Thyroid hormone-related quality control

Abstract

The present disclosure relates generally to the field of biological detection technology, and in particular to a quality control for thyroid hormone related assays. The quality control is a solution with serum matrix as the main solvent and contains an antigen coupled with T3 and / or T4, including both bound and free states, and a biomedical carrier material with a contact angle θ greater than 90°.

Description

Technical Field

[0001] This disclosure generally relates to the field of biodetection technology, and more specifically, to a quality control material related to thyroid hormones. Background Technology

[0002] Thyroid diseases are common and frequently occurring in endocrinology, encompassing various types including Hashimoto's thyroiditis, hyperthyroidism, hypothyroidism, nodular goiter, and thyroid tumors. Currently, the specific pathogenesis of thyroid diseases is not fully understood clinically. The causes are complex and diverse, including congenital defects, family history, medications, lifestyle habits, and age, with some factors being difficult to prevent and significantly impacting patients' daily lives. Fortunately, most thyroid diseases are curable, but some carry a risk of recurrence, necessitating early diagnosis and timely treatment.

[0003] Thyroid hormones are secreted by the thyroid gland and regulate various bodily functions, controlling growth and development, as well as the metabolism of carbohydrates, proteins, and fats. They also have significant effects on the nervous, endocrine, and cardiovascular systems. When the thyroid gland is diseased, thyroid hormone levels will change accordingly. Different thyroid diseases have different pathogenesis, resulting in variations in thyroid hormone expression. Therefore, timely and accurate assessment of a patient's thyroid function is crucial in clinical practice. Commonly used indicators for detecting thyroid function include thyroid-stimulating hormone (TSH), total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), and free thyroxine (FT4). TT4 exists in both bound and free forms. Most serum T4 exists in the blood bound to TBG, with only a very small portion existing in free form; therefore, serum FT4 levels are extremely low. The same applies to TT3. In addition, other indicators such as rT3 and TG are also clinically significant. Reverse triiodothyronine (rT3) is an isomer of T3 but lacks physiological activity and is often used to differentiate hypothyroidism from "low T3 syndrome." Thyroglobulin (TG) is a large glycoprotein synthesized by thyroid follicular epithelial cells. Under normal circumstances, only trace amounts of TG enter the bloodstream. Clinically, dynamic changes in TG are often observed to assess the surgical efficacy of thyroid cancer patients and monitor for recurrence after surgery in differentiated thyroid cancer. Accurate detection of thyroid hormones is crucial for the diagnosis, treatment monitoring, and prognostic assessment of thyroid diseases.

[0004] In the testing process, quality control materials are crucial for quality assurance. By using them, we can validate and calibrate analytical methods in the laboratory, ensuring the accuracy of test results. Quality control materials also help identify potential problems in the laboratory, such as instrument performance deviations and operator errors, allowing for timely correction and improvement. Using the same quality control materials across different laboratories allows for comparison of test results, assessing consistency and comparability between them. This effectively addresses discrepancies in test results between different laboratories, improving data reliability and comparability.

[0005] Furthermore, ideal quality control products generally possess good stability, minimal inter-vial variation, and appropriate quality control concentration levels. For combined thyroid hormone quality control products, it is necessary to simultaneously control the TT3 / TT4 and FT3 / FT4 values ​​to maintain them within the clinical sample concentration range. However, because FT4 / FT3 in serum binds to thyroid-binding protein (TBG), TT3 and TT4 are primarily in a bound state in serum, and the values ​​of FT4 and FT3 are affected by binding affinity. This results in low FT4 / FT3 levels in serum, failing to meet the concentration requirements for departmental internal quality control. Literature reports that the binding affinity of FT4 and FT3 to TBG is affected by factors such as temperature and pH. Adjusting the serum matrix temperature and pH to reduce the binding affinity of TBG to FT4 and FT3 can control the concentration of FT4 and FT3, but this often leads to protein loss in serum, affecting the matrix. Summary of the Invention

[0006] One objective of this disclosure is to provide a quality control product, which is a solution with a serum matrix as the main solvent, and comprises an antigen coupled with a biomedical carrier material having a contact angle θ greater than 90° and T3 and / or T4, wherein the T3 and / or T4 include both bound and free states.

[0007] Another object of this disclosure is to provide a kit comprising the quality control material as described above.

[0008] Another object of this disclosure is to provide a method for preparing the quality control material as described above, comprising: mixing the components and coupling the biomedical carrier material therein with T3 and / or T4.

[0009] Another object of this disclosure is to provide the application of the quality control material as described above in the determination of thyroid function.

[0010] Compared with the prior art, the beneficial effects of this disclosure are as follows:

[0011] Coupling T3 and / or T4 with biomedical carrier materials can increase the concentration of free T3 and / or T4, thereby enabling the TT3 / TT4 and FT3 / FT4 values ​​in the quality control material to remain within the range of clinical sample concentrations. Detailed Implementation

[0012] Reference will now be made to embodiments of this disclosure in detail, with one or more examples described below. Each example is provided for explanation and not for limitation of this disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to this disclosure without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0013] Unless otherwise stated, all terms used in the disclosure of this disclosure (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further guidance is provided below for a better understanding of the teachings of this disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure.

[0014] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0015] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this disclosure, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0016] The terms “containing,” “including,” and “comprise” as used in this disclosure are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0017] The range of values ​​represented by endpoints in this disclosure includes all values ​​and fractions contained within that range, as well as the endpoints referenced.

[0018] When this document uses the term "about" to refer to a value or parameter, it includes (and describes) an implementation of the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0019] The concentration values ​​mentioned in this disclosure include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may allow fluctuations within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are allowed. For example, 100mM may allow fluctuations within the ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations within ±10% are allowed.

[0020] In this disclosure, unless "v / v" or "m / v" is specifically specified before the percentage sign, it is determined according to the common understanding of those skilled in the art or the conventional weighing method. If the solute is a solid in its normal state, it is m / v; if it is a liquid, it is v / v. Normal state generally refers to room temperature and pressure, or the conditions under which the substance is usually handled (e.g., some substances generally require refrigeration, so normal state refers to their low-temperature state). Wherein m / v represents the mass-to-volume ratio, and m / v% represents the mass percentage contained in a given volume of the mixture. For example, a concentration of 5m / v% for substance A in a mixture means that 5 grams of substance A are contained in every 100 milliliters of the mixture. In this disclosure, v / v represents the volume ratio, and v / v% represents the volume percentage contained in a given volume of the mixture. For example, a concentration of 5v / v% for substance A in a mixture means that 5 milliliters of substance A are contained in every 100 milliliters of the mixture. Commonly used components in this disclosure are described in percentages according to their usual state, as exemplified below, usually expressed as m / v%: sucrose; usually expressed as v / v%: Proclin 300, mannitol, Tween 80.

[0021] In this disclosure, "quality control material" and "quality control product" can be used interchangeably, referring to a solution used for at least one of calibrators, quality control products and reference products when detecting target substances (including but not limited to thyroid hormones), which contains T3 and / or T4 antigens.

[0022] As used herein, "biomedical carrier material" refers to a controlled release / delivery system primarily used for drugs and bioactive substances (such as peptides, proteins and growth factors, nucleic acids, cells, etc.). In this context, it is typically a solid or semi-solid (e.g., gel) substance capable of coupling with T3 and / or T4 antigens and increasing their concentration through steric hindrance. Exemplary biomedical carrier materials include polymeric carrier materials, mainly divided into natural polymers and their derivatives and synthetic polymers. Natural polymers are primarily polysaccharides and proteins, while synthetic polymers include polyanhydrides, aliphatic polyesters represented by polylactic acid, polyphosphate esters, polyorthoesters, etc., as well as polyethylene glycol and its derivatives, polyamino acids, etc.

[0023] In this disclosure, "T3 and / or T4" includes bound and free T3 antigens, and / or bound and free T4 antigens. Depending on the context, it can also be used interchangeably with "T3 / T4 antigen" or "T3 and / or T4 antigen." "Bound state" refers to the state in which T3 and / or T4 antigens are bound to their binding proteins; "free state," in contrast to "bound state," refers to the state in which T3 and / or T4 antigens dissociate from their respective binding proteins, but "free state" does not mean that they are not coupled to biomedical carrier materials. When in a bound state, the portion coupled to the biomedical carrier material is still the T3 and / or T4 themselves, not their binding proteins.

[0024] As used herein, unless otherwise indicated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0025] In this disclosure, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0026] In this disclosure, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0027] In this disclosure, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments and should be understood not to limit the scope of protection of this disclosure. In this disclosure, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" options appear in a technical solution, unless otherwise specified and without contradictions or mutual constraints, each "optional" option is independent.

[0028] All documents mentioned in this disclosure are cited herein as if they were cited individually. Unless they conflict with the inventive purpose and / or technical solution of this disclosure, all cited documents are cited in their entirety and for all purposes. When citing documents in this disclosure, the definitions of relevant technical features, terms, nouns, phrases, etc., are also cited. When citing documents in this disclosure, examples and preferred embodiments of the relevant technical features may also be incorporated into this disclosure as reference, but only to the extent that they enable the implementation of this disclosure. It should be understood that when the cited content conflicts with the description in this disclosure, this disclosure shall prevail or modifications shall be made adaptively based on the description in this disclosure.

[0029] A first aspect of this disclosure provides a quality control product, which is a solution with a serum matrix as the main solvent, and comprises an antigen coupled with a biomedical carrier material having a contact angle θ greater than 90° and T3 and / or T4, wherein the T3 and / or T4 include both bound and free states.

[0030] Compared to existing technologies that improve the binding affinity of T3, T4, and binding proteins by adjusting temperature and pH, this disclosure offers a completely different solution: altering the binding affinity of T4, T3, and binding proteins by changing steric hindrance. In this approach, after T3 and / or T4 are coupled to a biomedical carrier material, the increased steric hindrance leads to a weakened binding affinity with binding proteins such as TBG. This results in fewer bound T3 / T4 and more free T3 and T4, thus ensuring that the TT3 / TT4 and FT3 / FT4 values ​​in the quality control sample remain within the clinical sample concentration range.

[0031] As used herein, the term "coupling" refers to covalent interactions, non-covalent interactions, and steric interactions, or combinations thereof. Covalent interactions are chemical bonds between two atoms or groups formed by sharing a single pair of electrons (single bond), two pairs of electrons (double bond), or three pairs of electrons (triple bond). Covalent interactions are also referred to in the art as electron pair interactions or electron pair bonds. Non-covalent interactions include, but are not limited to, van der Waals interactions, hydrogen bonds, weak chemical bonds (through short-range non-covalent forces), hydrophobic interactions, ionic bonds, etc. For a commentary on non-covalent interactions, see Alberts et al., *Molecular Biology of the Cell*, 3D edition, Garland Publishing, 1994. Stereo interactions are generally understood to include those in which the structure of the compound allows it to occupy a site through its three-dimensional structure, rather than any attractive force between the compound and the site. Couplings are typically tight and difficult to separate, and preferably, the coupling of T3 and / or T4 with a biomedical carrier material having a contact angle θ greater than 90° is at least partially covalent, and this connection can be a direct connection between the two or an indirect connection formed through a bridging agent.

[0032] "Biomedical carrier materials with a contact angle θ greater than 90°" can be approximately understood as hydrophilic biomedical carrier materials. The contact angle θ refers to the angle between the tangent at the gas-liquid interface at the gas-liquid-solid three-phase junction and the liquid-solid-liquid interface line; it is a measure of wettability. Generally, the more hydrophilic the material's chemical properties, the smaller the contact angle; the more hydrophobic the material's chemical properties, the larger the contact angle. In some embodiments, the biomedical carrier material includes polymers and / or carrier proteins. The biomedical carrier materials listed in the embodiments of this disclosure, such as BSA, KLH, OVA, and PEG, are all hydrophilic materials (e.g., *Principles and Applications of Biomedical Nanomagnetic Materials*, 2005, p. 423; *Natural Polymer Materials*, 2016, p. 156, edited by Duan Jiufang), and therefore their contact angles θ are all greater than 90°.

[0033] In some embodiments, the carrier protein includes at least one of bovine serum albumin (BSA), hemocyanin (KLH), and chicken ovalbumin (OVA).

[0034] In some embodiments, the polymer comprises PEG modified with linker groups; further, the linker groups include carboxyl or amino groups. The number of T3 / T4 antigens that PEG can link is one to two. In a preferred embodiment, each of the PEG-modified linker groups comprises one or two of the same type of linker group, all located at the end of the PEG molecule. This prevents antigen epitopes from masking each other. When used as a quality control material, it achieves higher antigen utilization and requires less antigen.

[0035] After PEG is pre-modified with amino / carboxyl groups, it can form amide bonds with the amino and / or carboxyl groups of T3 / T4 under the action of activators such as EDC, thus achieving relatively easy direct coupling.

[0036] In some embodiments, the polymer has a molecular weight of 2000 to 6000, for example 2500, 3000, 3500, 4000, 4500, 5000, and 5500.

[0037] In some embodiments, the quality control material further comprises the uncoupled biomedical carrier material, such as PEG modified with linker groups. In the quality control material, the molar ratio of the PEG modified with linker groups to T3 and / or T4 is (7–13):1, for example, 8:1, 9:1, 10:1, 11:1, or 12:1.

[0038] The quality control material can contain both PEG coupled with T3 and / or T4, and free PEG. When the carrier material is PEG, purification is preferably not performed after coupling with T3 and / or T4, leaving free PEG in the solution. This simplifies the entire preparation process, and the free PEG will not adversely affect the detection results when the quality control material is used. Furthermore, at this molar ratio, PEG is significantly in excess, ensuring that T3 or T4 is fully coupled to the antigen during preparation.

[0039] In some embodiments, the binding proteins of bound T3 and / or T4 include one or more of TBG, thyroxine transporters, and albumin. Under physiological conditions, approximately 99.98% of circulating T4 is normally bound to specific plasma proteins, including thyroxine-binding globulin (TBG, 60%–75%), thyroxine-binding prealbumin (TBPA, 15%–30%), and albumin (ALB, 10%); only 0.02% of circulating T4 is normally in a free state; 99.7% of circulating T3 specifically binds to TBG, with approximately 0.3% in a free state (FT3). Therefore, TBG binding is generally dominant, and TBG is the preferred binding protein.

[0040] In some implementations, it also includes thyroid function-related analytes.

[0041] In some embodiments, the thyroid function-related analytes include TSH, rT3, and TG.

[0042] In some embodiments, the thyroid function-related analytes include one or more of TSH, rT3, TG, thyroid-stimulating hormone receptor antibody, anti-thyroid microsomal antibody, and anti-thyroid peroxidase antibody.

[0043] In some embodiments, the quality control material also contains sex hormones.

[0044] In some embodiments, the sex hormones include at least one of luteinizing hormone, human chorionic gonadotropin (hCG), prolactin, progesterone, estradiol, testosterone, follicle-stimulating hormone (FSH), and anti-Müllerian hormone. Clinically, thyroid function-related analytes and sex hormones are often detected in combination. Thyroid hormones play a regulatory role in testicular and ovarian development, while abnormal sex hormone levels can also affect thyroid function. For example, estrogen can participate in the occurrence and development of hyperthyroidism through estrogen receptor β, and can also promote the proliferation of thyroid stem cells, leading to further proliferation of thyroid cells and the formation of nodules. Estrogen can also promote the proliferation of thyroid cancer cells through an estrogen receptor-dependent mechanism, and even participate in angiogenesis and metastasis, playing an important role in the outcome and prognosis of thyroid cancer. Furthermore, the synergistic detection of both can be used for the diagnosis of various diseases, such as chronic liver disease.

[0045] The T3 and / or T4 antigens, thyroid function-related analytes, and sex hormones contained in the quality control materials can be any of natural antigens, recombinant antigens, and synthetic antigens. For example, TG can be selected as a natural antigen, which can be extracted naturally, uses natural raw materials that are close to clinical samples, and can better reflect problems in the detection system; FT3, FT4, and rT3 can be selected as synthetic antigens, which are all small molecules that are not easy to extract naturally but are easy to synthesize; TSH antigen can be selected as a recombinant antigen, which is obtained by recombinant expression in mammalian cells and is close to natural raw materials.

[0046] In some embodiments, the quality control material further comprises at least one of a buffer component, a polyol, a preservative, a sugar, an amino acid, and a surfactant.

[0047] In this disclosure, the quality control materials preferably contain a buffering component. As used herein, the term "buffering component" refers to an aqueous solution or composition that resists pH changes and maintains a stable pH value when an acid or base is added to it. This resistance to pH changes is due to the presence of a buffering substance in such solutions. Therefore, a solution that can, to a certain extent, counteract or mitigate the effects of an external strong acid or base on the pH of a solution, thereby maintaining a relatively stable pH value, is called a buffer solution or buffered solution. Buffer solutions generally do not have an unlimited capacity to maintain the pH of a solution or composition. Instead, they are generally able to maintain a pH within a specific range, such as pH 6–pH 10, for example, pH = 6.5, pH = 7, pH = 8, pH = 9, pH = 9.5. Exemplary buffering components may include at least one of phosphate buffers, HEPES buffers, Tris-HCl buffers, glycine buffers, and borate buffers.

[0048] In some embodiments, the quality control material contains a polyol. Polyols have excellent stabilizing effects on the nonpolar surfaces of proteins, for example, they can keep the antigen surface moist, preventing it from becoming inactive due to water loss, and can also increase viscosity, reduce molecular motion, prevent protein aggregation, and have the effects of reducing wall adsorption and preventing freezing. The polyol includes at least one selected from sugar alcohols, glycerol, butylene glycol, inositol, and ethylene glycol. In some embodiments, the sugar alcohol may include one or more selected from sorbitol, mannitol, erythritol, maltitol, lactitol, and xylitol. More preferably, the amount of the polyol added is 0.1% to 10%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%.

[0049] In some embodiments, the quality control material contains a preservative. The preservative is added to prevent contamination of proteins and stabilizers, such as amino acids and sugars, by microorganisms in the air and water over extended storage periods. Exemplary preservatives may include ProClin 300, sodium azide, Bronidox, BND-10, PC-300, PC-950, BIT-10, and liquid preservative MB-1; ProClin 300 is preferred. Those skilled in the art can freely select the amount added based on the characteristics of the specific preservative; exemplary amounts are 0.01% to 0.5%, for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, and 0.4%.

[0050] In some embodiments, the quality control material contains sugar. Sugars are non-specific protein stabilizers with strong penetrating properties, capable of permeating and diffusing into the protein interior, preventing contact between the hydrophobic structures within the protein and water molecules, thereby improving stability. Sugars can be monosaccharides or polysaccharides, examples including at least one of glucose, fructose, lactose, sucrose, and trehalose. Exemplary amounts of sugar added are 0.1% to 20%, for example 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and 15%.

[0051] In some embodiments, the quality control material preferably contains amino acids. Amino acids can stabilize proteins through differences in free energy and surface tension, and amino acids such as lysine can enhance stability by forming electrostatic interactions with certain specific amino acids between peptide chains. Examples of amino acids include arginine, glycine, tryptophan, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, tyrosine, asparagine, lysine, and histidine, or salts of these amino acids (such as arginine hydrochloride and histidine hydrochloride). More preferably, the amount of the amino acid added is 0.01% to 5%, for example, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, and 4%.

[0052] In some embodiments, the quality control material contains a surfactant. Surfactants have dispersing and solubilizing effects; they can disrupt non-covalent bonds such as hydrogen bonds between proteins, depolymerize protein polymers, and prevent protein aggregation. Preferred surfactants include nonionic surfactants such as NP40, SLS, Triton (e.g., Triton X100, Triton X114), Tween (e.g., Tween 20, Tween 40, Tween 60, Tween 80), and C8APG (octyl glucoside). Exemplary amounts of surfactant added are 0.01% to 5%, for example, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, and 4%.

[0053] In some specific embodiments, the quality control materials include 0.05% to 0.15% Proclin 300, 2% to 8% sucrose, 1% to 5% mannitol, and 0.01% to 0.1% Tween 80.

[0054] In some embodiments, the serum matrix includes one or more of the following: hormone-free serum, healthy animal serum, and lipid-free serum.

[0055] Hormone-free serum refers to serum from which hormones such as T4, T3, FT3, FT4, FSH, and LH have been removed. Most commercial sera have high baseline levels of T4, FSH, and HCG, making them unsuitable for Level 1 (negative control) quality control. Therefore, hormone-free serum is necessary for Level 1 quality control. Lipid-free serum refers to serum from which lipids have been removed. When serum is stored at 2-8°C for extended periods, lipids can easily precipitate and cause turbidity due to pH changes. Lipid-free serum is beneficial for long-term storage and avoids interference from lipids.

[0056] When the quality control material contains TG, the serum matrix contains anti-TG antibody (TGA), which can bind to TG and catalyze its hydrolysis, leading to a reduction in the amount of TG to be detected, interfering with TG detection, and affecting the test results. Therefore, it is better to prepare the quality control material with a serum matrix free of TGA. In some specific embodiments, the serum matrix does not contain anti-TG antibody.

[0057] Methods for removing TGA from serum matrix are routine in the field, such as purification using one or more affinity chromatography columns, including A, L, and G columns. An A column is an affinity chromatography column where protein A is covalently coupled to an agarose medium. Protein A, derived from a strain of Staphylococcus aureus, contains five domains that specifically bind to the Fc region of antibody IgG molecules. Protein A, acting as an affinity ligand, is coupled to the agarose matrix, allowing it to specifically bind to antibody molecules in the sample while allowing other contaminating proteins to flow through, exhibiting extremely high selectivity. One protein A molecule can bind at least two IgG molecules. Besides IgG, protein A can also bind other immunoglobulins, such as IgA and IgM in certain species for purification. Recombinant protein A has a newly added cysteine ​​at its C-terminus, allowing for single-site directional coupling to the agarose matrix and reducing steric hindrance in antibody contact. The L-column refers to an affinity chromatography column where protein L is covalently coupled to agarose medium. Protein L is an immunoglobulin-binding protein that binds to the light chain of immunoglobulin K without affecting antigen binding. Compared to other antibody-binding proteins such as Protein A and Protein G, Protein L has a broader Ig and Ig subtype binding range: it can bind not only all Ig classes (IgG, IgM, IgA, IgE, and IgD), but also single-chain antibodies and Fab fragments. The G-column refers to an affinity chromatography column where protein G is covalently coupled to agarose medium. Protein G is a cell wall protein isolated from Gram-forming streptococci, with a molecular weight of 25 kDa. Compared to Protein A, Protein G has a higher affinity for most mammalian IgG, especially for IgG subunits such as human IgG3, mouse IgG1, and mouse IgG2a. Therefore, Protein G can be used to purify mammalian monoclonal and polyclonal IgG antibodies that do not bind well to Protein A.

[0058] Furthermore, in some preferred embodiments, the quality control materials provided in this disclosure exhibit excellent liquid stability. The increase / maintenance of stability can be evaluated through one or more dimensions, as is well known to those skilled in the art. Dimensions typically include the degree of disruption to the native structure and / or function of the protein caused by various physicochemical conditions (e.g., temperature, surface tension, oxidation, acid-base, deamination) and biological factors (e.g., protease hydrolysis). Common indicators of native structure include hydrogen bonds, ionic bonds, disulfide bonds, peptide bonds, and changes in three-dimensional structure; functions include enzyme activity, immunogenicity, and reactivity. In some cases, stability refers to conformational stability. The increase in stability is relative to the use of conventional preservation solutions under the same storage conditions. General storage conditions are typically sealed, short-term storage at 2°C to 8°C, and long-term freezing at -20°C; in special cases, freezing at -80°C or storage in liquid nitrogen is also possible. In some preferred embodiments of this disclosure, the unopened quality control material can be stably stored at 2-8°C for 1 year, and at 4°C for 30 days after opening; it can be stably stored at -20°C for 60 days after opening; the relative deviation of each analyte is within 15% after 7 days of accelerated freezing at 37°C; and the relative deviation of each analyte is within 8% after 3 repeated freeze-thaw cycles.

[0059] A second aspect of this disclosure provides a kit comprising the quality control material as described above.

[0060] In this disclosure, the term "kit" can refer to any article (e.g., packaging or container) that includes at least one device and incorporates quality control materials as described in this disclosure. The kit may further include instructions for use, supplementary reagents, and / or components or assemblies used in the methods or steps described in this disclosure. Components within the kit may be packaged in the form of solutions, solids, or test strips. Target substances, such as components required for thyroid hormone detection, may be provided in solid form, particularly various proteins, antibodies, peptides, and reaction buffer components. The solids include at least one of lyophilized microspheres, lyophilized cakes, lyophilized powders, and spots dependent on a solid medium.

[0061] In some embodiments, the kit is used to detect thyroid function. The kit can be used to detect a variety of thyroid-related disorders, such as hyperthyroidism (representative types include Graves' disease, toxic multinodular disease, and toxic nodules), hypothyroidism, thyroiditis, thyroid nodules, thyroid cancer, and occult cancers (e.g., subclinical hypothyroidism and subclinical hyperthyroidism).

[0062] In some embodiments, the kit is an immunoassay kit.

[0063] A third aspect of this disclosure provides a method for preparing the quality control material, comprising: mixing the components and coupling the biomedical carrier material therein with T3 and / or T4.

[0064] In this process, all components can be mixed first before coupling the biomedical carrier material with T3 and / or T4; alternatively, the biomedical carrier material can be coupled with T3 and / or T4 first, and then the coupled compound can be mixed with other components. There is no specific order of coupling.

[0065] In some embodiments, the serum matrix is ​​purified by affinity chromatography to remove antibodies therein, such as anti-TG antibodies;

[0066] In some embodiments, the biomedical carrier material is coupled to T3 and / or T4 under the action of an activator.

[0067] In some embodiments, the activator includes carbodiimide (which may include at least one of DCC, DIC, and EDC).

[0068] In some embodiments, the biomedical carrier material is PEG modified with amino or carboxyl groups, the activator is EDC, and the molar ratio of modified PEG to EDC is 1:(3-7); preferably 1:5.

[0069] In some embodiments, the biomedical carrier material is a carrier protein, and the molar ratio of T3 and / or T4 to the carrier protein is (15-25):1, preferably 20:1.

[0070] In some embodiments, the biomedical carrier material is PEG modified with linker groups, and the molar ratio of the PEG modified with linker groups to T3 and / or T4 is (7-13):1, preferably 10:1.

[0071] The fourth aspect of this disclosure provides the use of the quality control material described above in the determination of thyroid function.

[0072] The application of quality control materials / methods for the above-described kits is not particularly limited, as long as it is necessary to improve the stability of the quality control materials and / or improve the accuracy of thyroid detection. In some embodiments, immunoassays are used for the assay. Examples of platforms include magnetic particle chemiluminescence, fluorescence chromatography, and latex turbidimetry.

[0073] The description of the first aspect of this disclosure also applies to the second, third, and fourth aspects of this disclosure.

[0074] The embodiments of this disclosure will now be described in detail with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. For experimental methods in the following embodiments where specific conditions are not specified, reference should be made to the guidelines given in this disclosure, or to experimental manuals or conventional conditions in the art, or to other experimental methods known in the art, or to the conditions recommended by the manufacturer.

[0075] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0076] Example 1: Preparation method of coupling T3 and T4 with hydrophilic substances containing carboxyl or amino groups.

[0077] The carboxyl or amino groups on hydrophilic materials can undergo an amidation reaction with the amino / carboxyl groups of T3 / T4 to obtain PEG / BSA / OVA modified T3 / T4. The following is the coupling process (taking PEG with carboxyl groups as an example):

[0078] Step 1: Activate hydrophilic substances with EDC

[0079] Weigh an appropriate amount of PEG with a molecular weight of 2000-6000 and containing carboxyl groups, dissolve it in organic solvents such as DMF and DMSO, then add EDC, and react at room temperature for 8 hours to obtain a carboxyl-activated PEG solution (PEG:EDC molar ratio of 1:5).

[0080] Step 2: Coupling of hydrophilic substances with T3 / T4

[0081] An appropriate amount of T3 / T4 antigen was slowly added under magnetic stirring. The reaction was stopped after 16 hours at room temperature to obtain T3 / T4 antigen coupled with PEG.

[0082] Example 2: Preparation of composite quality control sample

[0083] The preparation method for compound quality control products, such as compound quality control products for thyroid hormones, is as follows:

[0084] Step 1: After thawing the serum matrix, store it at 2-8℃, and purify and filter it using affinity chromatography columns such as A, L and G columns;

[0085] Step 2: Add Proclin300 to the treated serum at a concentration of 0.05%-0.15%, then add 2%-8% sucrose, 1%-5% mannitol, and 0.01%-0.1% Tween 80. Stir thoroughly to obtain a clear matrix solution, and store it at 2-8℃.

[0086] Step 3: Add T3 (conjugated with PEG / BSA / OVA), T4 (conjugated with PEG / BSA / OVA), TSH, rT3, and TG antigens. Control the concentration of each analyte by adjusting the amount of antigen added to obtain low, medium, and high levels of thyroid hormone liquid composite quality control samples. Aliquot and store at 2–8°C. The concentrations of each analyte in the different levels of thyroid hormone liquid composite quality control samples are shown in Table 1 below.

[0087] Table 1. Addition amount and final concentration of each analyte in different levels of liquid composite quality control products for thyroid hormones (sample data)

[0088]

[0089] The normal reference range for TT4 is 5.00–13.00 μg / dL, the normal reference range for FT4 is 0.90–1.75 ng / dL, the normal reference range for TT3 is 0.75–2.10 ng / mL, and the normal reference range for FT3 is 2.00–4.20 pg / mL. The TT3 / TT4 and FT3 / FT4 values ​​in the above-prepared thyroid hormone compound quality control product can be maintained within the clinical sample concentration range.

[0090] Example 3: Effect of Coupling on the Concentrations of TT3 / FT3 and TT4 / FT4 in Composite Quality Control Samples

[0091] 1. Effects of PEG coupling with different molecular weights on the values ​​of TT3, FT3 / TT4, and FT4 in composite quality control samples.

[0092] 1.1 The specific preparation steps for PEG-conjugated T3 / T4 antigen are as follows:

[0093] Step 1: The molar ratio of PEG:T3 / T4 is 10:1. Weigh 0.0001 mol of PEG with a molecular weight of 2000-6000 that has been modified with carboxyl or amino groups (raw material purchased from Xi'an Ruixi Biotechnology Co., Ltd.) and dissolve it in 20 mL of DMF organic solvent. Then add 0.0005 mol (0.09585 g) of EDC and react at room temperature for 8 h to obtain a carboxyl-activated PEG solution.

[0094] Step 2: Under magnetic stirring, slowly add T3 / T4 antigen and PEG modified with carboxyl and / or amino groups according to the amounts shown in Table 2. After reacting at room temperature for 16 hours, stop the reaction to obtain T3 / T4 antigens coupled with PEG of different molecular weights.

[0095] Table 2. Preparation of T3 / T4 antigens and the amount of PEG modified with carboxyl or amino groups during the preparation of PEG-conjugated T3 / T4 with molecular weights of 2000-6000.

[0096]

[0097] 1.2 The steps for preparing quality control samples containing only TT3, FT3 / TT4, and FT4 after PEG coupling with T3 / T4 are as follows:

[0098] Step 1: Thaw 500 mL of serum matrix and store it at 2-8℃. Purify and filter it using an L-column affinity chromatography column.

[0099] Step 2: Add 0.1% Proclin 300 to the serum matrix obtained in Step 1, followed by 5% sucrose, 3% mannitol, and 0.05% Tween 80. Stir thoroughly to obtain a clear matrix solution, and store at 2-8℃.

[0100] Step 3: Add T3 / T4 antigens coupled with different molecular weight PEGs to the serum matrix obtained in Step 2, and detect the concentrations of TT3, FT3 / TT4, and FT4. By adjusting the amount of T3 / T4 antigens coupled with different molecular weight PEGs, the concentrations of each analyte are controlled to obtain high, medium, and low level thyroid hormone liquid composite quality control products, which are then aliquoted and stored at 2–8°C.

[0101] Introduction to the detection methods of TT3 and FT3 concentrations during the preparation of composite quality control products

[0102] The TT3 and FT3 chemiluminescence immunoassay reagents from New Industries Biomedical Engineering Co., Ltd. were used for detection. The TT3 reagent's detection principle is as follows: The principle is chemiluminescent immunoassay competition. The analysis procedure is as follows: The instrument automatically adds 40 μL of sample, 20 μL of buffer, and 40 μL of luminescent label. After incubation for 5 min, it is washed. Then, 20 μL of magnetic microspheres and 20 μL of buffer are automatically added. After incubation for 10 min, it is washed and detected. The FT3 reagent's detection principle is also as follows: The principle is chemiluminescent immunoassay competition. The analysis procedure is as follows: The instrument automatically adds 40 μL of sample, 20 μL of buffer, and 40 μL of luminescent label. After incubation for 5 min, it is washed. Then, 20 μL of magnetic microspheres and 80 μL of buffer are automatically added. After incubation for 10 min, it is washed and detected.

[0103] Methods for detecting TT4 and FT4 concentrations during the preparation of composite quality control products:

[0104] The TT4 and FT4 chemiluminescence immunoassay reagents from New Industries Biomedical Engineering Co., Ltd. were used for detection. The TT4 reagent's detection principle is as follows: The principle is chemiluminescent immunoassay competition. The analytical procedure is as follows: the instrument automatically adds 40 μL of sample, 40 μL of replacement agent, 40 μL of luminescent label, 80 μL of buffer, and 20 μL of magnetic microspheres. After incubation for 15 minutes, washing and detection are performed. The FT4 reagent's detection principle is also chemiluminescent immunoassay competition. The analytical procedure is as follows: the instrument automatically adds 40 μL of sample, 40 μL of buffer, 40 μL of luminescent label, and 20 μL of magnetic microspheres. After incubation for 15 minutes, washing and detection are performed.

[0105] 2. The effect of BSA / OVA coupling T3 / T4 on the values ​​of TT3, FT3 / TT4, and FT4 in composite quality control samples.

[0106] 2.1 The specific preparation steps for BSA / OVA conjugated with T3 / T4 antigen are as follows:

[0107] Step 1: The molar ratio of BSA / OVA:T3 / T4 antigens is 1:20. Weigh 0.00001mol BSA / OVA (0.6643g BSA, 0.45g OVA) (BSA manufacturer: Roche; OVA manufacturer: Roche) and dissolve it in (30)mL DMF organic solvent. Then add 0.0005mol (95.9mg) EDC and react at room temperature for 10h to obtain a carboxyl-activated BSA / OVA solution.

[0108] Step 2: Under magnetic stirring, slowly add T3 / T4 antigen according to the amounts shown in Table 3, and stop the reaction after reacting at room temperature for 18 hours.

[0109] Step 3: Purification: Transfer the reaction liquid to a dialysis bag of MwCO20000 and dialyze with PBS solution at room temperature for 8 hours, changing the solution every 2 hours to obtain BSA / OVA conjugated T3 / T4 antigen.

[0110] Table 3. Amounts of T3 / T4 antigen and BSA / OVA added during the preparation of BSA / OVA-modified T3 / T4.

[0111] Modification type T3 addition amount T4 addition amount BSA addition amount OVA addition amount Unmodified T3 antigen (control) 130.2mg / / / BSA-modified T3 antigen 130.2mg / 0.6643g / OVA-modified T3 antigen 130.2mg / / 0.45g Unmodified T4 antigen (control) / 155.4mg / / BSA-modified T4 antigen / 155.4mg 0.6643g / OVA-modified T4 antigen / 155.4mg / 0.45g

[0112] 2.2 Procedure for preparing quality control samples containing only TT3, FT3 / TT4, and FT4 after BSA / OVA coupling with T3 / T4:

[0113] Step 1: Thaw 500 mL of serum matrix and store it at 2-8℃. Purify and filter it using an L-column affinity chromatography column.

[0114] Step 2: Add 0.1% Proclin 300 to the serum matrix obtained in Step 1, followed by 5% sucrose, 3% mannitol, and 0.05% Tween 80. Stir thoroughly to obtain a clear matrix solution, and store at 2-8℃.

[0115] Step 3: Add BSA / OVA-conjugated T3 / T4 antigen to the serum matrix obtained in Step 2, and detect the concentrations of TT3, FT3 / TT4, and FT4. By adjusting the amount of BSA / OVA-conjugated T3 / T4 antigen added, the concentrations of each analyte are controlled to obtain high, medium, and low level thyroid hormone liquid composite quality control products, which are then aliquoted and stored at 2–8°C.

[0116] 2.3 The effect of PEG coupling with different molecular weights on the values ​​of TT3 and FT3 in the composite quality control sample containing only TT3 and FT3 is shown in Table 4.

[0117] Table 4

[0118]

[0119]

[0120] 2.4 The effect of PEG coupling with different molecular weights on the values ​​of TT4 and FT4 in the composite quality control product containing only TT4 and FT4 is shown in Table 5.

[0121] Table 5

[0122]

[0123] The effects of 2.5BSA / OVA-T3 coupling on the values ​​of TT3 and FT3 in composite quality control samples containing only TT3 and FT3 are shown in Table 6.

[0124] Table 6

[0125]

[0126] 2.6 The effect of BSA / OVA-T4 coupling on the values ​​of TT4 and FT4 in composite quality control samples containing only TT4 and FT4 is shown in Table 7.

[0127] Table 7

[0128]

[0129] As shown in Tables 4 to 7, FT3 and FT4 levels were low after T3 / T4 without PEG conjugation was added to human serum, and adding PEG alone to serum did not improve the situation. However, after T3 / T4 was conjugated with PEG of different molecular weights and added to serum, the FT3 and FT4 levels were significantly increased, which was closer to or better than the results of commercially available products. Similarly, T3 / T4 modified with OVA or BSA also had similar effects.

[0130] 3. Effects of conjugation on the values ​​of TT3, FT3 / TT4, and FT4 in a thyroid hormone complex quality control product containing five analytes.

[0131] 3.1 Preparation steps of a thyroid hormone composite quality control product containing 5 analytes:

[0132] Based on steps 1.2 or 2.2 above in this embodiment, the third step requires the addition of T4 / T3, TSH, rT3, and TG antigens. During the preparation process, the concentrations of TSH, rT3, and TG need to be detected. The methods for detecting the concentrations of each analyte are as follows:

[0133] TSH concentration detection method: The detection principle is chemiluminescent immunoassay sandwich method. The analysis procedure is as follows: the instrument automatically adds 100 μL of sample, 40 μL of luminescent label, 40 μL of buffer solution, and 20 μL of magnetic microspheres, incubates for 30 min, then washes and detects.

[0134] rT3 concentration detection method: The detection principle is chemiluminescent immunoassay. The analytical procedure is as follows: the instrument automatically adds 50 μL of sample, 80 μL of luminescent label, 80 μL of fluorescein label, and 20 μL of magnetic microspheres, incubates for 15 min, then washes and detects.

[0135] TG concentration detection method: The detection principle is chemiluminescent immunoassay sandwich method. The analysis procedure is as follows: the instrument automatically adds 40 μL of sample, 20 μL of luminescent label, 20 μL of fluorescein label, and 20 μL of magnetic microspheres, incubates for 15 min, then washes and detects.

[0136] 3.2 When the composite quality control contains TSH, rT3, TG, and the T3 / T4 compound in step 1.1 or 2.1 of this embodiment, the effects on the values ​​of TT3 and FT3 in the composite quality control sample are shown in Table 8.

[0137] Table 8

[0138]

[0139] 3.3 When the composite quality control contains TSH, rT3, TG, or the T3 / T4 compound mentioned in step 1.1 or 2.1 of this embodiment, the effects on the values ​​of TT4 and FT4 in the composite quality control sample are shown in Table 9.

[0140] Table 9

[0141]

[0142] Compared to the control, when using PEG, BSA, or OVA to conjugate T4 / T3 antigens to prepare quality controls, the FT4 / FT3 concentration increased significantly while the TT4 / TT3 concentration remained constant. This indicates that using T3 and T4 antigens conjugated with PEG, BSA, or OVA to prepare quality controls can effectively increase the concentrations of FT3 and FT4. When the composite quality control also includes TSH, rT3, and TG, the concentrations of TT3 and FT3, or TT4 and FT4, did not change significantly. This suggests that TSH, rT3, and TG have little impact on the values ​​of TT4 and FT4, or TT3 and FT4, in the composite quality control.

[0143] Example 4: Preparation of quality control samples at the same level; differences in dosage between BSA / OVA-T3 / T4 coupling and PEG-T3 / T4 coupling.

[0144] The results above show that PEG, OVA, and BSA conjugated with T3 / T4 antigens can effectively increase the FT3 / FT4 value when used to prepare composite quality control samples, thus obtaining quality control samples that meet the concentration requirements. Among these three conjugates, the amount of PEG-T3 / T4 conjugate used is less than that of BSA / OVA-T3 / T4 when preparing the same level of quality control sample. Specific results are shown in Table 10 below:

[0145] Table 10

[0146]

[0147] Example 5 Stability Verification

[0148] 1) Experimental methods for stability testing:

[0149] The following two methods can be selected for stability verification and evaluation:

[0150] Synchronous evaluation method: The forward synchronous method is adopted, in which a sufficient amount of quality control samples are placed in the control condition storage, and one bottle is taken at each test time point and placed in the experimental condition storage. At the end of the calibration period, all of them are taken out and tested synchronously with the quality control samples stored in the control condition. Alternatively, the reverse synchronous method is adopted, in which a sufficient number of quality control samples are placed in the experimental condition, and one bottle is taken at each test time point and placed in the control condition storage. At the end of the calibration period, all of them are taken out and tested synchronously.

[0151] Classical evaluation method: Place a sufficient amount of quality control sample in the control and experimental conditions for storage, and take out one bottle for simultaneous testing at each monitoring time point.

[0152] ① Unopened at 4℃ for one year: Unopened liquid quality control samples should be stored at 2-8℃ and evaluated at the following time points. Third-party quality control is required for reagent kit calibration.

[0153] Product shelf life Time point setting 24 months 0, 1, 2, 6, 12, 18, 20, 24, 26 months

[0154] At each time point, take one bottle and test each analyte once on the compatible detection system. The potency of each analyte must meet the following requirements: (1) potency decrease ≤ 15%; (2) the overall CV (SD / M) of each analyte is ≤ 15% (Note: If there is a fluctuation of more than ±15%, a retest is required to confirm whether the reagent has a value jump).

[0155] ② Open at 4℃, 30 days: Opened liquid quality control samples are stored in a refrigerator at 2-8℃ and evaluated using the forward / reverse simultaneous method and the classical method, with the following time points set:

[0156] Claiming school term Time point setting 30 days 0, 1, 4, 7, 9, 14, 20, 30, 32 days

[0157] Note: Special settings for special projects.

[0158] At each time point, take one bottle and test each analyte once on the compatible detection system. The potency of each analyte must meet the following requirements: (1) potency decrease ≤ 15%; (2) the overall CV (SD / M) of each analyte is ≤ 15% (Note: If there is a fluctuation of more than ±15%, a retest is required to confirm whether the reagent has a value jump).

[0159] ③-20℃, 60 days: After the opened liquid quality control samples are stored at -20℃, they are evaluated using the forward / reverse synchronous method. The monitoring time points can be set to 0, 1, 4, 7, 9, 14, 20, 30, and 60 days. Each analyte is tested once on the compatible detection system. The potency of each analyte must meet the following requirements: (1) Potency decrease ≤ 10% (2) Overall CV (SD / M) of each analyte ≤ 15% (Note: If there is a fluctuation exceeding ±15%, a retest is required to confirm whether the reagent has a value jump).

[0160] ④ 37℃, 7 days: Unopened quality control samples are stored on a shaker at 40℃ with the shaker set to 180 rpm. Evaluation is performed using a forward / reverse synchronous method. Each analyte is tested once on the compatible detection system, with the evaluation time points set as follows:

[0161] Product shelf life Acceleration time point settings 24 months 0, 1, 2, 3, 5, 7, 8 days

[0162] Note: Special settings for special projects.

[0163] The following conditions must be met for the detection of the potency of each analyte: (1) the potency decrease is ≤15%; (2) the overall CV (SD / M) of each analyte is ≤15% (Note: if the fluctuation exceeds ±15%, a retest is required to confirm whether the reagent has dropped in value).

[0164] Stability test calculation method: The formula for calculating the drop in test results at different temperatures is: Drop = (Detected concentration at corresponding time / Debugging result - 1) * 100%.

[0165] 2) Coefficient of variation between bottles

[0166] Liquid lyophilized products are tested on the claimed detection system using qualified kits to detect each analyte in the quality control product.

[0167] For qualitative quality control, 10 negative control bottles from the same batch are randomly selected, and each control bottle is tested once on the detection system. The test results of each analyte should be within the claimed acceptable range.

[0168] For qualitative positive level quality control and quantitative quality control, 10 bottles of each level quality control from the same batch are randomly selected (if it is a lyophilized product, different plates and different positions of the plates need to be selected) and randomly numbered 1 to 10. Each quality control bottle is tested for each analyte 3 times on the detection system, and the 3 measurements are performed in different orders, such as 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9. F and S are calculated according to formulas (1) to (11). bb S r and CV 瓶间 .

[0169]

[0170] SS 瓶内 =SS 总和 -SS 瓶间 (3)

[0171]

[0172] v1=α-1(7)

[0173] v2=N-α(8)

[0174]

[0175] In the formula:

[0176] SS – Variance;

[0177] x i —The measurement or calculation result of the specified parameter for the i-th time;

[0178] —Overall average;

[0179] n i —Number of repeated measurements for sample i;

[0180] xij —The j-th result of sample i;

[0181] MS—Mean Square;

[0182] v — Degrees of freedom;

[0183] F—F-test value;

[0184] n0 — Number of valid measurements;

[0185] α — Number of samples drawn;

[0186] N—Total number of measurements;

[0187] S bb —Standard deviation between bottles;

[0188] S r —Intra-bottle standard deviation (repeatability standard deviation).

[0189] When F≤1, with S r Replace S bb Calculate CV 瓶间 Homogeneity of each analyte (CV) 瓶间 All should be ≤10%.

[0190] When F≤F 0.05(v1，v2) When the test results showed no significant difference in homogeneity between bottles, the calculated CV between bottles and the homogeneity of each analyte (CV between bottles) should all be ≤10%.

[0191] When F>F 0.05(v1，v2) S bb When the value is ≤0.3δ, the homogeneity between bottles is considered to be good. The calculated CV between bottles and the homogeneity of each analyte (CV between bottles) should both be ≤10%.

[0192] When F>F 0.05(v1，v2) S bb When the value is >0.3δ, the homogeneity between bottles is considered poor, which does not meet the requirements for analyte homogeneity (CV). 瓶间 All should be ≤10%.

[0193] Note: δ is the target standard deviation.

[0194] The inter-bottle homogeneity (2 mL / bottle) and stability of the liquid composite quality control product prepared in this disclosure were evaluated using novel industrial biochemiluminescence reagents and instruments. The specific results are shown in Table 11:

[0195] Table 11

[0196]

[0197]

[0198] The above measurement data shows that the three-level quality control samples prepared in this disclosure exhibit excellent inter-bottle homogeneity and stability, with an inter-bottle coefficient of variation within 7%. Unopened samples can be stably stored for one year at 2-8℃, and stably for 30 days after opening at 4℃; after opening at -20℃...

[0199] It is stable for 60 days; after 7 days of accelerated freezing at 37°C, the relative deviation of each analyte is within 15%; after 3 freeze-thaw cycles, the relative deviation of each analyte is within 8%, and the coefficient of variation between vials is within 7%.

[0200] Summarize:

[0201] This disclosure utilizes macromolecular conjugation of T3 / T4 antigens. Using the conjugated T3 / T4 antigens to prepare quality control products can effectively increase the concentrations of FT3 and FT4 in the serum matrix, avoiding the problem of existing technologies where adjusting FT3 and FT4 concentrations significantly affects the matrix. Furthermore, the modified T3 / T4 antigens are used to prepare a highly stable liquid thyroid composite quality control product that can be stably stored at 2-8℃. During routine quality control in the department, no reconstitution is required, simplifying the operation and avoiding deviations caused by improper reconstitution.

[0202] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A thyroid hormone-related quality control product, which is a solution with a serum matrix as the main solvent, and comprises an antigen conjugated with a biomedical carrier material having a contact angle θ greater than 90° and T3 and / or T4, wherein the T3 and / or T4 include bound and free states; the biomedical carrier material having a contact angle θ greater than 90° and the antigen conjugated with T3 and / or T4 enable the TT3 / TT4 and FT3 / FT4 values ​​in the quality control product to be maintained within the clinical sample concentration range; The biomedical carrier material includes polymers and / or carrier proteins; The carrier protein includes at least one of bovine serum albumin (BSA) and chicken ovalbumin (OVA); The polymer includes PEG modified with linker groups.

2. The quality control material according to claim 1, wherein the linking group comprises a carboxyl group or an amino group.

3. The quality control product according to claim 1 or 2, wherein each of the modified PEG groups with linking groups is one or two, and is the same type of linking group, and the linking groups are all located at the end of the PEG molecule.

4. The quality control material according to claim 1, wherein the polymer has a molecular weight of 2000 to 6000.

5. The quality control material according to claim 1, wherein the quality control material further comprises PEG modified with a linking group in a non-coupled state; the molar ratio of the PEG modified with the linking group to T3 and / or T4 is (7~13):

1.

6. The quality control material according to any one of claims 1, 2, 4, and 5, wherein the binding protein bound to the bound state T3 and / or T4 includes one or more of TBG, thyroxine transporter, and albumin.

7. The quality control material according to any one of claims 1, 2, 4, and 5, further comprising thyroid function-related analytes.

8. The quality control material according to claim 7, wherein the thyroid function-related analytes include one or more of TSH, rT3, TG, thyroid-stimulating hormone receptor antibody, anti-thyroid microsomal antibody, and anti-thyroid peroxidase antibody.

9. The quality control material according to any one of claims 1, 2, 4, 5, and 8, wherein the quality control material further comprises gonadal hormones.

10. The quality control material according to claim 9, wherein the sex hormone comprises at least one of luteinizing hormone, human chorionic gonadotropin, prolactin, progesterone, estradiol, testosterone, follicle-stimulating hormone, and anti-Müllerian hormone.

11. The quality control material according to any one of claims 1, 2, 4, 5, 8, 10, wherein the quality control material further comprises at least one of a buffer component, a polyol, a preservative, a sugar, an amino acid, and a surfactant.

12. The quality control material according to claim 11, wherein the quality control material comprises 0.05% to 0.15% Proclin 300, 2% to 8% sucrose, 1% to 5% mannitol, and 0.01% to 0.1% Tween 80.

13. The quality control material according to claims 1, 2, 4, 5, 8, 10, and 12, wherein the serum matrix comprises one or more of hormone-free serum, healthy animal serum, and lipid-free serum.

14. The quality control material according to claim 13, wherein the serum matrix does not contain anti-TG antibodies.

15. A kit comprising the quality control material according to any one of claims 1-14.

16. The kit according to claim 15, wherein the kit is used for detecting thyroid function.

17. The kit according to claim 15, wherein it is an immunoassay kit.

18. A method for preparing the quality control material according to any one of claims 1-14, comprising: Mix all components thoroughly and couple the biomedical carrier material therein with T3 and / or T4.

19. The method for preparing the quality control material according to claim 18, wherein the serum matrix is ​​purified by affinity chromatography to remove antibodies therein.

20. The method for preparing the quality control material according to claim 19, wherein the removed antibody is selected from anti-TG antibodies.

21. The method for preparing the quality control material according to claim 18, wherein the biomedical carrier material is coupled to T3 and / or T4 under the action of an activator.

22. The method for preparing the quality control material according to claim 21, wherein the activator comprises carbodiimide.

23. The method for preparing the quality control material according to claim 21, wherein the biomedical carrier material is PEG modified with amino or carboxyl groups, the activator is EDC, and the molar ratio of modified PEG to EDC is 1:(3~7).

24. Use of the quality control material according to any one of claims 1-14 in the preparation of a kit for the determination of thyroid function.

25. The application according to claim 24, wherein thyroid function is measured using an immunoassay.

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