Thyroid hormone related quality control substances

Abstract

The invention relates to the technical field of biological detection, in particular to a thyroid hormone related quality control substance. The quality control substance is a solution taking a serum matrix as a main solvent, and comprises an antigen obtained by coupling a biomedical carrier material with a contact angle theta greater than 90 degrees and T3 and / or T4, and the T3 and / or T4 comprise a binding state and a free state.

Description

Technical Field

[0001] The present disclosure generally relates to the field of biological detection technology, and in particular, to a thyroid hormone-related quality control material. Background Art

[0002] Thyroid diseases are common and frequently occurring diseases in the endocrinology department, and there are many types of them, including Hashimoto's thyroiditis, hyperthyroidism, hypothyroidism, nodular goiter, and thyroid tumors. At present, the specific pathogenesis of thyroid diseases has not been clarified clinically. The causes of such diseases are complex and diverse, including congenital defects, family inheritance, drugs, lifestyle habits, age, etc., and some factors are difficult to prevent, which will have a great impact on patients' daily lives. Fortunately, most thyroid diseases can be cured, but some diseases have a risk of recurrence and need to be diagnosed and treated as soon as possible.

[0003] Thyroid hormones are secreted by thyroid tissues, which can regulate various functions of the body, control human growth and development, and the metabolism of carbohydrates, proteins, and fats, and have important effects on the nervous system, endocrine system, and cardiovascular system. If a disease occurs in the thyroid tissue, the thyroid hormone level will also change accordingly. Different thyroid diseases have different pathogenesis, and there are certain differences in the expression of thyroid hormone levels. Therefore, it is necessary to timely and accurately evaluate and judge the functional status of the patient's thyroid gland in clinical practice. At present, the commonly used indicators for clinical detection of thyroid function include thyroid stimulating hormone (TSH), total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), and free thyroxine (FT4). Among them, TT4 is divided into bound and free forms. Most of the serum T4 exists in the blood in the form of binding to TBG, and only a very small part of T4 exists in free form. Therefore, the content of FT4 in serum is extremely low, and the same is true for TT3. In addition, other indicators such as rT3 and TG are also clinically important. Reverse triiodothyronine (rT3) is an isomer of T3, but it is not physiologically active and is often used to differentiate hypothyroidism from "low T3 syndrome". Thyroglobulin (TG) is a macromolecular glycoprotein synthesized by thyroid follicular epithelial cells. Under normal circumstances, only a very small amount of TG enters the blood circulation. Clinically, the dynamic changes of TG are often observed to observe the surgical efficacy of thyroid cancer patients and monitor the recurrence of differentiated thyroid cancer after surgery. Accurate detection of thyroid hormones is very important for the diagnosis, treatment monitoring, and prognosis evaluation of thyroid diseases.

[0004] During the testing process, quality control products are one of the important substances for quality assurance. By using quality control products, we can verify and calibrate the laboratory's analytical methods to ensure the accuracy of the test results. Quality control products can also help discover potential problems in the laboratory, such as instrument performance deviations, operator errors, etc., so that they can be corrected and improved in a timely manner. By using the same quality control products between different laboratories, the test results of each laboratory can be compared to evaluate the consistency and comparability between laboratories. This can effectively solve the problem of differences in test results between different laboratories and improve the credibility and comparability of data.

[0005] In addition, ideal quality control products generally have good stability, small differences between bottles, and appropriate quality control concentration levels. For thyroid hormone composite quality control products, the values ​​of TT3 / TT4 and FT3 / FT4 need to be controlled at the same time to be kept within the clinical sample concentration range. However, since FT4 / FT3 in serum binds to thyroid binding protein (TBG), TT3 and TT4 are mainly in a bound state in serum, and the values ​​of FT4 and FT3 are affected by binding affinity, resulting in a low content of FT4 / FT3 in serum, which cannot meet the concentration requirements of the department's internal quality control. It has been reported in the literature that the binding affinity of FT4, FT3 and TBG is affected by factors such as temperature and pH. By adjusting the temperature and pH of the serum matrix to reduce the binding affinity of TBG with FT4 and FT3, the concentrations of FT4 and FT3 can be controlled, but this often causes the loss of protein in the serum and affects its matrix. Summary of the invention

[0006] One of the purposes of the present disclosure is to provide a quality control material, which is a solution with serum matrix as the main solvent, and comprises an antigen obtained by coupling a biomedical carrier material with a contact angle θ greater than 90° and T3 and / or T4, wherein the T3 and / or T4 include a bound state and a free state. .

[0007] Yet another object of the present disclosure is to provide a kit comprising the quality control substance as described above.

[0008] Another object of the present 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 the present disclosure is to provide a use of the quality control substance as described above in the determination of thyroid function.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] Coupling T3 and / or T4 with biomedical carrier materials can increase the concentration of free T3 and / or T4, so that the values ​​of TT3 / TT4 and FT3 / FT4 in quality control materials can be maintained within the concentration range of clinical samples. DETAILED DESCRIPTION

[0012] References to embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0013] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present disclosure is the same as that commonly understood by those of ordinary skill in the art to which the present disclosure belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0014] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0015] The terms "and / or", "or / and", and "and / or" used herein have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, wherein the any 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 the present disclosure, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0016] As used in this disclosure, the terms "comprising," "including," and "including" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0017]

[0013] Numerical ranges recited in this disclosure as endpoints include all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0018] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0019] The present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range. For example, fluctuations can be allowed within a corresponding precision range. For example, 2%, a fluctuation within the range of ±0.1% can be allowed. For values ​​that are large or do not require too fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. can be allowed. Involving molecular weight, the meaning is allowed to include fluctuations of ±10%.

[0020] In the present disclosure, if "v / v" or "m / v" is not specifically written before the percentage sign, it is determined according to the common understanding of those skilled in the art or the usual weighing method. If the solute is solid in the normal state, it is m / v; if it is liquid, it is v / v. The normal state is generally at room temperature and pressure, or under the normal use conditions of the substance (such as some substances generally need to be refrigerated, then the normal state is the state at low temperature). Wherein m / v represents the mass volume ratio, and m / v% represents the mass percentage contained in a certain volume of the mixed system. For example, the concentration of substance A in the mixed system is 5m / v%, which means that 5 grams of substance A are contained in every 100 ml of the mixed system. In the present disclosure, v / v represents the volume ratio, and v / v% represents the volume percentage contained in a certain volume of the mixed system. For example, the concentration of substance A in the mixed system is 5v / v%, which means that 5 ml of substance A are contained in every 100 ml of the mixed system. The commonly used components in the present disclosure are described in percentage according to their usual states, for example, as follows, usually expressed as m / v %: sucrose; usually expressed as v / v %: Proclin 300, mannitol, Tween 80.

[0021] In the present disclosure, "quality control material" and "quality control material" and "quality control product" can be used interchangeably, and refer to a solution used for at least one of the purposes of a calibrator, quality control product and reference product when detecting a target substance (including but not limited to thyroid hormone), which contains T3 and / or T4 antigens.

[0022] As used herein, "biomedical carrier material" refers to a controlled release / delivery system mainly used for drugs and bioactive substances (such as polypeptides, proteins and growth factors, nucleic acids, cells, etc.). Herein, it is usually a solid or semi-solid (e.g., gel-like) substance that can couple T3 and / or T4 antigens and increase their concentration through steric hindrance. Exemplary biomedical carrier materials include: polymer carrier materials, which are mainly divided into natural polymers and derivatives and artificial synthetic polymer materials. Natural polymer materials are mainly polysaccharides and proteins, and synthetic polymer materials include polyanhydrides, aliphatic polyesters represented by polylactic acid, polyphosphates, polyorthoesters, etc., in addition to polyethylene glycol and its derivatives, polyamino acids, etc.

[0023] In the present disclosure, "T3 and / or T4" includes T3 antigens in bound and free states, and / or T4 antigens in bound and free states. Depending on the context, it can also be replaced 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 binding proteins; "free state" is relative to "bound state", and refers to the state in which T3 and / or T4 antigens are dissociated from their respective binding proteins, but "free state" does not mean that they are not coupled to biomedical carrier materials. When they are in a bound state, the part coupled to the biomedical carrier material is still T3 and / or T4 itself, not its binding protein.

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

[0025] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0026] In the present disclosure, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0027] In the present disclosure, "preferred", "better", "more preferred", and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0028] All documents mentioned in the present disclosure are cited as references in the present disclosure, just as each document is cited as a reference individually. Unless they conflict with the invention objectives and / or technical solutions of the present disclosure, the cited documents involved in the present disclosure are cited with all their contents and all their purposes. When the present disclosure involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure involves cited documents, the examples and preferred methods of the cited relevant technical features may also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure shall prevail or be modified adaptively according to the description of the present disclosure.

[0029] The first aspect of the present disclosure provides a quality control material, which is a solution with serum matrix as the main solvent, and comprises an antigen obtained by coupling a biomedical carrier material with a contact angle θ greater than 90° and T3 and / or T4, wherein the T3 and / or T4 include a bound state and a free state. .

[0030] Compared with the prior art method of improving the binding affinity of T3, T4 and binding proteins from the perspective of temperature and pH, the present disclosure provides a completely different solution, that is, by changing the steric hindrance, the binding affinity of T4, T3 and binding proteins is changed. In this solution, after T3 and / or T4 are coupled with the biomedical carrier material, the steric hindrance increases, resulting in a weakened binding affinity with binding proteins such as TBG, thereby resulting in a decrease in bound T3 / T4 and an increase in free T3 and T4, so that the values ​​of TT3 / TT4 and FT3 / FT4 in the quality control product can be maintained within the clinical sample concentration range.

[0031] The term "coupling" as used herein refers to covalent interactions, non-covalent interactions and steric interactions, or a combination thereof. Covalent interactions are chemical connections between two atoms or groups formed by sharing a pair of electrons (single bonds), two pairs of electrons (double bonds) or three pairs of electrons (triple bonds). Covalent interactions are also referred to as electron pair interactions or electron pair bonds in the art. 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 reviews of non-covalent interactions, see Alberts et al., Molecular Biology of the Cell, 3D edition, Garland Publishing, 1994. Steric interactions are generally understood to include those in which the structure of the compound enables it to occupy a site through its three-dimensional structure, rather than any attraction between the compound and the site. Coupling is usually tight and difficult to separate, and preferably the coupling of T3 and / or T4 to 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 bridge.

[0032] "Biomedical carrier materials with contact angle θ greater than 90°" can be roughly understood as hydrophilic biomedical carrier materials. The contact angle θ refers to the angle between the tangent of the gas-liquid interface at the intersection of the gas, liquid and solid phases passing through the liquid and the solid-liquid boundary line. It is a measure of the degree of wettability. Generally speaking, the more hydrophilic the chemical properties of the material, the smaller the contact angle; the more hydrophobic the chemical properties, the larger the contact angle. In some embodiments, the biomedical carrier material includes a polymer and / or a carrier protein. Among them, the biomedical carrier materials listed in the embodiments of the present disclosure, such as BSA, KLH, OVA, PEG, etc., are all hydrophilic materials (for example, "Principles and Applications of Biomedical Nanomagnetic Materials" 2005.P423, Duan Jiufang, ed. "Natural Polymer Materials" 2016.P156), so 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 includes PEG modified with a linking group; further, the linking group includes a carboxyl group or an amino group. The number of T3 / T4 antigens that can be connected to PEG is 1 to 2. In a preferred embodiment, each of the PEG modified with a linking group has one or two modified groups, and they are the same type of linking groups, and the linking groups are all located at the ends of the PEG molecule. In this way, the antigen epitopes will not shield each other. When used as a quality control product, a higher antigen utilization rate can be achieved, and the amount of antigen used is also less.

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

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

[0037] In some embodiments, the quality control material further comprises the biomedical carrier material in a non-conjugated state, such as PEG modified with a linking group. In the quality control material, the molar ratio of the PEG modified with a linking group to the T3 and / or T4 is (7-13):1, such as 8:1, 9:1, 10:1, 11:1, 12:1.

[0038] The quality control material may contain both PEG coupled to T3 and / or T4 and free PEG. When the carrier material is PEG, it is preferably not purified after coupling with T3 and / or T4. The solution contains free PEG, which makes the entire preparation process simpler, and the free PEG will not adversely affect the test results when the quality control material is used. In addition, at this molar ratio, PEG is significantly excessive, which can ensure that T3 or T4 fully forms a coupled antigen during the preparation process.

[0039] In some embodiments, the binding proteins to which the bound T3 and / or T4 are bound include one or more of TBG, thyroxine transporter and albumin. Under physiological conditions, about 99.98% of T4 in circulation is usually bound to specific plasma proteins, including thyroxine binding globulin (TBG, accounting for 60% to 75%), thyroxine binding prealbumin (TBPA, accounting for 15% to 30%) and albumin (ALB, accounting for 10%); only 0.02% of T4 in circulation is usually in a free state; 99.7% of T3 in circulation is specifically bound to TBG, and about 0.3% is in a free state (FT3). Therefore, the binding of TBG is usually the main one, and the preferred binding protein is TBG.

[0040] In some embodiments, it further comprises a thyroid function-related analyte.

[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 antibodies, anti-thyroid microsomal antibodies, and anti-thyroid peroxidase antibodies.

[0043] In some embodiments, the quality control further comprises gonadal hormones.

[0044] In some embodiments, the gonadal hormone includes at least one of luteinizing hormone, human chorionic gonadotropin, prolactin, progesterone, estradiol, testosterone, follicle stimulating hormone and anti-Mullerian hormone. Clinically, thyroid function-related analytes and gonadal hormones are often detected together. Thyroid hormones have a certain regulatory effect on the development of testicles and ovaries, and 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, and thyroid cells further proliferate to form nodules. Estrogen can also promote the proliferation of thyroid cancer cells through estrogen receptor-dependent mechanisms, and even participate in angiogenesis and metastasis processes, which plays an important role in the outcome and prognosis of thyroid cancer. In addition, the coordinated detection of the two can also 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 gonadal hormones contained in the quality control materials can be any of natural antigens, recombinant antigens, and synthetic antigens. For example, the natural antigens selected are TG, which can be extracted naturally. The use of natural raw materials is close to clinical samples and can better reflect the problems existing in the detection system; the synthetic antigens selected are FT3, FT4, and rT3. These antigens are small molecules, not easy to extract naturally, but easy to synthesize; the recombinant antigens include TSH antigens, which are obtained by recombinant expression of mammalian cells and are 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 the present disclosure, it is preferred that the quality control material contains a buffer component. As used herein, the term "buffer component" refers to an aqueous solution or composition that resists changes in pH and maintains a stable pH value when an acid or base is added to the solution. This resistance to pH changes is due to the presence of a buffer substance in such a solution. Therefore, a solution that can offset and reduce the effect of an external strong acid or strong base on the pH of a solution to a certain extent, thereby maintaining a relatively stable pH value of the solution, is referred to as a buffer or buffer solution. Buffers generally do not have the ability to maintain the pH of a solution or composition infinitely. Instead, they are generally able to maintain a pH within a specific range, such as pH 6–pH 10, such as pH=6.5, pH=7, pH=8, pH=9, pH=9.5. As exemplary buffer components, at least one of phosphate buffer, HEPES buffer, Tris-HCl buffer, glycine buffer and borate buffer can be mentioned.

[0048] In some embodiments, the quality control material contains polyols. Polyols have a good stabilizing effect on the non-polar surface of proteins, for example, they can keep the surface of antigens moist so that they will not be inactivated due to water loss, increase viscosity, reduce molecular motion, avoid protein aggregation, and have the effect of reducing wall absorption and antifreeze. The polyols include at least one of sugar alcohols, glycerol, butanediol, inositol, and ethylene glycol. In some embodiments, the sugar alcohols may include one or more of sorbitol, mannitol, erythritol, maltitol, lactitol, and xylitol. It is further preferred that the addition amount of the polyols is 0.1% to 10%, for example 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%.

[0049] In some embodiments, the quality control material contains a preservative. Preservatives are added to prevent organic substances such as amino acids and sugars in proteins and stabilizers from being contaminated by microorganisms in the air and water as the storage time increases. Exemplary preservatives can be ProClin 300, sodium azide, Bronidox, Niusha BND-10, Niusha PC-300, Niusha PC-950, Niusha BIT-10, liquid preservative MB-1; preferably ProClin 300. Those skilled in the art can freely choose the amount of addition according to the characteristics of the specific preservative, and an exemplary amount of addition is 0.01% to 0.5%, such as 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 osmotic effects. They can penetrate and diffuse into the interior of the protein to prevent the hydrophobic segment structure inside the protein from contacting water molecules, thereby improving stability. Sugars can be monosaccharides or polysaccharides, examples of which include at least one of glucose, fructose, lactose, sucrose and trehalose. Exemplary added amounts of sugar are 0.1% to 20%, for example 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%.

[0051] In some embodiments, it is preferred that the quality control material contains amino acids. Amino acids can stabilize proteins through free energy differences and surface tension, and amino acids such as lysine can form electrostatic interactions with certain specific amino acids between peptide chains to improve stability. 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, histidine hydrochloride). It is further preferred that the amount of amino acids added is 0.01% to 5%, for example 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%.

[0052] In some embodiments, the quality control material contains a surfactant. Surfactants have dispersing and solubilizing effects; they can destroy non-covalent bonds such as hydrogen bonds between proteins, depolymerize protein aggregates, and prevent aggregation between proteins. Preferred surfactants include non-ionic 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). An exemplary addition amount of the surfactant is 0.01% to 5%, such as 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, and 4%.

[0053] In some specific embodiments, the quality control substances 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 hormone-free serum, healthy animal serum, and delipidated serum.

[0055] Hormone-free serum refers to serum from which hormones such as T4, T3, FT3, FT4, FSH, LH, etc. are removed. The basic values ​​of T4, FSH, HCG, etc. in most commercial sera are too high and cannot be used for quality control of level 1 (negative quality control). Therefore, hormone-free serum is required for quality control of level 1. Delipidated serum refers to serum from which lipid substances are removed. When serum is stored at 2-8°C for a long time, lipid substances are easily precipitated and cause turbidity due to changes in serum pH and other reasons. Delipidated serum is conducive to long-term storage and avoids interference from lipid substances.

[0056] When the quality control material contains TG, since the serum matrix contains anti-TG antibodies (TGA), TGA can bind to TG and catalyze the hydrolysis of TG, resulting in a reduction in the TG to be measured, interfering with the detection of TG and affecting the test results. Therefore, it is better to prepare the quality control material with a serum matrix that removes TGA. In some specific embodiments, the serum matrix does not contain anti-TG antibodies.

[0057] The method of removing TGA from serum matrix is ​​conventional in the art, for example, it can be purified by one or more affinity chromatography columns such as A column, L column and G column. A column refers to an affinity chromatography column that covalently couples protein A to an agarose medium. Protein A is derived from a strain of Staphylococcus aureus and contains 5 domains that can specifically bind to the Fc segment of antibody IgG molecules. Protein A is coupled to the agarose matrix as an affinity ligand, which can specifically bind to the antibody molecules in the sample and allow other impurities to flow through, with extremely high selectivity. One Protein A molecule can bind to at least 2 IgGs. In addition to IgG, Protein A can also bind to other immunoglobulins, such as IgA and IgM used for purification of certain species. A new Cysteine ​​is added to the C-terminus of recombinant Protein A, which can be coupled to the agarose matrix in a single site-directed manner to reduce the steric hindrance of contact with the antibody. L column refers to an affinity chromatography column that covalently couples protein L to agarose medium. Protein L is an immunoglobulin binding protein that can bind to immunoglobulin K light chain without affecting antigen binding. Compared with other antibody binding proteins such as Protein A and Protein G, Protein L has a wider range of Ig and Ig subtype binding: it can not only bind to all Ig classes (IgG, IgM, IgA, IgE and IgD), but also single-chain antibodies and Fab fragments. G column refers to an affinity chromatography column that covalently couples protein G to agarose medium. Protein G is a cell wall protein isolated from G-type Streptococci with a molecular weight of 25kDa. Compared with 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 that cannot bind well to Protein A.

[0058] In addition, in some preferred embodiments, the quality control material provided by the present disclosure has good liquid stability. The increase / maintenance of stability can be evaluated by one or more dimensions, which is familiar to those skilled in the art. Dimensions generally include the degree of destruction of the natural structure and / or function of the protein caused by various physical and chemical conditions (such as temperature, surface tension, oxidation, acid-base, deamination) and biological factors (such as protease hydrolysis). Common indicators of natural structure include changes in hydrogen bonds, ionic bonds, disulfide bonds, peptide bonds, three-dimensional structures, etc., and functions such as enzyme activity, immunogenicity, reactivity, etc. In some cases, stability refers to conformational stability. The increase in stability refers to the use of conventional preservation solutions under the same storage conditions. The general conditions for storage are usually sealed, short-term 2°C to 8°C, long-term -20°C freezing; in special cases, it can also be frozen at -80°C, or stored in liquid nitrogen. In some preferred embodiments of the present disclosure, the quality control material can be stably stored at 2-8°C for 1 year without opening, and can be stable for 30 days after opening at 4°C; it can be stable for 60 days after opening at -20°C; the relative deviation of each analyte is within 15% after accelerated storage at 37°C for 7 days; and the relative deviation of each analyte is within 8% after repeated freezing and thawing for 3 times.

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

[0060] The term "kit" in the present disclosure may refer to any article (e.g., package or container) comprising at least one device, which includes a quality control as described in the present disclosure. The kit may further include instructions for use, supplementary reagents and / or components or assemblies used in the methods or steps thereof described in the present disclosure. The components in the kit may be packaged in the form of solutions, solids or test strips. The target substance, such as components required for thyroid hormone detection, particularly various proteins, antibodies, peptides, and reaction buffer components, etc., may be provided in solid form. The solid includes at least one of freeze-dried microspheres, freeze-dried cakes, freeze-dried powders, and spots that rely on the presence of a solid medium.

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

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

[0063] The third aspect of the present 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] Among them, all the components can be mixed first and then the biomedical carrier material and T3 and / or T4 can be coupled; or the biomedical carrier material and T3 and / or T4 can be coupled first and then the conjugate is mixed with other components. There is no order of precedence.

[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 the T3 and / or T4 under the action of an activating agent.

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

[0068] In some embodiments, the biomedical carrier material is PEG modified with an amino group or a carboxyl group, the activator is EDC, and the molar ratio of the 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 the 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 a linking group, and the molar ratio of the PEG modified with a linking group to the T3 and / or T4 is (7-13):1, preferably 10:1.

[0071] A fourth aspect of the present disclosure provides use of the quality control substance described above in the determination of thyroid function.

[0072] The method of using the quality control material / the above kit is not particularly limited, as long as it is necessary to improve the stability of the quality control material and / or improve the accuracy of thyroid detection. In some embodiments, the determination is performed using an immunoassay. Examples include magnetic particle chemiluminescence, fluorescence chromatography, latex turbidimetry and other platforms.

[0073] The description of the first aspect of the present disclosure is equally applicable to the second, third and fourth aspects of the present disclosure.

[0074] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be based on the experimental manuals or conventional conditions in the art, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0075] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

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

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

[0078] Step 1: Activate hydrophilic substances with EDC

[0079] An appropriate amount of PEG with a molecular weight of 2000-6000 and a carboxyl group is weighed and dissolved in an organic solvent such as DMF or DMSO. EDC is then added and reacted at room temperature for 8 hours to obtain a carboxyl-activated PEG solution (PEG:EDC molar ratio is 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, and 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 product

[0083] The preparation method of composite quality control products such as thyroid hormone composite quality control products is as follows:

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

[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 well to obtain a clear matrix solution, and store at 2-8°C;

[0086] Step 3: Add T3 coupled with PEG / BSA / OVA, T4 coupled with PEG / BSA / OVA, TSH, rT3, and TG antigens, and 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 products, which are packaged and stored in a refrigerator at 2-8°C. The concentrations of each analyte in different levels of thyroid hormone liquid composite quality control products are shown in Table 1:

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

[0088]

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

[0090] Example 3 Effect of coupling on the concentration of TT3 / FT3 and TT4 / FT4 in composite quality control products

[0091] 1. The effect of different molecular weight PEG coupling on the values ​​of TT3, FT3 / TT4, and FT4 in composite quality control products.

[0092] 1.1 The specific preparation steps of PEG-coupled 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 hours to obtain a carboxyl-activated PEG solution.

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

[0095] Table 2 The amount of T3 / T4 antigen and PEG modified with carboxyl or amino group added during the preparation of T3 / T4 coupled with PEG with molecular weight 2000-6000

[0096]

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

[0098] Step 1: Thaw 500 mL of serum matrix and store it at 2-8°C, purify it using an L-column affinity chromatography column, and filter it;

[0099] Step 2: Add 0.1% Proclin 300 to the serum matrix obtained in the first step, then add 5% sucrose, 3% mannitol, and 0.05% Tween 80, stir and mix thoroughly to obtain a clear matrix solution, and store at 2-8°C;

[0100] Step 3: Add T3 / T4 antigens coupled with PEG of different molecular weights to the serum matrix obtained in the second step, detect the concentrations of TT3, FT3 / TT4, and FT4, and control the concentration of each analyte by adjusting the amount of T3 / T4 antigens coupled with PEG of different molecular weights to obtain high, medium, and low levels of liquid composite quality control products of thyroid hormones, which are then packaged and stored in a refrigerator at 2 to 8°C.

[0101] Introduction to the detection method of TT3 and FT3 concentration in the process of preparing composite quality control products

[0102] The TT3 and FT3 chemiluminescent detection reagents of New Industry Biomedical Engineering Co., Ltd. were used for detection. The detection principle of TT3 reagent is as follows: the detection principle is chemiluminescent immunocompetitive method, and the analysis procedure is: the instrument automatically adds 40μL sample, 20μL buffer, 40μL luminescent marker, incubates for 5 minutes and then washes, and then automatically adds 20μL magnetic microspheres and 20μL buffer, incubates for 10 minutes and then washes and detects. The detection principle of FT3 reagent is: the detection principle is chemiluminescent immunocompetitive method, and the analysis procedure is: the instrument automatically adds 40μL sample, 20μL buffer, 40μL luminescent marker, incubates for 5 minutes and then washes, and then automatically adds 20μL magnetic microspheres and 80μL buffer, incubates for 10 minutes and then washes and detects.

[0103] Introduction to the detection method of TT4 and FT4 concentrations in the preparation of composite quality control products:

[0104] The TT4 and FT4 chemiluminescent detection reagents of New Industry Biomedical Engineering Co., Ltd. were used for detection. The detection principle of the TT4 reagent is as follows: the detection principle is the chemiluminescent immunocompetitive method, and the analysis procedure is: the instrument automatically adds 40μL sample, 40μL replacement agent, 40μL luminescent marker, 80μL buffer, and 20μL magnetic microspheres. After incubation for 15min, wash and detect. The detection principle of the FT4 reagent is the chemiluminescent immunocompetitive method, and the analysis procedure is: the instrument automatically adds 40μL Xiangben, 40μL buffer, 40μL luminescent marker, and 20μL magnetic microspheres, and after incubation for 15min, wash and detect.

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

[0106] 2.1 The specific preparation steps of BSA / OVA-coupled T3 / T4 antigen are as follows:

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

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

[0109] Step 3: Purification: The reaction liquid was transferred to a dialysis bag of MwCO20000 and dialyzed with PBS solution at room temperature for 8 hours, with the solution replaced every 2 hours to obtain BSA / OVA-coupled T3 / T4 antigens.

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

[0111] Modification Type T3 Addition T4 Addition 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 Steps for preparing quality control products containing only TT3, FT3 / TT4, and FT4 after coupling BSA / OVA with T3 / T4:

[0113] Step 1: Thaw 500 mL of serum matrix and store it at 2-8°C, purify it using an L-column affinity chromatography column, and filter it;

[0114] Step 2: Add 0.1% Proclin 300 to the serum matrix obtained in the first step, then add 5% sucrose, 3% mannitol, and 0.05% Tween 80, stir and mix thoroughly to obtain a clear matrix solution, and store at 2-8°C;

[0115] Step 3: Add BSA / OVA-coupled T3 / T4 antigens to the serum matrix obtained in step 2, detect the concentrations of TT3, FT3 / TT4, and FT4, and control the concentration of each analyte by adjusting the amount of BSA / OVA-coupled T3 / T4 antigen added to obtain high, medium, and low levels of thyroid hormone liquid composite quality control products, which are then packaged and stored in a refrigerator at 2 to 8°C.

[0116] 2.3 The effects of different molecular weight PEG coupling on the values ​​of TT3 and FT3 in the composite quality control product containing only TT3 and FT3 are shown in Table 4.

[0117] Table 4

[0118]

[0119]

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

[0121] Table 5

[0122]

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

[0124] Table 6

[0125]

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

[0127] Table 7

[0128]

[0129] It can be seen from Tables 4 to 7 that after T3 / T4 without PEG coupling was added into human serum, FT3 and FT4 were low and adding PEG alone into the serum had no improvement effect. However, after T3 / T4 was coupled with PEG of different molecular weights and added into serum, FT3 and FT4 results increased significantly, which were closer to or better than the results of commercially available products. Similar effects were also achieved by modifying T3 / T4 with OVA or BSA.

[0130] 3. The effect of coupling on the values ​​of TT3, FT3 / TT4, and FT4 in a thyroid hormone composite quality control product containing five analytes.

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

[0132] Based on the above step 1.2 or 2.2 of this embodiment, the third step also 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 the chemiluminescent immunosandwich method. The analysis procedure is: the instrument automatically adds 100 μL sample, 40 μL luminescent marker, 40 μL buffer, 20 μL magnetic microspheres, incubates for 30 minutes, then washes and detects.

[0134] rT3 concentration detection method: The detection principle is chemiluminescent immunocompetitive method. The analysis procedure is: the instrument automatically adds 50μL sample, 80μL luminescent marker, 80μL fluorescent marker, 20μL magnetic microspheres, incubates for 15 minutes, then washes and detects.

[0135] TG concentration detection method: The detection principle is the chemiluminescent immunosandwich method. The analysis procedure is: the instrument automatically adds 40μL sample, 20μL luminescent marker, 20μL fluorescent marker, 20μL magnetic microspheres, incubates for 15 minutes, then washes and detects.

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

[0137] Table 8

[0138]

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

[0140] Table 9

[0141]

[0142] Compared with the control, when PEG, BSA or OVA were used to couple T4 antigen / T3 antigen to prepare quality control, the concentration of FT4 / FT3 increased significantly when the concentration of TT4 / TT3 remained unchanged, indicating that the concentration of FT3 and FT4 can be effectively increased by using T3 and T4 antigens coupled with PEG, BSA or OVA to prepare quality control products. When TSH, rT3 and TG were included in the composite quality control, the concentration of TT3 and FT3 or TT4 and FT4 did not change significantly, indicating that TSH, rT3 and TG had little effect on the values ​​of TT4, FT4 or TT3 and FT4 in the composite quality control products.

[0143] Example 4 Preparation of the same level of quality control products, BSA / OVA-T3 / T4 coupling and PEG-T3 / T4 coupling preparation dosage difference

[0144] From the above results, it can be seen that PEG, OVA, and BSA conjugated T3 / T4 antigens can effectively increase the FT3 / FT4 value when used to prepare composite quality control products, and obtain quality control products that meet the concentration requirements. Among these three conjugates, the amount of PEG-T3 / T4 conjugated preparation is less than that of BSA / OVA-T3 / T4 when preparing the same level of quality control products. The specific results are shown in Table 10 below:

[0145] Table 10

[0146]

[0147] Example 5 Stability Verification

[0148] 1) Experimental method for stability test:

[0149] There are two options for each stability verification and evaluation method:

[0150] Synchronous method evaluation method: Use the forward synchronization method, place sufficient quality control products under control conditions for storage, take out 1 bottle at each test time point and place it under experimental conditions for storage, take out all of them at the end of the calibration period, and test them synchronously with the quality control products stored under control conditions; or use the reverse synchronization method, take a sufficient number of quality control products and place them under experimental conditions, take out 1 bottle at each test time point and place it under control conditions for storage, take out all of them at the end of the calibration period for synchronous testing.

[0151] Classical evaluation method: Store sufficient quality control products under control and experimental conditions, and take out one bottle for simultaneous testing at each monitoring time point.

[0152] ① Unopened at 4°C, one year: Unopened liquid quality control products are stored at 2-8°C and evaluated at the following time points. A third-party quality control is required to calibrate the kit.

[0153] Product Expiration Date 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 adapted detection system. The titer of each analyte must meet the following requirements: (1) the titer drop is ≤15%; (2) the overall CV (SD / M) of each analyte is ≤15% (Note: if there is a fluctuation exceeding ±15%, retesting is required to confirm whether the reagent has a jump value).

[0155] ②Open bottle at 4℃, 30 days: The opened liquid quality control products are stored in a refrigerator at 2-8℃ and evaluated using the forward / reverse synchronous method and the classical method. The time points are set as follows:

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

[0157] Note: Special project specific settings.

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

[0159] ③-20℃, 60 days: After the opened bottles of liquid quality control products 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 adapted detection system. The potency of each analyte must meet the following requirements: (1) The potency drop is ≤10%; (2) The overall CV (SD / M) of each analyte is ≤15% (Note: If there is a fluctuation exceeding ±15%, retesting is required to confirm whether the reagent has a jump value).

[0160] ④37℃, 7 days: Unopened quality control products are stored in a 40℃ shaker with the shaker set to 180rpm. The forward / reverse synchronous method is used for evaluation. Each analyte is tested once on the adapted detection system. The evaluation time points are set as follows:

[0161] Product Expiration Date Acceleration time point setting 24 months 0, 1, 2, 3, 5, 7, 8 days

[0162] Note: Special project specific settings.

[0163] The titer of each analyte must meet the following requirements: (1) the titer drop is ≤15%; (2) the overall CV (SD / M) of each analyte is ≤15% (Note: if there is a fluctuation exceeding ±15%, retesting is required to confirm whether the reagent has a jump value).

[0164] Calculation method for stability test: Test results at different temperatures - calculation formula for reduction: reduction = (corresponding time detection concentration / debugging result - 1) * 100%.

[0165] 2) Coefficient of variation between bottles

[0166] The liquid lyophilized product is tested for each analyte in the quality control product using a qualified kit on the claimed detection system.

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

[0168] For qualitative positive horizontal quality control and quantitative quality control, randomly select (if it is freeze-dried products, different plate layers and different positions of the plate layers need to be selected) 10 bottles of each horizontal quality control from the same batch and randomly number them 1 to 10. Each bottle of quality control 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. Calculate F and S 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)

[0172]

[0173] v1=α-1(7)

[0175] v2=N-α(8)

[0177]

[0178] Where:

[0179] SS – variance;

[0180] x i ——the measurement or calculation result of the specified parameter for the i-th time;

[0181] —— overall average value;

[0182] n i ——Number of repeated measurements of sample i;

[0183] xij ——the jth result of sample i;

[0184] MS – mean square;

[0185] v——degrees of freedom;

[0186] F——F test value;

[0187] n0——effective measurement times;

[0188] α——the number of samples drawn;

[0189] N——total number of measurements;

[0190] S bb ——Standard deviation between bottles;

[0191] S r ——Standard deviation within the bottle (repeatability standard deviation).

[0192] When F≤1, S r Instead of S bb Calculate CV 瓶间 , homogeneity of each analyte (CV 瓶间 ) should be ≤10%.

[0193] When F≤F 0.05(v1，v2) When the test results show that there is no significant difference in the uniformity between bottles, the calculated result is CV between bottles, and the uniformity of each analyte (CV between bottles) should be ≤10%.

[0194] When F>F 0.05(v1，v2) , S bb When ≤0.3δ, the uniformity between bottles is considered good, and the calculated result is CV between bottles. The uniformity of each analyte (CV between bottles) should be ≤10%.

[0195] When F>F 0.05(v1，v2) , S bb When >0.3δ, the inter-bottle uniformity is considered poor and does not meet the analyte uniformity (CV 瓶间 ) should be ≤10%.

[0196] Note: δ is the target standard deviation.

[0197] The uniformity between bottles (2 mL / bottle) and stability of the liquid composite quality control product prepared in the present disclosure were evaluated using the New Industry Biochemiluminescence Reagent and Instrument. The specific results are shown in Table 11:

[0198] Table 11

[0199]

[0200]

[0201] It can be seen from the above test data that the three-level quality control products prepared in the present disclosure have very good uniformity and stability among bottles, with a coefficient of variation within 7%. They can be stably stored at 2-8°C for 1 year without opening, and can be stable for 30 days after opening at 4°C; and can be stable for 1 year after opening at -20°C.

[0202] It is stable for 60 days; the relative deviation of each analyte after accelerated freezing and thawing for 7 days is within 15%; the relative deviation of each analyte after repeated freezing and thawing for 3 times is within 8%, and the coefficient of variation between bottles is within 7%.

[0203] Summarize:

[0204] The present invention discloses a macromolecular coupling of T3 / T4 antigens, and the use of the coupled T3 / T4 antigens to prepare quality control can effectively increase the concentrations of FT3 and FT4 in the serum matrix, thereby avoiding the problem of the prior art that the adjustment of FT3 and FT4 concentrations has a significant impact on the matrix; in addition, the modified T3 / T4 antigens are used to prepare a thyroid liquid composite quality control product with excellent stability that can be stably stored at 2-8°C. When the department performs daily quality control, no re-dissolution is required, the operation is simple, and the deviation caused by improper re-dissolution operation is avoided.

[0205] The above-mentioned embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A quality control material, which is a solution with serum matrix as the main solvent, and comprises an antigen obtained by coupling a biomedical carrier material with a contact angle θ greater than 90° and T3 and / or T4, wherein the T3 and / or T4 include a bound state and a free state.

2. The quality control object according to claim 1, wherein the biomedical carrier material comprises a polymer and / or a carrier protein; Preferably, the carrier protein comprises at least one of bovine serum albumin (BSA), hemocyanin (KLH), and chicken ovalbumin (OVA); Preferably, the polymer includes PEG modified with a linking group (more preferably, the linking group includes a carboxyl group or an amino group); preferably, each of the PEG modified with a linking group has one or two, and are the same type of linking group, and the linking groups are all located at the end of the PEG molecule; preferably, the molecular weight of the polymer is 2000 to 6000.

3. The quality control material according to claim 2, further comprising PEG modified with a linking group in a non-coupled state; the molar ratio of the PEG modified with a linking group to the T3 and / or T4 is (7-13):

1.

4. The quality control material according to claim 1 or 2, wherein the binding protein to which the bound T3 and / or T4 is bound comprises one or more of TBG, thyroxine transporter and albumin.

5. The quality control material according to any one of claims 1 to 3, further comprising thyroid function-related analytes; preferably comprising one or more of TSH, rT3, TG, thyroid stimulating hormone receptor antibodies, anti-thyroid microsomal antibodies, and anti-thyroid peroxidase antibodies; Preferably, the quality control substance further comprises gonadal hormones, such as at least one of luteinizing hormone, human chorionic gonadotropin, prolactin, progesterone, estradiol, testosterone, follicle stimulating hormone and anti-Mullerian hormone.

6. The quality control material according to any one of claims 1 to 3, further comprising at least one of a buffer component, a polyol, a preservative, a sugar, an amino acid, and a surfactant; preferably comprising 0.05% to 0.15% Proclin 300, 2% to 8% sucrose, 1% to 5% mannitol, and 0.01% to 0.1% Tween 80.

7. The quality control material according to any one of claims 1 to 3, wherein the serum matrix comprises one or more of hormone-free serum, healthy animal serum and delipidated serum; and the serum matrix preferably does not contain anti-TG antibodies.

8. A kit comprising the quality control material according to any one of claims 1 to 7; the kit is preferably used for detecting thyroid function; more preferably, it is an immunoassay kit.

9. The method for preparing the quality control material according to any one of claims 1 to 7, comprising: Mixing the components and coupling the biomedical carrier material and T3 and / or T4 therein; Preferably, the serum matrix is ​​purified by affinity chromatography to remove antibodies therein, such as anti-TG antibodies; Preferably, the biomedical carrier material is coupled with the T3 and / or T4 under the action of an activator; preferably, the activator comprises carbodiimide; More preferably, the biomedical carrier material is PEG modified with an amino group or a carboxyl group, the activator is EDC, and the molar ratio of the modified PEG to EDC is 1:(3-7).

10. Use of the quality control substance according to any one of claims 1 to 7 in the determination of thyroid function; preferably the determination is carried out by immunoassay.

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