Denaturation buffer solution, rheumatoid factor kit as well as preparation method and application of rheumatoid factor kit

Abstract

The invention relates to the technical field of kit materials, in particular to a denaturation buffer solution, a rheumatoid factor kit and a preparation method and application of the denaturation buffer solution and the rheumatoid factor kit. The denaturation buffer solution comprises a buffer solution and a surfactant, the buffer solution comprises a buffer solute and a buffer solvent, the amount of substance n1 of the buffer solute and the volume V1 of the buffer solvent meet the relational expression that n1: V1 = (10-100): 1, and if the unit of n1 is mmol, the unit of V1 is L; the mass m1 of the surfactant and the volume V1 of the buffer solvent meet the relational expression: m1: V1 = (0.1-0.5): 100, and if the unit of m1 is g, the unit of V1 is mL. According to the buffer solution, the pH of the solution is maintained to be stable and the surface property of IgG molecules is changed through the composition and concentration ratio of the buffer solution and the action of the surfactant, so that the aggregation tendency of the IgG molecules is reduced, and the dispersion degree of IgG in the thermal denaturation process of rheumatoid factor kit detection is improved.

Description

Technical Field

[0001] The present application relates to the technical field of kit materials, and in particular to a denaturing buffer, a rheumatoid factor kit, and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is one of the autoimmune diseases, which is mainly manifested as symmetrical, erosive polyarthritis lesions. RA can cause joint deformities and loss of function, and at the same time damage multiple organs of the body, which will seriously affect the patient's health. In addition, if RA patients are not treated in time or are improperly treated, serious consequences such as joint deformities may occur. Therefore, early diagnosis and timely treatment of RA are of great significance. However, the early symptoms of RA patients are atypical, especially when there are only intermittent joint pain or asymmetric early arthritis lesions, the diagnosis of RA is more difficult. At present, the clinical diagnosis of RA mostly relies on disease manifestations, X-ray equipment and other methods. These detection methods have atypical characteristics and are prone to misdiagnosis and missed diagnosis. Therefore, it is necessary to find more effective methods in clinical practice to improve the accuracy of early diagnosis of rheumatoid arthritis. At present, serological indicators can be used as the basis for the diagnosis of RA. Common serological indicators include rheumatoid factor, anti-cyclic citrullinated peptide antibody, etc. Among them, rheumatoid factor (RF) is an autoantibody with the Fc fragment of denatured IgG as the target antigen. According to the type of immunoglobulin, RF can be divided into IgA-RF, IgG-RF, IgM-RF, IgE-RF and IgD-RF. Among these immunoglobulins, IgM-RF is the most common. In addition, based on the characteristics of high agglutination and easy precipitation of IgM-RF, RA is mainly detected clinically by measuring IgM-RF. At present, rheumatoid factor is mostly detected clinically by latex agglutination, ELISA and latex immunoturbidimetry. Among them, the latex agglutination method has the advantages of simple operation and no need for special instruments, but its sensitivity is low and it is easily affected by many factors. In addition, the latex agglutination method can only be detected qualitatively or semi-quantitatively, which has certain limitations for the observation of the efficacy of clinical patients. The accuracy and sensitivity of ELISA method are higher than those of latex agglutination method, but ELISA method has more steps than latex immunoturbidimetry, and the operation is complicated and time-consuming, and the detection process is prone to poor repeatability. Latex immunoturbidimetry can avoid the shortcomings of the above two methods, and latex immunoturbidimetry can be realized in large quantities through automatic biochemical analyzer. It has the advantages of simple operation, short detection time, good repeatability and accurate results, and is the most commonly used detection method in clinical practice.

[0003] However, most of the existing rheumatoid factor antigens are obtained by denaturing human IgG. Currently, there are two denaturation methods: chemical denaturation and physical denaturation. Chemical denaturation will introduce chemical denaturants, and the introduction of chemical denaturants will affect the preparation of rheumatoid factor kits. Physical denaturation mainly uses thermal denaturation methods, but the thermal denaturation process is prone to cause self-aggregation of IgG, resulting in batch differences and poor stability of different rheumatoid factor antigens obtained, which in turn brings many problems to the application of rheumatoid factor kits, such as batch differences in reagents prepared after coupling with latex, and unsatisfactory specificity and stability. Summary of the invention

[0004] The present application provides a denaturation buffer, a rheumatoid factor kit, and a preparation method and application thereof to solve the following technical problem: how to improve the dispersion degree of IgG during the thermal denaturation process detected by the rheumatoid factor kit.

[0005] In the first aspect, an embodiment of the present application provides a denaturing buffer, which includes a buffer and a surfactant, and the buffer includes a buffer solute and a buffer solvent. The amount n1 of the buffer solute and the volume V1 of the buffer solvent satisfy the relationship: n1:V1=(10~100):1, if the unit of n1 is mmol, then the unit of V1 is L; the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy 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 type of buffer solute includes at least one of the following: phosphate buffer, hydroxyethylpiperazineethanesulfonic acid and 2-(N-morpholine)ethanesulfonic acid; and / or

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

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

[0009] In a second aspect, the present application provides a method for preparing the denaturing buffer according to the first aspect, the method comprising:

[0010] Mixing a buffer solute and a buffer solvent to obtain a buffer;

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

[0012] In a third aspect, the present application provides a rheumatoid factor kit, wherein the kit comprises the denaturing buffer described in the first aspect.

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

[0014] Optionally, the kit further comprises an activator, and the mass concentration of the activator is 1 mg / mL to 10 mg / mL.

[0015] In a fourth aspect, the present application provides a method for denaturing a rheumatoid factor antigen, the denaturation method comprising:

[0016] diluting the rheumatoid factor antigen using the denaturing buffer described in the first aspect to obtain a dilution;

[0017] The dilution is subjected to thermal polymerization to obtain a polymerized rheumatoid factor antigen.

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

[0019] Optionally, 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.

[0020] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0021] A denaturing buffer provided in an embodiment of the present application is formed by adding a surfactant to a buffer, and the amount n1 of the buffer solute and the volume V1 of the buffer solvent satisfy the relationship: n1:V1=(10-100):1, which enables the buffer to have a sufficient amount of buffer solute. Based on the characteristic that the buffer 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 m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=(0.1-0.5):100, which enables the denaturing buffer to have a sufficient amount of surfactant. Based on the hydrophilic and hydrophobic groups of the surfactant, a sufficient amount of surfactant can wrap around the IgG molecules or interact with them during the thermal denaturation stage, reduce the mutual attraction between IgG molecules during the thermal denaturation process, and reduce the aggregation tendency, thereby improving the dispersion degree of IgG during the thermal denaturation process detected by the rheumatoid factor kit. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0024] Figure 1 A schematic flow chart of a method for preparing the denaturing buffer is provided for the embodiment of the present application;

[0025] Figure 2 A schematic flow chart of a method for denaturing a rheumatoid factor antigen is provided for the present application example. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0027] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the 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., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited number (fractional or integer) within the indicated range.

[0028] In this document, the terms including "including" and "including" mean "including but not limited to". Relational terms such as "first" and "second" are only used 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 association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "plurality" means two or more; "at least one", "at least one of the following" or similar expressions, refers 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 all 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" such as parts by weight and parts by mass indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula in the order of description, and the proportional numbers should be understood as the second term of the proportional formula. For example, if the mass ratio of substance A, substance B and substance C is 1:2:3, then substance A, substance B and substance C should correspond to the proportional numbers in the proportional formula 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, various 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, in view of the problems existing in the prior art in the background technology, the inventors found that: in order to alleviate the technical problems of IgG self-agglutination during the denaturation process in the prior art, the polymer after denaturation is unstable. Reduce the batch difference of rheumatoid factor antigen preparation, and then solve the batch difference, specificity and stability of the rheumatoid factor assay kit at the application end. Adding a surfactant substance of suitable type and concentration to the IgG denaturation buffer can prevent the IgG molecules from self-agglutination during the thermal denaturation process, and the denatured IgG molecules obtained are more uniform and have smaller batch differences. Afterwards, the denatured IgG molecule antigen is coupled to latex microspheres of suitable particle size to prepare a rheumatoid factor assay kit. The rheumatoid factor antigen prepared by this method has smaller batch difference, smaller batch difference of rheumatoid factor reagent, higher specificity, and stability can be up to 24 months.

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

[0032] It should be noted that the value of the amount n1 of the buffer solute 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 the present application provides a denaturing buffer, the principle of which is to reduce the aggregation of rheumatoid factor IgG by using a specific denaturing buffer, which helps to maintain the pH stability of the solution through its composition and concentration ratio, and may change the surface properties of IgG molecules through the action of surfactants, thereby reducing their aggregation tendency; the detailed explanation is as follows:

[0035] 1. The role of buffer:

[0036] A buffer is a solution consisting of a weak acid and its conjugate base or a weak base and its conjugate acid that slows down changes in pH when a certain amount of another substance is added. This property is essential for maintaining the stability of biomolecules, as changes in pH may affect the structure and function of the molecules.

[0037] In the present application, the buffer solution is composed of a buffer solute and a buffer solvent, and the amount and volume of the substances satisfy a specific ratio relationship. This ratio relationship may help to 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 can reduce the surface tension of liquids. They usually have hydrophilic and hydrophobic groups. In solution, surfactant molecules will be oriented, with the hydrophilic groups facing the water phase and the hydrophobic groups facing the air or other non-aqueous phase.

[0040] In the present application, the volume of the surfactant and the buffer solvent satisfies a specific ratio. This ratio may allow the surfactant to form an appropriate concentration in the solution, thereby changing the surface properties of the IgG molecules. For example, the surfactant may reduce the mutual attraction between molecules by wrapping the IgG molecules or interacting with them, thereby reducing the aggregation tendency.

[0041] 3. Comprehensive effect:

[0042] Through the combined action of the buffer and the surfactant, the denaturing buffer may be able to maintain the pH of the solution and change the surface properties of the IgG molecules. These actions together reduce the aggregation tendency of the IgG molecules, thereby helping to reduce the aggregation of rheumatoid factor IgG.

[0043] In summary, the principle of reducing the aggregation of rheumatoid factor IgG lies in the use of a specific denaturing buffer of the present application, which, through its composition and concentration ratio as well as the action of a surfactant, jointly maintains the pH stability of the solution and changes the surface properties of IgG molecules, thereby reducing their aggregation tendency.

[0044] In some optional embodiments, the type of buffer solute includes at least one of the following: phosphate buffer, hydroxyethylpiperazineethanesulfonic acid and 2-(N-morpholine)ethanesulfonic acid; and / or

[0045] The type of the surfactant includes at least one of the following: Tween, Triton, polyoxyethylene lauryl ether and Kao series surfactant;

[0046] In these embodiments, the types of buffers include: at least one of phosphate buffer, hydroxyethylpiperazineethanesulfonic acid and 2-(N-morpholine)ethanesulfonic acid, which can cover most biological buffers, thereby increasing the scope of application of the denaturing buffer; the types of surfactants can include at least one of Tween, Triton, polyoxyethylene lauryl ether and Kao series surfactants, which can cover most biological surfactants, thereby increasing the scope of application of the surfactants.

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

[0048] HEPES (Hydroxyethylpiperazineethanesulfonic acid) is a zwitterionic buffer with a stable molecular structure and neutral pH value, suitable for a wide pH range. Its advantages include excellent buffering properties, high biocompatibility and low cost. HEPES has a large solubility in water and a small difference in solubility at different pH values, making it suitable for fields such as drug development and cell culture;

[0049] MES (2-(N-morpholino)ethanesulfonic acid) is a zwitterionic buffer containing amino and carboxyl groups and is 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 may be Tween-20 or Tween-80, Triton may be Triton-100, polyoxyethylene lauryl ether may be polyoxyethylene lauryl ether-35, and the Kao-based surfactant may be Kao-A90.

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

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

[0053] Figure 1 The present application provides a schematic flow chart of a method for preparing the denaturing buffer;

[0054] Based on a general inventive concept, such as Figure 1 As shown, the present application embodiment provides a method for preparing the denaturing buffer, the method comprising:

[0055] S1. mixing a buffer solute and a buffer solvent to obtain a buffer;

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

[0057] The system is a method for preparing the above-mentioned denaturing buffer. The specific composition of the denaturing buffer can refer to the above-mentioned embodiment. Since the system adopts part or all of the technical solutions of the above-mentioned embodiment, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be repeated here one by one.

[0058] Based on a general inventive concept, an embodiment of the present application provides a rheumatoid factor kit, which includes the denaturing buffer.

[0059] The rheumatoid factor kit is implemented based on the above-mentioned denaturing buffer. The specific composition of the denaturing buffer can be referred to the above-mentioned embodiment. Since the rheumatoid factor kit adopts part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

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

[0061] Based on latex immunoturbidimetry, denatured IgG molecules are cross-linked to latex particles. On the surface of latex particles, denatured IgG molecules will meet with rheumatoid factors in the sample to be tested in the liquid phase to form an antigen-antibody complex, and the weight content of the complex formed is proportional to the turbidity of the solution formed within the linear range. Therefore, at a wavelength of 600nm, by detecting the turbidity data of the solution, and then comparing the detected turbidity data with the turbidity data of the calibrator treated in the same way, the content of rheumatoid factor in the sample can be calculated.

[0062] In some optional embodiments, the kit further comprises a dialysis buffer and a storage buffer, the dialysis buffer comprises 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 storage buffer comprises 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;

[0063] In these embodiments, the kit may further include a dialysis buffer, and the dialysis buffer includes a first phosphate buffer and a first sodium azide, and 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, so that the dialysis buffer has a sufficient amount of the first phosphate buffer and the first sodium azide. The dialysis buffer may maintain the stability of the multimeric denatured rheumatoid factor antigen and extend the service life of the denatured IgG molecule, thereby preventing the multimeric denatured rheumatoid factor antigen from being contaminated. In addition, the kit may further include a storage buffer, and the storage buffer may include a second phosphate buffer and a second sodium azide, and 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, so that the storage buffer has a sufficient amount of the second phosphate buffer and the second sodium azide. The storage buffer may maintain the stability of the multimeric denatured rheumatoid factor antigen and extend the service life of the multimeric denatured rheumatoid factor antigen, thereby avoiding contamination of the multimeric denatured rheumatoid factor antigen.

[0064] The molar concentration of the first phosphate buffer may 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 may 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 comprises an activator, and the mass concentration of the activator is 1 mg / mL to 10 mg / mL;

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

[0070] The mass 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 The present application example provides a schematic flow chart of a method for denaturing a rheumatoid factor antigen;

[0072] Based on a general inventive concept, such as Figure 2 As shown, the present application embodiment provides a method for denaturing a rheumatoid factor antigen, the denaturation method comprising:

[0073] S1. diluting the rheumatoid factor antigen using the denaturing buffer to obtain a dilution;

[0074] S2. subjecting the diluted material to a thermal polymerization reaction to obtain a polymerized rheumatoid factor antigen.

[0075] The denaturation method is implemented based on the above-mentioned denaturation buffer. The specific composition of the denaturation buffer can refer to the above-mentioned embodiment. Since the denaturation method adopts part or all of the technical solutions of the above-mentioned embodiment, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be described one by one here.

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

[0077] In these embodiments, the target mass concentration of dilution can be 1 mg / mL to 10 mg / mL, so that the rheumatoid factor antigen can be within the optimal mass concentration range. Under the joint action of the surfactant and the buffer of the denaturing buffer, the rheumatoid factor antigen of sufficient mass concentration can undergo thermal polymerization to obtain evenly dispersed denatured rheumatoid factor antigen, and the evenly dispersed denatured rheumatoid factor antigen can effectively meet with the rheumatoid factor of the sample to be tested in the liquid phase and combine to form an antigen-antibody complex, so as to reduce the batch difference of the rheumatoid factor assay kit and improve 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 30min to 60min;

[0079] In these embodiments, the temperature of the thermal polymerization reaction can be 56°C to 63°C, and the time of the thermal polymerization reaction can be 30 min to 60 min. Under the joint action of the surfactant of the denaturing buffer and the buffer, the rheumatoid factor antigen is prompted to fully undergo thermal polymerization, and a uniformly dispersed denatured rheumatoid factor antigen can be obtained. The uniformly dispersed denatured rheumatoid factor antigen can effectively meet with the rheumatoid factor of the sample to be tested in the liquid phase and combine to form an antigen-antibody complex, so as to reduce the batch difference of the rheumatoid factor assay kit and improve the specificity and stability of the rheumatoid factor assay kit.

[0080] The temperature of the thermal polymerization reaction can be 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C or 63°C.

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

[0082] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured in accordance with national standards / industry standards; if there are no corresponding national standards / industry standards, they are measured in accordance with common international standards, conventional conditions or conditions recommended by the manufacturer.

[0083] Implementation Cases

[0084] 1. Materials used include:

[0085] IgG: purchased from Sinopharm Wuhan Biopharmaceutical Co., Ltd.;

[0086] Latex microspheres: purchased from Beijing Borma Biotechnology Co., Ltd., the particle size of the microspheres is 100nm~200nm, and the solid content is 5%.

[0087] 2. The preparation method of the rheumatoid factor comprises:

[0088] (1) Denaturation buffer:

[0089] The denaturing buffer comprises a buffer and a surfactant, wherein the amount n1 of the buffer solute and the volume V1 of the buffer solvent satisfy the relationship: n1:V1=10mmol / 1L-100mmol / 1L; the types of the buffer solute include at least one of the following: phosphate buffer (PBS buffer), hydroxyethylpiperazine ethanesulfonic acid (HEPES) and 2-(N-morpholine)ethanesulfonic acid (MES); the pH of the buffer is 7.0-7.5. The types of the surfactant include at least one of the following: Tween, Triton, polyoxyethylene lauryl ether and Kao series surfactant; the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=(0.1-0.5)g:100mL.

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

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

[0092] diluting the rheumatoid factor antigen using a denaturing buffer to obtain a dilution;

[0093] The dilution is subjected to thermal polymerization to obtain a polymerized rheumatoid factor antigen.

[0094] The obtained multimeric rheumatoid factor antigen is cooled at 2°C to 8°C for 4 hours.

[0095] The target mass concentration of the dilution is 1 mg / mL to 10 mg / mL.

[0096] 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.

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

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

[0099] The dialysis buffer comprises 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, 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 comprises 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 R1 component: 0.1 mol / L MES flushing liquid, 0.08 mol / L to 0.30 mol / L sodium chloride, 2 g / L to 10 g / L polyethylene glycol lauryl ether (Thesit), and sodium azide with a mass concentration of 0.1%, and the pH of R1 component is 6.0 to 7.0;

[0104] 2) R2 storage buffer: 25mmol / L to 50mmol / L HEPES buffer, 0.1% EDTA-2Na, 5% sucrose, 0.1% PC-300 preservative, 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, the pH of the activation buffer is 5.5 to 6.5.

[0106] 4) Coupling buffer: 20 mmol / L to 50 mmol / L HEPES buffer, the pH of the coupling buffer is 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 for use immediately.

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

[0109] (2) Preparation of latex reagent:

[0110] 1) 1 mL of latex microspheres with a particle size of 100 nm to 200 nm and an activation buffer with a pH of 5.5 to 6.5 are mixed at a volume ratio of 1:3 until uniform, to obtain an activation buffer containing latex microspheres;

[0111] 2) adding 0.2 mL of an activator to the activation buffer containing the latex microspheres, mixing well, and then placing in a constant temperature shaker to react for 15 min to 25 min to obtain activated latex microspheres; the operating temperature of the constant temperature shaker is 30° C. to 37° C.;

[0112] 3) diluting the rheumatoid factor antigen to 1 mg / mL with a coupling buffer, then adding 2 mL to 5 mL of the 1 mg / mL diluted denatured IgG molecules to the activated latex microspheres, mixing them evenly, and then placing them in a constant temperature shaker for reaction for 2 h to 3 h to obtain a reaction solution; the operating temperature of the constant temperature shaker is 30° C. to 37° C.;

[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 a blocking product; the operating temperature of the constant temperature shaker is 30° C. to 37° C.;

[0114] 5) After the reaction is completed, the blocked product is centrifuged and the supernatant is removed to obtain a centrifugal precipitate; the centrifugal speed is 16000 rpm and the centrifugal time is 50 min.

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

[0116] Example 1

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

[0118] The amount of the buffer solute n1 and the volume of the buffer solvent V1 satisfy the relationship: n1:V1=50mmol / 1L; the types of buffer solutes are phosphate buffer, hydroxyethylpiperazineethanesulfonic acid and 2-(N-morpholine)ethanesulfonic 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 the buffer solvent V1 satisfy the relationship:

[0119] m1:V1=0.1g:100mL.

[0120] The dialysis buffer comprises a first phosphate buffer and a first sodium azide, the molar concentration of the first phosphate buffer is 50 mmol / L, the mass concentration of the first sodium azide is 1.0 g / L; and the pH of the dialysis buffer is 7.4.

[0121] The storage buffer comprises 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 value of the storage buffer is 7.4.

[0122] The target concentration of the dilution was 5 mg / mL.

[0123] The temperature of the thermal polymerization reaction is 60°C, and the time of the thermal polymerization reaction is 30 minutes.

[0124] (1) Solution preparation

[0125] 1) Preparation of R1 component: 0.1 mol / L MES flushing liquid, 0.15 mol / L sodium chloride, 5 g / L polyethylene glycol lauryl ether (Thesit), sodium azide with a mass concentration of 0.1%, and the pH of R1 component is 6.5;

[0126] 2) R2 storage buffer: 50 mmol / L HEPES buffer, 0.1% EDTA-2Na, 5% sucrose, 0.1% PC-300 preservative, the pH of R2 storage buffer is 7.4.

[0127] 3) Activation buffer: 50 mmol / L MES buffer, the pH of the activation buffer is 6.0.

[0128] 4) Coupling buffer: 50 mmol / L HEPES buffer, the pH of the coupling buffer is 7.6.

[0129] 5) Activator: Weigh ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) at a concentration of 5 mg / mL and prepare it for use immediately.

[0130] 6) Blocking solution: 0.8 mol / L glycine, pH of the blocking solution is 8.0, and the mass concentration is 10% bovine serum albumin (BSA).

[0131] (2) Preparation of latex reagent:

[0132] 1) 1 mL of latex microspheres with a particle size of 180 nm was mixed with an activation buffer at a volume ratio of 1:3 until uniform, to obtain an activation buffer containing latex microspheres;

[0133] 2) adding 0.2 mL of an activator to the activation buffer containing the latex microspheres, mixing well, and then placing in a constant temperature shaker for reaction for 25 minutes to obtain activated latex microspheres; the operating temperature of the constant temperature shaker is 37° C.;

[0134] 3) diluting the rheumatoid factor antigen to 1 mg / mL with a coupling buffer, then adding 4 mL of the 1 mg / mL diluted denatured IgG molecules to the activated latex microspheres, mixing them evenly, and then placing them in a constant temperature shaker for reaction for 2 hours to obtain a reaction solution; the operating temperature of the constant temperature shaker is 37° C.;

[0135] 4) Add 3 mL of blocking solution to the reaction solution, mix well, and then place in a constant temperature shaker for 1 hour to obtain a blocking product; the operating temperature of the constant temperature shaker is 37°C;

[0136] 5) After the reaction is completed, the blocked product is centrifuged and the supernatant is removed to obtain a centrifugal precipitate; the centrifugal speed is 16000 rpm and the centrifugal time is 50 min.

[0137] 6) Use 60 mL of R2 component buffer to repeatedly dissolve the centrifugal precipitate to resuspend the latex microspheres. During the resuspension process, a cell disruptor can be used to assist in the resuspension.

[0138] Example 2

[0139] Based on the contents disclosed in Example 1, the following modifications are further made:

[0140] The type of surfactant is Tween-80, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.2g:100mL.

[0141] Example 3

[0142] Based on the contents disclosed in Example 1, the following modifications are further made:

[0143] The type of surfactant is Triton-100, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.3g:100mL.

[0144] Example 4

[0145] Based on the contents disclosed in Example 1, the following modifications are further made:

[0146] The type of surfactant is Brij-35, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.4g:100mL.

[0147] Example 5

[0148] Based on the contents disclosed in Example 1, the following modifications are further made:

[0149] The type of surfactant is Kao A90, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.5g:100mL.

[0150] Example 6

[0151] Based on the contents disclosed in Example 1, the following modifications are further made:

[0152] The temperature of the thermal polymerization reaction is 56°C, and the time of the thermal polymerization reaction is 30 minutes.

[0153] Example 7

[0154] Based on the contents disclosed in Example 1, the following modifications are further made:

[0155] The type of surfactant is Tween-80, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.5g:100mL.

[0156] The temperature of the thermal polymerization reaction is 56°C, and the time of the thermal polymerization reaction is 50 minutes.

[0157] Example 8

[0158] Based on the contents disclosed in Example 1, the following modifications are further made:

[0159] The type of surfactant is Tween-80, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.5g:100mL.

[0160] The temperature of the thermal polymerization reaction is 60°C, and the time of the thermal polymerization reaction is 60 minutes.

[0161] Example 9

[0162] Based on the contents disclosed in Example 1, the following modifications are further made:

[0163] The type of surfactant is Triton-100, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.5g:100mL.

[0164] The temperature of the thermal polymerization reaction is 63°C, and the time of the thermal polymerization reaction is 50 minutes.

[0165] Comparative Example 1

[0166] Based on the contents disclosed in Example 1, the following modifications are further made:

[0167] No surfactant was added.

[0168] Comparative Example 2

[0169] Based on the contents disclosed in Example 1, the following modifications are further made:

[0170] The type of surfactant is Triton-100, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=1.0 g:100 mL.

[0171] Comparative Example 3

[0172] Based on the contents disclosed in Example 1, the following modifications are further made:

[0173] The type of surfactant is Brij-35, and the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=0.05g:100mL.

[0174] Example 4

[0175] Based on the contents disclosed in Example 1, the following modifications are further made:

[0176] The temperature of the thermal polymerization reaction is 50°C, and the time of the thermal polymerization reaction is 30 minutes.

[0177] Example 5

[0178] Based on the contents disclosed in Example 1, the following modifications are further made:

[0179] The temperature of the thermal polymerization reaction is 65°C, and the time of the thermal polymerization reaction is 30 minutes.

[0180] Related experiments and effect data:

[0181] Effect evaluation:

[0182] Instrument selection: HITACHI7180, using the reagents prepared in each embodiment and comparative example to test the sensitivity, specificity, batch difference and stability. The specific evaluation method is as follows:

[0183] (1) Sensitivity: the absorbance difference (ΔA) when the concentration of rheumatoid factor is 20 IU / mL;

[0184] (2) Specificity: Measure the third-party quality control product and calculate the deviation from the target value, Bias = (measured mean value - target value) / target value ╳ 100%;

[0185] (3) Batch difference: Three batches of rheumatoid factor antigen were prepared repeatedly, and the antigen was used to prepare rheumatoid factor assay kits. Calibrators of different concentrations were measured respectively, and the differences in absorbance of each batch of reagents were compared;

[0186] (4) Stability: Observe the storage stability of the reagent. Test the reagent at the same calibration high value (calibrator for this test: 100 IU / mL) at the 0th, 6th, 12th, 18th, and 24th month. Measure three times at each time point and calculate the CV of all data.

[0187] The above test results are shown in Tables 1 to 6.

[0188] Table 1 Sensitivity data results of various embodiments and comparative examples

[0189]

[0190]

[0191] Table 2 Specific data results of each embodiment and comparative example

[0192]

[0193] Table 3 Results of batch difference data of various embodiments and comparative examples

[0194]

[0195]

[0196]

[0197] Table 4 Stability data results of each example

[0198]

[0199] Table 5 Stability data results of each comparative example

[0200]

[0201]

[0202] Table 6 Comprehensive comparison of sensitivity, specificity, batch difference and stability of various embodiments and comparative examples

[0203]

[0204] It can be seen from Tables 1 to 5 that although the sensitivity of Comparative Examples 1, 3 and 5 is good, the specificity, batch difference and stability are poor. Although the batch difference and stability of Comparative Examples 2 and 4 meet the requirements, the sensitivity and specificity are poor. In addition, it can be seen from the comparison between Comparative Example 1 and the embodiment that when no surfactant is added to the denaturing buffer, the batch difference, specificity and stability of the rheumatoid antigen obtained by the denaturing method are poor; and when a surfactant of a suitable concentration is added to the denaturing buffer, the sensitivity, specificity, batch difference and stability of the rheumatoid antigen obtained by the denaturing method are good. In addition, according to Comparative Examples 4 and 5, when the temperature of the thermal polymerization reaction of the denaturing method is lower than 56°C, the denaturation efficiency of the IgG antigen is low, and the sensitivity and specificity of the reagent are also poor. When the denaturation temperature of the thermal polymerization reaction of the denaturing method is higher than 63°C, the thermal polymerization reaction of the IgG antigen will be excessively carried out. Although the sensitivity of the reagent is high, the specificity and stability are poor.

[0205] In summary, the embodiments of the present application provide a denaturation buffer, which, through its composition and concentration ratio and the action of a surfactant, jointly maintains the pH stability of the solution and changes the surface properties of IgG molecules, thereby reducing their aggregation tendency, so as to improve the dispersion degree of IgG during the thermal denaturation process of the rheumatoid factor kit detection.

[0206] In addition, the embodiment of the present application provides a rheumatoid factor kit, the antigen prepared by the rheumatoid factor kit and the kit itself have high sensitivity, specificity, stability and low batch difference, which can meet the needs of clinical use, and the preparation process of the kit is simple and can be scaled up for production.

[0207] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown in the present application, but will conform to the widest range consistent with the principles and novel features applied for by the present application.

Claims

1. A denaturing buffer, comprising a buffer and a surfactant, wherein the buffer comprises a buffer solute and a buffer solvent, wherein the amount n1 of the buffer solute and the volume V1 of the buffer solvent satisfy the relationship: n1:V1=(10-100):1, and if the unit of n1 is mmol, the unit of V1 is L; the mass m1 of the surfactant and the volume V1 of the buffer solvent satisfy the relationship: m1:V1=(0.1-0.5):100, and if the unit of m1 is g, the unit of V1 is mL.

2. The denaturing buffer according to claim 1, wherein the type of buffer solute comprises at least one of the following: phosphate buffer, hydroxyethylpiperazineethanesulfonic acid and 2-(N-morpholine)ethanesulfonic acid; and / or The types of the surfactant include at least one of the following: Tween, Triton, polyoxyethylene lauryl ether and Kao series surfactants.

3. The denaturing buffer according to claim 1, wherein the pH of the buffer is 7.0 to 7.

5.

4. A method for preparing the denaturing buffer according to any one of claims 1 to 3, the method comprising: Mixing a buffer solute and a buffer solvent to obtain a buffer; The surfactant and the buffer are mixed to obtain a denaturing buffer.

5. A rheumatoid factor kit, comprising the denaturing buffer according to any one of claims 1 to 3.

6. The kit according to claim 5, further comprising a dialysis buffer and a storage buffer, the dialysis buffer comprising a first phosphate buffer and a first sodium azide, the molar concentration of the first phosphate buffer being 10 mmol / L to 100 mmol / L, and the mass concentration of the first sodium azide being 0.5 g / L to 2 g / L; the storage buffer comprising a second phosphate buffer and a second sodium azide, the molar concentration of the second phosphate buffer being 10 mmol / L to 100 mmol / L, and the mass concentration of the second sodium azide being 0.5 g / L to 2 g / L.

7. The kit according to claim 4, further comprising an activator, wherein the mass concentration of the activator is 1 mg / mL to 10 mg / mL.

8. A method for denaturing a rheumatoid factor antigen, the method comprising: diluting the rheumatoid factor antigen using the denaturing buffer according to any one of claims 1 to 3 to obtain a dilution; The dilution is subjected to thermal polymerization to obtain a polymerized rheumatoid factor antigen. 9 . The denaturation method according to claim 8 , wherein the target mass concentration of the dilution is 1 mg / mL to 10 mg / mL.

10. The denaturation method according to claim 8, wherein 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.

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