Determining a free thyroid hormone

Abstract

The present invention relates to a method for determining a free thyroid hormone, comprising combining a blood sample with a capture reagent which comprises a magnetic particle conjugate having a magnetic particle and a specific binder for the thyroid hormone to be determined; adding a detector reagent which comprises a thyronine derivative tracer of formula (II); and inducing and quantifying a chemiluminescence. The invention further relates to a thyronine derivative tracer of formula (II) and to a kit for a heterogeneous immunoassay for determining a free thyroid hormone, comprising a capture reagent and a detector reagent.

Description

[0001] P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0002] DETERMINATION OF FREE THYROID HORMONE

[0003] Field of invention

[0004] The present invention relates to a method for determining free thyroid hormone, comprising combining a blood sample with a capture reagent comprising a magnetic particle conjugate with a magnetic particle and a specific binder of the thyroid hormone to be determined; adding a detector reagent comprising a thyronine derivative tracer of formula (II); and triggering and quantifying chemiluminescence. The invention further relates to a thyronine derivative tracer of formula (II) and a kit for a heterogeneous immunoassay for determining free thyroid hormone, comprising a capture and a detector reagent.

[0005] Background of the invention

[0006] Thyroid disorders are particularly widespread in industrialized countries; in the USA, for example, 10% of the population is affected. To diagnose a thyroid disorder, clinical diagnostics primarily involve a test for thyroid-stimulating hormone (TSH). In many cases, one or both thyroid hormone parameters, free thyroxine (fT4) and free triiodothyronine (fT3), or alternatively, total triiodothyronine (TT3), are also measured, either additionally or subsequently.

[0007] The thyroid hormones triiodothyronine (T3), thyroxine (T4) and their hormonally inactive metabolite reverse triiodothyronine (rT3) are poorly water-soluble and are strongly protein-bound for transport in the bloodstream.

[0008] The three proteins thyroxine-binding globulin (TBG), prealbumin (transthyretin), and albumin, collectively known as T4-binding proteins, are responsible for the reversible binding of T3 and T4, with decreasing proportions in the aforementioned protein order. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0009] Due to the protein binding described, T4 is present in free form in the blood only to about 0.03% and T3 only to about 0.3%.

[0010] Since only the free fraction of thyroid hormones can penetrate the cells to exert their hormonal activity, the free concentration of a thyroid hormone is generally considered more clinically meaningful than its total concentration (i.e., the sum of protein-bound thyroid hormone and free thyroid hormone) in the blood (Thienpont et al., Best Practice & Research Clinical Endocrinology & Metabolism 27 (2013) 689-700; Spencer, Thyroid 33(4) (2023) 407-419).

[0011] Accordingly, fT4 is preferred as a laboratory parameter over the determination of total T4 (TT4), even though in the case of fT4 the analyte concentration is 3000 times lower and the determination here presents a significantly greater challenge.

[0012] In the case of T3, from the theoretical perspective described above, fT3 would also be the better laboratory parameter. However, the fT3 concentration in the blood is three times lower than that of fT4, and existing assays for fT3 have a comparatively high susceptibility to error. Because of this, there are differing opinions in the literature as to whether fT3 or total T3 (TT3) should currently be used as the laboratory parameter (Van Uytfanghe et al., Thyroid 33(9) (2023) 1013-1028; Favresse et al., Endocrine Reviews 39(5) (2018) 830-850).

[0013] That there is still potential for improvement in existing fT4 and fT3 immunoassays, particularly regarding reducing their susceptibility to error, is well documented in the literature. (Külz et al., Clinical Chemistry and Laboratory Medicine 60(6) (2022) 877-885; Westbye et al., Clinical Biochemistry 121-122 (2023) 110676; Favresse et al., Endocrine Reviews 39(5) (2018) 830-850; Thienpont et al., Best Practice & Research Clinical Endocrinology & Metabolism 27 (2013) 689-700). The laboratory parameters fT3, fT4, TT3, and TT4 are almost exclusively determined in routine clinical diagnostics using immunoassays on fully automated testing platforms.

[0014] It is also possible to detect these hormones using LC-MS methods, although in the case of free thyroid hormones, a dialysis or ultrafiltration step must be performed beforehand. However, these methods are not suitable due to the high technical requirements. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0015] They are far from being suitable for routine clinical diagnostics due to the effort involved. However, they are already used as reference methods, e.g., for the standardization of routine immunoassay methods. The IFCC Committee for the Standardization of Thyroid Function Tests (C-STFT) has established a laboratory network that offers these analyses for reference measurements (Vesper et al., Clinica Chimica Acta 519 (2021) 183-186).

[0016] Several assay formats and procedures are used for routine immunoassays of free thyroid hormones, which are well described in the literature (Christophides: Free Analyte Immunoassay, 123-137 from Wild, The Immunoassay Handbook, 2013, fourth edition, Elsevier; Spencer, Thyroid 33(4) (2023) 407-419). A basic distinction can be made between two-step and one-step assays.

[0017] In the two-step assay, the blood sample is first mixed with a reagent containing the anti-thyroid hormone antibody bound to a solid phase. After an incubation period, this assay format includes a wash step that removes the mixture of unbound reagent and sample, leaving only the antibody bound to the solid phase and the substances attached to it. In a second step, a conjugate consisting of a thyroid hormone analog and a tracer substance is added and the sample is incubated again. After this incubation, the sample is washed once more to remove unbound tracer, and the tracer bound to the solid phase is quantified.

[0018] One-step assays typically involve only one wash step after incubation is complete. There are two main ways to combine the assay reagents: the labeled analog tracer method, where the signal-generating component is bound to the thyroid hormone analog, and the labeled antibody method, where the signal-generating component is bound to the antibody. Typically, in a one-step assay, the reagent containing the thyroid hormone analog is added at a later time.

[0019] All three assay formats mentioned have their advantages and disadvantages. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0020] Typically, one-step methods exhibit higher sensitivity and somewhat higher precision due to the fewer assay process steps, the generally longer incubation time of the assay reagents with each other, and, compared to the labeled antibody method, a more homogeneous distribution of the antibody in the solution. However, the disadvantage of the one-step assay is that during assay incubation, the reagent containing the thyroid hormone analog comes into contact with the sample and thus with the T4-binding proteins it contains. These T4-binding proteins naturally have a residual affinity—higher or lower depending on the reagent design—for the thyroid hormone analog of the assay, thereby reducing its activity. This is critical because patient samples can contain very different concentrations of T4-binding proteins. For example, the plasma of pregnant women in the third week of pregnancy contains...During the third trimester of pregnancy, the concentration of the most important T4-binding protein, TBG, is approximately three times higher. Furthermore, hereditary abnormalities in a patient, such as those affecting the albumin sequence, can lead to an altered affinity of this protein for the tracer in that specific patient sample (e.g., familial dysalbuminemic hyperthyroxinemia or analbuminemia). Alternatively, the activity of the analog conjugate can be influenced by autoantibodies present in the sample (Spencer, Thyroid 33(4) (2023) 407-419). Modulation of tracer activity by samples with T4-binding protein concentrations / affinities that deviate from the typical value can lead to an altered assay signal in a 1-step assay, independent of the analyte concentration, and thus to a falsification of the measured value.

[0021] The last-mentioned error mechanism is eliminated in the 2-step assay because the sample, containing its T4-binding proteins, is washed away after the first incubation before the thyroid hormone analog tracer is added. In general, this effectively prevents interference caused by the interaction of sample components with the analog tracer in the 2-step assay.

[0022] Many approaches to preventing the interaction between thyroid hormone analog reagent and T4-binding proteins in the sample can be found in the patent literature for 1-step assay formats. One main route is the steric hindrance of the thyroid hormone analog by binding to a protein, e.g., albumin or HRP, or to the solid phase of the assay. However, according to the authors, these measures only reduce the intensity of the interaction and do not eliminate it completely. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0023] One problem with currently used routine assays is that they employ reagents that can only be reproduced with very limited reproducibility and identical properties. This applies particularly to the two active assay components involved in the formation of the immune complex: the binder and the thyroid analog conjugate.

[0024] On the antibody / binder side, the majority of clinical assays used for the determination of free thyroid hormones employ polyclonal antibodies. Polyclonal antibodies have a disadvantage compared to monoclonal antibodies, for example, that a new animal must be immunized when producing a new batch, and the resulting product will have different properties due to the individually varying immune response in the new organism.

[0025] On the thyroid-analog conjugate side, commercial reagents or reagent precursors are available in which the thyroid analog, usually T3, is covalently linked to a protein, e.g., albumin or HRP. This approach solves two problems at once. First, the solubility of the hormone analogs and the dye used is ensured by the binding to the large protein; second, the thyroid hormone is sterically protected from excessive binding by T4-binding proteins by the binding to the protein. Typically, several T3 groups per protein are non-selectively coupled to some of the existing functional groups, e.g., lysines, of the protein. This method creates an entire population of, for example, (T3) in a single lot of reagents. xAlbumin molecules, in which each individual tracer molecule has a different molar ratio of T3 to protein, and in which the T3 groups are also bound to individual lysine groups of the protein. Since, for example, human serum albumin (HSA) has 59 lysine groups, one can imagine that there are many possible coupling points. To produce a tracer, several dye groups must additionally be coupled to the resulting T3 albumin, so that this heterogeneous diversity also arises for the coupled dye molecules. For a tracer produced in this way, it is not possible to produce an identical product in a new batch. In clinical diagnostics, however, it is particularly important that the reagents have the same properties in each batch, as otherwise costly revalidation procedures would be necessary for each production run.P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH.

[0026] If such revalidation is omitted for new batches that cannot be produced reproducibly, unexpected errors in clinical use are to be expected.

[0027] Given the situation described, there is a need to further improve existing fT4 and fT3 assays. These assays should utilize cost-effective and reproducibly manufactured active components (especially binders and thyroid analog conjugates), exhibit low susceptibility to interference, and deliver high analytical and clinical performance (e.g., sensitivity, accuracy, reproducibility, stability).

[0028] Brief description of the invention

[0029] The present invention relates to a thyronine derivative tracer of formula (II):

[0030] CL-Y-TX

[0031] (II).

[0032] CL stands for a chemiluminescence group.

[0033] Y represents a linear peptide with 3 to 11 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids.

[0034] TX represents a group of formula (III) or (IV):

[0035] Xi, X2, X3 and X4 stand independently for I, H, Br, CI or CN.

[0036] Z1 stands for OH or NH2, and Z2 stands for H or C(O)CH3.

[0037] The present invention also relates to a method for the quantitative determination of a free thyroid hormone. The method comprises providing a blood sample. The method further comprises combining the blood sample with a capture reagent in a reaction vessel to obtain a first mixture, wherein the capture reagent comprises a magnetic particle conjugate, and wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the thyroid hormone to be determined. The method further comprises processing the first mixture. The method further comprises adding a detector reagent to the reaction vessel to obtain a second mixture, wherein the detector reagent comprises a thyronine derivative tracer of formula (II) described herein. The method further comprises processing the second mixture.The procedure further includes inducing chemiluminescence by adding at least one preparation reagent and at least one initiation reagent, and quantifying the chemiluminescence.

[0038] The work-up of the first mixture includes incubating the first mixture, immobilizing the magnetic particle conjugate on the wall of the reaction vessel, removing the liquid phase, and washing the solid phase by repeatedly adding and taking up a wash buffer.

[0039] The work-up of the second mixture includes resuspending the particles of the magnetic particle conjugate in the detector reagent, incubating the second mixture, immobilizing the magnetic particle conjugate on the wall of the reaction vessel, removing the liquid phase, and washing the solid phase by repeatedly dispensing and taking up a wash buffer.

[0040] The present invention also relates to a kit for a heterogeneous immunoassay for the determination of a free thyroid hormone. The kit comprises a capture reagent comprising a magnetic particle conjugate, wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the corresponding thyroid hormone, and a detector reagent comprising a thyronine derivative tracer of the formula (II) described herein.

[0041] Detailed description of the invention

[0042] Within the scope of the present invention, the terms "thyroid hormones" or "thyroid hormones" refer to the iodine-containing hormones 3,3',5-triiodo-L-thyronine (triiodothyronine, T3) and L-thyroxine (thyroxine, T4) produced in the follicular epithelial cells of the thyroid gland (thyrocytes). The terms "thyroid hormones" and "thyroid hormones" are used within the scope of the present invention analogously to P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH. T3 and T4 are present in the blood to a high degree in protein-bound form. Their free fraction (non-protein-bound fraction) in the blood is referred to as free triiodothyronine (fT3) and free thyroxine (fT4), respectively.

[0043] In the context of the present invention, "amino acids" are understood to mean α-amino acids, i.e., organic compounds with an amino group that is directly adjacent to a terminal carboxyl group at the calcium atom. Preferred "amino acids" in the context of the present invention are proteinogenic amino acids, particularly preferably canonical amino acids. "Acidic amino acids" are understood to mean amino acids that have more carboxyl groups than amino groups. Preferred "acidic amino acids" in the context of the present invention are the proteinogenic amino acids aspartic acid (D) and glutamic acid (E). "Basic amino acids" are understood to mean amino acids that have more basic groups than carboxyl groups. Preferred "basic amino acids" in the context of the present invention are the proteinogenic amino acids lysine (K) and arginine (R).

[0044] The term "peptide" refers to a chemical compound formed by linking several amino acids. The individual amino acids of a peptide are linked together via peptide bonds between their α-NH₂ groups and α-carboxy groups. Using the common one-letter symbols for amino acids, the N-terminal amino acid is on the far left, meaning it has a free α-NH₂ group. This amino acid, with its α-carboxy group, is fused via a peptide bond to the α-amino group of the following amino acid, and so on. The last amino acid in this chain, which then has a free α-carboxy group, is called the C-terminal amino acid. For example, the peptide EEEEE, which consists of five fused glutamic acids, has the following chemical structural formula: P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0045] Within the scope of the present invention, the term "tracer abbreviation" refers to the encoding of the chemical structure of the tracers CL-Y-TX (II) according to the invention, as described below. The tracer abbreviation begins with the abbreviation of the chemiluminescent group used, e.g., "NSPSA," which is also defined in the detailed description. The amino acid sequence of peptide Y is described in the tracer abbreviation by the single-letter symbols of the amino acids it contains, the structure and linkage of which are explained under the term "peptide." The bond between chemiluminescent group CL and peptide Y, as well as the bond between thyronine derivative TX and peptide Y, are peptide bonds.

[0046] It is fundamentally possible that the chemiluminescent group is bound to an amino group of peptide Y (preferably the N-terminal amino acid of peptide Y) via a carboxyl group. Similarly, it is fundamentally possible that the thyronine derivative TX is bound to an amino group of peptide Y (preferably the N-terminal amino acid of peptide Y) via a carboxyl group, or that the thyronine derivative TX is bound to a carboxyl group of linear peptide Y (preferably the C-terminal amino acid of peptide Y) via an amino group.

[0047] Preferably, the chemiluminescent group is linked to the α-amino group of the N-terminal amino acid of peptide Y via a peptide bond and a carboxyl group. Similarly, the C-terminal amino acid of linear peptide Y is preferably linked to the thyronine derivative TX via a peptide bond.

[0048] The exact structure of TX is defined in the tracer short formula by specifying the formula group used (for example, (IIIa) to (Ille) or (IVa) to (IVe)), which are defined in the detailed description of the invention. The TX structures of type

[0049] (III) are substituted with a Zi group, which TX structures of type (IV) are substituted with a Z2 group.

[0050] Type (III) TX groups are preferentially linked via a peptide bond to the α-carboxy group of the C-terminal amino acid of peptide Y. Type (III) TX groups

[0051] (IV) are preferentially linked via a peptide bond to an NH2 group of the side chain of the C-terminal amino acid (for lysine, the E-NH2 group) of the linear peptide Y. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0052] Within the scope of the present invention, the Zi and Z2 groups are also specified in the tracer abbreviations. For example, (1llc)OH represents a TX group of the formula (Ille), where Z1 represents OH.

[0053] For illustrative purposes, the structural formulas of two tracer abbreviations are shown as examples:

[0054] - Structural formula for the tracer abbreviation NSPSA-EEEEEEE-(1llc)OH:

[0055] - Structural formula for the tracer abbreviation NSPSA-EKEK-(IVa)C(O)CH3:

[0056] The term "half" refers to one of two equal parts of a whole. For an absolute value, half is obtained by dividing the value by 2. Accordingly, "at least half" of an absolute value means half of that value or more. In the case of an odd integer, the value is rounded up within the scope of the invention P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH. For example, for a value x = 8, "at least half" is 4 or more. For example, for a value x = 9, "at least half" is 5 or more.

[0057] According to the present invention, the term "specific binders" refers to compounds with a high binding affinity to the thyroid hormone to be determined, i.e., either T3 or T4. Within the scope of the present invention, the term "specific binders" includes antibodies, antibody fragments (such as F(ab')2 or Fab fragments), nanobodies, receptors, and aptamers, provided they exhibit a high binding affinity to the thyroid hormone to be determined.

[0058] A thyroid assay according to the present invention specifically detects only one of the two hormones, either T3 or T4. Therefore, for example, in the case of an fT4 assay, the binder should have a high binding affinity to T4, but a significantly lower binding affinity (e.g., at least twice as strong, preferably at least five times as strong, more preferably at least 20 times as strong) to T3. Similarly, in the case of an fT3 assay, the binder should have a high binding affinity to T3, but a significantly lower binding affinity (e.g., at least twice as strong, preferably at least five times as strong, more preferably at least 20 times as strong) to T4.

[0059] Within the scope of the present invention, the term "tracer" refers to active compounds of the immunoassay that contain a component, e.g., a dye, which generates the signal in the immunoassay. Within the scope of the present invention, this dye is a chemiluminescent dye.

[0060] In the context of this invention, ‘chemiluminescent dye’ refers to a chemical compound which can be stimulated to chemiluminescence by the addition of further chemicals, for example compounds from the group of acridinium esters and acridinium sulfonamides.

[0061] According to the present invention, a “high bond strength” is defined as a bond with an affinity constant of at least 1*10 9 l / mol, preferably at least 1*10 1 ° l / mol, further preferably at least 2*10 10 l / mol understood. The affinity constant can be determined, for example, using a Scatchard plot or surface plasmon resonance (e.g., with a Biacore instrument). P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0062] The term "size" in relation to particles refers to the average diameter of the particles used, as measured, for example, by electron microscopy.

[0063] In the context of the present invention, "approximately" means a deviation of less than 20%, e.g., less than 10% or less than 5%, particularly preferably less than 2%. In the context of the present invention, "approximately spherical" means, with regard to the shape of particles, that the largest diameter and the smallest diameter of a particle do not differ from each other by more than 20% (e.g., less than 10% or less than 5%, particularly preferably less than 2%).

[0064] “Quantitative” or “quantitative determination” describes the determination of the amount or concentration of a component, e.g. a thyroid hormone, in a sample.

[0065] The present invention relates to a thyronine derivative tracer of formula (II):

[0066] CL-Y-TX (II).

[0067] CL stands for a chemiluminescence group.

[0068] Y represents a linear peptide with 3 to 11 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids.

[0069] TX represents a group of formula (III) or (IV):

[0070] Xi, X2, X3 and X4 stand independently for I, H, Br, CI or CN.

[0071] Z1 stands for OH or NH2, and Z2 stands for H or C(O)CH3. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0072] In formula (II), Y represents a linear peptide with 3 to 12 amino acids. The linear peptide comprises at least 3, for example, at least 4, at least 5, or at least 6 amino acids. The linear peptide comprises no more than 12, for example, no more than 11, no more than 10, no more than 9, or no more than 8 amino acids. Preferably, the linear peptide comprises 4 to 10 amino acids, and particularly preferably 5 to 8 amino acids.

[0073] Suitable amino acids within the scope of the present invention are L-amino acids and D-amino acids. For example, the amino acids of the linear peptide can be D-amino acids, or the amino acids of the linear peptide can be L-amino acids, or the linear peptide can contain both D- and L-amino acids. Preferably, the amino acids of the linear peptide are D-amino acids. For example, at least one amino acid of the linear peptide can be a D-amino acid. Two, three, or at least half of the amino acids of the linear peptide can be D-amino acids.

[0074] At least half of the amino acids in the linear peptide are acidic amino acids. Preferably, at least half of the amino acids in the linear peptide are glutamic or aspartic acids. For example, more than half of the amino acids in the linear peptide can be glutamic or aspartic acids, or all of the amino acids in the linear peptide can be glutamic or aspartic acids.

[0075] It may also be preferred that the linear peptide is formed from acidic and basic amino acids. Preferably, lysine is included as the basic amino acid.

[0076] The linear peptide can contain 1 to 6 lysines, e.g., 1-5, 1-4, or 1-3 lysines. The linear peptide can, for example, contain at least 2 or at least 3 lysines. The linear peptide can, for example, contain no more than 5, no more than 4, or no more than 3 lysines. The linear peptide can, for example, contain 1, 2, 3, 4, 5, or 6 lysines. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0077] Examples of linear peptides are EKE, EKEKE, EKEKEKE, EKEKEKEKE, EEE, EEEEE, EEEEEEE, EKEKEKEKEKE, EKEKEEKEKEK, EEEEEEEEK, eee, eeeee and eeeeeee, where E stands for L-glutamic acid, e for D-glutamic acid and K for L-lysine.

[0078] In this document, Xi, X2, X3 and X4 independently represent I, H, Br, CI or CN.

[0079] For example, one of Xi, X2, X3, and X4 represents H. Preferably, X2 represents H. For example, two of Xi, X2, X3, and X4 represent H, e.g., X2 represents H and X4 represents H.

[0080] Preferably, at least one of Xi, X2, X3 and X4 represents I, more preferably at least two of Xi, X2, X3 and X4 represent I, and even more preferably three of Xi, X2, X3 and X4 represent I.

[0081] Within the scope of the present invention, it is preferred that Xi, X3 and X4 stand for I and X2 stands for H.

[0082] One or more than one of Xi, X2, X3, and X4 can represent Br, CI, or CN. Preferably, no more than one of Xi, X2, X3, and X4 represents Br, CI, or CN.

[0083] Z1 represents OH or NH2, and Z2 represents H or C(O)CH3. Preferably, Z1 represents NH2.

[0084] Suitable TX groups are, for example, groups of the formula (11)-(11) and (14)-(14): P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0085] In formula (II), CL represents a chemiluminescence group. The chemiluminescence group is based on a chemiluminescent dye that can be linked to the linear peptide via a suitable chemical bond. Preferably, the chemiluminescence group is bound to the N-terminal end of the linear peptide, preferably via an amide bond.

[0086] Suitable chemiluminescent dyes are preferably selected from the group consisting of acridinium ester dyes and acridinium sulfonamide dyes.

[0087] Examples of suitable chemiluminescent dyes are given below, either the chemiluminescent dyes themselves or their N-hydroxysuccinimide (NHS) active esters suitable for coupling: 3-(9-((3-Carboxypropyl)(tosyl)carbamoyl)acridinium-10-yl)propane-1-sulfonate (NSPSA, CAS No. 211106-69-3, published in US5468646), 9-((4-(((2,5-Dioxopyrrolidin-1-yl)oxy)carbonyl)-2,6-dimethylphenoxy)carbonyl)-10-methylacridin-10-ium methyl sulfate (DMAE-NHS, CAS No. 115853-74-2), P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0088] 2',6'-Dimethylcarbonylphenyl-10-sulfopropylacridinium-9-carboxylate-4'-NHS ester (NSP-DMAE-NHS, published in US5656426),

[0089] 3-(9-((4-((23-((2,5-dioxopyrrolidin-1-yl)oxy)-19,23-dioxo-3,6,9,12,15-pentaoxa-18- azatricosyl)carbamoyl)-2,6-dimethylphenoxy)carbonyl)acridin-10-ium-10-yl)propan-1- sulfonat (NSP-DMAE-HEG-Glu-NHS, veröffentlicht in US6664043B2), 2',6'-Dimethyl-4'-carboxyphenyl-2,7-bis[O-methoxyhexa(ethylen)glycol]-10-N-sulfopropyl- acridinium-9-carboxylat (NSP-2,7-(OMHEG)2-DMAE, veröffentlicht in US2005221390A1 / US7309615B2), 2',6'-Dimethyl-4'-carboxyphenyl-2,7-bis[O-methoxyhexa(ethylen)glycol]-10-N-methyl- acridinium-9-carboxylat (2,7-(OMHEG)2-DMAE, veröffentlicht in US2005221390A1 / US7309615B2), 2',6'-Dimethyl-4'-carboxyphenyl-2,7-bis[O-methoxytri(ethylen)glycol]-10-N-sulfopropyl- acridinium-9-carboxylat (NSP-2,7-(OMTEG)2-DMAE, veröffentlicht in US2005221390A1 / US7309615B2), 2'6'-Dimethyl-4'-N-succinimidyl-oxyglutarylamidohexa(ethylen)-glycolamido- carbonylphenyl-2,7-bis[O-methoxytri(ethylen)glycol]-10-sulfopropyl-acridinium-9- carboxylat (NSP-2,7-(OMTEG)2-DMAE-HEG-glutarate-NHS,veröffentlicht in US2005221390A1 / US7309615B2), 2',6'-Dimethyl-4'-N-succinimidyl-oxycaproylamidocarbonylphenyl-2,7-bis[O- methoxy(hexa)ethylenglycol]-10-N-sulfopropyl-acridinium-9-carboxylat (NSP-2,7- (OMHEG)2-DMAE-AC-NHS, veröffentlicht in US2005221390A1 / US7309615B2), 2',6'-Dimethyl-4'-N-succinimidyloxy- glutarylamidohexa(ethylen)glycolamidocarbonylphenyl-2,7-bis[O-methoxy- hexa(ethylen)glycol]-10-sulfopropyl-acridinium-9-carboxylat (NSP-2,7-(OMHEG)2-DMAE- HEG-glutarate-NHS, veröffentlicht in US2005221390A1 / US7309615B2),

[0090] 4-(2-Succinimidyl-oxycarbonylethyl)-phenyl-10-acridinium-9-carboxylat- trifluormethylsulfonat (Acridinium C2-NHS, CAS-Nr. 177332-37-5),

[0091] 9-Acridincarbonsäure-4-[3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl]phenylester (Desmethyl Acridinium-NHS, CAS-Nr. 87198-87-6), 3-(9-((6-(2,5-Dioxopyrrolidin-1-yloxy)-6-oxohexyl)(tosyl)carbamoyl)acridinium-10- yl)propan-1-sulfonat (FD106521-NHS, CAS-Nr. 866366-12-3),

[0092] 10-Methyl-N-butyl-Np-toluenesulfonyl-9-acridinium carboxamide (published in US5468646), 10-Methyl-N-(5-carboxypentyl)-Np-nitrobenzenesulfonyl-9-acridinium carboxamide (published in US5468646), P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0093] 10-(3-Sulfopropyl)-N-tosyl-N-(3-sulfopropyl)-9-acridinium carboxamide (published in

[0094] (NSP-DMAE-Z-NHS, published in US8778624B2).

[0095] The chemiluminescence group is particularly preferably based on 3-(9-((3-

[0096] Carboxypropyl)(tosyl)carbamoyl)acridinium-10-yl)propane-1-sulfonate (NSPSA).

[0097] Examples of T3-based thyronine derivative tracers of formula (II) are given below in the form of the tracer short formula:

[0098] NSPSA-EEEE-(lllc)OH, NSPSA-EEEEEE-(lllc)OH, NSPSA-EEEEEE-(lllc)OH, NSPSA- EEEEEEE-(lllc)OH, NSPSA-EEEEEE-(lllc)OH, NSPSA-EEEE-(lllc)NH2, NSPSA- EEEEE-(lllc)NH2, NSPSA-EEEEEE-(I IIC)NH2, NSPSA-EEEEEE-(II IC)NH2, NSPSA- EEEEEEEE-(II IC)NH2, NSPSA-eeee-(lllc)NH2, NSPSA-eeeeee-(lllc)NH2, NSPSA-eeeeee-(lllc)NH2, NSPSA-eeeeee-(lllc)NH2, NSPSA-eeeeee-(lllc)NH2, NSPSA-eeeeee-(lllc)NH2, NSPSA-eeeeeeee-(lllc)NH2, NSPSA-eeeeeeee-(lllc)NH2, NSPSA-eeeeeeee-(lllc)NH2, NSPSA-eeeeeeee-(lllc)NH2, NSPSA-eeeeeeee-(lllc)NH2, NSPSA-eeeeeeee-(lllc)NH2, NSPSA-eeeeeeee-(lllc)OH, NSPSA-eeeeee-(lllc)OH, NSPSA- eeeeeee-(lllc)OH, NSPSA-eeeeeeee-(lllc)OH, NSPSA-eeeeeeeeee-(lllc)OH, NSPSA-HEG-Glu-EEEE-(lllc)OH, NSP-DMAE-HEG-Glu-EEEEEE-(lllc)OH, NSP- DMAE-HEG-Glu-EEEEEE-(lllc)OH, NSP- DMAE-HEG-Glu-EEEEEE-(lllc)OH, NSP- DMAE-HEG-Glu-EEEEEEEE-(lllc)OH, NSP- DMAE-HEG-Glu-EEEEEE-(lllc)NH2, NSP- DMAE-HEG-Glu-EEEEEE-(lllc)NH2, NSP- DMAE-HEG-GIU-EEEEEE-(I IIC)NH2, NSP- DMAE-HEG-GIU-EEEEEEEE-(II IC)NH2, NSP- DMAE-HEG-GIU-EEEEEEEE-(II IC)NH2, P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0099] NSP-DMAE-HEG-Glu-eeee-(lllc)NH2, NSP-DMAE-HEG-Glu-eeee-(lllc)NH2, NSP-

[0100] DMAE-HEG-Glu-eeeeee-(lllc)NH2, NSP-DMAE-HEG-Glu-eeeeee-(lllc)NH2, NSP-

[0101] DMAE-HEG-Glu-eeeeeeee-(lllc)NH2, NSP-DMAE-HEG-Glu-eeeeeeee-(lllc)NH2, NSP-DMAE-HEG-Glu-eeee-(lllc)OH, NSP-DMAE-HEG-Glu-eeeeee-(lllc)OH, NSP-DMAE- HEG-Glu-eeeeee-(lllc)OH, NSP-DMAE- HEG-Glu-eeeeee-(lllc)OH, NSP-DMAE-HEG-Glu-eeeeee-(lllc)OH, NSP-DMAE-HEG- Glu-eeeeeeee-(lllc)OH, NSP-DMAE-HEG-Glu-eeeeeeee-(lllc)OH, NSP-DMAE-EEEE-(lllc)OH, NSP-DMAE-EEEEEE-(lllc)OH, NSP-DMAE-EEEEEE-(lllc)OH, NSP-DMAE-EEEEEE-(lllc)OH, NSP- DMAE-EEEE-(lllc)NH2, NSP-DMAE-EEEEE-(I IIC)NH2, NSP-DMAE-EEEEEE-(II IC)NH2, NSP-DMAE-EEEEEEEE-(II IC)NH2, NSP-DMAE-EEEEEEEE-(II IC)NH2,

[0102] NSP-DMAE-eeee-(lllc)NH2, NSP-DMAE-eeeeee-(lllc)NH2, NSP-DMAE-eeeeee-(lllc)NH2, NSP-DMAE-eeeeee-(lllc)NH2, NSP-DMAE-eeeeee-(lllc)NH2, NSP-DMAE- eeeeeee-(lllc)NH2, NSP-DMAE-eeee-(lllc)NH2, NSP-DMAE-eeee-(lllc)OH, NSP-DMAE-eeee-(lllc)OH, NSP-DMAE-eeeeee-(lllc)OH, NSP-DMAE-eeeeee-(lllc)OH, NSP-DMAE-eeeeee-(lllc)OH, NSP-DMAE-eeeeeeee-(lllc)OH, NSP-DMAE-eeeeee-(lllc)OH, NSP-DMAE-2,7-(OMHEG)2-DMAE-EEEE-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-EEEE-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE- EEEEEEE-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(II IC)NH2, NSP- 2,7-(OMHEG)2-DMAE-EEEEEE-(II IC)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(I IIC)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(I IIC)NH2, NSP-2,7-(OMHEG)2-DMAE-eeee-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeeeee-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeeeee-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE- eeeeeeee-(lllc)NH2, NSP-2,7-(OMHEG)2-DMAE-eeee-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllc)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeeeee-(l I lc)OH, NSPSA-EEEEEK-(IVc)H, NSPSA-EEEEEEEK-(IVc)H, NSPSA-EEEEEEEK-(IVc)H, NSPSA-EEEEEEEK-(IVc)H, NSPSA-eeeeK-(IVc)C(O)CH3, NSPSA-eeeeeeK-(IVc)C(O)CH3, NSPSA-eeeeeeK-(IVc)C(O)CH3, NSPSA-eeeeeeK-(IVc)C(O)CH3, NSPSA-eeeeeeeeK-(IVc)C(O)CH3, NSPSA-eeeeeeeeK-(IVc)C(O)CH3, P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH,

[0103] NSPSA-eeeeK-(IVc)H, NSPSA-eeeeeK-(IVc)H, NSPSA-eeeeeeK-(IVc)H, NSPSA- eeeeeeeK-(IVc)H, NSPSA-eeeeeeeeK-(IVc)H, NSPSA-eeeeeeeeeK-(IVc)H, NSP-DMAE-HEG-Glu-EEEEK-(IVc)H, NSP-DMAE-HEG-Glu-EEEEEK-(IVc)H, NSP- DMAE-HEG-Glu-EEEEEEK-(IVc)H, NSP-DMAE-HEG-Glu-EEEEEEEK-(IVc)H, NSP- DMAE-HEG-Glu-EEEEEEEEK-(IVc)H, NSP-DMAE-HEG-Glu-eeeeK-(IVc)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeK-(IVc)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeK-

[0104] (IVc)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVc)C(O)CH3, NSP-DMAE-HEG-Glu- eeeeeeeeK-(IVc)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVc)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeK-(IVc)H, NSP-DMAE-HEG-Glu-eeeeeK-(IVc)H, NSP-DMAE- HEG-Glu-eeeeeeK-(IVc)H, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVc)H, NSP-DMAE-HEG- Glu-eeeeeeeeK-(IVc)H, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVc)H, NSP-DMAE-EEEEK-(IVc)H, NSP-DMAE-EEEEEK-(IVc)H, NSP-DMAE-EEEEEEK- (IVc)H, NSP-DMAE-EEEEEEEK-(IVc)H, NSP-DMAE-EEEEEEEEK-(IVc)H, NSP-DMAE- eeeeK-(IVc)C(O)CH3, NSP-DMAE-eeeeeK-(IVc)C(O)CH3, NSP-DMAE-eeeeeeK- (IVc)C(O)CH3, NSP-DMAE-eeeeeeeK-(IVc)C(O)CH3, NSP-DMAE-eeeeeeeeK- (IVc)C(O)CH3, NSP-DMAE-eeeeeeeeeK-(IVc)C(O)CH3, NSP-DMAE-eeeeK-(IVc)H, NSP-DMAE-eeeeeK-(IVc)H, NSP-DMAE-eeeeeeK-(IVc)H, NSP-DMAE-eeeeeeeK-(IVc)H, NSP-DMAE-eeeeeeeeK-(IVc)H, NSP-DMAE- eeeeeeeeeK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE-EEEEK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEK- (IVc)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE- EEEEEEEK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEEK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE-eeeeK-(IVc)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeK-,

[0105] (IVc)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVc)C(O)CH3, NSP-2,7- (OMHEG)2-DMAE-eeeeeeeK-(IVc)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK- (IVc)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeeK-(IVc)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeK- (IVc)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK-(IVc)H, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeeeK-(IVc)H.

[0106] Examples of rT3-based thyronine derivative tracers of formula (II) are given below in the form of the tracer short formula:

[0107] NSPSA-EEEE-(111)OH, NSPSA-EEEEEE-(111)OH, NSPSA-EEEEEE-(111)OH, NSPSA- EEEEEEE-(111)OH, NSPSA-EEEEEEEE-(111)OH, NSPSA-EEEE-(111)NH2, NSPSA- P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0108] EEEEE-(llld)NH2, NSPSA-EEEEEE-(llld)NH2, NSPSA-EEEEEEE-(llld)NH2, NSPSA- EEEEEEEE-(llld)NH2, NSPSA-eeee-(llld)NH2, NSPSA-eeeee-(llld)NH2, NSPSA-eeeeee- (llld)NH2, NSPSA-eeeeeee-(llld)NH2, NSPSA-eeeeeeee-(llld)NH2, NSPSA-eeeeeeeee- (llld)NH2, NSPSA-eeee-(llld)OH, NSPSA-eeeee-(llld)OH, NSPSA-eeeeee-(llld)OH, NSPSA- eeeeeee-(llld)OH, NSPSA-eeeeeeee-(llld)OH, NSPSA-eeeeeeeee-(llld)OH, NSP-DMAE-HEG-Glu-EEEE-(llld)OH, NSP-DMAE-HEG-Glu-EEEEE-(llld)OH, NSP- DMAE-HEG-Glu-EEEEEE-(llld)OH, NSP-DMAE-HEG-Glu-EEEEEEE-(llld)OH, NSP- DMAE-HEG-Glu-EEEEEEEE-(llld)OH, NSP-DMAE-HEG-Glu-EEEE-(llld)NH2, NSP- DMAE-HEG-Glu-EEEEE-(llld)NH2, NSP-DMAE-HEG-Glu-EEEEEE-(llld)NH2, NSP- DMAE-HEG-Glu-EEEEEEE-(llld)NH2, NSP-DMAE-HEG-Glu-EEEEEEEE-(llld)NH2, NSP-DMAE-HEG-Glu-eeee-(llld)NH2, NSP-DMAE-HEG-Glu-eeeee-(llld)NH2, NSP- DMAE-HEG-Glu-eeeeee-(llld)NH2, NSP-DMAE-HEG-Glu-eeeeeee-(llld)NH2, NSP- DMAE-HEG-Glu-eeeeeeee-(llld)NH2, NSP-DMAE-HEG-Glu-eeeeeeeee-(llld)NH2, NSP-DMAE-HEG-Glu-eeee-(llld)OH,NSP-DMAE-HEG-Glu-eeeee-(llld)OH, NSP-DMAE- HEG-Glu-eeeeee-(llld)OH, NSP-DMAE-HEG-Glu-eeeeeee-(llld)OH, NSP-DMAE-HEG- Glu-eeeeeeee-(llld)OH, NSP-DMAE-HEG-Glu-eeeeeeeee-(llld)OH, NSP-DMAE-EEEE-(llld)OH, NSP-DMAE-EEEEE-(llld)OH, NSP-DMAE-EEEEEE- (llld)OH, NSP-DMAE-EEEEEEE-(llld)OH, NSP-DMAE-EEEEEEEE-(llld)OH, NSP- DMAE-EEEE-(llld)NH2, NSP-DMAE-EEEEE-(llld)NH2, NSP-DMAE-EEEEEE-(llld)NH2, NSP-DMAE-EEEEEEE-(llld)NH2, NSP-DMAE-EEEEEEEE-(llld)NH2, NSP-DMAE-eeee-(llld)NH2, NSP-DMAE-eeeee-(llld)NH2, NSP-DMAE-eeeeee- (llld)NH2, NSP-DMAE-eeeeeee-(llld)NH2, NSP-DMAE-eeeeeeee-(llld)NH2, NSP- DMAE-eeeeeeeee-(llld)NH2, NSP-DMAE-eeee-(llld)OH, NSP-DMAE-eeeee-(llld)OH, NSP-DMAE-eeeeee-(llld)OH, NSP-DMAE-eeeeeee-(llld)OH, NSP-DMAE-eeeeeeee-(llld)OH, NSP-DMAE-eeeeeeeee- (llld)OH, NSP-2,7-(OMHEG)2-DMAE-EEEE-(llld)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEE- (llld)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(llld)OH, NSP-2,7-(OMHEG)2-DMAE- EEEEEEE-(llld)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(llld)OH, NSP-2,7- (OMHEG)2-DMAE-EEEEEE-(llld)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(llld)NH2, NSP- 2,7-(OMHEG)2-DMAE-EEEEEE-(llld)NH2, NSP-2.7-(OMHEG)2-DMAE-EEEEEEEE-(llld)NH2, NSP-2.7-(OMHEG)2- DMAE-eeee-(llld)NH2, NSP-2.7-(OMHEG)2-DMAE-eeeee-(llld)NH2, NSP-2.7- (OMHEG)2-DMAE-eeeeee-(llld)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeeee-(llld)NH2, P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH,

[0109] NSP-2,7-(OMHEG)2-DMAE-eeeeeeee-(llld)NH2, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeee-(llld)NH2, NSP-2,7-(OMHEG)2-DMAE-eeee-(llld)OH, NSP-2,7-(OMHEG)2-DMAE-eeeee-(llld)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llld)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeeee- (llld)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeeeee-(llld)OH, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeee-(l I ld)OH , NSPSA-EEEEK-(IVd)H, NSPSA-EEEEEK-(IVd)H, NSPSA-EEEEEEK-(IVd)H, NSPSA- EEEEEEEK-(IVd)H, NSPSA-EEEEEEEEK-(IVd)H, NSPSA-eeeeK-(IVd)C(O)CH3, NSPSA-eeeeeK-(IVd)C(O)CH3, NSPSA-eeeeeeK-(IVd)C(O)CH3, NSPSA-eeeeeeeK- (IVd)C(O)CH3, NSPSA-eeeeeeeeK-(IVd)C(O)CH3, NSPSA-eeeeeeeeeK-(IVd)C(O)CH3, NSPSA-eeeeK-(IVd)H, NSPSA-eeeeeK-(IVd)H, NSPSA-eeeeeeK-(IVd)H, NSPSA- eeeeeeeK-(IVd)H, NSPSA-eeeeeeeeK-(IVd)H, NSPSA-eeeeeeeeeK-(IVd)H, NSP-DMAE-HEG-Glu-EEEEK-(IVd)H, NSP-DMAE-HEG-Glu-EEEEEK-(IVd)H, NSP- DMAE-HEG-Glu-EEEEEEK-(IVd)H, NSP-DMAE-HEG-Glu-EEEEEEEK-(IVd)H, NSP- DMAE-HEG-Glu-EEEEEEEEK-(IVd)H, NSP-DMAE-HEG-Glu-eeeeK-(IVd)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeK-(IVd)C(O)CH3,NSP-DMAE-HEG-Glu-eeeeeeK-,

[0110] (IVd)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVd)C(O)CH3, NSP-DMAE-HEG-Glu- eeeeeeeeK-(IVd)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVd)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeK-(IVd)H, NSP-DMAE-HEG-Glu-eeeeeK-(IVd)H, NSP-DMAE- HEG-Glu-eeeeeeK-(IVd)H, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVd)H, NSP-DMAE-HEG- Glu-eeeeeeeeK-(IVd)H, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVd)H, NSP-DMAE-EEEEK-(IVd)H, NSP-DMAE-EEEEEK-(IVd)H, NSP-DMAE-EEEEEEK- (IVd)H, NSP-DMAE-EEEEEEEK-(IVd)H, NSP-DMAE-EEEEEEEEK-(IVd)H, NSP-DMAE- eeeeK-(IVd)C(O)CH3, NSP-DMAE-eeeeeK-(IVd)C(O)CH3, NSP-DMAE-eeeeeeK- (IVd)C(O)CH3, NSP-DMAE-eeeeeeeK-(IVd)C(O)CH3, NSP-DMAE-eeeeeeeeK- (IVd)C(O)CH3, NSP-DMAE-eeeeeeeeeK-(IVd)C(O)CH3, NSP-DMAE-eeeeK-(IVd)H, NSP-DMAE-eeeeeK-(IVd)H, NSP-DMAE-eeeeeeK-(IVd)H, NSP-DMAE-eeeeeeeK-(IVd)H, NSP-DMAE-eeeeeeeeK-(IVd)H, NSP-DMAE- eeeeeeeeeK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE-EEEEK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEK- (IVd)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE- EEEEEEEK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEEK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE-eeeeK-(IVd)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeK-,

[0111] (IVd)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVd)C(O)CH3, NSP-2,7- P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0112] (OMHEG)2-DMAE-eeeeeeeK-(IVd)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK- (IVd)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeeK-(IVd)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeK- (IVd)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK-(IVd)H, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeeeK-(IVd)H.

[0113] Examples of T2-based thyronine derivative tracers of formula (II) are given below in the form of the tracer short formula:

[0114] NSPSA-EEEE-(llla)OH, NSPSA-EEEEEE-(llla)OH, NSPSA-EEEEEE-(llla)OH, NSPSA- EEEEEEE-(llla)OH, NSPSA-EEEEEEEE-(llla)OH, NSPSA-EEEE-(llla)NH2, NSPSA- EEEEE-(llla)NH2, NSPSA-EEEEEE-(llla)NH2, NSPSA-EEEEEE-(llla)NH2, NSPSA-EEEEEE-(llla)NH2, NSPSA-eeee-(llla)NH2, NSPSA-eeeeee-(llla)NH2, NSPSA-eeeeee-(llla)NH2, NSPSA-eeeeee-(llla)NH2, NSPSA-eeeeee-(llla)NH2, NSPSA-eeeeee-(llla)NH2, NSPSA-eeeeeeee-(llla)NH2, NSPSA-eeeeeeee-(llla)NH2, NSPSA-eeeeeeee-(llla)NH2, NSPSA-eeeeeeee-(llla)NH2, NSPSA-eeeeeeee-(llla)NH2, NSPSA-eeeeeeee-(llla)NH2, NSPSA-eeeeeeee-(llla)OH, NSPSA-eeeeee-(llla)OH, NSPSA- eeeeeee-(llla)OH, NSPSA-eeeeeeee-(llla)OH, NSPSA-eeeeeeee-(llla)OH, NSP-DMAE-HEG-Glu-EEEE-(llla)OH, NSP-DMAE-HEG-Glu-EEEEEE-(llla)OH, NSP-

[0115] DMAE-HEG-Glu-EEEEEE-(llla)OH, NSP-DMAE-HEG-Glu-EEEEEE-(llla)OH, NSP-

[0116] DMAE-HEG-Glu-EEEEEEEE-(llla)OH, NSP-DMAE-HEG-Glu-EEEE-(llla)NH2, NSP-

[0117] DMAE-HEG-Glu-EEEEE-(llla)NH2, NSP-DMAE-HEG-Glu-EEEEEE-(llla)NH2, NSP-

[0118] DMAE-HEG-Glu-EEEEEE-(llla)NH2, NSP-DMAE-HEG-Glu-EEEEEEEE-(llla)NH2, NSP-DMAE-HEG-Glu-eeee-(llla)NH2, NSP-DMAE-HEG-Glu-eeee-(llla)NH2, NSP-

[0119] DMAE-HEG-Glu-eeeeee-(llla)NH2, NSP-DMAE-HEG-Glu-eeeeee-(llla)NH2, NSP-

[0120] DMAE-HEG-Glu-eeeeeeee-(llla)NH2, NSP-DMAE-HEG-Glu-eeeeeeee-(llla)NH2, NSP-DMAE-HEG-Glu-eeee-(llla)OH, NSP-DMAE-HEG-Glu-eeeeee-(llla)OH, NSP-DMAE- HEG-Glu-eeeeee-(llla)OH, NSP-DMAE- HEG-Glu-eeeeee-(llla)OH, NSP-DMAE-HEG-Glu-eeeeee-(llla)OH, NSP-DMAE-HEG- Glu-eeeeeeee-(llla)OH, NSP-DMAE-HEG-Glu-eeeeeeee-(llla)OH, NSP-DMAE-EEEE-(llla)OH, NSP-DMAE-EEEEEE-(llla)OH, NSP-DMAE-EEEEEE-(llla)OH, NSP-DMAE-EEEEEE-(llla)OH, NSP-DMAE-EEEEEE-(llla)OH, NSP- DMAE-EEEE-(llla)NH2, NSP-DMAE-EEEEEE-(llla)NH2, NSP-DMAE-EEEEEE-(llla)NH2, NSP-DMAE-EEEEEE-(llla)NH2, NSP-DMAE-EEEEEEEE-(llla)NH2, NSP-DMAE-eeee-(llla)NH2, NSP-DMAE-eeee-(llla)NH2, NSP-DMAE-eeeeee-(llla)NH2, NSP-DMAE-eeeeee-(llla)NH2, NSP-DMAE-eeeeee-(llla)NH2, NSP-DMAE-eeeeee-(llla)NH2, NSP-DMAE-eeeeeeee-(llla)NH2, NSP- DMAE-eeeeeeee-(llla)NH2, P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0121] NSP-DMAE-eeee-(llla)OH, NSP-DMAE-eeeeee-(llla)OH, NSP-DMAE-eeeeee-(llla)OH, NSP-DMAE-eeeeee-(llla)OH, NSP-DMAE-eeeeeeee-(llla)OH, NSP-DMAE-eeeeeeee-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-EEEE-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(llla)OH, NSP-2,7-(OMHEG)2-DMAE- EEEEEEE-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-EEEE-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEE-(llla)NH2, NSP- 2,7-(OMHEG)2-DMAE-EEEEEE-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-eeee-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE- eeeeeeee-(llla)NH2, NSP-2,7-(OMHEG)2-DMAE-eeee-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(llla)OH,NSP-2,7-(OMHEG)2-DMAE- eeeeeeeee-(l I la)OH ,

[0122] NSPSA-EEEEK-(IVa)H, NSPSA-EEEEEK-(IVa)H, NSPSA-EEEEEEEK-(IVa)H, NSPSA- EEEEEEEK-(IVa)H, NSPSA-EEEEEEEK-(IVa)H, NSPSA-EEEEEEEK-(IVa)H, NSPSA-eeeeK-(IVa)C(O)CH3, NSPSA-eeeeeeK-(IVa)C(O)CH3, NSPSA-eeeeeeK-(IVa)C(O)CH3, NSPSA-eeeeeeeeK-(IVa)C(O)CH3, NSPSA-eeeeeeeeK-(IVa)C(O)CH3, NSPSA-eeeeeeeeK-(IVa)H, NSPSA-eeeeeeK-(IVa)H, NSPSA-eeeeeeK-(IVa)H, NSPSA-eeeeeeK-(IVa)H, NSPSA-eeeeeeK-(IVa)H, NSP-DMAE-HEG-Glu-EEEEK-(IVa)H, NSP-DMAE-HEG-Glu-EEEEEEK-(IVa)H, NSP- DMAE-HEG-Glu-EEEEEEK-(IVa)H, NSP-DMAE-HEG-Glu-EEEEEEK-(IVa)H, NSP- DMAE-HEG-Glu-EEEEEEEEK-(IVa)H, NSP-DMAE-HEG-Glu-eeeeK-(IVa)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeK-(IVa)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeK-

[0123] (IVa)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVa)C(O)CH3, NSP-DMAE-HEG-Glu- eeeeeeeeK-(IVa)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVa)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeK-(IVa)H, NSP-DMAE-HEG-Glu-eeeeeK-(IVa)H, NSP-DMAE- HEG-Glu-eeeeeeK-(IVa)H, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVa)H, NSP-DMAE-HEG- Glu-eeeeeeeeK-(IVa)H, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVa)H, NSP-DMAE-EEEEK-(IVa)H, NSP-DMAE-EEEEEK-(IVa)H, NSP-DMAE-EEEEEEK-(IVa)H, NSP-DMAE-EEEEEEEK-(IVa)H, NSP-DMAE-EEEEEEEK-(IVa)H, NSP-DMAE- P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH eeeeK-(IVa)C(O)CH3, NSP-DMAE-eeeeeK-(IVa)C(O)CH3, NSP-DMAE-eeeeeeK- (IVa)C(O)CH3, NSP-DMAE-eeeeeeeK-(IVa)C(O)CH3, NSP-DMAE-eeeeeeeK-(IVa)C(O)CH3, NSP-DMAE-eeeeeeeeK- (IVa)C(O)CH3, NSP-DMAE-eeeeeeeeeK-(IVa)C(O)CH3, NSP-DMAE-eeeeK-(IVa)H, NSP-DMAE-eeeeeK-(IVa)H, NSP-DMAE-eeeeeeK-(IVa)H, NSP-DMAE-eeeeeeeK-(IVa)H, NSP-DMAE-eeeeeeeeK-(IVa)H, NSP-DMAE- eeeeeeeeeK-(IVa)H, NSP-2,7-(OMHEG)2-DMAE-EEEEK-(IVa)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEK-(IVa)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEK-(IVa)H, NSP-2,7-(OMHEG)2-DMAE- EEEEEEEK-(IVa)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEEK-(IVa)H, NSP-2,7- (OMHEG)2-DMAE-eeeeK-(IVa)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeK-,

[0124] (IVa)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVa)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVa)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK-(IVa)C(O)CH3, , NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVa)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK-(IVa)H, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeeeK-(IVa)H, NSPSA-EEEE-(lllb)OH, NSPSA-EEEE-(lllb)OH, NSPSA-EEEEEE-(lllb)OH, NSPSA- EEEEEEE-(lllb)OH, NSPSA-EEEEEE-(lllb)OH, NSPSA-EEEE-(lllb)NH2, NSPSA- EEEEE-(lllb)NH2, NSPSA-EEEEEE-(lllb)NH2, NSPSA-EEEEEEEE-(lllb)NH2, NSPSA- EEEEEEEE-(lllb)NH2, NSPSA-eeeeee-(lllb)NH2, NSPSA-eeeeee-(lllb)NH2, NSPSA-eeeeee-(lllb)NH2, NSPSA-eeeeee-(lllb)NH2, NSPSA-eeeeee-(lllb)NH2, NSPSA-eeeeeeee-(lllb)NH2, NSPSA-eeee-(lllb)OH, NSPSA-eeeeee-(lllb)OH, NSPSA-eeeeee-(lllb)OH, NSPSA- eeeeeee-(lllb)OH, NSPSA-eeeeeeee-(lllb)OH, NSPSA-eeeeeeee-(lllb)OH, NSPSA-eeeeeee-(lllb)OH, NSPSA-DMAE-HEG-Glu-EEEE-(lllb)OH,, NSP-DMAE-HEG-Glu-EEEEEE-(lllb)NH2, NSP- DMAE-HEG-Glu-EEEEEEE-(lllb)NH2, NSP-DMAE-HEG-Glu-EEEEEEEE-(lllb)NH2,

[0125] NSP-DMAE-HEG-Glu-eeee-(lllb)NH2, NSP-DMAE-HEG-Glu-eeeeee-(lllb)NH2, NSP- DMAE-HEG-Glu-eeeeee-(lllb)NH2, NSP-DMAE-HEG-Glu-eeeeee-(lllb)NH2, NSP- DMAE-HEG-Glu-eeeeeeee-(lllb)NH2, NSP- DMAE-HEG-Glu-eeeeeeee-(lllb)NH2, NSP- DMAE-HEG-Glu-eeeeeeee-(lllb)NH2, P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0126] NSP-DMAE-HEG-Glu-eeee-(lllb)OH, NSP-DMAE-HEG-Glu-eeeee-(lllb)OH, NSP-DMAE- HEG-Glu-eeeeee-(lllb)OH, NSP-DMAE-HEG-Glu-eeeeeee-(lllb)OH, NSP-DMAE-HEG- Glu-eeeeeeee-(lllb)OH, NSP-DMAE-HEG-Glu-eeeeeeeee-(lllb)OH, NSP-DMAE-EEEE-(lllb)OH, NSP-DMAE-EEEEE-(lllb)OH, NSP-DMAE-EEEEEE- (lllb)OH, NSP-DMAE-EEEEEEE-(lllb)OH, NSP-DMAE-EEEEEEEE-(lllb)OH, NSP- DMAE-EEEE-(lllb)NH2, NSP-DMAE-EEEEE-(lllb)NH2, NSP-DMAE-EEEEEE-(lllb)NH2, NSP-DMAE-EEEEEEE-(lllb)NH2, NSP-DMAE-EEEEEEEE-(lllb)NH2, NSP-DMAE-eeee-(lllb)NH2, NSP-DMAE-eeeee-(lllb)NH2, NSP-DMAE-eeeeee- (lllb)NH2, NSP-DMAE-eeeeeee-(lllb)NH2, NSP-DMAE-eeeeeeee-(lllb)NH2, NSP- DMAE-eeeeeeeee-(lllb)NH2, NSP-DMAE-eeee-(lllb)OH, NSP-DMAE-eeeee-(lllb)OH, NSP-DMAE-eeeeee-(lllb)OH, NSP-DMAE-eeeeeee-(lllb)OH, NSP-DMAE-eeeeeeee-(lllb)OH, NSP-DMAE-eeeeeeeee- (lllb)OH, NSP-2,7-(OMHEG)2-DMAE-EEEE-(lllb)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEE- (lllb)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEE-(lllb)OH, NSP-2,7-(OMHEG)2-DMAE- EEEEEEE-(lllb)OH, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(lllb)OH,NSP-2,7- (OMHEG)2-DMAE-EEEE-(lllb)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEE-(lllb)NH2, NSP- 2,7-(OMHEG)2-DMAE-EEEEEE-(lllb)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEEE- (lllb)NH2, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEE-(lllb)NH2, NSP-2,7-(OMHEG)2- DMAE-eeee-(lllb)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeee-(lllb)NH2, NSP-2,7- (OMHEG)2-DMAE-eeeeee-(lllb)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeeee-(lllb)NH2, NSP-2,7-(OMHEG)2-DMAE-eeeeeeee-(lllb)NH2, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeee-(lllb)NH2, NSP-2,7-(OMHEG)2-DMAE-eeee-(lllb)OH, NSP-2,7-(OMHEG)2-DMAE-eeeee-(lllb)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeee-(lllb)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeeee- (lllb)OH, NSP-2,7-(OMHEG)2-DMAE-eeeeeeee-(lllb)OH, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeee-(l I lb)OH, NSPSA-EEEEK-(IVb)H, NSPSA-EEEEEK-(IVb)H, NSPSA-EEEEEEK-(IVb)H, NSPSA- EEEEEEEK-(IVb)H, NSPSA-EEEEEEEEK-(IVb)H, NSPSA-eeeeK-(IVb)C(O)CH3, NSPSA-eeeeeK-(IVb)C(O)CH3, NSPSA-eeeeeeK-(IVb)C(O)CH3, NSPSA-eeeeeeeK- (IVb)C(O)CH3, NSPSA-eeeeeeeeK-(IVb)C(O)CH3, NSPSA-eeeeeeeeeK-(IVb)C(O)CH3, NSPSA-eeeeK-(IVb)H,NSPSA-eeeeeK-(IVb)H, NSPSA-eeeeeeK-(IVb)H, NSPSA- eeeeeeeK-(IVb)H, NSPSA-eeeeeeeeK-(IVb)H, NSPSA-eeeeeeeeeK-(IVb)H, NSP-DMAE-HEG-Glu-EEEEK-(IVb)H, NSP-DMAE-HEG-Glu-EEEEEK-(IVb)H, NSP-DMAE-HEG-Glu-EEEEEEK-(IVb)H, NSP-DMAE-HEG-Glu-EEEEEEEK-(IVb)H, NSP-P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH,

[0127] DMAE-HEG-Glu-EEEEEEEEK-(IVb)H, NSP-DMAE-HEG-Glu-eeeeK-(IVb)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeK-(IVb)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeK-

[0128] (IVb)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVb)C(O)CH3, NSP-DMAE-HEG-Glu- eeeeeeeeK-(IVb)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVb)C(O)CH3, NSP-DMAE-HEG-Glu-eeeeK-(IVb)H, NSP-DMAE-HEG-Glu-eeeeeK-(IVb)H, NSP-DMAE- HEG-Glu-eeeeeeK-(IVb)H, NSP-DMAE-HEG-Glu-eeeeeeeK-(IVb)H, NSP-DMAE-HEG- Glu-eeeeeeeeK-(IVb)H, NSP-DMAE-HEG-Glu-eeeeeeeeeK-(IVb)H, NSP-DMAE-EEEEK-(IVb)H, NSP-DMAE-EEEEEK-(IVb)H, NSP-DMAE-EEEEEEK- (IVb)H, NSP-DMAE-EEEEEEEK-(IVb)H, NSP-DMAE-EEEEEEEEK-(IVb)H, NSP-DMAE- eeeeK-(IVb)C(O)CH3, NSP-DMAE-eeeeeK-(IVb)C(O)CH3, NSP-DMAE-eeeeeeK- (IVb)C(O)CH3, NSP-DMAE-eeeeeeeK-(IVb)C(O)CH3, NSP-DMAE-eeeeeeeeK- (IVb)C(O)CH3, NSP-DMAE-eeeeeeeeeK-(IVb)C(O)CH3, NSP-DMAE-eeeeK-(IVb)H, NSP-DMAE-eeeeeK-(IVb)H, NSP-DMAE-eeeeeeK-(IVb)H, NSP-DMAE-eeeeeeeK-(IVb)H, NSP-DMAE-eeeeeeeeK-(IVb)H, NSP-DMAE- eeeeeeeeeK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE-EEEEK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEK- (IVb)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE- EEEEEEEK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE-EEEEEEEEK-(IVb)H, NSP-2,7- (OMHEG)2-DMAE-eeeeK-(IVb)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeK-,

[0129] (IVb)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVb)C(O)CH3, NSP-2,7- (OMHEG)2-DMAE-eeeeeeeK-(IVb)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK- (IVb)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeeK-(IVb)C(O)CH3, NSP-2,7-(OMHEG)2-DMAE-eeeeK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeK- (IVb)H, NSP-2,7-(OMHEG)2-DMAE-eeeeeeeeK-(IVb)H, NSP-2,7-(OMHEG)2-DMAE- eeeeeeeeeK-(IVb)H.

[0130] The present invention relates to a method for the quantitative determination of a free thyroid hormone. The method comprises: a. providing a blood sample (human serum, human plasma); b. combining the blood sample with a capture reagent in a reaction vessel to obtain a first mixture, wherein the capture reagent comprises a magnetic particle conjugate, and wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the thyroid hormone to be determined (either T3 or T4); c. processing the first mixture; d. adding a detector reagent to the reaction vessel to obtain a second mixture, wherein the detector reagent comprises a thyronine derivative tracer of formula (II); e. processing the second mixture; f.Triggering the chemiluminescence of the thyroid derivative tracer bound to the magnetic particle conjugate by adding at least one preparation reagent and at least one initiation reagent; g. measuring the chemiluminescence; and h. quantitatively determining the free thyroid hormone.

[0131] A thyronine derivative tracer of formula (II) has the structure

[0132] CL-Y-TX

[0133] (II).

[0134] It says

[0135] CL for a chemiluminescence group,

[0136] Y for a linear peptide with 3 to 11 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids, and

[0137] TX for a group of formula (III) or (IV)

[0138] In this, Xi, X2, X3 and X4 stand independently for I, H, Br, CI or CN, Z1 for OH or NH2 and Z2 for H or C(O)CHs .

[0139] The method according to the present invention enables the quantitative determination of a free thyroid hormone, i.e., fT3 or fT4. The method is a heterogeneous two-step immunoassay. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0140] Step a.

[0141] The blood sample is preferably human serum or human plasma. The blood sample may be cell-free, in particular cell-free serum or plasma samples. Cell-free serum samples can be obtained by drawing a blood sample, initiating coagulation (e.g., by adding a coagulation activator or using a blood collection tube containing coagulation activators), incubating, centrifuging, and separating the liquid phase. Plasma samples can be obtained by drawing a blood sample, preventing coagulation (e.g., by adding an anticoagulant such as EDTA or using an EDTA or heparin blood collection tube), centrifuging, and separating the liquid phase.

[0142] Step b.

[0143] The blood sample is combined with a capture reagent in a reaction vessel to obtain an initial mixture.

[0144] The capture reagent is preferably in liquid form. Methods for combining two components present in a liquid medium are well known to those skilled in the art. Preferably, the two components are pipetted one after the other into a reaction vessel and then mixed, for example by moving the reaction vessel by vortexing, swirling, or shaking, or by mixing the contained liquid volume by stirring or repeated aspiration and discharge.

[0145] The capture reagent comprises a magnetic particle conjugate. The magnetic particle conjugate includes a magnetic particle and a specific binder of the thyroid hormone T3 or T4 to be determined. By combining the blood sample with the capture reagent, the thyroid hormone T3 or T4 to be determined binds to the specific binder of the magnetic particle conjugate.

[0146] The specific binder exhibits a high binding affinity for the thyroid hormone being measured – either T3 or T4. The binding affinity is considered high if the affinity constant of the binder for the thyroid hormone being measured is greater than or equal to 1 x 10⁻⁶. 9 l / mol. Preferably, the specific binder has an affinity constant for the thyroid hormone to be determined of at least 1*10 1 ° l / mol, further preferably of at least 2*10 10 l / mol. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0147] Preferably, the specific binder of the thyroid hormone T3 or T4 to be determined is selected from the group consisting of antibodies, antibody fragments, nanobodies, receptors, and aptamers. Particularly preferably, the specific binder is an antibody.

[0148] Both monoclonal and polyclonal antibodies are suitable. A monoclonal antibody is preferred. Examples of suitable antibodies include the monoclonal anti-T4 antibody MAT02-525 (clone 204-14525) distributed by Meridian Bioscience and the monoclonal antibody T5460-01G (clone 9L720) distributed by US Biological Life Sciences. Both products cite Bellisario, R., et al., (2000), Clinical Chemistry, 46 (9), 1422-1424 in their data sheets. Examples of antibody fragments include F(ab')2 fragments or Fab fragments.

[0149] Preferably, the specific binder is coupled to the magnetic particle via a spacer.

[0150] For example, the specific binder has a first binding unit and the magnetic particle a second binding unit, wherein the first and second binding units can bond to each other. The binding units can bond to each other covalently or non-covalently, preferably non-covalently.

[0151] The specific binder can be covalently linked to the first binding unit, preferably via a linker. Preferably, the specific binder is covalently linked to biotin via a linker. The linker can, for example, consist of one or more PEG units and can optionally also include a dye that, after coupling, allows the determination of the average number of linkers per binder. Examples of suitable biotin-linker-dye conjugates that are NHS-activated for coupling to the antibody are the products Sulfo ChromaLink Biotin (Catalog # B-1007) and ChromaLink Biotin (Catalog # B-1001), such as those offered by Vector Laboratories.

[0152] The magnetic particle can be coated with the second bonding unit. Preferably, the magnetic particles have a surface coated with streptavidin. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0153] The specific binder is preferably coupled to the magnetic particle via a non-covalent biotin-streptavidin bond.

[0154] The magnetic particles and the specific binders are preferably present in the magnetic particle conjugate in a mass ratio of 25:1 to 2500:1. For example, the magnetic particles and the specific binders are present in a mass ratio of at least 30:1, at least 50:1, at least 75:1, at least 100:1, or at least 125:1. For example, the magnetic particles and the specific binders are present in a mass ratio of not more than 2000:1, at least 1500:1, at least 1000:1, at least 500:1, or at least 400:1. Particularly preferably, the magnetic particles and the specific binders are present in a mass ratio of 150:1 to 350:1.

[0155] The magnetic particles can have a size of 0.5 to 8 pm, e.g. from 1 to 5 pm, preferably from 2 to 3 pm.

[0156] The magnetic particles can have any shape. They are preferably approximately spherical.

[0157] Preferably, the capture reagent comprises a buffer. Suitable buffers are, for example, phosphate-buffered salt solutions (PBS buffers). The buffer concentration can be in the range of 5 to 200 mM. Suitable pH values ​​are, for example, in the range of 6.5 to 8.2, preferably 6.8 to 7.8, and particularly preferably 7.0 to 7.4.

[0158] The capture reagent may additionally contain a preservative, such as sodium azide, as well as an immunoglobulin. Optionally, it may also contain one or more detergents and / or one or more proteins, such as albumin.

[0159] Preferably, the capture reagent comprises an albumin, e.g., human serum albumin (HSA), bovine serum albumin (BSA), recombinant albumin, delipidated albumin, or albumin purified with activated charcoal; more preferably, recombinant albumin (rHSA) or albumin purified with activated charcoal. The detector reagent most preferably comprises recombinant HSA (rHSA). P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0160] Step c.

[0161] The first mixture is worked up. The work-up of the first mixture comprises at least i. incubating the first mixture; ii. immobilizing the magnetic particle conjugate on the wall of the reaction vessel; iii. removing the liquid phase; and iv. washing the solid phase by repeatedly depositing and taking up a wash buffer.

[0162] According to step i., the first mixture is incubated. Preferably, the first mixture is incubated at 37°C. During incubation, the first mixture can either be left at rest or kept in motion, for example, by stirring, shaking, swirling, or vortexing. Stirring, shaking, or vortexing can be performed continuously or intermittently.

[0163] Preferably the incubation time of the first mixture is 1 min to 60 min, e.g. 5 to 40 min or 10 to 30 min, particularly preferably 15 to 20 min.

[0164] Incubation can, in principle, be carried out under various light conditions, for example, in complete darkness, with ambient light, or with irradiation. Incubation in complete darkness is preferred.

[0165] According to step ii, the magnetic particle conjugate is immobilized on the wall of the reaction vessel. This can be done, for example, by applying a magnetic field to the wall of the reaction vessel.

[0166] According to step iii, the liquid phase is removed. In principle, the liquid phase can be removed by any method known to those skilled in the art, for example by pipetting or suction. Preferably, the liquid phase is removed by suction.

[0167] According to step iv., the solid phase is washed by repeated addition and removal of a wash buffer. For example, washing is performed 3x, 4x, 5x, or 6x. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0168] The washing buffer used is preferably suitable for removing components that are not part of the magnetic particle conjugate to which the optionally determined thyroid hormone T3 or T4 is bound. Examples include components from the sample, including the thyroid hormones T3 and T4 not bound to the magnetic particle conjugate, especially in protein-bound form, the T4-binding proteins contained in the sample, and components of the capture reagent that are not part of the magnetic particle conjugate.

[0169] Preferably, the wash buffer is a phosphate buffer or a PBS buffer. The preferred pH value of the wash buffer is in the range of 7.0 to 7.5.

[0170] Preferably, the washing buffer comprises a detergent. The detergent can be anionic, cationic, non-ionic, or zwitterionic. Detergents are well known to those skilled in the art. Examples of anionic detergents are alkylbenzenesulfonates with an alkyl group having 8 to 24 carbon atoms, alkylsulfonates with an alkyl group having 8 to 24 carbon atoms, and alkali salts of fatty acids. Examples of cationic detergents are tetraalkylammonium salts with at least one alkyl group having 8 to 24 carbon atoms (e.g., distearyldimethylammonium chloride or cetyltrimethylammonium bromide) and esterquats based on quaternary triethanol-methylammonium or quaternary diethanol-dimethylammonium compounds esterified with fatty acids. Examples of non-ionic detergents are esters or ethers made from polyoxyethylenes and aliphatic alcohols or carboxylic acids, e.g., polyoxyethylene-23-lauryl ether.Examples of zwitterionic detergents are CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) and CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate). The wash buffer preferably comprises a non-ionic detergent.

[0171] The washing buffer may also include other components, such as preservatives.

[0172] Washing the solid phase according to step iv. may include resuspending and reimmobilizing the particles of the magnetic particle conjugate in the wash buffer. This may, for example,

[0173] - by removing the magnetic field from the wall of the reaction vessel, mixing the solid phase in the washing buffer (e.g. by vortexing, shaking or swirling) and P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0174] - a magnetic field is reapplied to the wall of the reaction vessel.

[0175] Step d.

[0176] A detector reagent is added to the reaction vessel, resulting in a second mixture.

[0177] The detector reagent comprises a thyronine derivative tracer of formula (II). The preferred and exemplary features of the thyronine derivative tracer of formula (II) described above apply accordingly to the thyronine derivative tracer of formula (II) used in this procedure.

[0178] Preferably, Xi, X2, X3, and X4 represent I or H independently of one another. Preferably, at least one of Xi, X2, X3, and X4 represents I; more preferably, at least two of Xi, X2, X3, and X4 represent I. Particularly preferably, Xi, X3, and X4 represent I and X2 represents H.

[0179] The thyronine derivative tracer of formula (II) can be selected depending on the thyroid hormone to be determined; preferably, the thyronine derivative of the thyronine derivative tracer has one fewer iodine substituent than the thyroid hormone to be determined. For example, a T3- or rT3-based tracer, i.e., a tracer of the TX group of one of the formulas described above (Ille), (II Id), (IVc), or (IVd), is preferred for the determination of fT4, and a T2-based tracer, i.e., a tracer of the TX group of one of the formulas described above (Illa), (Illb), (IVa), or (IVb), is preferred for the determination of fT3.

[0180] Preferably, the detector reagent comprises an albumin, e.g., human serum albumin (HSA), bovine serum albumin (BSA), recombinant albumin, or albumin purified with activated charcoal; more preferably, recombinant albumin (rHSA) or albumin purified with activated charcoal. Particularly preferably, the detector reagent comprises recombinant HSA (rHSA).

[0181] The detector reagent contains albumin (preferably rHSA), preferably in an amount of 0.1 to 5 wt.%, particularly preferably in an amount of 0.5 to 2 wt.%, in each case based on the total mass of the detector reagent. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0182] Preferably, the albumin contained in the detector reagent, the capture reagent or the detector and capture reagent is recombinant albumin or albumin purified with activated carbon.

[0183] The detector reagent is preferably in liquid form. Preferably, the detector reagent comprises a buffer. Suitable buffers are, for example, phosphate-buffered salt solutions (PBS buffer) or tris(hydroxymethyl)aminomethane buffer (TRIS buffer). The buffer concentration can be in the range of 5 to 200 mM. Suitable pH values ​​are, for example, in the range of 6.5 to 9, preferably 6.8 to 8.0, and particularly preferably 7.0 to 7.5.

[0184] The detector reagent contains the thyronine derivative tracer of formula (II) preferably in a concentration of 0.1 to 400 ng / mL, e.g. from 1 to 200 ng / mL or from 5 to 100 ng / mL or from 10 to 70 ng / mL or from 12 to 50 ng / mL, particularly preferably from 15-25 ng / mL.

[0185] The detector reagent preferably contains at least one additive. Examples of suitable additives are stabilizers, antioxidants, detergents, mono- or disaccharides, or combinations thereof. The detector reagent preferably comprises a stabilizer, particularly preferably EDTA. The detector reagent preferably comprises an antioxidant, particularly preferably 4-methoxyphenol. The detector reagent preferably comprises a stabilizer and an antioxidant, particularly preferably EDTA and 4-methoxyphenol. The detector reagent may additionally contain a preservative, such as sodium azide, and an immunoglobulin.

[0186] Preferably, the detector reagent is provided as a two-component system. In such a two-component system, at least the thyronine derivative tracer and the albumin are spatially separated from each other before the addition of the detector reagent in step d.

[0187] The thyronine derivative tracer and the albumin can each be provided in buffers. For example, the thyronine derivative tracer and the albumin can be provided in buffers of different pH values. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0188] In this case, the thyronine derivative tracer can be prepared in a buffer with a slightly acidic pH. The thyronine derivative tracer exhibits higher stability in slightly acidic medium than in basic medium.

[0189] In this case, the albumin can be provided in a buffer with a neutral or slightly alkaline pH. When combining the components, a pH value in the range of 7.0 to 8.5, e.g., 7.0 to 8.0, is preferably achieved. One of the two components may have a higher buffering capacity, so that this significantly influences the resulting pH value after mixing the components.

[0190] Step e.

[0191] The second mixture is worked up. The work-up of the second mixture comprises at least i) resuspending the particles of the magnetic particle conjugate in the detector reagent; ii) incubating the second mixture; iii) immobilizing the magnetic particle conjugate on the wall of the reaction vessel; iv) removing the liquid phase; and v) washing the solid phase by repeated addition and removal of a wash buffer.

[0192] According to step i), the particles of the magnetic particle conjugate are resuspended in the detector reagent. This can be achieved by removing the magnetic field from the wall of the reaction vessel and subsequently mixing the components within the vessel. Mixing can be accomplished, for example, by vortexing, agitating, or shaking the reaction vessel, by stirring, or by repeatedly aspirating and dispensing the second mixture. Stirring, shaking, agitating, or vortexing can be performed continuously or intermittently.

[0193] According to step ii), the second mixture is incubated. Preferably, the second mixture is incubated at 37 °C. During incubation, the second mixture can, in principle, be at rest or kept in motion, for example, stirred, shaken, vortexed, or swirled. Stirring, shaking, swirling, or vortexing can be performed continuously or intermittently. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0194] Preferably, the incubation time of the second mixture is 1 min to 60 min, e.g. 2 to 20 min or 2 to 10 min, particularly preferably 3 to 5 min.

[0195] Incubation can, in principle, be carried out under various light conditions, for example, in complete darkness, with ambient light, or with irradiation. Incubation in complete darkness is preferred.

[0196] According to step iii), the magnetic particle conjugate is immobilized on the wall of the reaction vessel. This can be done, for example, by applying a magnetic field to the wall of the reaction vessel.

[0197] According to step iv), the liquid phase is removed. In principle, the liquid phase can be removed by any method known to those skilled in the art, for example by pipetting or suction. Preferably, the liquid phase is removed by suction.

[0198] According to step v), the solid phase is washed by repeatedly adding and taking up a wash buffer. For example, it is washed 3x, 4x, 5x, or 6x.

[0199] The washing buffer used is preferably suitable for removing components that are not part of the magnetic particle conjugate to which the thyroid hormone T3 or T4 to be determined and the thyronine derivative tracer are bound. Examples include unbound components of the detector reagent, in particular unbound thyronine derivative tracer.

[0200] Preferably, the wash buffer is a phosphate buffer or a PBS buffer. The preferred pH value of the wash buffer is in the range of 7.0 to 7.5.

[0201] Preferably, the washing buffer comprises a detergent. The detergent can be anionic, cationic, non-ionic, or zwitterionic. Detergents are well known to those skilled in the art. Examples of anionic detergents are alkylbenzenesulfonates with an alkyl group having 8 to 24 carbon atoms, alkylsulfonates with an alkyl group having 8 to 24 carbon atoms, and alkali salts of fatty acids. Examples of cationic detergents are tetraalkylammonium salts with at least one alkyl group having 8 to 24 carbon atoms (e.g., distearyldimethylammonium chloride or cetyltrimethylammonium bromide) and esterquats based on fatty acid-esterified quaternary triethanol-methylammonium or quaternary diethanol-dimethylammonium compounds. Examples of non-ionic detergents are esters or ethers made from polyoxyethylenes and aliphatic alcohols or carboxylic acids, e.g., polyoxyethylene-23-lauryl ether.Examples of zwitterionic detergents are CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) and CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate). The wash buffer preferably comprises a non-ionic detergent.

[0202] Washing the solid phase according to step v) may involve resuspending and reimmobilizing the particles of the magnetic particle conjugate in the wash buffer. This can be done, for example, by

[0203] Removing the magnetic field from the wall of the reaction vessel,

[0204] Mixing the solid phase in the washing buffer (e.g. by vortexing, shaking or swirling, or by stirring up when releasing the liquid volume) and

[0205] - a magnetic field is reapplied to the wall of the reaction vessel.

[0206] Step f.

[0207] The determination of free thyroid hormone in the sample is carried out by measuring the chemiluminescence of the thyroid derivative tracer bound to the magnetic particle conjugate. Chemiluminescence is triggered by adding at least one preparation reagent and at least one initiation reagent to the solid phase obtained in step e.

[0208] Preferably, the preparation reagent comprises an aqueous solution containing an acid (e.g., HNO3) and H2O2. Preferably, the H2O2 is present in a significant molar excess compared to the thyronine derivative tracer used.

[0209] Preferably, the initiation reagent comprises an aqueous solution containing a base (e.g., NaOH) and a detergent. Suitable detergents include, for example, those described in the context of the wash buffer. Preferably, the initiation reagent is also present in a significant molar excess compared to the thyronine derivative tracer used. Preferably, the amount of base in the initiation reagent is present in a significant molar excess compared to the amount of acid in the preparation reagent. Preferably, the pH value after addition of the P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0210] Initiation reagent in strongly basic solutions, e.g. at pH above 10 or pH above 11, particularly preferably in a pH range of 12 to 13.

[0211] The mechanism of triggering chemiluminescence can be illustrated by example as in Natrajan (Org. Biomol. Chem. 12 (2014) 3887-3901) for NSP-containing acridinium esters:

[0212] The presence of a detergent can increase the signal strength and accelerate the response.

[0213] Preferably, the initiation of chemiluminescence comprises: a. the addition of the preparation reagent, b. the mixing and resuspension of the magnetic particle conjugate in the preparation reagent and P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH c. the addition of the initiation reagent.

[0214] For example, mixing while resuspending the magnetic particle conjugate in the preparation reagent can be achieved by

[0215] Removal of the magnetic field from the wall of the reaction vessel and mixing of the magnetic particle conjugate in the preparation reagent (e.g. by rapid addition of the preparation reagent, vortexing, shaking, swirling or repeated aspiration and dispensing with a pipette).

[0216] Mixing preferably takes place over a period of 0.1 s to 20 s, e.g. more than 0.1 s or more than 0.5 s or more than 1 s, e.g. up to 20 s or up to 10 s or up to 5 s or up to 3 s.

[0217] It is assumed that the bond between the thyronine derivative tracer and the magnetic particle conjugate dissolves in the acidic preparation reagent environment, so that the thyronine derivative tracer is no longer bound to the magnetic particle. Optionally, the magnetic particles can be re-immobilized on the wall of the reaction vessel by applying a magnetic field. By removing the magnetic particles from the light path to the detector, the signal strength of the chemiluminescence can be increased.

[0218] The solution of the initiation reagent is preferably added as quickly as possible, for example in a fast stream, to ensure rapid mixing of the components.

[0219] Step q.

[0220] Chemiluminescence is measured using a light detector, preferably a photomultiplier.

[0221] Chemiluminescence is preferably measured for a limited period, beginning immediately after the addition of the initiation reagent. Chemiluminescence can be measured within 20 s, e.g., within 10 s, preferably within 5 s, after the addition of the initiation reagent. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0222] Step h.

[0223] Preferably, the free thyroid hormone is quantified by comparison with a calibration curve.

[0224] To determine the calibration curve, several calibrator samples are measured, which, due to their different free thyroid hormone concentrations, generate different chemiluminescence signals in the described procedure.

[0225] Determining the reference concentrations of calibrator solutions is more complex for free hormones than for many other analytes, as the free hormone concentration depends on the thyroid hormone concentration in the blood, the concentration of individual T4-binding proteins, and other potential factors, such as the presence of other substances that interact with the binding sites of these proteins. The IFCC Committee for the Standardization of Thyroid Function Tests (C-STFT) addresses this problem and presents possible solutions (https: / / www.ifcc-cstft.org / , De Grande, Clinical Chemistry 63(10) (2017) 1642-1652). Furthermore, cost-effective calibration matrices for clinical use do not always behave identically to clinical samples.Therefore, the manufacturer must assign reference values ​​to the calibrators that ensure that clinical samples read from the calibration curve yield correct clinical, ideally standardized, results for their assay.

[0226] Typically, up to 10 calibrators, preferably 2 to 6 calibrators, and more preferably 2 or 6 calibrators, are measured within the measuring range of the free thyroid assay for assay calibration. Each calibrator exhibits different, assigned free thyroid concentrations distributed across the assay's measuring range.

[0227] A typical calibration range for fT4 is, for example, 0-100 pmol / l fT4. A typical calibration range for fT3 is, for example, 0-50 pmol / l fT3. However, the lower limit of the calibration range can also be higher, and the upper limit of the calibration range can also be higher or lower.

[0228] After measuring the calibrators with the free thyroid assay, the pairs of values ​​for the corresponding concentrations and the assay signal can be plotted graphically, and a curve can be fitted across all calibrators. Typically, 4PL curves (Four Parameter Logistic Regression) are used for the non-linear fit. Since a non-linear relationship must be assumed, the typical calibration function must also be known when using only 2 or 3 calibrators and is then scaled based on the 2-3 measured pairs of values.

[0229] To quantitatively determine the corresponding free thyroid hormone in a sample, the measured chemiluminescence of the sample can be quantified into the corresponding free thyroid hormone concentration using a calibration curve determined in this way.

[0230] Therefore, the quantitative determination of free thyroid hormone preferably includes

[0231] (i) the determination of chemiluminescence in two to ten calibrator solutions, wherein the calibrator solutions contain the thyroid hormone to be determined in different concentrations and at least one T4-binding protein, and wherein the calibrator solutions have been assigned a suitable reference value for the free thyroid hormone to be determined for this assay, for example as described above;

[0232] (ii) Creating a calibration curve based on the associated free hormone concentrations of the calibrator solutions and the determined chemiluminescence; and

[0233] (iii) Quantifying the thyroid hormone by comparing the chemiluminescence of a blood sample measured according to step g. and the calibration curve prepared under (ii).

[0234] The procedure described here, or one or more of its steps a. to h., can be carried out semi-automatically or fully automatically.

[0235] The present invention also relates to a kit for a heterogeneous immunoassay for the determination of a free thyroid hormone. The kit comprises i. a capture reagent comprising a magnetic particle conjugate, wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the corresponding thyroid hormone; and ii. a detector reagent comprising a thyronine derivative tracer of formula (II) as described above. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0236] The preferred and exemplary embodiments of the capture and detector reagent described in the procedure also apply accordingly to the capture and detector reagent contained in the kit.

[0237] The preferred and exemplary features of the thyronine derivative tracer of formula (II) described above apply accordingly to the thyronine derivative tracer of formula (II) used in this procedure.

[0238] Preferably, Xi, X2, X3, and X4 represent I or H independently of one another. Preferably, at least one of Xi, X2, X3, and X4 represents I; more preferably, at least two of Xi, X2, X3, and X4 represent I. Particularly preferably, Xi, X3, and X4 represent I and X2 represents H.

[0239] Preferably, the detector reagent is a 2-component system as described in the procedure.

[0240] Preferably, the kit further comprises a washing buffer. The washing buffer preferably comprises a detergent. The preferred and exemplary embodiments of the washing buffer described in the process also apply accordingly to the washing buffer contained in the kit.

[0241] Preferably, the kit also includes a calibration component. A calibration curve can be generated using the calibration component. The calibration component contains calibrator stages containing the thyroid hormone to be determined in various concentrations. Each calibrator stage of the calibration component corresponds to a free concentration of the thyroid hormone to be determined. The calibration component can be provided in liquid or lyophilized form. The calibrator stages of the calibration component preferably contain a calibration fluid in which at least the thyroid hormone to be determined is present in various concentrations in a liquid medium, e.g., a serum-based matrix or an aqueous buffer containing at least one of the T4-binding proteins. If the calibration component is in lyophilized form, it must be reconstituted with a suitable aqueous medium, e.g., a serum-based matrix or an aqueous buffer containing at least one of the T4-binding proteins, before use.Water, to be resuspended. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH.

[0242] Preferably, the calibration solutions with the different free thyroid concentrations are available in ready-to-use form.

[0243] P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0244] Exemplary embodiments

[0245] 1. Thyronine derivative tracer of formula (II)

[0246] CL-Y-TX

[0247] (H) where

[0248] CL stands for a chemiluminescence group,

[0249] Y represents a linear peptide consisting of 3 to 12 amino acids, where at least half of the amino acids of the linear peptide are acidic.

[0250] Amino acids are, and

[0251] TX for a group of formula (III) or (IV)

[0252] (Hl) (IV) stands where Xi, X2, X3 and X4 independently represent I, H, Br, CI or CN,

[0253] Z1 stands for OH or NH2 and

[0254] Z2 stands for H or C(O)CH3.

[0255] 2. The thyronine derivative tracer according to embodiment 1, wherein the amino acids

[0256] D-Amino acids are.

[0257] 3. The thyronine derivative tracer according to one of the preceding embodiments, wherein the linear peptide comprises 5 to 8 amino acids. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0258] 4. The thyronine derivative tracer according to one of the preceding embodiments, wherein at least half of the amino acids of the linear peptide are glutamic acids or aspartic acids.

[0259] 5. The thyronine derivative tracer according to one of the preceding embodiments, wherein the linear peptide comprises 1 to 6 lysines.

[0260] 6. The thyronine derivative tracer according to one of the foregoing embodiments, wherein Xi, X2, X3 and X4 independently represent I or H.

[0261] 7. The thyronine derivative tracer according to one of the preceding embodiments, wherein at least one of Xi, X2, X3 and X4 represents I, preferably at least two of Xi, X2, X3 and X4 represent I.

[0262] 8. The thyronine derivative tracer according to one of the foregoing embodiments, wherein Xi, X3 and X4 represent I and X2 represents H.

[0263] 9. The thyronine derivative tracer according to one of the foregoing embodiments, wherein Z1 represents NH2.

[0264] 10. The thyronine derivative tracer according to one of the foregoing embodiments, wherein CL is a chemiluminescence group based on a chemiluminescence dye selected from the group consisting of acridinium ester dyes and acridinium sulfonamide dyes.

[0265] 11. The thyronine derivative tracer according to one of the preceding embodiments, wherein CL and the N-terminal amino acid of the linear peptide Y are linked by a peptide bond, and wherein TX and the C-terminal amino acid of the linear peptide Y are linked by a peptide bond.

[0266] 12. The thyronine derivative tracer according to one of the preceding embodiments, wherein the C-terminal amino acid of the linear peptide Y is linked to TX via the C-terminal carboxyl group or via an E-NH2 group by means of a peptide bond. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0267] 13. Method for the quantitative determination of a free thyroid hormone, comprising: a. providing a blood sample; b. combining the blood sample with a capture reagent in a reaction vessel to obtain a first mixture, wherein the capture reagent comprises a magnetic particle conjugate, and wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the thyroid hormone to be determined; c. working up the first mixture, comprising at least: i. incubating the first mixture; ii. immobilizing the magnetic particle conjugate on the wall of the reaction vessel; iii. removing the liquid phase; and iv. washing the solid phase by repeated addition and removal of a wash buffer; d. adding a detector reagent to the reaction vessel to obtain a second mixture, wherein the detector reagent comprises a thyronine derivative tracer of formula (II) CL-Y-TX

[0268] (H) includes, wherein

[0269] CL stands for a chemiluminescence group,

[0270] Y represents a linear peptide consisting of 3 to 12 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids, and

[0271] TX for a group of formula (III) or (IV)

[0272] (Hl) (IV) stands, P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH where Xi, X2, X3 and X4 independently stand for I, H, Br, CI or CN,

[0273] Z1 stands for OH or NH2 and

[0274] Z2 stands for H or C(O)CH3; e. Work-up of the second mixture comprising at least i) resuspending the particles of the magnetic particle conjugate in the detector reagent; ii) incubating the second mixture; iii) immobilizing the magnetic particle conjugate on the wall of the reaction vessel; iv) removing the liquid phase; and v) washing the solid phase by repeated addition and removal of a wash buffer; f. initiating the chemiluminescence of the thyroid derivative tracer bound to the magnetic particle conjugate by adding at least one preparation reagent and at least one initiation reagent; g. measuring the chemiluminescence; and h. quantitatively determining the free thyroid hormone.

[0275] 14. The method according to embodiment 13, wherein the thyronine derivative tracer is a thyronine derivative tracer according to one of embodiments 1 to 12.

[0276] 15. The method according to one of embodiments 13 or 14, wherein Xi, X2, X3 and X4 independently represent I or H.

[0277] 16. The method according to one of embodiments 13 to 15, wherein at least one of Xi, X2, X3 and X4 represents I, preferably at least two of Xi, X2, X3 and X4 represent I.

[0278] 17. The method according to one of embodiments 13 to 16, wherein the detector reagent comprises albumin, preferably recombinant albumin or albumin purified with activated carbon. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0279] 18. The method according to embodiment 17, wherein the detector reagent comprises albumin in an amount of 0.1 to 5% based on the total mass of detector reagent.

[0280] 19. The method according to one of embodiments 13 to 18, wherein the specific binder of the thyroid hormone to be determined is selected from the group consisting of an antibody, an antibody fragment, a nanobody, a receptor and an aptamer.

[0281] 20. The method according to one of embodiments 13 to 19, wherein the detector reagent is provided as a 2-component system.

[0282] 21. The method according to one of embodiments 13 to 20, wherein the specific binder is a monoclonal antibody.

[0283] 22. The method according to one of embodiments 13 to 21, wherein the specific binder is coupled to the magnetic particle via a spacer.

[0284] 23. The method according to embodiment 22, wherein the specific binder is coupled to the magnetic particle via a non-covalent biotin-streptavidin bond.

[0285] 24. The method according to one of embodiments 13 to 23, wherein the magnetic particles have a surface coated with streptavidin.

[0286] 25. The method according to one of embodiments 13 to 24, wherein the specific binder is covalently bound to biotin via a linker, preferably consisting of PEG units, optionally further comprising a dye for determining the binding ratio of the linker to the antibody.

[0287] 26. The method according to one of embodiments 13 to 25, wherein the magnetic particles and the specific binders are present in a mass ratio of 25:1 to 2500:1 in the magnetic particle conjugate.

[0288] 27. The method according to one of embodiments 13 to 26, wherein the magnetic particles are approximately spherical. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0289] 28. The method according to one of embodiments 13 to 27, wherein the magnetic particles have a size of 0.5 to 8 pm.

[0290] 29. The method according to one of embodiments 13 to 28, wherein the capture reagent comprises an albumin, preferably recombinant albumin or albumin purified with activated carbon.

[0291] 30. The method according to embodiment 29, wherein the capture reagent comprises 0.1 to 5 wt.% albumin, based on the total mass of capture reagent.

[0292] 31. The method according to one of embodiments 13 to 30, wherein the capture reagent comprises a buffer.

[0293] 32. The method according to one of embodiments 13 to 31, wherein the capture reagent has a pH value in the range of 6.5 to 8.2.

[0294] 33. The method according to one of embodiments 13 to 32, wherein the detector reagent comprises a buffer.

[0295] 34. The method according to one of embodiments 13 to 33, wherein the thyronine derivative tracer is contained in the detector reagent in a concentration of 0.1 to 400 ng / mL.

[0296] 35. The method according to one of embodiments 13 to 34, wherein the detector reagent comprises at least one additive.

[0297] 36. The method according to one of embodiments 13 to 35, wherein the incubation time according to step i. is 1 min to 60 min.

[0298] 37. The method according to one of embodiments 13 to 36, wherein the incubation time according to step ii) is 30 s to 10 min P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0299] 38. The method according to one of embodiments 13 to 37, wherein the liquid phase is removed according to step iii. or iv) or both by pipetting or by suction.

[0300] 39. The method according to one of embodiments 13 to 38, wherein the washing buffer according to step iv. or v) or both comprises a detergent.

[0301] 40. The method according to one of embodiments 13 to 39, wherein the washing of the solid phase according to step iv. or v) comprises resuspending and reimmobilizing the particles of the magnetic particle conjugate in the washing buffer.

[0302] 41. The method according to one of embodiments 13 to 40, wherein the preparation reagent is an aqueous solution comprising HNO3 and H2O2.

[0303] 42. The method according to one of embodiments 13 to 41, wherein the initiation reagent is an aqueous solution comprising NaOH and a detergent.

[0304] 43. The method according to one of embodiments 13 to 42, wherein the initiation of chemiluminescence comprises: a. adding the preparation reagent; b. mixing while resuspending the magnetic particle conjugate in the preparation reagent; and c. adding the initiation reagent.

[0305] 44. The method according to one of embodiments 13 to 43, wherein the chemiluminescence is quantified using a photomultiplier.

[0306] 45. The method according to one of embodiments 13 to 44, wherein the free thyroid hormone to be determined is fT4.

[0307] 46. ​​The method according to one of embodiments 13 to 44 wherein the free thyroid hormone to be determined is fT3.

[0308] 47. The method according to one of embodiments 13 to 46, wherein the quantitative determination of free thyroid hormone according to step h. comprises: P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0309] (i) Determining the chemiluminescence in 2 to 10 calibrator solutions, wherein the calibrator solutions contain the thyroid hormone to be determined in different concentrations and at least one T4-binding protein, and wherein a concentration of the free thyroid hormone to be determined is assigned to the calibrator solutions;

[0310] (ii) Creating a calibration curve based on the associated free thyroid hormone concentrations of the calibrator solutions and the determined chemiluminescence; and

[0311] (iii) Quantifying the thyroid hormone by comparing the chemiluminescence of a blood sample measured according to step g. and the calibration curve prepared under (ii).

[0312] 48. Kit for a heterogeneous immunoassay for the determination of a free thyroid hormone comprising i. a capture reagent comprising a magnetic particle conjugate, wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the corresponding thyroid hormone; and ii. a detector reagent comprising a thyronine derivative tracer of formula (II)

[0313] CL-Y-TX

[0314] (H) where

[0315] CL stands for a chemiluminescence group,

[0316] Y represents a linear peptide consisting of 3-12 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids, and

[0317] TX for a group of formula (III) or (IV)

[0318] (Hl) (IV) P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH stands, where Xi, X2, X3 and X4 independently stand for I, H, Br, CI or CN,

[0319] Z1 stands for OH or NH2 and

[0320] Z2 stands for H or C(O)CH3.

[0321] 49. The kit according to embodiment 48, further comprising iii. a washing buffer, wherein the washing buffer comprises a detergent.

[0322] 50. The kit according to embodiment 48 or 49, wherein the thyronine derivative tracer is a thyronine derivative tracer according to one of embodiments 1 to 12.

[0323] 51. The kit according to one of embodiments 48 to 50, wherein the detector reagent is a 2-component system.

[0324] 52. The kit according to one of embodiments 48 to 51, wherein Xi, X2, X3 and X4 independently represent I or H.

[0325] 53. The kit according to one of embodiments 48 to 52, wherein at least one of Xi, X2, X3 and X4 represents I, preferably at least two of Xi, X2, X3 and X4 represent I.

[0326] 54. The kit according to one of embodiments 48 to 53, further comprising iv. at least one calibration component containing the thyroid hormone to be determined, wherein the calibration component is associated with a free thyroid hormone concentration of the hormone to be determined.

[0327] P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0328] Examples

[0329] The examples show the execution of a method according to the invention for the quantitative determination of fT4. This assay is a quantitative, heterogeneous, competitive immunoassay, which is carried out as a two-step assay.

[0330] The chemiluminescence measurement was performed on the Mithras LB 943 multimode microplate reader.

[0331] All immunoassay tests were performed on a fully automated immunoassay laboratory analyzer. Unless otherwise specified, all assay steps and incubation times were carried out at 37°C.

[0332] Production of capture reaction and magnetic particle conjugate

[0333] In the examples, only the anti-T4 antibody MAT02-525 (clone 204-14525) distributed by Meridian Bioscience was used as the antibody. It was covalently conjugated to biotin via a PEG linker containing a UV dye using Sulfo ChromaLink Biotin (SCLB) (catalog # B-1007), such as that distributed by Vector Laboratories. For this purpose, the antibody was diluted with PBS to a concentration of 2 mg / mL, and Sulfo ChromaLink Biotin in anhydrous DMSO was added in a 26-fold molar excess. The mixture was then incubated in the dark for 1 hour. A 1000-fold molar excess of glycine, based on SCLB, was added as a stopping solution, and the antibody-SCLB conjugate was subsequently purified using a gel filtration column (HiTrap Desalting type, 5 ml, GE Healthcare Bio-Sciences AB). An Äkta Purifier 10 from Amersham Pharmacia Biotech was used as the chromatography system.

[0334] In the examples, only Dynabeads M-270 Streptavidin from Invitrogen were used as magnetic particles. These magnetic particles have a particle size of 2.8 pm (± < 5%) and are functionalized on their surface with carboxylic acids to which streptavidin is covalently bound.

[0335] The magnetic particles were washed twice with PBS buffer and then incubated in PBS buffer with the antibody-SCLB conjugate for 1.5 h at room temperature, shaken. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0336] The concentration of the magnetic particles and the antibody-SCLB conjugate at this coupling step corresponded to the later final concentration in the reagent recipe.

[0337] As noted in the example, the saturation of the beads with biotin was carried out after the coupling step. Biotin was added to the incubation buffer at a final concentration of 0.3 pg / mL per bead concentration of 1 mg / mL (e.g., 0.075 pg / mL biotin for a bead concentration of 0.25 mg / mL) and shaken again for 1.5 h at room temperature. The magnetic particle conjugate was then washed three times with PBS buffer and subsequently reconstituted in the capture reagent buffer. The prepared capture reagent was homogenized and filled into the reagent containers intended for the immunoassay laboratory analyzer.

[0338] Preparation of detector reagent and thyronine derivative tracer

[0339] All peptide thyronine derivative tracers were synthesized by solid-phase synthesis by Biosynth (Lelystad, Netherlands) and characterized by UPLC-MS and UPLC-UV. Table 1 shows the thyronine derivative tracers used in the examples, including their tracer formulas and purity.

[0340] To prepare the comparator tracer T3-BSA-NSPSA, T3-BSA from Biospacific (catalog no. 19000) was covalently coupled to the chemiluminescent dye NSPSA by adding NSPSA-NHS (3-[9-(((3-(N-succinimidyloxycarboxypropyl)[4-methylphenyl]sulfonyl)amine)carboxyl]-10-acridiniumyl)-1-propanesulfonate; CAS no. 199293-83-9). For this purpose, T3-BSA was diluted with PBS to a concentration of 1.85 mg / mL, and NSPSA-NHS was added in anhydrous DMF in a 20-fold molar excess. The mixture was then incubated in the dark for 25 minutes. The product was subsequently purified using a gel filtration column (HiTrap Desalting type, GE Healthcare Bio-Sciences AB). An Äkta Purifier 10 from Amersham Pharmacia Biotech was used as the chromatography system. By evaluating the peak areas of the chromatogram for detection at 370 nm, a molar ratio of NSPSA dye to BSA of 6.1 was determined for the T3-BSA-NSPSA conjugate.

[0341] The tracer intended for the respective reagent was diluted to the specified concentration with the indicated detector reagent buffer. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0342] The finished detector reagent was filled into the reagent containers intended for the immunoassay laboratory analyzer.

[0343] CT- = Comparison Tracer

[0344] T- = Tracer according to the invention

[0345] Example 1: T4-based thyronine derivative tracers

[0346] The immunoassay was performed in two steps according to the invention.

[0347] In the first assay step, 50 pL of sample was added to 50 pL of capture reagent.

[0348] The capture reagent consisted of 0.25 mg / mL magnetic particles (Dynabeads M270-streptavidin from invitrogen) coupled to 1 pg / ml MAT02-525-SCLB conjugate in 50 mM PBS buffer with 1% recombinant HSA (SIGMA A9731), 0.1% mouse IgG (from Meridian Bioscience) and 0.05% NalXh at pH 7.4.

[0349] The mixture was incubated for 18 min at 37°C, magnetically separated by applying a magnetic field to the wall of the reaction vessel, and washed 3 times with wash buffer.

[0350] In the second assay step, the magnetic particles were remobilized from the wall of the reaction vessel by removing the magnetic field, stirred up with 50 pL of detector reagent (44 ng / mL thyronine derivative tracer according to Table 2 or 3600 ng / mL T3-BSA-NSPSA each in 50 P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH mM Tris buffer, 0.15 M NaCl, 0.3 % Tween 20, 0.05 % NalXh, pH 8.0) and incubated for 4 min at 37°C.

[0351] The magnetic particles were separated again magnetically by applying a magnetic field to the wall of the reaction vessel and washed again 3 times with wash buffer.

[0352] 100 pL of preparation reagent solution (0.075% HNO3, 1.32% H2O2, 0.027% Triton X-100) and subsequently 300 pL of initiation reagent solution (1.39% NaOH, 2% Triton X-100) were added to trigger the chemiluminescence signal. The chemiluminescence signal was recorded with a photomultiplier.

[0353] The results are shown in Table 2. The results for the determination of chemiluminescence after dilution by a factor of 15 million (direct addition of pretrigger and trigger to the diluted solution without performing an immunoassay) are shown as a percentage of the results of the reference CT-A and per unit of NSPSA concentration.

[0354] The LOD (Limit of Detection) value describes the sensitivity of the immunoassay. It corresponds to twice the standard deviation calculated from a 10-fold determination of the zero matrix measurement divided by the magnitude of the slope of the calibration function in the lower concentration range.

[0355] To determine the non-specific binding to the magnetic particle, a second capture reagent, 'F-0', was prepared in the same manner, but without the addition of the antibody-SCLB conjugate. Since this capture reagent F-0 does not contain a specific binding element for the tracer, the tracer, in combination with this capture reagent, can only bind non-specifically to the beads. Therefore, to calculate the non-specific binding fraction of the assay, the signal of the assays with capture reagent F-0 was compared as a percentage to the assay signal with the standard capture reagent, using a euthyroid serum pool as the sample.

[0356] Table 2: Performance of T4-based thyronine derivative tracer compared to the

[0357] Comparison tracer T3-BSA-NSPSA (CT-A) P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0358] 1 Dilution matrix: 10 mM PBS + 0.05% Tween 20 + 0.5% BSA, pH 7.4

[0359] It is shown that, compared to the commercially available tracer (T3-BSA-NSPSA), the chemiluminescence intensity and sensitivity of the assay are lower, but non-specific binding to the magnetic particles can be drastically reduced (see example CT-A vs. example TA). It is assumed that the replacement of the macromolecule BSA (molecular mass of BSA approximately 66,000 Da) by the low-molecular-weight, strongly negatively charged linker of TA (molecular mass of TA approximately 1,990 Da) is responsible for the significantly reduced non-specific binding in the assay from CT-A to TA. However, it must be assumed that binding rivalry with the anti-T4 antibody is higher for T4-based tracers (TA) than for T3-based tracers (CT-A), which explains the lower sensitivity in example TA.

[0360] It is shown that, with comparable sensitivities, the chemiluminescence strength is significantly lower and the non-specific binding to the magnetic particles is higher when a short basic peptide linker (CT-B) is used instead of a short acidic peptide linker (TA).

[0361] Example 2: T3-based thyronine derivative tracers

[0362] The immunoassay was performed in two steps according to the invention.

[0363] In the first assay step, 50 pL of sample was added to 50 pL of capture reagent.

[0364] The capture reagent consisted of 0.25 mg / mL magnetic particles (Dynabeads M270-streptavidin from Invitrogen) coupled to 1 pg / ml MAT02-525-SCLB conjugate in 100 mM PBS buffer with 1% recombinant HSA (SIGMAA9731), 0.1% mouse IgG (from Meridian), and 0.05% NaN3 at pH 7.0. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0365] The mixture was incubated for 18 min at 37°C, magnetically separated by applying a magnetic field to the wall of the reaction vessel, and washed 3 times with wash buffer.

[0366] In the second assay step, the magnetic particles were remobilized from the wall of the reaction vessel by removing the magnetic field, stirred up with 50 pL of detector reagent (40 ng / mL thyronine derivative tracer according to Table 3 or 1200 ng / mL T3-BSA-NSPSA in 60 mM barbital buffer with 0.15 M NaCl, 1% recombinant HSA (SIGMA A9731), 0.1% mouse IgG (from Meridian), 0.05% NalXh, pH 8.6) and incubated for 4 min at 37°C.

[0367] The magnetic particles were magnetically separated by applying a magnetic field to the wall of the reaction vessel and washed again 3 times with wash buffer.

[0368] 100 pL of the preparation reagent solution (0.027% HNO3, 1.32% H2O2, 0.027% Triton X-100) and subsequently 300 pL of the initiation reagent solution (1.39% NaOH, 2% Triton X-100) were added to trigger the chemiluminescence signal. The chemiluminescence signal was recorded with a photomultiplier.

[0369] The results are shown in Table 3. To determine the correlation coefficient r 2 Sixty euthyroid serum samples from potentially healthy human donors were analyzed using both the immunoassays described here and the Cobas fT4 assay as a reference system. The results of each assay described here were correlated with the Cobas fT4 assay results using least-squares fit, and the correlation coefficient r was calculated. 2 calculated.

[0370] The offset (also called upper measurement limit offset (OMG)) is the ratio of the signal of the calibrator with the highest fT4 concentration to the signal of the zero calibrator. A high offset (especially >30%) may indicate that the tracer added in assay step 2 significantly displaces antigen from the antibody binding sites, which can negatively affect assay performance. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0371] Table 3: Performance of T3-based thyronine derivative tracer

[0372] The results show a low percentage offset when using low-molecular-weight T3-based tracers according to the present invention, which is also significantly lower compared to commercially available T3-albumin conjugates. Furthermore, higher sensitivity can be achieved when using acidic peptide linkers (e.g., TB). While mixed linkers (with acidic and basic amino acids, e.g., TC) show desirablely low offset values, the sensitivity and the correlation coefficient r decrease. 2 The results are worse than when using acidic peptide linkers. The use of basic linkers does not show satisfactory results.

[0373] When the T3 group is linked to the peptide via its carboxy group using an acidic linker (example: TD), results with slightly lower sensitivity and correlation coefficients are observed. However, it is suspected that whether linking the TX group via the amino or carboxy group is more effective depends on the antibody used.

[0374] Example 3: Influence of rHSA

[0375] The assay was performed analogously to Example 2. A 50 mM PBS containing 0.1% mouse IgG and 0.05% NaN3 and rHSA variant-dependent (according to Table 4) at pH 7.7 was used as the detector buffer. The tracer concentration in the detector reagent was 15 ng / mL. The influence of different amounts of rHSA in the detector reagent was determined. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0376] The results are shown in Table 4. The LOQ (Limit of Quantitation) describes the concentration at which the test can determine a sample with at least a specified precision (here <10%) and was determined by measuring 5 samples with fT4 concentrations in the range of 3–10 pmol / L in 10-fold measurements. The coefficient of variation of the concentration determinations was calculated for each sample, and regression was used to determine the fT4 concentration at which the coefficient of variation averaged 10%.

[0377] Table 4: Influence of rHSA in the detector reagent

[0378] The presence of 1% rHSA in the detector reagent leads to a significant performance improvement in the case of LOQ and a slight performance improvement in the case of correlation to the reference system.

[0379] Example 4: Saturation of the beads with biotin

[0380] The assay is performed analogously to Example 2.

[0381] The capture reagent consisted of 0.25 mg / mL magnetic particles (Dynabeads M270-streptavidin from Invitrogen) coupled to 1 pg / mL MAT02-525-SCLB conjugate in 100 mM PBS buffer containing 1% recombinant HSA (SIGMAA9731), 0.1% mouse IgG (from Meridian), and 0.05% NaNs at pH 7.0. For one reagent variant (see Table 5), the beads were saturated with 0.075 pg / mL biotin after coupling the MAT02-525-SCLB conjugate (additional 1.5 h incubation time), as described above. This saturation step was not performed for the other reagent variant. The amount of biotin used to saturate the beads corresponded to approximately 90% of the biotin-binding sites remaining free after coupling of the antibody-SCLB conjugate in the M270-streptavidin beads. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH

[0382] A 50 mM PBS solution containing 1% rHSA and 0.05% NaN3 at pH 7.5 was used as the detector buffer. The TB tracer concentration in the detector reagent was 25 ng / mL.

[0383] The results are shown in Table 5. To determine the biotin interference of the assay, 1200 ng / mL of biotin was added to a human serum pool sample (a stock solution of 0.2 mg / mL biotin in purified water). The reference sample was a mixture of the same serum pool sample and purified water in the same volume ratio.

[0384] For each assay variant, both the biotin-containing sample and the reference sample were measured alternately in seven separate determinations. The percentage difference between the mean determined fT4 concentration of the biotin-containing sample and the reference sample was calculated.

[0385] Table 5: Influence of biotin saturation of the magnetic particle conjugate on the biotin interference of the assay

[0386] The results show that the significant biotin interference of the assay can be almost completely eliminated by saturating the beads with biotin.

[0387] Example 5: Tracer with D-amino acid linker and ZI=NH2

[0388] The assay is performed analogously to Example 2.

[0389] The detector reagent buffer used was 50 mM PBS with 1% rHSA, 0.02% 4-methoxyphenol, 0.5 mM EDTA, and 0.05% NalXh at pH 7.3. Either 15 ng / mL TB or 25 ng / mL TE were used as tracers.

[0390] The capture reagent contained 0.44 mg / mL M270-streptavidin beads coupled to 1.75 g / mL MAT02-525-SCLB conjugate and was saturated with biotin as described above by P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH. The capture reagent buffer consisted of 100 mM PBS, 1% rHSA, 0.1% mouse IgG, and 0.05% NaN3 at pH 7.0.

[0391] The results are shown in Table 6. To determine calibration stability, the reagents to be tested were stored on the immunoassay analyzer for 43 days, 24 hours a day, during which time they were continuously shaken and stored at 4–8 °C. The reagents were calibrated on day 0. On days 0, 4, 11, 21, 31, and 43, aliquots of an fT4 sample with approximately 8 pmol / L fT4 and a sample with approximately 16 pmol / L fT4 were thawed (stored at -20 °C) and measured in quadruplicate. The change in sample concentration during the measurement period was averaged using least squares fit and extrapolated to a period of 10 days.

[0392] Table 6: Influence of tracer type on calibration stability

[0393] It is evident that the measured sample concentration remains more stable during the measurement period when using tracer TE, and therefore tracer TE has a higher calibration stability than tracer TB.

Claims

P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH Patent claims 1. Thyronine derivative tracer of formula (II) where CL stands for a chemiluminescence group, Y represents a linear peptide consisting of 3 to 12 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids, and TX for a group of formula (III) or (IV) (Hl) (IV) stands where Xi, X2, X3 and X4 independently represent I, H, Br, CI or CN, Z1 stands for OH or NH2 and Z2 stands for H or C(O)CH3.

2. The thyronine derivative tracer according to claim 1, wherein the amino acids D-Amino acids are.

3. The thyronine derivative tracer according to any one of the preceding claims, wherein Xi, X3 and X4 stand for I and X2 stands for H. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH 4. The thyronine derivative tracer according to any one of the preceding claims, wherein Zi represents NH2.

5. Method for the quantitative determination of a free thyroid hormone, comprising: a. providing a blood sample; b. combining the blood sample with a capture reagent in a reaction vessel to obtain a first mixture, wherein the capture reagent comprises a magnetic particle conjugate, and wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the thyroid hormone to be determined; c. working up the first mixture, comprising at least: i. incubating the first mixture; ii. immobilizing the magnetic particle conjugate on the wall of the reaction vessel; iii. removing the liquid phase; and iv. washing the solid phase by repeated addition and removal of a wash buffer; d. adding a detector reagent to the reaction vessel to obtain a second mixture, wherein the detector reagent comprises a thyronine derivative tracer of formula (II) CL-Y-TX (H) includes, wherein CL stands for a chemiluminescence group, Y represents a linear peptide consisting of 3 to 12 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids, and TX for a group of formula (III) or (IV) P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH where Xi, X2, X3 and X4 independently represent I, H, Br, CI or CN, Z1 stands for OH or NH2 and Z2 stands for H or C(O)CH3; e. Work-up of the second mixture comprising at least i) resuspending the particles of the magnetic particle conjugate in the detector reagent; ii) incubating the second mixture; iii) immobilizing the magnetic particle conjugate on the wall of the reaction vessel; iv) removing the liquid phase; and v) washing the solid phase by repeated addition and removal of a wash buffer; f. initiating the chemiluminescence of the thyroid derivative tracer bound to the magnetic particle conjugate by adding at least one preparation reagent and at least one initiation reagent; g. measuring the chemiluminescence; and h. quantitatively determining the free thyroid hormone.

6. The method according to claim 5, wherein the detector reagent comprises an albumin, optionally in an amount of 0.1 to 5% based on the total mass of the detector reagent, and / or wherein the capture reagent comprises an albumin, optionally in an amount of 0.1 to 5% based on the total mass of the capture reagent. P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH 7. The method according to claim 6, wherein the albumin contained in the detector reagent, the capture reagent or the detector and capture reagent is recombinant albumin or albumin purified with activated carbon.

8. The method according to any one of claims 5 to 7, wherein the detector reagent is provided as a 2-component system.

9. The method according to any one of claims 5 to 8, wherein the thyronine derivative tracer is contained in the detector reagent in a concentration of 0.1 to 400 ng / mL.

10. The method according to any one of claims 5 to 9, wherein the free thyroid hormone to be determined is fT4 or fT3.

11. The method according to any one of claims 5 to 10, wherein the quantitative determination of the free thyroid hormone according to step h. comprises: (i) Determining the chemiluminescence in 2 to 10 calibrator solutions, wherein the calibrator solutions contain the thyroid hormone to be determined in different concentrations and at least one T4-binding protein, and wherein a concentration of the free thyroid hormone to be determined is assigned to the calibrator solutions; (ii) Creating a calibration curve based on the associated free thyroid hormone concentrations of the calibrator solutions and the determined chemiluminescence; and (iii) Quantifying the thyroid hormone by comparing the chemiluminescence of a blood sample measured according to step g. and the calibration curve prepared under (ii).

12. Kit for a heterogeneous immunoassay for the determination of a free thyroid hormone comprising i. a capture reagent comprising a magnetic particle conjugate, wherein the magnetic particle conjugate comprises a magnetic particle and a specific binder of the corresponding thyroid hormone; and ii. a detector reagent comprising a thyronine derivative tracer of formula (II) P80139WO pes Gesellschaft für medizinische Diagnosesysteme mbH CL-Y-TX (H) where CL stands for a chemiluminescence group, Y represents a linear peptide consisting of 3-12 amino acids, where at least half of the amino acids of the linear peptide are acidic amino acids, and TX for a group of formula (III) or (IV) (Hl) (IV) stands where Xi, X2, X3 and X4 independently represent I, H, Br, CI or CN, Z1 stands for OH or NH2 and Z2 stands for H or C(O)CH3.

13. The kit according to claim 12, further comprising iii. a washing buffer, wherein the washing buffer comprises a detergent.

14. The kit according to one of claims 12 or 13, wherein the detector reagent is a 2-component system.

15. The kit according to one of claims 12 to 14, further comprising iv. at least one calibration component containing the thyroid hormone to be determined, wherein the calibration component is associated with a free thyroid hormone concentration of the hormone to be determined.

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