Denatured IgG for determining rheumatoid factors, preparation method thereof, detection reagent and kit

Abstract

The invention discloses denatured IgG for determining rheumatoid factors, a preparation method of the denatured IgG, a detection reagent and a kit, and relates to the technical field of rheumatoid factor detection. The preparation method of the denatured IgG for measuring the rheumatoid factors comprises the following steps: carrying out thermal denaturation treatment on IgG to obtain first denatured IgG; mixing the first denatured IgG with basic amino acid to obtain second denatured IgG; mixing the second denatured IgG with a denaturant to obtain third denatured IgG; and mixing the third denatured IgG with a cross-linking agent, and carrying out a cross-linking reaction to obtain the denatured IgG for measuring rheumatoid factors. According to the present invention, the antigen epitope is completely exposed, and the antigen reaction activity is enhanced, such that the RF detection sensitivity is improved, the linear range is widened, and the HOOK ability is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of rheumatoid factor detection, in particular to a denatured IgG for determining rheumatoid factor and a preparation method, a detection reagent and a kit thereof. BACKGROUND

[0002] Rheumatoid factor (RF) is a specific antibody produced by the human body against the antigenic determinant on the Fc segment of the denatured immunoglobulin (IgG) molecule in the body, mainly secreted and produced by B lymphocytes. The quantitative detection of rheumatoid factor is mainly used for serological diagnosis of rheumatoid arthritis, and is also one of the diagnostic indicators of protein metabolism abnormalities, other autoimmune diseases such as systemic lupus erythematosus, Sjogren's syndrome, mixed connective tissue disease, toxic diffuse goiter, hepatitis, tuberculosis, and hyperglobulinemia. In 1987, the American College of Rheumatology (ACR) made RF one of the diagnostic criteria for rheumatoid arthritis (RA), which has been used until now. RF plays an important role in differentiating rheumatoid arthritis and other rheumatic diseases.

[0003] At present, the methods for detecting rheumatoid factor mainly include ELISA method, chemiluminescence method and immunosorbent turbidity method. The immunosorbent turbidity method is a relatively common detection method because it can realize automatic operation on the instrument and reduce the influence of human factors on the detection results. However, this method has the following problems: (1) narrow linear range: the detection range of the traditional kit is usually 5-640 IU / mL, and high-concentration samples need to be diluted for detection, which is complicated and easy to introduce errors; (2) significant hook effect: when the RF concentration exceeds 640 IU / mL, the signal is falsely reduced due to excessive antigen, and the missed detection rate is as high as 30%; (3) insufficient sensitivity and specificity: the sensitivity of the traditional reagent is about 70-75%, and the specificity is about 75-80%. Therefore, a rheumatoid factor detection method with wide detection range, high sensitivity and high specificity still needs to be developed. SUMMARY

[0004] The main purpose of the present application is to provide a denatured IgG for determining rheumatoid factor and a preparation method, a detection reagent and a kit thereof, which aims to solve the problems of narrow detection range, low sensitivity and low specificity in the process of determining rheumatoid factor by the existing immunosorbent turbidity method.

[0005] To achieve the above-mentioned purpose, the present application provides a preparation method of a denatured IgG for determining rheumatoid factor, comprising the following steps:

[0006] S1, heat denaturation treatment is performed on IgG to obtain first denatured IgG;

[0007] S2. Mix the first denatured IgG with basic amino acids to obtain the second denatured IgG;

[0008] S3. Mix the second denatured IgG with the denaturing agent to obtain the third denatured IgG;

[0009] S4. The third denatured IgG and the cross-linking agent are mixed and a cross-linking reaction is carried out to obtain the denatured IgG used for the determination of rheumatoid factor; wherein:

[0010] The basic amino acid includes arginine;

[0011] The denaturing agent includes guanidine thiocyanate;

[0012] The crosslinking agent includes at least one of a first crosslinking agent and a second crosslinking agent, wherein the first crosslinking agent includes succinimide 3,3'-dithiodipropionate and the second crosslinking agent includes dimethylpimedomide ester.

[0013] In one embodiment, in step S1, IgG is subjected to thermal denaturation by heating in a buffer solution, wherein the buffer solution includes PBS buffer or PB buffer, and the pH of the buffer solution is 7.2–7.6; the heating conditions include heating at 50–56°C for 10–15 min; and / or,

[0014] In step S2, the first denatured IgG is cooled to 0-4°C within 1-5 minutes, and then the cooled first denatured IgG is mixed with basic amino acids for 25-35 minutes; and / or,

[0015] In step S3, the second denatured IgG and the denaturing agent are mixed at 22–27°C for 15–20 min.

[0016] In one embodiment, in step S1, the concentration of the IgG is 10 mg / mL;

[0017] In step S2, the basic amino acids are added to the first denatured IgG at a final concentration of 0.05 to 0.15 M for mixing.

[0018] In step S3, the denaturing agent is added to the second denatured IgG at a final concentration of 0.9 to 2.1 M for mixing.

[0019] In one embodiment, the crosslinking agent includes a first crosslinking agent and a second crosslinking agent, and step S4 includes:

[0020] S41. The third denatured IgG and the first cross-linking agent are mixed to carry out the first cross-linking reaction to obtain the fourth denatured IgG;

[0021] S42. The fourth denatured IgG and the second cross-linking agent are mixed to carry out a second cross-linking reaction to obtain the denatured IgG used for the determination of rheumatoid factor.

[0022] In one embodiment, in step S41, the first crosslinking agent is added to the third denatured IgG at a final concentration of 2-3 mM for mixing; in step S42, the second crosslinking agent is added to the fourth denatured IgG at a final concentration of 0.45-1.5 mM for mixing; and / or,

[0023] The pH value of the first crosslinking reaction is 6.4–6.6; and / or,

[0024] The temperature of the first crosslinking reaction is 22–27°C; and / or,

[0025] The first crosslinking reaction takes 50–70 min; and / or,

[0026] The pH value of the second crosslinking reaction is 7.5–8.5; and / or,

[0027] The temperature of the second crosslinking reaction is 22–27°C; and / or,

[0028] The second cross-linking reaction takes 25 to 35 minutes.

[0029] This invention proposes a denatured IgG for measuring rheumatoid factor, wherein the denatured IgG for measuring rheumatoid factor is prepared according to the preparation method of denatured IgG for measuring rheumatoid factor described in the foregoing technical solution.

[0030] This invention proposes a detection reagent for measuring rheumatoid factor. The detection reagent comprises microspheres conjugated with rheumatoid factor antigen, wherein the rheumatoid factor antigen in the microspheres conjugated with rheumatoid factor antigen includes the denatured IgG for measuring rheumatoid factor described in the aforementioned technical solution.

[0031] In one embodiment, the microspheres coupled with rheumatoid factor antigen are prepared by the following steps:

[0032] An activation buffer, activator, carboxyl microspheres, protectant, and denatured IgG for the determination of rheumatoid factor as described in the aforementioned technical solution are provided. The carboxyl microspheres, activator, and activation buffer are mixed and activated to obtain an activated carboxyl microsphere suspension.

[0033] The activated carboxyl microsphere suspension was mixed with denatured IgG for the determination of rheumatoid factor and a coupling reaction was carried out. After the reaction was completed, a protective agent was added to obtain the microspheres coupled with rheumatoid factor antigen.

[0034] In one embodiment, the activation buffer comprises 50 mM 2-morpholine ethanesulfonate buffer, and the activator comprises a mixed solution of 0.4 mg / mL sulfonated N-hydroxysuccinimide and 0.1 mg / mL carbodiimide; the activated carboxyl microsphere suspension has a mass concentration of 0.1%; and / or,

[0035] The concentration of the denatured IgG used for determining rheumatoid factor is 10 mg / mL, and the volume ratio of the denatured IgG used for determining rheumatoid factor to the activated carboxyl microsphere suspension is (3-20):(980-997); and / or,

[0036] The protective agent comprises histidine at a final concentration of 5–10 mM.

[0037] This invention proposes a kit for measuring rheumatoid factor, the kit comprising the detection reagents described in the foregoing technical solution.

[0038] In the technical solution of this invention, IgG is first subjected to gradient denaturation in three stages: pre-denaturation, controlled renaturation, and deep denaturation. Pre-denaturation is achieved through thermal denaturation, causing the Fc segment of IgG to partially unfold, resulting in first-denatured IgG. Subsequently, basic amino acids are added for renaturation, yielding second-denatured IgG. Since the first-denatured IgG exposes hydrophobic regions and negatively charged groups, the basic amino acids, with their cationic properties, can neutralize charge repulsion, thereby reducing protein aggregation. Simultaneously, the basic amino acids can reversibly bind to the hydrophobic regions of the denatured protein, guiding the denatured IgG to gradually fold back to its native conformation, preferentially restoring the α-helix structure of the RF-targeted Fc segment, thus enhancing epitope specificity. Furthermore, the addition of basic amino acids can adjust the pH and ionic strength of the solution, thereby stabilizing the partially folded intermediate state of the denatured IgG, preventing misfolding, and providing a suitable microenvironment to promote the gradual restoration of the native structure of IgG. Finally, deep denaturation is performed using a chemical denaturing agent to enhance epitope exposure, thereby enhancing antigen reactivity. Therefore, IgG after gradient denaturation fully exposes the antigenic epitopes and enhances antigenic reactivity, thereby improving the detection sensitivity of RF, broadening the linear range, and enhancing the hooking ability.

[0039] Secondly, the technical solution of this invention further involves a cross-linking reaction between the denatured IgG after gradient denaturation treatment and a cross-linking agent. By attaching the cross-linking agent to the denatured IgG, the stability of the denatured state is maintained, and refolding is prevented. Specifically, the first cross-linking agent introduces disulfide bonds to limit the exposure of hydrophobic residues and reduces non-specific intermolecular interactions through steric hindrance, thereby effectively inhibiting the aggregation of denatured IgG. The second cross-linking agent covalently bridges the two denatured IgG molecules by forming amide bonds, fixing the local conformation of IgG through covalent bonding, reducing the exposure of hydrophobic residues, and preferentially forming intramolecular cross-links rather than intermolecular connections, thus effectively inhibiting aggregation, fixing the conformation of key epitopes, and preventing IgG from reverting from the denatured state to its naturally folded state, which is beneficial for stabilizing the three-dimensional structure of the protein. Therefore, after the cross-linking reaction, the aggregation between denatured IgG molecules is reduced, which helps the denatured IgG to fully expose antigenic epitopes, enhances the reactivity of the antigen, and thus further improves the detection sensitivity of the detection reagent, broadens the linear range, enhances HOOK capability, and improves diagnostic efficacy. Detailed Implementation

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0042] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0043] Rheumatoid factor (RF) is a specific antibody produced by the human body against the antigenic determinant of the Fc fragment of the denatured immunoglobulin (IgG) molecule, primarily secreted by B lymphocytes. Quantitative detection of RF is mainly used for the serological diagnosis of rheumatoid arthritis, and it is also one of the diagnostic indicators for protein metabolism abnormalities, other autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome, mixed connective tissue disease, toxic diffuse goiter, as well as hepatitis, tuberculosis, and hyperglobulinemia. In 1987, the American College of Rheumatology (ACR) included RF as one of the diagnostic criteria for rheumatoid arthritis (RA), and this criterion remains in use today. RF plays an important role in differentiating rheumatoid arthritis from other rheumatic diseases.

[0044] Currently, the main methods for detecting rheumatoid factor include ELISA, chemiluminescence immunoassay, and immunoturbidimetric assay. Immunoturbidimetric assay is a commonly used method because it can be automated on the instrument, reducing the influence of human factors on the test results. However, this method has the following problems: (1) Narrow linear range: The detection range of traditional reagent kits is usually 5-640 IU / mL. High-concentration samples need to be diluted before detection, which is cumbersome and prone to errors; (2) Significant hook effect: When the RF concentration exceeds 640 IU / mL, the signal is falsely reduced due to excessive antigen, and the false negative rate is as high as 30%; (3) Insufficient sensitivity and specificity: The sensitivity of traditional reagents is about 70-75%, and the specificity is about 75-80%. Therefore, a rheumatoid factor detection method with a wide detection range, high sensitivity, and high specificity still needs to be developed.

[0045] The antigen raw materials used in conventional rheumatoid factor test reagents are mostly denatured IgG monomers or Fc fragments prepared by thermal denaturation methods, and the binding site of rheumatoid factor is singular; even if thermally polymerized IgG prepared by thermal polymerization methods has multiple binding epitopes, its structure is unstable under low-temperature storage conditions, making it difficult to control batch-to-batch differences.

[0046] IgG that has undergone thermal and chemical denaturation still has the problem of easy aggregation, mainly due to the following reasons: (1) The hydrophobic amino acid residues of IgG are usually wrapped inside the molecule to maintain its water solubility. Denaturation destroys the structure and exposes the hydrophobic regions. The hydrophobic parts tend to combine with each other and form aggregates through hydrophobic interactions; (2) IgG relies on intra- and inter-chain disulfide bonds to maintain stability. If the disulfide bonds are destroyed, the subunits separate and the structure becomes loose, increasing the chance of binding with other molecules; (3) Changes in surface charge: Denaturation can lead to changes in the distribution of surface charge. When approaching the isoelectric point (PI), the net charge decreases and the electrostatic repulsion weakens, promoting aggregation; (4) Enhanced intermolecular forces: After denaturation, the structure unfolds and more residues are exposed, which enhances the intermolecular hydrogen bonds or van der Waals forces, forming aggregates; (5) Effects of glycosylation: The Fc segment of IgG glycans usually enhances solubility and stability. After the glycan structure is damaged, it loses its protective effect and promotes aggregation.

[0047] Based on the above background, this invention proposes a method for preparing denatured IgG for the determination of rheumatoid factor, comprising the following steps:

[0048] S1. IgG is subjected to heat denaturation treatment to obtain first denatured IgG;

[0049] S2. Mix the first denatured IgG with basic amino acids to obtain the second denatured IgG;

[0050] S3. Mix the second denatured IgG with the denaturing agent to obtain the third denatured IgG;

[0051] S4. The third denatured IgG and the cross-linking agent are mixed and a cross-linking reaction is carried out to obtain the denatured IgG used for the determination of rheumatoid factor; wherein:

[0052] The basic amino acid includes arginine;

[0053] The denaturing agent includes guanidine thiocyanate;

[0054] The crosslinking agent includes at least one of a first crosslinking agent and a second crosslinking agent, wherein the first crosslinking agent includes succinimide 3,3'-dithiodipropionate and the second crosslinking agent includes dimethylpimedomide ester.

[0055] In the technical solution of this invention, IgG is first subjected to gradient denaturation in three stages: pre-denaturation, controlled renaturation, and deep denaturation. Pre-denaturation is achieved through thermal denaturation, causing the Fc segment of IgG to partially unfold, resulting in first-denatured IgG. Subsequently, basic amino acids are added for renaturation, yielding second-denatured IgG. Since the first-denatured IgG exposes hydrophobic regions and negatively charged groups, the basic amino acids, with their cationic properties, can neutralize charge repulsion, thereby reducing protein aggregation. Simultaneously, the basic amino acids can reversibly bind to the hydrophobic regions of the denatured protein, guiding the denatured IgG to gradually fold back to its native conformation, preferentially restoring the α-helix structure of the RF-targeted Fc segment, thus enhancing epitope specificity. Furthermore, the addition of basic amino acids can adjust the pH and ionic strength of the solution, thereby stabilizing the partially folded intermediate state of the denatured IgG, preventing misfolding, and providing a suitable microenvironment to promote the gradual restoration of the native structure of IgG. Finally, deep denaturation is performed using a chemical denaturing agent to enhance epitope exposure, thereby enhancing antigen reactivity. Therefore, IgG after gradient denaturation fully exposes the antigenic epitopes and enhances antigenic reactivity, thereby improving the detection sensitivity of RF, broadening the linear range, and enhancing the hooking ability.

[0056] Secondly, the technical solution of this invention further involves a cross-linking reaction between the denatured IgG after gradient denaturation treatment and a cross-linking agent. By attaching the cross-linking agent to the denatured IgG, the stability of the denatured state is maintained, and refolding is prevented. Specifically, the first cross-linking agent introduces disulfide bonds to limit the exposure of hydrophobic residues and reduces non-specific intermolecular interactions through steric hindrance, thereby effectively inhibiting the aggregation of denatured IgG. The second cross-linking agent covalently bridges the two denatured IgG molecules by forming amide bonds, fixing the local conformation of IgG through covalent bonding, reducing the exposure of hydrophobic residues, and preferentially forming intramolecular cross-links rather than intermolecular connections, thus effectively inhibiting aggregation, fixing the conformation of key epitopes, and preventing IgG from reverting from the denatured state to its naturally folded state, which is beneficial for stabilizing the three-dimensional structure of the protein. Therefore, after the cross-linking reaction, the aggregation between denatured IgG molecules is reduced, which helps the denatured IgG to fully expose antigenic epitopes, enhances the reactivity of the antigen, and thus further improves the detection sensitivity of the detection reagent, broadens the linear range, enhances HOOK capability, and improves diagnostic efficacy.

[0057] It should be noted that the technical solution of this application proceeds in the order of pre-denaturation, reversion and deep denaturation. This arrangement is beneficial to solving the problem of insufficient epitope exposure.

[0058] It is understood that the crosslinking agent includes at least one of the first crosslinking agent and the second crosslinking agent, which means that the crosslinking agent can be either the first crosslinking agent or the second crosslinking agent, or the first crosslinking agent and the second crosslinking agent can be used simultaneously as crosslinking agents, and the detection effect when using two crosslinking agents simultaneously is better than that when using a single crosslinking agent.

[0059] In an embodiment of the present invention, IgG is subjected to thermal denaturation treatment by heating in a buffer solution, wherein the buffer solution includes PBS buffer or PB buffer, and the pH of the buffer solution is 7.2 to 7.6; the heating conditions include heating at 50 to 56°C for 10 to 15 minutes.

[0060] The Fc segment of IgG is partially unfolded through a heat denaturation process to obtain first-denatured IgG. Considering that excessively high heating temperature or excessively long heating time during heat denaturation can lead to over-denaturation, resulting in the destruction of key epitopes or changes in spatial conformation, and that over-denatured IgG may also expose non-specific hydrophobic regions, allowing it to bind to other non-target proteins; and that excessively low heating temperature or excessively short heating time can lead to insufficient denaturation, the technical solution of this invention selects to heat IgG at 50-56°C for 10-15 minutes.

[0061] In an embodiment of the present invention, in step S2, the first denatured IgG is cooled to 0-4°C within 1-5 minutes, and then the cooled first denatured IgG and basic amino acids are mixed for 25-35 minutes.

[0062] It should be noted that cooling the first denatured IgG to 0-4°C within 1-5 minutes is for rapid cooling. Rapid cooling reduces the time the protein remains at intermediate temperatures, thereby reducing the risk of aggregation due to hydrophobic interactions. The present invention does not impose specific limitations on the cooling method. Exemplarily, the first denatured IgG sample can be transferred to an ice bath or pre-cooling device to complete the cooling within 1-5 minutes.

[0063] Excessively high mixing temperatures of the first denatured IgG and basic amino acids may lead to increased aggregation, structural damage, and weakened effects of the basic amino acids; excessively low mixing temperatures may result in slowed molecular motion, significantly reduced folding, and a risk of freezing of the solution. If the mixing time of the first denatured IgG and basic amino acids is too long, the basic amino acids may decompose over time, affecting the refolding environment; if the mixing time is too short, incomplete refolding may occur.

[0064] In an embodiment of the present invention, the concentration of the IgG is 10 mg / mL;

[0065] In step S2, the basic amino acids are added to the first denatured IgG at a final concentration of 0.05 to 0.15 M for mixing.

[0066] In step S3, the denaturing agent is added to the second denatured IgG at a final concentration of 0.9 to 2.1 M for mixing.

[0067] Basic amino acids can preferentially restore the α-helical structure of the Fc segment targeted by RF, which helps improve epitope specificity. In the technical solution of this invention, the amount of basic amino acids has a significant impact on the final detection reagent results. Too little amount will lead to insufficient buffering capacity, pH fluctuations, and protein precipitation or misfolding; too much amount will result in high ionic strength, which will interfere with protein-solvent interactions and inhibit renaturation. Considering the denaturation effect, sensitivity requirements, and specificity requirements, the technical solution of this invention sets the amount of basic amino acids within the above-mentioned range. Excessive denaturant will lead to over-denaturation, while too little denaturant will lead to incomplete denaturation. Setting the amount of denaturant within the above-mentioned range can control the degree of denaturation and reduce the adverse effects of over-denaturation.

[0068] In an embodiment of the present invention, in step S3, the second denatured IgG and the denaturing agent are mixed at 22–27°C for 15–20 min. Excessively high mixing temperatures may lead to increased aggregation and structural damage; excessively low mixing temperatures may result in slow molecular motion and reduced denaturation efficiency. Prolonged mixing times may trigger unnecessary side reactions, while insufficient mixing times may prevent adequate interaction with the denatured IgG, leading to incomplete denaturation. Considering both the denaturation effect and the reduction of unnecessary side reactions, the present invention selects mixing the second denatured IgG and the denaturing agent at 22–27°C for 15–20 min.

[0069] In some embodiments of the present invention, after step S3, the method further includes: removing guanidine thiocyanate using size exclusion chromatography (SEC). Specifically, after denaturing the second denatured IgG, the resulting solution is loaded onto a size exclusion chromatography column, and the IgG is separated from guanidine thiocyanate by utilizing the difference in molecular weight. The eluent corresponding to the protein peak that appears first during the elution process is collected, thereby obtaining an IgG sample free of guanidine thiocyanate.

[0070] In an embodiment of the present invention, the crosslinking agent includes a first crosslinking agent and a second crosslinking agent, and step S4 includes:

[0071] S41. The third denatured IgG and the first cross-linking agent are mixed to carry out the first cross-linking reaction to obtain the fourth denatured IgG;

[0072] S42. The fourth denatured IgG and the second cross-linking agent are mixed to carry out a second cross-linking reaction to obtain the denatured IgG used for the determination of rheumatoid factor.

[0073] In this invention, crosslinking is performed sequentially using a first crosslinking agent and a second crosslinking agent. Considering that both the first and second crosslinking agents react with the amino groups of denatured IgG, simultaneous addition of both may lead to interference between the crosslinking agents, forming a non-uniform crosslinked network. Furthermore, if the second crosslinking agent is used first, followed by the first, the second crosslinking agent may excessively occupy sites, preventing the first crosslinking agent from effectively regulating the dynamic results and reducing stability. Therefore, this invention employs a stepwise crosslinking reaction, sequentially crosslinking the first denatured IgG with the first and second crosslinking agents. This approach avoids competitive binding, ensures crosslinking efficiency, and allows for preliminary fixation of the molecular conformation through crosslinking with the first crosslinking agent, followed by crosslinking with the second crosslinking agent, enabling more precise stabilization of specific regions.

[0074] In an embodiment of the present invention, in step S41, the first crosslinking agent is added to the third denatured IgG at a final concentration of 2-3 mM for mixing; in step S42, the second crosslinking agent is added to the fourth denatured IgG at a final concentration of 0.45-1.5 mM for mixing. Setting the amounts of the first and second crosslinking agents within the above ranges can maintain high stability of the denatured IgG while controlling costs.

[0075] In embodiments of the present invention, the pH value of the first cross-linking reaction is 6.4 to 6.6. The first cross-linking reaction is carried out under weakly acidic conditions. The sulfonic acid groups of DTSSP can maintain high solubility and stability under weakly acidic conditions, reducing spontaneous hydrolysis. Furthermore, weakly acidic conditions can slow down the reaction rate, avoiding protein aggregation or non-specific binding caused by rapid cross-linking.

[0076] In embodiments of the present invention, the temperature of the first cross-linking reaction is 22–27°C. High temperatures may accelerate the hydrolysis of the cross-linking agent or cause protein denaturation, while low temperatures significantly reduce reaction efficiency. Conducting the reaction at room temperature is beneficial for balancing the reaction rate and protein stability.

[0077] In an embodiment of the present invention, the first crosslinking reaction takes 50 to 70 minutes. This setting ensures sufficient but not excessive crosslinking.

[0078] In an embodiment of the present invention, the pH value of the second crosslinking reaction is 7.5 to 8.5. This setting allows the second crosslinking agent DMP to maintain high stability in an alkaline environment.

[0079] In embodiments of the present invention, the temperature of the second cross-linking reaction is 22–27°C. High temperatures may accelerate the hydrolysis of the cross-linking agent or cause protein denaturation, while low temperatures significantly reduce reaction efficiency. Conducting the reaction at room temperature is beneficial for balancing the reaction rate and protein stability.

[0080] In an embodiment of the present invention, the second crosslinking reaction takes 25–35 minutes. The second crosslinking agent, DMP, maintains high stability under alkaline conditions and reacts rapidly. Setting the second crosslinking reaction time within the above range allows for a complete reaction.

[0081] This invention proposes a denatured IgG for measuring rheumatoid factor, wherein the denatured IgG for measuring rheumatoid factor is prepared according to the preparation method of denatured IgG for measuring rheumatoid factor described in the foregoing technical solution.

[0082] This invention proposes a detection reagent for measuring rheumatoid factor. The reagent comprises microspheres conjugated with rheumatoid factor antigen, wherein the rheumatoid factor antigen in the microspheres includes the denatured IgG described in the foregoing technical solutions for measuring rheumatoid factor. The detection kit incorporates all the technical solutions described for measuring denatured IgG of rheumatoid factor, and therefore possesses all the beneficial effects of the aforementioned method for measuring denatured IgG of rheumatoid factor. These advantages will not be elaborated upon further here.

[0083] In an embodiment of the present invention, the microspheres coupled with rheumatoid factor antigen are prepared by the following steps:

[0084] An activation buffer, activator, carboxyl microspheres, protectant, and denatured IgG for the determination of rheumatoid factor as described in the aforementioned technical solution are provided. The carboxyl microspheres, activator, and activation buffer are mixed and activated to obtain an activated carboxyl microsphere suspension.

[0085] The activated carboxyl microsphere suspension was mixed with denatured IgG for the determination of rheumatoid factor and a coupling reaction was carried out. After the reaction was completed, a protective agent was added to obtain the microspheres coupled with rheumatoid factor antigen.

[0086] In this invention, a protective agent is added after the conventional coupling process as an Fc fragment protectant to reduce antibody Fab fragment inactivation and improve the reactivity of the prepared microspheres coupled with rheumatoid factor antigen. It should be noted that since the coupling reaction typically relies on the binding of activated chemical groups on the microsphere surface to specific groups of IgG, adding the protective agent prematurely might competitively occupy the microsphere activation sites, leading to a decrease in coupling efficiency. Therefore, this invention chooses to add the protective agent after the coupling reaction is complete.

[0087] In an embodiment of the present invention, the activation buffer comprises 50 mM 2-morpholine ethanesulfonate buffer, and the activator comprises a mixed solution of 0.4 mg / mL sulfonated N-hydroxysuccinimide solution and 0.1 mg / mL carbodiimide solution; in the activated carboxyl microsphere suspension, the mass concentration of the activated carboxyl microspheres is 0.1%.

[0088] The specific preparation process can be carried out according to the following steps:

[0089] Prepare a 50 mM pH 6.0 2-morpholinoethanesulfonic acid (MES) buffer solution. Use the MES buffer to prepare a 40 mg / mL sulfonated N-hydroxysuccinimide (sNHS) solution and a 20 mg / mL N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) solution.

[0090] Mix 100 μL of carboxyl microspheres, 100 μL of 40 mg / mL sNHS solution and 50 μL of 20 mg / mL EDAC solution, bring the volume to 10 mL with MES buffer, and then incubate at room temperature with shaking for 30 min to obtain an activated carboxyl microsphere suspension.

[0091] N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) and sulfonated N-hydroxysuccinimide (sNHS) were used as activators to activate carboxyl microspheres. EDAC activates the carboxyl groups on the microsphere surface into intermediates, which are then converted into more stable active esters by sNHS. Insufficient activator will result in some carboxyl groups remaining unactivated, leading to reduced coupling efficiency; excessive activator may cause cross-linking between microspheres, resulting in increased particle size or even precipitation, while also increasing cost. The above technical solution ensures sufficient activation of the carboxyl groups on the microspheres and suppresses side reactions.

[0092] In an embodiment of the present invention, the concentration of the denatured IgG used to determine rheumatoid factor is 10 mg / mL, and the volume ratio of the denatured IgG to the activated carboxyl microspheres is (3-20):(980-997). Setting the amounts of the denatured IgG and the activated carboxyl microspheres within the above range yields a better coupling effect.

[0093] In embodiments of the present invention, the protective agent comprises histidine at a final concentration of 5–10 mM. The various beneficial properties of histidine (such as buffering capacity, metal ion chelation, direct protection, and antioxidant activity) are utilized to protect the Fc portion of IgG from further damage, thereby maintaining its structural stability and biological function, and reducing antibody Fab fragment inactivation.

[0094] In an embodiment of the present invention, the microspheres are latex microspheres with a particle size of 180 nm. The present invention uses conventional latex microspheres to prepare the microspheres coupled with rheumatoid factor antigen.

[0095] In an embodiment of the present invention, the sealing process includes:

[0096] The second coupling reaction product was centrifuged, the supernatant was discarded, and then a blocking solution was added and reacted for 60 min. After centrifugation, the supernatant was discarded. The blocking solution was prepared by mixing 50 mM glycine, 0.1 mM ethanolamine and 5% bovine serum albumin.

[0097] In an embodiment of the present invention, after the microspheres conjugated with rheumatoid factor antigen are prepared, a storage solution is added to the microspheres conjugated with rheumatoid factor antigen for storage. The storage solution is prepared by mixing 50 mM PBS buffer, 0.2% bovine serum albumin and 0.02% preservative.

[0098] In embodiments of the present invention, the detection reagent further includes a buffer solution and a sample diluent. Wherein:

[0099] The buffer solution is prepared according to the following steps: the following materials are added sequentially to 1L of pure water: 11.9155g HEPES, 30g polyethylene glycol 6000, 8.766g NaCl, 5mL Triton X-100, and 1mL P300. After the materials are completely dissolved, the pH is adjusted to 7.4 with concentrated hydrochloric acid or sodium hydroxide solution, and then filtered.

[0100] The sample diluent was prepared according to the following steps: the following materials were added sequentially to 1L of pure water: 9g of NaCl and 1mL of Proclin30 (P300). After the materials were completely dissolved, the solution was filtered.

[0101] This invention provides a kit for determining rheumatoid factor, the kit comprising the detection reagents described in the foregoing technical solutions. The kit includes all the technical solutions of the detection reagents, and therefore possesses all the beneficial effects of the detection reagents, which will not be elaborated upon further here.

[0102] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0103] Example 1

[0104] A detection reagent comprising a buffer solution, microspheres conjugated with rheumatoid factor antigen, and a sample diluent, wherein:

[0105] (a) The preparation steps of microspheres coupled with rheumatoid factor antigen are as follows:

[0106] 1. A method for preparing denatured IgG for the determination of rheumatoid factor, comprising the following steps:

[0107] (1) Mix 100 mg of purified human IgG with PBS buffer (pH 7.4, total volume 10 mL), then heat in a 52 °C water bath for 10 min to perform heat denaturation treatment to obtain the first denatured IgG;

[0108] (2) Cool 8.75 mL of the first denatured IgG obtained in step (1) to 4°C in 1–5 min, then add 1.25 mL of 0.8 M arginine and mix for 30 min to obtain the second denatured IgG;

[0109] (3) Mix 7.5 mL of the second denatured IgG obtained in step (2) with 2.5 mL of 6 M guanidine thiocyanate (GuHCl), shake at room temperature for 15 min, and then pass the treated mixed sample through size exclusion chromatography (SEC). Collect the first protein peak that appears during the elution process to obtain the third denatured IgG.

[0110] (4) Add 0.2 mL of 0.1 M DTSSP (3,3'-dithiodipropionate succinimide) to 9.8 mL of the third denatured IgG obtained in step (3), and carry out the first cross-linking reaction at pH 6.5 for 1 h to obtain the fourth denatured IgG.

[0111] (5) Add 0.333 mL of 30 mM DMP (dimethyl pimecrolimide) to 9.667 mL of the fourth denatured IgG obtained in step (3), and carry out the second cross-linking reaction at pH 8.0 for 30 min to obtain the denatured IgG used for the determination of rheumatoid factor.

[0112] 2. A method for preparing microspheres conjugated with rheumatoid factor antigen, comprising the following steps:

[0113] (1) Solution preparation: Carbodiimide (EDAC) powder and sulfonated N-hydroxysuccinimide (sNHS) powder were removed from a sealed bag containing desiccant at 4°C and allowed to equilibrate to room temperature. A 40 mg / mL sNHS solution and a 20 mg / mL EDAC solution were prepared using 50 mM 2-morpholine ethanesulfonic acid (MES) pH 6.0 (Buffer 1).

[0114] (2) Microsphere activation: Mix 100 μL of carboxyl microspheres (particle size: 180 nm), 100 μL of 40 mg / mL sNHS solution and 50 μL of 20 mg / mL EDAC solution, and bring the volume to 10 mL with MES buffer. Then, place the mixture at room temperature and shake for 30 min to activate the microspheres to obtain the activated carboxyl microsphere suspension.

[0115] (3) Coupling reaction: The concentration of the denatured IgG used for the determination of rheumatoid factor is 10 mg / mL. 80 μL of the prepared denatured IgG used for the determination of rheumatoid factor is mixed with 9.92 mL of activated carboxyl microsphere suspension and the mixture is shaken at room temperature for 2 h to carry out the coupling reaction and obtain the first coupling reaction product. The coupling process is sonicated once for 30 min. The final concentration of the denatured IgG used for the determination of rheumatoid factor is 80 μg / mL.

[0116] (4) Targeted coupling optimization: Add 37.5 μL of histidine at a concentration of 0.2 M to the first coupling reaction product to obtain the second coupling reaction product.

[0117] (5) Blocking: Centrifuge the product of the second coupling reaction, discard the supernatant, add blocking solution (50mM histidine + 0.1mM ethanolamine + 5% bovine serum albumin), react for 60min, centrifuge, discard the supernatant, and obtain the microspheres coupled with rheumatoid factor antigen.

[0118] (6) Storage: Add storage solution (50mM PBS + 0.2% bovine serum albumin + 0.02% preservative).

[0119] (II) Preparation of buffer solution:

[0120] Add the following materials sequentially to 1L of pure water: 11.9155g HEPES, 30g polyethylene glycol 6000, 8.766g NaCl, 5mL Triton X-100, and 1mL P300. After the materials are completely dissolved, adjust the pH to 7.4 with concentrated hydrochloric acid or sodium hydroxide solution and filter.

[0121] (III) Preparation of sample dilution buffer:

[0122] Add the following materials sequentially to 1L of pure water: 9g NaCl, 1mL P300. After the materials are completely dissolved, filter.

[0123] Example 2

[0124] Compared with Example 1, the difference is that step (5) was not performed in the preparation steps of the denatured IgG for the determination of rheumatoid factor, that is, the cross-linking step of DMP (dimethyl hemiphthalimide ester) was not performed.

[0125] Example 3

[0126] Compared with Example 1, the difference is that step (4) was not performed in the preparation steps of the denatured IgG for the determination of rheumatoid factor, that is, the cross-linking step of DTSSP (3,3'-dithiodipropionate succinimide ester) was not performed.

[0127] Comparative Example 1

[0128] The difference from Example 1 is that step (2) is not performed in the process of preparing the denatured IgG for the determination of rheumatoid factor.

[0129] Comparative Example 2

[0130] Compared with Example 1, the difference is that in the step of preparing the denatured IgG for the determination of rheumatoid factor, step (3) is performed first, and then step (2) is performed. That is, after heat denaturation, denaturation is performed first with a denaturing agent, and then with basic amino acids.

[0131] Comparative Example 3

[0132] The difference from Example 1 is that histidine is not added in the step of preparing microspheres coupled with rheumatoid factor antigen.

[0133] Comparative Example 4

[0134] Compared with Example 1, the difference is that in the preparation of microspheres coupled with rheumatoid factor antigen, step (5) is performed first and then step (4). That is, after thermal denaturation, DMP (dimethyl pimecrolimide ester) is used for cross-linking first, and then DTSSP (3,3'-dithiodipropionate succinimide ester) is added for cross-linking.

[0135] Comparative Example 5

[0136] Compared with Example 1, the difference is that the preparation steps of the denatured IgG used to determine rheumatoid factor are as follows: steps (4) and (5) are not performed, that is, the cross-linking steps of DMP (dimethyl hempyrimidine ester) and DTSSP (3,3'-dithiodipropionate succinimide ester) are not performed.

[0137] Comparative Example 6

[0138] Compared with Example 1, the difference is that in the preparation steps of the denatured IgG for the determination of rheumatoid factor, steps (2) to (5) were not performed, and only the thermal denaturation process of step (1) was performed.

[0139] Performance testing

[0140] The test reagents used in the test examples and comparative examples were used to test and verify the reaction, sensitivity, and specificity.

[0141] Reaction principle:

[0142] This reagent uses immunoturbidimetry. The microspheres conjugated with rheumatoid factor antigen can agglutinate with rheumatoid factor (RF) in the sample, forming an antigen-antibody complex and resulting in turbidity. The change in turbidity is positively correlated with the RF content in the sample. By measuring the reaction change at a specific wavelength using immunoturbidimetry and referring to a multi-point calibration curve, the RF content in the sample can be calculated.

[0143] The testing process is as follows:

[0144] (a) Start-up check: Check whether the instrument's purified water, cleaning solution, power cord, etc. are properly connected, whether the waste liquid container is not full, and whether the reagent bottle is placed inside the instrument and the cap is open.

[0145] (b) After powering on, once the instrument's power-on fluid circuit maintenance is complete, enter the login interface, enter your username and password, and then enter the test interface;

[0146] (c) Reading the card: On the online status interface, click Reading the card, place the reagent card with the reagent kit in the magnetic card sensing area of ​​the instrument, and remove the magnetic card after successful reading. Click Return.

[0147] (d) Loading reagents: Place the reagents into the reagent tray. On the online status interface, click Load Reagents. If loading is successful, the reagent tray information will be automatically updated. If the barcode is damaged, you can manually load the reagents.

[0148] (e) Measurement: On the main measurement interface, enter the sample information (number, sample type, tube type, pre-dilution ratio), select the test item RF; place the sample on the test tube rack and put it into the track sample inlet; start the test, the fully automated specific protein analyzer will perform the following operations in sequence: ① The sample processing system will transfer the sample rack to the aspiration position, the sample needle will aspirate 20 μL of sample to the dilution position and mix, at the same time the reagent needle will aspirate 240 μL of buffer to the reaction cup, the sample needle will aspirate 20 μL of diluted sample to the buffer, and the sample needle will shake to mix; ② The reagent needle will aspirate 60 μL of antiserum to the mixture of sample and buffer, the reagent needle will mix, and incubate in the reaction plate for 3 min; ③ The photometric reaction system will send each reaction cup to the signal acquisition position to collect signals for calculating the AD value; ④ Calculate the content of rheumatoid factor in the sample;

[0149] (f) After the test is completed, the results viewing interface can display the sample, quality control, and calibration test results for the day.

[0150] The test results are as follows:

[0151] 1. Gradient calibrator test

[0152] Table 1. Amplitude value test results of the detection reagents in Examples 1-3 and Comparative Examples 1-6

[0153]

[0154]

[0155] As shown in Table 1, the test results of the gradient calibrators indicate that the curve trends of Comparative Examples 1-6 are worse than those of Examples 1-3.

[0156] 2. Linear range test:

[0157] (1) Experimental method:

[0158] High-value RF raw materials were added to the sample to prepare a low-concentration target serum matrix sample L1 (concentration: 0.5 IU / mL) with a concentration less than or equal to the lower limit of the expected linear range (concentration: 1 IU / mL).

[0159] High-value RF raw materials were added to sample L1 to prepare a high-concentration serum matrix sample L11 (concentration: 1690 IU / mL) with a concentration 30% higher than the upper limit of the expected linear interval (concentration: 1300 IU / mL). The high-concentration sample with a concentration 30% higher than the upper limit of the expected linear interval was mixed with the corresponding low-concentration sample with a concentration less than or equal to the lower limit of the expected linear interval in a certain proportion, and 11 linear interval establishment samples were prepared according to Table 2.

[0160] Table 2. Sample preparation and dilution ratios for linearity testing:

[0161] Sample No. Formulation Ratio Dilution L1 Low Concentration Sample (L) 0 L2 0.985L + 0.015H 0.015 L3 0.95L + 0.05H 0.05 L4 0.9L + 0.1H 0.1 L5 0.8L + 0.2H 0.2 L6 0.6L + 0.4H 0.4 L7 0.4L + 0.6H 0.6 L8 0.3L + 0.7H 0.7 L9 0.2L + 0.8H 0.8 L10 0.1L + 0.H 0.9 L11 High Concentration Sample (H) 1

[0162] Each sample was tested using three batches of kits, with each sample tested four times per batch, and the average concentration was calculated. The linear deviation was calculated using relevant formulas to investigate the linear range of the kits.

[0163] The criteria for judging relative or absolute deviation requirements are shown in Table 3.

[0164] Table 3 Relative or Absolute Deviation Requirements

[0165] Concentration Range (IU / mL) Relative Bias (B) Absolute Bias (D) [5,40] -- ± 4 IU / mL (40,1300] ±10％ --

[0166] (2) Data statistical analysis methods:

[0167] (a) Outlier test:

[0168] The Grubbs test was used to analyze outliers, and the Grubbs' interval was calculated according to formula (1):

[0169] Grubbs' interval = Mean ± G × SD ……………………………(1);

[0170] In the formula:

[0171] Mean — the average of the sample test results;

[0172] SD – Standard deviation of sample test results;

[0173] Based on the total number of tests N for each sample, the significance level α is set to 0.05, and the corresponding G value is obtained by consulting the Grubbs critical value table.

[0174] Any test result outside the Grubbs' interval is defined as an outlier. Each sample can have no more than one outlier, and the total number of outliers in all samples can not exceed two.

[0175] (b) Linearity judgment:

[0176] Perform linear data analysis according to the following steps:

[0177] 1) Multinomial regression analysis was performed on the data sets using statistical software (SPSS Statistics Version 25.0) to obtain first-, second-, and third-order polynomials. The mathematical models of the first-, second-, and third-order polynomials are shown in Table 4:

[0178] Table 4. Mathematical Models of First, Second, and Third Order Polynomials

[0179] Order Polynomial Regression Degrees of Freedom (Rdf) First Order [Y = b0 + b1X] 2 Second Order [Y = b0 + b1X + b2X 2 ]] 3 Third Order [Y = b0 + b1X + b2X 2 +b3X 3 ]]> 4

[0180] 2) Perform a linearity test on the regression equation.

[0181] In the multiple regression equation, b i The coefficients represented are regression coefficients. In the second and third order equations, b2 and b3 are nonlinear coefficients. A t-test is performed on each nonlinear coefficient to determine whether the regression coefficient is significantly different from zero.

[0182] Test b2 and b3 according to formula (2) and calculate the statistic t;

[0183]

[0184] In the formula:

[0185] SE i —The standard error of the slope for each nonlinear coefficient.

[0186] Calculate the degrees of freedom according to formula (3):

[0187] df=L×R-Rdf………………………………(3);

[0188] In the formula:

[0189] L – Concentration level number;

[0190] R — The number of determinations at each concentration level;

[0191] Rdf — The regression degrees of freedom, that is, the number of coefficients (including b0) in the regression equation.

[0192] According to the degrees of freedom value, look up the t critical value in the t value table (two-sided test, α = 0.05). If the non-linear coefficient is not significant (t < t critical value), the data set is considered linear. At this time, the precision test can be carried out on the data set. The specific method is as follows. When the precision meets the linear judgment requirements, the data analysis ends, and the conclusion is that the data set has statistical linearity or first-order linearity. If any non-linear coefficient is statistically significant, the data set is considered non-linear. At this time, it is necessary to judge the degree of non-linearity. If the degree of non-linearity meets the acceptance criteria, it is determined as clinically acceptable non-linearity. If it does not meet the acceptance criteria, reduce the concentration points until an acceptable linear range is established.

[0193] 3) Judgment of the degree of non-linearity:

[0194] Linear evaluation serves clinical practice and only has practical value when combined with clinical actual situations. Polynomial regression analysis emphasizes using statistical methods to judge linearity and does not consider that a certain deviation is allowed clinically. Therefore, a judgment of the degree of non-linearity of the statistical standard is proposed.

[0195] Calculate the linear deviation DL according to formula (4) i :

[0196]

[0197] In the formula:

[0198] p(xi) — The fitting value of the optimal fitting second-order or third-order equation;

[0199] b0 + b1xi — The fitting value of the fitting first-order equation;

[0200] xi — The sample theoretical concentration.

[0201] (3) Test results:

[0202] Table 5 Test results of theoretical concentration

[0203]

[0204] Table 6 Results of outlier analysis

[0205] <000048​​​​​

[0208]

[0209] Table 8 Deviation Analysis Results

[0210]

[0211] According to the results in Tables 5 to 8, the optimal fitting equation is a first-order equation. There is no significant difference between the non-linear coefficients of the second-order and third-order equations and zero (t < t critical value). Between the expected measurement range of 1 IU / mL to 1300 IU / mL, the deviations all meet the acceptance criteria.

[0212] 3. Construct a ROC curve (Receiver Operating Characteristic Curve) through the IVDstatistics software to verify the diagnostic efficacy of the detection reagents in Example 1, Comparative Example 2, Comparative Example 4, and Comparative Example 5. The test results are shown in Table 9.

[0213] (1) Optimal cutoff: The optimal cutoff refers to the threshold that can maximize the model performance.

[0214] (2) AUC (Area Under the Curve): AUC is the area under the ROC curve, which measures the ability of the model to distinguish positive and negative examples. The AUC value ranges from 0 to 1, and the value closer to 1 indicates better discrimination ability of the model. When AUC is between 0.5 and 0.7, there is low diagnostic accuracy; when between 0.7 and 0.9, there is certain diagnostic accuracy, and when AUC is above 0.9, there is high diagnostic accuracy.

[0215] (3) Sensitivity: The ability of an in vitro diagnostic test procedure to identify the presence of a target biomarker related to a specific disease or condition.

[0216] (4) Specificity: The ability of an in vitro diagnostic test procedure to identify the absence of a target biomarker related to a specific disease or condition.

[0217] (5) Youden index: The Youden index is an index that comprehensively considers sensitivity and specificity. The calculation formula is: Youden index = sensitivity + specificity - 1. The larger the Youden index, the better the model performance under this cutoff value, indicating higher diagnostic value.

[0218] (6) P value: The P value is used to test hypotheses and judge whether the results are statistically significant.

[0219] Table 9 Diagnostic Efficacy Results of the Detection Reagents in Example 1, Comparative Example 2, Comparative Example 4, and Comparative Example 5

[0220]

[0221] As shown in Table 9, the p-values ​​for all cutoff values ​​were less than 0.05, indicating that these results were statistically significant. Among them, the model corresponding to Example 1 had the best performance, with the highest diagnostic accuracy and diagnostic value. The AUCs of Comparative Examples 2, 4, 5, and Example 1 were all greater than 0.7, and the AUC of Example 1 was greater than 0.9, indicating that Example 1 had high diagnostic accuracy. When used for diagnosis, Example 1 had higher sensitivity and specificity, with sensitivity and specificity of 90.00% and 89.00%, respectively, at the optimal cutoff value.

[0222] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A method for preparing denatured IgG for the determination of rheumatoid factor, characterized in that, Includes the following steps: S1. IgG is subjected to heat denaturation treatment to obtain first denatured IgG; S2. Mix the first denatured IgG with basic amino acids to obtain the second denatured IgG; S3. Mix the second denatured IgG with the denaturing agent to obtain the third denatured IgG; S4. The third denatured IgG and the cross-linking agent are mixed and a cross-linking reaction is carried out to obtain the denatured IgG used for the determination of rheumatoid factor; wherein: The basic amino acid includes arginine; The denaturing agent includes guanidine thiocyanate; The crosslinking agent includes at least one of a first crosslinking agent and a second crosslinking agent, wherein the first crosslinking agent includes succinimide 3,3'-dithiodipropionate and the second crosslinking agent includes dimethylpimedomide ester.

2. The method for preparing denatured IgG for determining rheumatoid factor as described in claim 1, characterized in that, In step S1, IgG is subjected to thermal denaturation by heating in a buffer solution, wherein the buffer solution includes PBS buffer or PB buffer, and the pH of the buffer solution is 7.2–7.6; the heating conditions include heating at 50–56°C for 10–15 min; and / or, In step S2, the first denatured IgG is cooled to 0-4°C within 1-5 minutes, and then the cooled first denatured IgG is mixed with basic amino acids for 25-35 minutes; and / or, In step S3, the second denatured IgG and the denaturing agent are mixed at 22–27°C for 15–20 min.

3. The method for preparing denatured IgG for determining rheumatoid factor as described in claim 1, characterized in that, In step S1, the concentration of the IgG is 10 mg / mL; In step S2, the basic amino acids are added to the first denatured IgG at a final concentration of 0.05 to 0.15 M for mixing. In step S3, the denaturing agent is added to the second denatured IgG at a final concentration of 0.9 to 2.1 M for mixing.

4. The method for preparing denatured IgG for determining rheumatoid factor as described in claim 3, characterized in that, The crosslinking agent includes a first crosslinking agent and a second crosslinking agent, and step S4 includes: S41. The third denatured IgG and the first cross-linking agent are mixed to carry out the first cross-linking reaction to obtain the fourth denatured IgG; S42. The fourth denatured IgG and the second cross-linking agent are mixed to carry out a second cross-linking reaction to obtain the denatured IgG used for the determination of rheumatoid factor.

5. The method for preparing denatured IgG for determining rheumatoid factor as described in claim 4, characterized in that, In step S41, the first cross-linking agent is added to the third denatured IgG at a final concentration of 2-3 mM for mixing; in step S42, the second cross-linking agent is added to the fourth denatured IgG at a final concentration of 0.45-1.5 mM for mixing; and / or, The pH value of the first crosslinking reaction is 6.4–6.6; and / or, The temperature of the first crosslinking reaction is 22–27°C; and / or, The first crosslinking reaction takes 50–70 min; and / or, The pH value of the second crosslinking reaction is 7.5–8.5; and / or, The temperature of the second crosslinking reaction is 22–27°C; and / or, The second cross-linking reaction takes 25 to 35 minutes.

6. A denatured IgG for determining rheumatoid factor, characterized in that, The denatured IgG for determining rheumatoid factor is prepared according to any one of claims 1 to 5 by the method for preparing denatured IgG for determining rheumatoid factor.

7. A detection reagent, characterized in that, For the determination of rheumatoid factor, the detection reagent comprises microspheres conjugated with rheumatoid factor antigen, wherein the rheumatoid factor antigen in the microspheres conjugated with rheumatoid factor antigen comprises the denatured IgG for the determination of rheumatoid factor as described in claim 6.

8. The detection reagent as described in claim 7, characterized in that, The method for preparing the microspheres coupled with rheumatoid factor antigen includes the following steps: An activation buffer, activator, carboxyl microspheres, protectant, and denatured IgG for determining rheumatoid factor as described in claim 6 are provided. The carboxyl microspheres, activator, and activation buffer are mixed and activated to obtain an activated carboxyl microsphere suspension. The activated carboxyl microsphere suspension was mixed with denatured IgG for the determination of rheumatoid factor and a coupling reaction was carried out. After the reaction was completed, a protective agent was added to obtain the microspheres coupled with rheumatoid factor antigen.

9. The detection reagent as described in claim 8, characterized in that, The activation buffer comprises 50 mM 2-morpholine ethanesulfonate buffer, and the activator comprises a mixed solution of 0.4 mg / mL sulfonated N-hydroxysuccinimide and 0.1 mg / mL carbodiimide; the activated carboxyl microsphere suspension contains 0.1% by mass; and / or, The concentration of the denatured IgG used for determining rheumatoid factor is 10 mg / mL, and the volume ratio of the denatured IgG used for determining rheumatoid factor to the activated carboxyl microsphere suspension is (3-20):(980-997); and / or, The protective agent comprises histidine at a final concentration of 5–10 mM.

10. A reagent kit, characterized in that, For the determination of rheumatoid factor, the kit includes the detection reagent as described in any one of claims 7 to 9.

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