and plasma. ® cobas analyzer (Roche Diagnostics GmbH) according to the manufacturer's instructions. The Elecsys Toxo IgG assay is a sandwich immunoassay for the quantitative determination of IgG antibodies to Toxoplasma gondii in human serum and plasma. ® cobas is a registered trademark of the Roche Group.

[0107] The assay was performed according to the sandwich principle (IgG antibodies sandwiched between two Toxo-p30 antigens). In the first incubation of 10 μΙ sample, a biotinylated recombinant Toxoplasma gondii specific antigen and a Toxoplasma gondii specific recombinant antigen labeled with a ruthenium complex form a sandwich complex. In the second step, streptavidin-coated microparticles are added, which bind the immunocomplex of sample antibodies and Toxo-antigen to the solid phase via the interaction of biotin and streptavidin. Next, the reaction mixture is pipetted into the measuring chamber, where the microparticles are magnetically captured onto the surface of the electrodes. Unbound substances are then removed with ProCell / ProCell M (a buffer solution containing tripropyl amine necessary for signal generation). Then, a voltage is applied to the electrodes, which induces the emission of chemiluminescence, which is measured by a photomultiplier. The result is determined via a calibration curve, which is specifically generated by a 2-point calibration (Cal1 = negative calibrator containing anti-toxo negative human serum; Cal2 = positive calibrator containing human serum reactive to anti-toxo IgG).

[0108] In detail, two differently labeled recombinant Toxo-p30 antigen species were used, one labeled with BP-Ru and the other with sulfo-Ru (chemical name of the label, see Example 1). The two recombinant Toxo-p30 antigen species were used to generate 3 different versions of the Toxo IgG assay: Assay 1 : 100% of the recombinant Toxo-p30 antigen used in R2 was labeled with sulfo-Ru Assay 2: 100% of the recombinant Toxo-p30 antigen used in R2 was labeled with BP-Ru Determination 3: A 1+1 mixture of Determination 1 R2 and Determination 2 R2 (meaning a mixture of BP-Ru and sulfonyl-Ru) was used. The signal contributions of BP-Ru and sulfonyl-Ru to the overall signal were similar.

[0109] According to the manufacturer's instructions, results <1 IU / mL are interpreted as non-reactive; results ≥ 1 to < 3 IU / mL are indeterminate; and results ≥ 3 IU / mL are reactive. Results using calibrators and standard samples are shown in Table 3. Furthermore, several anti-sulfonyl-Ru and anti-BP-Ru interference samples that caused false negatives or false indeterminates in their respective assay variations were detected using all three assays (Table 4). As expected, anti-sulfonyl-Ru interference samples were false negatives or false indeterminates in assay 1, but true indeterminates or true positives in assay 2. The reverse was also true for anti-BPRu interference samples interfering with assay 2. Interestingly, in assay 3, which contained a mixture of BP-Ru and sulfonyl-Ru markers, all samples were found to be true indeterminates or true negatives, indicating a significant increase in sensitivity.

[0110] 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), Ch. 5.3 - Interferences in Immunoassay, in The Immunoassay Handbook (4th Edition), 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 (ed.) WO 2016097116 A1 WO 2017093271 A1。 < / hav> < / hav>

Claims

1. A method for reducing interference from an anti-analyte compound from a sample comprising: a) contacting the sample with at least a first and a second detector compound; b) determining the amount of complex comprising at least one detector compound; and, c) determining the analyte in the sample based on the results of step b), wherein the first detector compound comprises a first binding moiety and a first label, and the second detector compound comprises a second binding moiety and a second label, and wherein the first label and the second label are different, and the label in the first detector compound comprises Ru(bpy)2-bpyCO-OSu (CAS Registry Number 137323-76-3) and / or wherein the label in the second detector compound comprises Sulfo-BP Ru NHS ester (CAS Registry Number 482618-42-8).

2. The method of claim 1, wherein the first and second labels provide the same detectable property.

3. The method of claim 1 or 2, wherein the detectable property provided by the first and / or second detector compound is a radiation property, in one embodiment a luminescence property, in a further embodiment a chemiluminescence property.

4. The method of any one of claims 1 to 3, wherein determining the amount of complex in step b) comprises detecting the detectable property of the first detector compound and the second detector compound, in one embodiment comprises detecting the detectable property of the first detector compound and the second detector compound simultaneously.

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

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

7. The method of any one of claims 1 to 6, wherein the analyte is an antigen from a pathogenic organism, in one embodiment a viral antigen or a bacterial antigen.

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

9. Use of a composition comprising at least a first detector compound and a second detector compound for detecting an analyte, wherein the first detector compound comprises a first binding moiety and a first label, and the second detector compound comprises a second binding moiety and a second label, and wherein the first label and the second label are different, and the label in the first detector compound comprises Ru(bpy)2-bpyCO-OSu (CAS Registry Number 137323-76-3) and / or wherein the label in the second detector compound comprises Sulfo-BP Ru NHS ester (CAS Registry Number 482618-42-8).

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