A denaturation method for rheumatoid factor antigen

Abstract

This application relates to the field of reagent kit materials technology, and more particularly to a method for denaturing rheumatoid factor antigen. The denaturation buffer comprises a buffer solution and a surfactant. The buffer solution comprises a buffer solute and a buffer solvent. The amount of the buffer solute, n1, and the volume of the buffer solvent, V1, satisfy the relationship: n1:V1 = (10~100):1. If the unit of n1 is mmol, then the unit of V1 is L. The mass of the surfactant, m1, and the volume of the buffer solvent, V1, satisfy the relationship: m1:V1 = (0.1~0.5):100. If the unit of m1 is g, then the unit of V1 is mL. This buffer solution, through its composition and concentration ratio, and the action of the surfactant, jointly maintains the pH stability of the solution and alters the surface properties of IgG molecules, thereby reducing their aggregation tendency and improving the dispersion of IgG during the thermal denaturation process detected by the rheumatoid factor reagent kit.

Description

Technical Field

[0001] This application relates to the field of reagent kit materials technology, and more particularly to a method for denaturing rheumatoid factor antigen. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by symmetrical, erosive polyarticular joint lesions. RA can cause joint deformities and loss of function, and simultaneously damage multiple organs, severely impacting the patient's health. Furthermore, if RA is not treated promptly or is treated improperly, it can lead to serious consequences such as joint deformities. Therefore, early diagnosis and timely treatment of RA are of great significance. However, early symptoms of RA are often atypical, especially when only intermittent joint pain or asymmetrical early-stage arthritis is present, making diagnosis challenging. Currently, clinical diagnosis of RA largely relies on disease manifestations and X-ray equipment. These methods suffer from atypical characteristics, leading to misdiagnosis and missed diagnosis. Therefore, clinical practice needs to find more effective methods to improve the accuracy of early diagnosis of rheumatoid arthritis. Currently, serological markers can be used as diagnostic criteria for rheumatoid arthritis (RA). Common serological markers include rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies. RF is an autoantibody targeting the Fc fragment of denatured IgG. Based on the type of immunoglobulin, RF can be classified into IgA-RF, IgG-RF, IgM-RF, IgE-RF, and IgD-RF. Among these immunoglobulins, IgM-RF is the most common. Furthermore, due to the high agglutination and easy precipitation characteristics of IgM-RF, it is mainly used clinically to diagnose RA. Currently, rheumatoid factor is mostly detected clinically using latex agglutination, ELISA, and latex immunoturbidimetry. Latex agglutination has the advantages of being simple to operate and requiring no special instruments, but its sensitivity is low, easily affected by various factors, and it can only perform qualitative or semi-quantitative detection, which limits its application in observing the treatment efficacy of clinical patients. While ELISA offers higher accuracy and sensitivity than latex agglutination assays, it involves more steps, is more complex and time-consuming, and is prone to poor reproducibility. Latex immunoturbidimetry, on the other hand, avoids these drawbacks. Furthermore, it can be automated on a large scale using a fully automated biochemical analyzer, offering advantages such as ease of operation, short testing time, good reproducibility, and accurate results, making it the most commonly used clinical testing method.

[0003] However, most existing rheumatoid factor antigens are obtained by denaturing human IgG. Currently, there are two denaturation methods: chemical denaturation and physical denaturation. Chemical denaturation introduces chemical denaturing agents, which can affect the preparation of rheumatoid factor kits. Physical denaturation mainly uses thermal denaturation, but thermal denaturation can easily cause IgG to aggregate, resulting in batch-to-batch differences and poor stability of different rheumatoid factor antigens. This leads to many problems in the application of rheumatoid factor kits, such as batch-to-batch differences in reagents prepared after coupling with latex, and unsatisfactory specificity and stability. Summary of the Invention

[0004] This application provides a method for denaturing rheumatoid factor antigen to solve the following technical problem: how to improve the dispersion of IgG during the thermal denaturation process detected by rheumatoid factor kits.

[0005] In a first aspect, embodiments of this application provide a denaturing buffer solution, the denaturing buffer solution comprising a buffer solution and a surfactant, the buffer solution comprising a buffer solute and a buffer solvent, the amount of the buffer solute n1 and the volume of the buffer solvent V1 satisfying the relationship: n1:V1=(10~100):1, if the unit of n1 is mmol, then the unit of V1 is L; the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfying the relationship: m1:V1=(0.1~0.5):100, if the unit of m1 is g, then the unit of V1 is mL.

[0006] Optionally, the buffer solute may include at least one of the following: phosphate buffer, hydroxyethylpiperazine ethanethiolic acid, and 2-(N-morpholino)ethanethiolic acid; and / or

[0007] The surfactants include at least one of the following: Tween, Triton, polyoxyethylene lauryl ether, and Kao surfactants.

[0008] Optionally, the pH of the buffer solution is 7.0 to 7.5.

[0009] Secondly, this application provides a method for preparing the denaturing buffer solution described in the first aspect, the method comprising:

[0010] The buffer solute and buffer solvent are mixed to obtain a buffer solution;

[0011] The surfactant and the buffer solution are mixed to obtain a denaturing buffer solution.

[0012] Thirdly, this application provides a rheumatoid factor kit, the kit comprising the denaturing buffer described in the first aspect.

[0013] Optionally, the kit further includes a dialysis buffer and a storage buffer. The dialysis buffer comprises a first phosphate buffer and a first sodium azide, wherein the molar concentration of the first phosphate buffer is 10 mmol / L to 100 mmol / L and the mass concentration of the first sodium azide is 0.5 g / L to 2 g / L. The storage buffer comprises a second phosphate buffer and a second sodium azide, wherein the molar concentration of the second phosphate buffer is 10 mmol / L to 100 mmol / L and the mass concentration of the second sodium azide is 0.5 g / L to 2 g / L.

[0014] Optionally, the kit may further include an activator having a mass concentration of 1 mg / mL to 10 mg / mL.

[0015] Fourthly, this application provides a method for denaturing rheumatoid factor antigen, the denaturation method comprising:

[0016] The rheumatoid factor antigen was diluted using the denaturing buffer solution described in the first aspect to obtain a diluted product;

[0017] The diluted material was subjected to a thermal polymerization reaction to obtain polymorphic rheumatoid factor antigen.

[0018] Optionally, the target concentration of the dilution is 1 mg / mL to 10 mg / mL.

[0019] Optionally, the temperature of the thermal polymerization reaction is 56℃~63℃, and the time of the thermal polymerization reaction is 30min~60min.

[0020] The technical solutions provided in this application have the following advantages compared with the prior art:

[0021] This application provides a denaturing buffer solution, which is formed by adding a surfactant to a buffer solution. The amount of buffer solute n1 and the volume of buffer solvent V1 satisfy the relationship: n1:V1=(10~100):1, ensuring that the buffer solution has a sufficient amount of buffer solute. Based on the characteristic that the buffer solution is composed of a weak acid and its conjugate base or a weak base and its conjugate acid, a sufficient amount of buffer solute can stabilize the antigen distribution of IgG during thermal denaturation and improve the dispersion uniformity of denatured IgG. In addition, the mass of surfactant m1 and the volume of buffer solvent V1 satisfy the relationship: m1:V1=(0.1~0.5):100, ensuring that the denaturing buffer solution has a sufficient amount of surfactant. Based on the hydrophilic and hydrophobic groups of the surfactant, a sufficient amount of surfactant can encapsulate or interact with IgG molecules during the thermal denaturation stage, reducing the mutual attraction between IgG molecules during thermal denaturation and reducing the tendency to aggregate, thereby improving the dispersion degree of IgG during the thermal denaturation process detected by the rheumatoid factor kit. Attached Figure Description

[0022] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This application provides a schematic flowchart of a method for preparing the denaturing buffer solution.

[0025] Figure 2 This application provides a schematic flowchart of a method for denaturing rheumatoid factor antigen. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0027] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range; in addition, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0028] In this document, terms such as “comprising” mean “including but not limited to”. Relational terms such as “first” and “second” are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. “And / or” describes the relationship between related objects, indicating that there can be three relationships, for example, A and / or B can mean: A alone, A and B simultaneously, or B alone; where A and B can be singular or plural. “At least one” means one or more, “more” means two or more; “at least one,” “at least one of the following,” or similar expressions refer to any combination of these items, including any combination of single or plural items; for example, “at least one of a, b, or c,” or “at least one of a, b, and c,” can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple. "Parts representation" such as parts by weight or parts by mass indicates the proportional relationship between components. In the proportional relationships discussed in this article, the parameters that need to be described by proportion should be understood as the first term of the proportion in the order of description, and the proportion figures should be understood as the second term of the proportion. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substances A, B, and C should correspond one-to-one with the proportion figures in the proportion in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0030] It should be noted that, addressing the problems existing in the prior art, the inventors discovered that, in order to alleviate the technical issues of IgG self-aggregation during denaturation and the instability of the denatured polymer, the batch-to-batch variation in rheumatoid factor antigen preparation can be reduced, thereby solving the batch-to-batch variation, specificity, and stability of rheumatoid factor assay kits in application applications. Adding suitable types and concentrations of surfactants to the IgG denaturation buffer can prevent IgG molecules from self-aggregating during thermal denaturation, resulting in more uniform denatured IgG molecules and smaller batch-to-batch variation. Subsequently, the denatured IgG antigen molecules are coupled to latex microspheres of appropriate particle size to prepare a rheumatoid factor assay kit. The rheumatoid factor antigen prepared by this method has smaller batch-to-batch variation, smaller batch-to-batch variation in the rheumatoid factor reagent, higher specificity, and stability up to 24 months.

[0031] This application provides a denaturing buffer solution, which includes a buffer solution and a surfactant. The buffer solution includes a buffer solute and a buffer solvent. The amount of the buffer solute, n1, and the volume of the buffer solvent, V1, satisfy the following relationship: n1:V1 = (10~100):1. If the unit of n1 is mmol, then the unit of V1 is L. The mass of the surfactant, m1, and the volume of the buffer solvent, V1, satisfy the following relationship: m1:V1 = (0.1~0.5):100. If the unit of m1 is g, then the unit of V1 is mL.

[0032] It should be noted that the amount of buffer solute n1 can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100.

[0033] It should be noted that the mass m1 of the surfactant can be 0.1, 0.2, 0.3, 0.4 or 0.5.

[0034] It should be noted that this application provides a denaturing buffer solution. The principle behind this denaturing buffer solution reducing the aggregation of rheumatoid factor IgG lies in the use of a specific denaturing buffer solution. This buffer solution, through its composition and concentration ratio, helps maintain the pH stability of the solution and may, through the action of surfactants, alter the surface properties of IgG molecules, thereby reducing their aggregation tendency. A detailed explanation follows:

[0035] 1. The role of buffer solution:

[0036] A buffer solution is a solution composed of a weak acid and its conjugate base or a weak base and its conjugate acid, which can slow down pH changes when a certain amount of other substances are added. This property is crucial for maintaining the stability of biomolecules, as changes in pH can affect the structure and function of molecules.

[0037] In this application, the buffer solution consists of a buffer solute and a buffer solvent, and their amounts and volumes satisfy a specific ratio. This ratio may help optimize the buffering capacity of the buffer solution, thereby better maintaining the pH stability of the solution.

[0038] 2. The role of surfactants:

[0039] Surfactants are substances that reduce the surface tension of liquids; they typically possess both hydrophilic and hydrophobic groups. In solution, surfactant molecules align themselves with each other, with the hydrophilic groups facing the aqueous phase and the hydrophobic groups facing the air or other non-aqueous phases.

[0040] In this application, the volumes of the surfactant and the buffer solvent satisfy a specific ratio. This ratio may allow the surfactant to reach an appropriate concentration in the solution, thereby altering the surface properties of the IgG molecules. For example, the surfactant may reduce the intermolecular attraction by encapsulating or interacting with IgG molecules, thus reducing their aggregation tendency.

[0041] 3. Comprehensive effects:

[0042] Through the combined action of buffer and surfactant, denaturing buffer may be able to maintain pH stability in the solution and alter the surface properties of IgG molecules. These effects collectively reduce the tendency of IgG molecules to aggregate, thereby helping to reduce the aggregation of rheumatoid factor IgG.

[0043] In summary, the principle of reducing rheumatoid factor IgG aggregation lies in the use of the denaturing buffer solution specifically described in this application. This buffer solution, through its composition and concentration ratio, as well as the action of surfactants, works together to maintain the pH stability of the solution and alter the surface properties of IgG molecules, thereby reducing their aggregation tendency.

[0044] In some alternative embodiments, the buffer solute includes at least one of the following: phosphate buffer, hydroxyethylpiperazine ethanethiolic acid, and 2-(N-morpholino)ethanethiolic acid; and / or

[0045] The surfactants include at least one of the following: Tween, Triton, polyoxyethylene lauryl ether, and Kao surfactants;

[0046] In these embodiments, the buffer solution includes at least one of phosphate buffer, hydroxyethylpiperazine ethanethioic acid, and 2-(N-morpholino)ethanethioic acid, which can cover most biological buffer solutions, thereby increasing the applicability of the denaturing buffer solution; the surfactant can include at least one of Tween, Triton, polyoxyethylene lauryl ether, and Kao surfactants, which can cover most biological surfactants, thereby increasing the applicability of the surfactant.

[0047] It should be noted that PBS (phosphate-buffered saline) is a widely used buffer solution, whose main components include Na₂HPO₄, KH₂PO₄, NaCl, and KCl. It has a mild pH and good ionic strength, making it suitable for experiments such as cell culture and tissue sectioning. The osmotic pressure and ion concentration of PBS are similar to those of human body fluids, therefore it is often used to maintain normal cellular physiological functions.

[0048] HEPES (hydroxyethylpiperazine ethanethiol) is a zwitterionic buffer with a stable molecular structure and neutral pH, making it suitable for a wide pH range. Its advantages include excellent buffering performance, high biocompatibility, and low cost. HEPES has high solubility in water, with minimal variation in solubility across different pH values, making it suitable for applications such as drug development and cell culture.

[0049] MES (2-(N-morpholino)ethanesulfonic acid) is a zwitterionic buffer containing amino and carboxyl groups, suitable for a wide pH range. It is widely used in experiments such as plant cell culture and protein electrophoresis. However, MES is expensive and may be cytotoxic.

[0050] It should be noted that Tween can be Tween-20 or Tween-80, Triton can be Triton-100, polyoxyethylene lauryl ether can be polyoxyethylene lauryl ether-35, and Kao surfactants can be Kao-A90.

[0051] In some optional embodiments, the pH of the buffer solution is 7.0 to 7.5;

[0052] In these embodiments, the pH of the buffer solution can be 7.0 to 7.5, so that the buffer solution is in a neutral range, so that the surfactant can fully disperse the IgG molecules, thereby reducing the mutual attraction between IgG molecules and thus reducing the tendency of denatured IgG antigens to aggregate.

[0053] Figure 1 An exemplary embodiment of this application provides a schematic flowchart of a method for preparing the denaturing buffer solution;

[0054] Based on a general inventive concept, such as Figure 1 As shown in the embodiments of this application, a method for preparing the denaturing buffer solution is provided, the method comprising:

[0055] S1. Mix the buffer solute and buffer solvent to obtain a buffer solution;

[0056] S2. Mix the surfactant and the buffer solution to obtain a denaturing buffer solution.

[0057] This system is for the preparation method of the denaturing buffer solution described above. The specific composition of the denaturing buffer solution can be referred to in the above embodiments. Since this system adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0058] Based on a general inventive concept, embodiments of this application provide a rheumatoid factor kit, the kit comprising the denaturing buffer solution.

[0059] This rheumatoid factor kit is based on the denaturing buffer described above. The specific composition of the denaturing buffer can be referred to in the above embodiments. Since this rheumatoid factor kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0060] It should be noted that the detection principle of this rheumatoid factor kit is as follows:

[0061] Based on latex immunoturbidimetry, denatured IgG molecules are cross-linked onto latex microparticles. These denatured IgG molecules on the surface of the latex microparticles encounter rheumatoid factor in the sample during liquid chromatography, forming an antigen-antibody complex. The weight concentration of this complex is linearly proportional to the turbidity of the resulting solution. Therefore, by detecting the turbidity of this solution at a wavelength of 600 nm and comparing it with the turbidity of a calibrated sample treated in the same way, the content of rheumatoid factor in the sample can be calculated.

[0062] In some optional embodiments, the kit further includes a dialysis buffer and a storage buffer. The dialysis buffer comprises a first phosphate buffer and a first sodium azide, wherein the molar concentration of the first phosphate buffer is 10 mmol / L to 100 mmol / L and the mass concentration of the first sodium azide is 0.5 g / L to 2 g / L. The storage buffer comprises a second phosphate buffer and a second sodium azide, wherein the molar concentration of the second phosphate buffer is 10 mmol / L to 100 mmol / L and the mass concentration of the second sodium azide is 0.5 g / L to 2 g / L.

[0063] In these embodiments, the kit may further include a dialysis buffer, which comprises a first phosphate buffer and a first sodium azide. The molar concentration of the first phosphate buffer may be 10 mmol / L to 100 mmol / L, and the mass concentration of the first sodium azide may be 0.5 g / L to 2 g / L. This ensures that the dialysis buffer contains sufficient amounts of the first phosphate buffer and the first sodium azide. The dialysis buffer helps maintain the stability of the multipolymerized rheumatoid factor antigen and prolongs the lifespan of the denatured IgG molecules, while preventing contamination of the multipolymerized rheumatoid factor antigen. Additionally, the kit may include a storage buffer, which may include a second phosphate buffer and a second sodium azide. The molar concentration of the second phosphate buffer may be 10 mmol / L to 100 mmol / L, and the mass concentration of the second sodium azide may be 0.5 g / L to 2 g / L. This ensures that the storage buffer contains sufficient amounts of the second phosphate buffer and the second sodium azide. The storage buffer can maintain the stability of the multimodal rheumatoid factor antigen and extend its usage period, while preventing contamination of the multimodal rheumatoid factor antigen.

[0064] The molar concentration of the first phosphate buffer can be 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, or 100 mmol / L.

[0065] The mass concentration of the first sodium azide can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2.0 g / L.

[0066] The molar concentration of the second phosphate buffer can be 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, or 100 mmol / L.

[0067] The mass concentration of the second sodium azide can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, or 2.0 g / L.

[0068] In some optional embodiments, the kit further includes an activator having a mass concentration of 1 mg / mL to 10 mg / mL;

[0069] In these embodiments, the kit may further include an activator, and the mass concentration of the activator may be 1 mg / mL to 10 mg / mL, ensuring that the kit has a sufficient amount of activator. The sufficient activator can effectively activate the latex microspheres, enabling the latex microspheres to effectively bind to denatured IgG molecules, so as to facilitate subsequent antibody-antigen immunization with rheumatoid factor of the sample to be tested.

[0070] The concentration of the activator can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL.

[0071] Figure 2 An exemplary schematic diagram of a method for denaturing rheumatoid factor antigen provided in this application is shown.

[0072] Based on a general inventive concept, such as Figure 2 As shown in the embodiments of this application, a method for denaturing rheumatoid factor antigen is provided, the denaturation method comprising:

[0073] S1. The rheumatoid factor antigen is diluted using the denaturing buffer solution to obtain a diluted product;

[0074] S2. The diluted material is subjected to a thermal polymerization reaction to obtain polymorphic rheumatoid factor antigen.

[0075] The denaturation method is based on the denaturation buffer solution described above. The specific composition of the denaturation buffer solution can be referred to in the above embodiments. Since the denaturation method adopts some or all of the technical solutions in the above embodiments, it has at least all the beneficial effects brought about by the technical solutions in the above embodiments, which will not be elaborated here.

[0076] In some optional embodiments, the target concentration of the dilution is 1 mg / mL to 10 mg / mL;

[0077] In these embodiments, the target concentration of dilution can be from 1 mg / mL to 10 mg / mL, allowing rheumatoid factor antigen to be within the optimal concentration range. Under the combined action of the surfactant in the denaturing buffer and the buffer solution, a sufficient amount of rheumatoid factor antigen undergoes a thermal polymerization reaction to obtain uniformly dispersed denatured rheumatoid factor antigen. The uniformly dispersed denatured rheumatoid factor antigen can effectively meet with the rheumatoid factor in the test sample in the liquid phase and bind to form an antigen-antibody complex, thereby reducing the batch-to-batch variation of the rheumatoid factor assay kit and improving the specificity and stability of the rheumatoid factor assay kit.

[0078] In some optional embodiments, the temperature of the thermal polymerization reaction is 56°C to 63°C, and the time of the thermal polymerization reaction is 30 min to 60 min;

[0079] In these embodiments, the temperature of the thermal polymerization reaction can be 56°C to 63°C, and the reaction time can be 30 min to 60 min. Under the combined action of the surfactant in the denaturing buffer and the buffer solution, the rheumatoid factor antigen undergoes a thorough thermal polymerization reaction, resulting in a uniformly dispersed denatured rheumatoid factor antigen. This uniformly dispersed denatured rheumatoid factor antigen can effectively meet and bind with the rheumatoid factor in the test sample in the liquid phase, forming an antigen-antibody complex. This reduces the batch-to-batch variation of the rheumatoid factor assay kit and improves its specificity and stability.

[0080] The temperature for this thermal polymerization reaction can be 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃ or 63℃.

[0081] The time for the thermal polymerization reaction can be 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min.

[0082] The present application is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national / industry standards; if there is no corresponding national / industry standard, they are performed according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0083] Implementation Cases

[0084] 1. The materials used include:

[0085] IgG: Purchased from Wuhan Biopharmaceutical Co., Ltd., a subsidiary of Sinopharm Group;

[0086] Latex microspheres: Purchased from Beijing Bomei Biotechnology Co., Ltd., with a particle size of 100nm-200nm and a solid content of 5%.

[0087] 2. The preparation method of this rheumatoid factor includes:

[0088] (1) Denaturing buffer:

[0089] The denaturing buffer comprises a buffer solution and a surfactant. The amount of the buffer solute, n1, and the volume of the buffer solvent, V1, satisfy the relationship: n1:V1 = 10 mmol / L to 100 mmol / L. The buffer solute includes at least one of the following: phosphate buffer (PBS), hydroxyethylpiperazine ethanesulfonic acid (HEPES), and 2-(N-morpholino)ethanesulfonic acid (MES). The pH of the buffer solution is 7.0 to 7.5. The surfactant includes at least one of the following: Tween, Triton, polyoxyethylene lauryl ether, and Kao surfactants. The mass of the surfactant, m1, and the volume of the buffer solvent, V1, satisfy the relationship: m1:V1 = (0.1 to 0.5) g: 100 mL.

[0090] (2) Temperature and time of the thermal polymerization reaction:

[0091] like Figure 1 As shown, a method for denaturing rheumatoid factor antigen includes:

[0092] The rheumatoid factor antigen was diluted using denaturing buffer to obtain a diluted solution.

[0093] The diluted material was subjected to a thermal polymerization reaction to obtain polymorphic rheumatoid factor antigen.

[0094] The obtained polymorphic rheumatoid factor antigen was cooled at 2℃~8℃ for 4 hours.

[0095] The target concentration for dilution is 1 mg / mL to 10 mg / mL.

[0096] The temperature of the thermal polymerization reaction is 56℃~63℃, and the reaction time is 30min~60min.

[0097] (3) Dialysis, concentration and storage:

[0098] Select appropriate dialysis equipment, filter the multimodal rheumatoid factor antigen using dialysis buffer to remove impurities, concentrate it using an ultrafiltration cup or hollow fiber column, and then dilute the multimodal rheumatoid factor antigen to a suitable concentration using storage buffer and store it. The concentration can be diluted to 5 mg / mL to 20 mg / mL. It can be stored at 4°C for short-term storage or -20°C for long-term storage.

[0099] The dialysis buffer consists of a first phosphate buffer and a first sodium azide. The molar concentration of the first phosphate buffer is 10 mmol / L to 100 mmol / L, and the mass concentration of the first sodium azide is 0.5 g / L to 2 g / L. The pH of the dialysis buffer is 7.4.

[0100] The storage buffer consists of a second phosphate buffer and a second sodium azide. The molar concentration of the second phosphate buffer is 10 mmol / L to 100 mmol / L, and the mass concentration of the second sodium azide is 0.5 g / L to 2 g / L. The pH of the storage buffer is 7.4.

[0101] 3. Preparation of Rheumatoid Factor Assay Kit

[0102] (1) Solution preparation

[0103] 1) Preparation of component R1: 0.1 mol / L MES solution, 0.08 mol / L to 0.30 mol / L sodium chloride, 2 g / L to 10 g / L polyethylene glycol lauryl ether (Thesit), 0.1% sodium azide, and the pH of component R1 is 6.0 to 7.0.

[0104] 2) R2 storage buffer: 25 mmol / L to 50 mmol / L HEPES buffer, 0.1% EDTA-2Na, 5% sucrose, 0.1% PC-300 preservative, and the pH of R2 storage buffer is 7.0 to 7.5.

[0105] 3) Activation buffer: 20 mmol / L to 50 mmol / L MES buffer, with a pH of 5.5 to 6.5.

[0106] 4) Coupling buffer: 20 mmol / L to 50 mmol / L HEPES buffer, with a pH of 7.4 to 7.8.

[0107] 5) Activator: Weigh ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) at a concentration of 1 mg / mL to 10 mg / mL, and prepare it immediately before use.

[0108] 6) Blocking solution: 0.5 mol / L to 1.0 mol / L glycine, pH 8.0, and 10% bovine serum albumin (BSA).

[0109] (2) Preparation of latex reagent:

[0110] 1) Take 1 mL of latex microspheres with a particle size of 100 nm to 200 nm and mix them with an activation buffer solution with a pH of 5.5 to 6.5 at a volume ratio of 1:3 until homogeneous to obtain an activation buffer solution containing latex microspheres.

[0111] 2) Add 0.2 mL of activator to the activation buffer containing latex microspheres, mix well, and then place in a constant temperature shaker for 15 min to 25 min to obtain activated latex microspheres; the working temperature of the constant temperature shaker is 30℃ to 37℃.

[0112] 3) Dilute rheumatoid factor antigen to 1 mg / mL with coupling buffer, then add 2 mL to 5 mL of 1 mg / mL diluted denatured IgG molecules to activated latex microspheres, mix well, and then place in a constant temperature shaker for 2 to 3 hours to obtain the reaction solution; the working temperature of the constant temperature shaker is 30℃ to 37℃.

[0113] 4) Add 1 mL to 5 mL of blocking solution to the reaction solution, mix well, and then place in a constant temperature shaker to react for 0.5 h to 1 h to obtain the blocking product; the working temperature of the constant temperature shaker is 30℃ to 37℃.

[0114] 5) After the reaction is complete, centrifuge the blocked product and remove the supernatant to obtain a centrifuged precipitate; the centrifugation speed is 16000 rpm and the centrifugation time is 50 min.

[0115] 6) Use 60 mL of R2 component buffer to repeatedly dissolve the centrifuged precipitate to resuspend the latex microspheres. A cell disruptor can be used to assist in the resuspension process.

[0116] Example 1

[0117] Based on the content disclosed in the implementation case, the following modifications were made:

[0118] The amount of buffer solute, n1, and the volume of buffer solvent, V1, satisfy the following relationship: n1:V1 = 50 mmol / L; the types of buffer solutes are phosphate buffer, hydroxyethylpiperazine ethanethioic acid, and 2-(N-morpholino)ethanethioic acid; the pH of the buffer is 7.5. The type of surfactant is Tween-20, and the mass of the surfactant, m1, and the volume of buffer solvent, V1, satisfy the following relationship: m1:V1 = 0.1 g: 100 mL.

[0119] The dialysis buffer consists of a first phosphate buffer and a first sodium azide. The molar concentration of the first phosphate buffer is 50 mmol / L, and the mass concentration of the first sodium azide is 1.0 g / L. The pH of the dialysis buffer is 7.4.

[0120] The storage buffer consists of a second phosphate buffer and a second sodium azide. The molar concentration of the second phosphate buffer is 50 mmol / L, and the mass concentration of the second sodium azide is 1.0 g / L. The pH of the storage buffer is 7.4.

[0121] The target concentration for dilution is 5 mg / mL.

[0122] The temperature of the thermal polymerization reaction is 60℃, and the reaction time is 30 minutes.

[0123] (1) Solution preparation

[0124] 1) Preparation of component R1: 0.1 mol / L MES solution, 0.15 mol / L sodium chloride, 5 g / L polyethylene glycol lauryl ether (Thesit), 0.1% sodium azide, and pH of component R1 is 6.5;

[0125] 2) R2 storage buffer: 50 mmol / L HEPES buffer, 0.1% EDTA-2Na, 5% sucrose, 0.1% PC-300 preservative, and the pH of R2 storage buffer is 7.4.

[0126] 3) Activation buffer: 50 mmol / L MES buffer, pH 6.0.

[0127] 4) Coupling buffer: 50 mmol / L HEPES buffer, pH 7.6.

[0128] 5) Activator: Weigh ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) at a concentration of 5 mg / mL, and prepare it immediately before use.

[0129] 6) Blocking solution: 0.8 mol / L glycine, pH 8.0, and 10% bovine serum albumin (BSA).

[0130] (2) Preparation of latex reagent:

[0131] 1) Take 1 mL of latex microspheres with a particle size of 180 nm and mix them with the activation buffer at a volume ratio of 1:3 until homogeneous to obtain the activation buffer containing latex microspheres;

[0132] 2) Add 0.2 mL of activator to the activation buffer containing latex microspheres, mix well, and then place in a constant temperature shaker for 25 min to obtain activated latex microspheres; the working temperature of the constant temperature shaker is 37℃.

[0133] 3) Dilute rheumatoid factor antigen to 1 mg / mL with coupling buffer, then add 4 mL of 1 mg / mL diluted denatured IgG molecules to activated latex microspheres, mix well, and then place in a constant temperature shaker for 2 h to obtain the reaction solution; the working temperature of the constant temperature shaker is 37℃.

[0134] 4) Add 3 mL of blocking solution to the reaction solution, mix well, and then place it in a constant temperature shaker to react for 1 h to obtain the blocking product; the working temperature of the constant temperature shaker is 37℃.

[0135] 5) After the reaction is complete, centrifuge the blocked product and remove the supernatant to obtain a centrifuged precipitate; the centrifugation speed is 16000 rpm and the centrifugation time is 50 min.

[0136] 6) Use 60 mL of R2 component buffer to repeatedly dissolve the centrifuged precipitate to resuspend the latex microspheres. A cell disruptor can be used to assist in the resuspension process.

[0137] Example 2

[0138] Based on the content disclosed in Example 1, the following modifications are made:

[0139] The surfactant is Tween-80, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=0.2g:100mL.

[0140] Example 3

[0141] Based on the content disclosed in Example 1, the following modifications are made:

[0142] The surfactant is Triton-100, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=0.3g:100mL.

[0143] Example 4

[0144] Based on the content disclosed in Example 1, the following modifications are made:

[0145] The surfactant is Brij-35, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=0.4g:100mL.

[0146] Example 5

[0147] Based on the content disclosed in Example 1, the following modifications are made:

[0148] The surfactant is Kao A90. The mass of the surfactant, m1, and the volume of the buffer solvent, V1, satisfy the relationship: m1:V1=0.5g:100mL.

[0149] Example 6

[0150] Based on the content disclosed in Example 1, the following modifications are made:

[0151] The temperature of the thermal polymerization reaction was 56℃, and the reaction time was 30 minutes.

[0152] Example 7

[0153] Based on the content disclosed in Example 1, the following modifications are made:

[0154] The surfactant is Tween-80, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=0.5g:100mL.

[0155] The temperature of the thermal polymerization reaction was 56℃, and the reaction time was 50 min.

[0156] Example 8

[0157] Based on the content disclosed in Example 1, the following modifications are made:

[0158] The surfactant is Tween-80, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=0.5g:100mL.

[0159] The temperature of the thermal polymerization reaction is 60℃, and the reaction time is 60 minutes.

[0160] Example 9

[0161] Based on the content disclosed in Example 1, the following modifications are made:

[0162] The surfactant is Triton-100, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=0.5g:100mL.

[0163] The temperature of the thermal polymerization reaction was 63℃, and the reaction time was 50 minutes.

[0164] Comparative Example 1

[0165] Based on the content disclosed in Example 1, the following modifications are made:

[0166] No surfactants are added.

[0167] Comparative Example 2

[0168] Based on the content disclosed in Example 1, the following modifications are made:

[0169] The surfactant is Triton-100, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=1.0g:100mL.

[0170] Comparative Example 3

[0171] Based on the content disclosed in Example 1, the following modifications are made:

[0172] The surfactant is Brij-35, and the mass of the surfactant m1 and the volume of the buffer solvent V1 satisfy the relationship: m1:V1=0.05g:100mL.

[0173] Example 4

[0174] Based on the content disclosed in Example 1, the following modifications are made:

[0175] The temperature of the thermal polymerization reaction is 50℃, and the reaction time is 30 minutes.

[0176] Example 5

[0177] Based on the content disclosed in Example 1, the following modifications are made:

[0178] The temperature of the thermal polymerization reaction is 65℃, and the reaction time is 30 minutes.

[0179] Relevant experimental and effect data:

[0180] Effect evaluation:

[0181] Instrument selection: HITACHI 7180. Reagents prepared for each example and comparative example were used for detection. Sensitivity, specificity, batch-to-batch variation, and stability were evaluated using the following methods:

[0182] (1) Sensitivity: The difference in absorbance (ΔA) when the concentration of rheumatoid factor is 20 IU / mL of the analyte.

[0183] (2) Specificity: Measure the third-party quality control sample and calculate the deviation from the target value: Bias = (Measured mean - Target value) / Target value × 100%;

[0184] (3) Inter-batch difference: Three batches of rheumatoid factor antigen were prepared repeatedly, and rheumatoid factor assay kits were prepared using the antigen. Calibrators of different concentrations were measured, and the difference in absorbance of each batch of reagents was compared.

[0185] (4) Stability: Observe the storage stability of the reagent. Test the same calibration high value (calibrator for this test: 100 IU / mL) at 0 months, 6 months, 12 months, 18 months and 24 months respectively. Measure 3 times at each time point and calculate the CV of all data.

[0186] The test results are shown in Tables 1 to 6.

[0187] Table 1. Sensitivity data results for each embodiment and comparative example.

[0188]

[0189] Table 2. Specific data results for each embodiment and comparative example.

[0190]

[0191] Table 3. Results of batch-to-batch difference data for each embodiment and comparative example.

[0192]

[0193] Table 4. Stability data results for each embodiment.

[0194]

[0195] Table 5 shows the stability data results for each comparative example.

[0196]

[0197] Table 6 provides a comprehensive comparison of sensitivity, specificity, batch-to-batch variation, and stability for each embodiment and comparative example.

[0198]

[0199] As shown in Tables 1 to 5, although Comparative Examples 1, 3, and 5 exhibited good sensitivity, their specificity, batch-to-batch variation, and stability were poor. While Comparative Examples 2 and 4 met the requirements for batch-to-batch variation and stability, their sensitivity and specificity were still poor. Furthermore, a comparison between Comparative Example 1 and the examples shows that without surfactant added to the denaturing buffer, the rheumatoid antigen obtained by the denaturation method exhibited poor batch-to-batch variation, specificity, and stability; however, with an appropriate concentration of surfactant added to the denaturing buffer, the rheumatoid antigen obtained by the denaturation method showed better sensitivity, specificity, batch-to-batch variation, and stability. Moreover, according to Comparative Examples 4 and 5, when the thermal polymerization reaction temperature of the denaturation method was below 56°C, the denaturation efficiency of the IgG antigen was low, and the sensitivity and specificity of the reagent were also poor. When the denaturation temperature of the thermal polymerization reaction of the denaturation method was above 63°C, the thermal polymerization reaction of the IgG antigen would proceed excessively; although the reagent sensitivity was high, its specificity and stability were poor.

[0200] In summary, the embodiments of this application provide a denaturing buffer solution. This buffer solution, through its composition, concentration ratio, and the action of surfactants, jointly maintains the pH stability of the solution and alters the surface properties of IgG molecules, thereby reducing their aggregation tendency and improving the dispersion of IgG during the thermal denaturation process detected by the rheumatoid factor kit.

[0201] In addition, the rheumatoid factor kit provided in this application has high sensitivity, specificity, stability and low batch-to-batch variation in the antigen prepared by the rheumatoid factor kit and the kit itself, which can meet the needs of clinical use. Moreover, the preparation process of the kit is simple and can be scaled up for production.

[0202] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed in this application.

Claims

1. A method for denaturing rheumatoid factor antigen, the denaturation method comprising: The rheumatoid factor antigen was diluted using denaturing buffer to obtain a diluted solution. The diluted material was subjected to a thermal polymerization reaction to obtain a polymer-modified rheumatoid factor antigen. The denaturing buffer comprises a buffer solution and a surfactant. The buffer solution comprises a buffer solute and a buffer solvent. The amount of the buffer solute, n1, and the volume of the buffer solvent, V1, satisfy the relationship: n1:V1 = (10~100):

1. If the unit of n1 is mmol, then the unit of V1 is L. The mass of the surfactant, m1, and the volume of the buffer solvent, V1, satisfy the relationship: m1:V1 = (0.1~0.5):

100. If the unit of m1 is g, then the unit of V1 is mL. The buffer solutes include at least one of the following: phosphate buffer, hydroxyethylpiperazine ethanethioic acid, and 2-(N-morpholino)ethanethioic acid; The surfactants include at least one of the following: Tween, Triton, polyoxyethylene lauryl ether, and Kao-A90; The pH of the buffer solution is 7.0 to 7.

5.

2. The denaturation method according to claim 1, wherein the target concentration of the dilution is 1 mg / mL to 10 mg / mL.

3. The denaturation method according to claim 1, wherein the temperature of the thermal polymerization reaction is 56℃~63℃, and the time of the thermal polymerization reaction is 30min~60min.

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