High-stability immunodetection reagent and kit

Abstract

The invention relates to the field of in-vitro diagnostic detection reagents, in particular to a high-stability immunoreagent and a kit, a specific protective agent combination is added into a diagnostic reagent or / and a calibrator to stabilize interchain disulfide bonds of multi-subunit antigen protein and antibody molecules and improve the stability of the multi-subunit antigen protein and antibody molecules, so that the reactivity of the reagent within the validity period is ensured, and the detection accuracy is improved. Meanwhile, the detection accuracy and precision can be improved.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents, specifically to a highly stable immunoassay reagent and kit. Background Technology

[0002] Immunoassay, based on the specific binding reaction between antigens and antibodies, is a widely used method for detecting analytes in in vitro samples. Depending on the detection signal, it is divided into classic immunoturbidimetric assays and emerging chemiluminescence assays. The principle of immunoturbidimetry is that when antigens and antibodies meet in solution, if the ratio is appropriate, they will form insoluble antigen-antibody complexes. The size of the complex particles affects the intensity of scattered light, leading to a change in turbidity in the solution. The magnitude of turbidity is related to the concentration of the analyte. By measuring the turbidity of the reaction solution and comparing it with a calibrator, the content of the analyte in the sample can be calculated. Chemiluminescence assays, on the other hand, involve binding an enzyme-labeled antibody to an antigen, removing the unbound free enzyme-labeled antibody, adding a luminescent substrate, and calculating the content of the analyte in the sample by detecting the luminescence intensity.

[0003] Immunoturbidimetric assay kits typically consist of Reagent 1 (R1), Reagent 2 (R2, containing detection antibody or antigen), and calibrator (containing a reference for the analyte). R1 ​​provides the reaction environment, R2 provides the specific recognition environment, and the calibrator, using the same detection system and methods, ensures consistent results under different conditions. These three components work together to quantitatively detect the analyte in the sample. The stability of the reagents and calibrators is crucial for ensuring the accuracy and repeatability of the test results. However, it has been found that with prolonged storage or opening time, the test results of some kits gradually deviate from the acceptable range. Several factors affect reagent stability, which may stem from inherent defects in the chemical components of the reagents themselves or from the local environment within the reagent compartment after opening. One important factor is the alteration of the molecular structure of the multi-subunit antibody or antigen protein in Reagent R2 or the calibrator, leading to reduced reactivity. This type of problem also exists with the antibody reagent R2 and calibrator in chemiluminescence assay kits.

[0004] The inventors discovered that there are various changes in the antigen protein or antibody molecule used for detection in reagent R2 or calibrator, including oxidation, reduction or other modifications of amino acid residues, which lead to changes in hydrogen bonds, charge interactions and hydrophilic-hydrophobic interactions, and changes in intra-chain or inter-chain linkages, resulting in changes in molecular conformation, thereby affecting the reactivity with antigens or antibodies. Among these changes, changes in inter-chain disulfide bonds are an important factor. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention adds specific protective agents to diagnostic reagents and / or calibrators to stabilize the interchain disulfide bonds of multi-subunit antigen proteins and antibody molecules, thereby improving their stability and ensuring the accuracy and precision of detection within the reagent's shelf life.

[0006] The first aspect of this invention provides an immunoassay reagent. The technical solution is as follows: An immunoassay reagent, the key of which is that it includes: An antibody or antigen protein, wherein the antibody or antigen protein molecule has one or more interchain disulfide bonds; A protective agent combination comprising at least two of the following: a metal ion chelating agent, a small molecule oxidant of NADPH, thioredoxin, and a protein disulfide isomerase.

[0007] In one embodiment, the protective agent combination may be a combination of a metal ion chelating agent and a small molecule oxidant of NADPH, or a combination of a metal ion chelating agent and thioredoxin, or a combination of a metal ion chelating agent and protein disulfide isomerase, or a combination of a small molecule oxidant of NADPH and thioredoxin, or a combination of a small molecule oxidant of NADPH and protein disulfide isomerase, or a combination of thioredoxin and protein disulfide isomerase.

[0008] In one embodiment, the metal ion chelating agent is ethylenediaminetetraacetic acid or its sodium salt, and the small molecule NADPH oxidant is H2O2 or L-cysteine.

[0009] In one embodiment, the content of ethylenediaminetetraacetic acid or its sodium salt in the immunoassay reagent is 0.01-3‰.

[0010] In one embodiment, the hydrogen peroxide content in the immunoassay reagent is 0.01-0.5‰.

[0011] In one embodiment, the L-cysteine ​​content in the immunoassay reagent is 0.5-20‰.

[0012] In one embodiment, the thioredoxin content in the immunoassay reagent is 0.5-5‰.

[0013] In one embodiment, the content of protein disulfide isomerase in the immunoassay reagent is 1-5 KU / L.

[0014] In one embodiment, the antibody or antigen protein is a protein molecule containing multiple subunits; preferably, the subunits of the antibody or antigen protein molecule have interchain disulfide bonds.

[0015] In one embodiment, the antigen protein is selected from complement C1q, complement C3, complement C4, or D-dimer protein.

[0016] In one embodiment, the antibody is selected from IgM or IgA.

[0017] In one embodiment, the molecular weight of the antibody or antigen protein is above 100 kDa.

[0018] In one embodiment, the immunoassay reagent is used for immunoturbidimetry; preferably, it is used for conventional immunoturbidimetry.

[0019] In one embodiment, the immunoassay reagent further contains a coagulant. The coagulant may be a common PEG, i.e., polyethylene glycol.

[0020] In another embodiment, the immunoassay reagent is used in chemiluminescent immunoassay.

[0021] In one embodiment, the antibody or antigen protein molecule is an enzyme-labeled antibody or enzyme-labeled antigen, or an antibody or antigen coated with magnetic beads.

[0022] In one embodiment, the immunoassay reagent further contains a buffer solution; preferably, it also contains a surfactant or preservative.

[0023] A second aspect of the present invention provides a kit using the immunoassay reagents described above. The technical solution is as follows: A kit comprising the immunoassay reagents described above.

[0024] In one embodiment, the diagnostic reagent or calibrator in the kit is an immunoassay reagent as described above, or both the diagnostic reagent and the calibrator in the kit are immunoassay reagents as described above. The composition of the diagnostic reagent and the calibrator differs. The diagnostic reagent is a reagent containing antibodies or antigens that react immunely with the analyte, typically an R2 reagent.

[0025] In one embodiment, the kit is used for immunoturbidimetric detection.

[0026] Optionally, the diagnostic reagent contains a coagulant. The coagulant may be a common PEG, i.e., polyethylene glycol.

[0027] In another embodiment, the kit is used for luminescent immunoassay.

[0028] The diagnostic reagents in the kit are the immunoassay reagents described above, and the antibody or antigen protein molecules are enzyme-labeled antibodies or enzyme-labeled antigens or magnetic bead-coated antibodies or magnetic bead-coated antigens.

[0029] A third aspect of this invention provides the use of a protective agent in the preparation of immunoassay reagents. The technical solution is as follows: The application of a protective agent combination in the preparation of an immunoassay reagent, wherein the protective agent combination comprises at least two of the following: a metal ion chelating agent, a small molecule oxidant of NADPH, a thioredoxin, and a protein disulfide isomerase; the immunoassay reagent contains an antibody or an antigen protein, wherein the antibody or antigen protein molecule has one or more interchain disulfide bonds.

[0030] As described above, the protective agent combination may be a combination of a metal ion chelating agent and a small molecule oxidant of NADPH, or a combination of a metal ion chelating agent and thioredoxin, or a combination of a metal ion chelating agent and protein disulfide isomerase, or a combination of a small molecule oxidant of NADPH and thioredoxin, or a combination of a small molecule oxidant of NADPH and protein disulfide isomerase, or a combination of thioredoxin and protein disulfide isomerase.

[0031] In the applications described above, the metal ion chelating agent is ethylenediaminetetraacetic acid or its sodium salt, and the small molecule NADPH oxidant is H2O2 or L-cysteine.

[0032] In the application described above, the antigen protein is selected from at least one of complement C1q, complement C3, complement C4, and D-dimer protein, and the antibody is selected from IgM or IgA.

[0033] A fourth aspect of this invention provides a method for improving the stability of immunoassay reagents. The technical solution is as follows: A method for improving the stability of immunoassay reagents involves adding the aforementioned combination of protective agents when preparing reagents containing antibodies or antigens for detection.

[0034] The beneficial effects of this invention are as follows: By adding a combination of protective agents to immunoassay reagents containing antigen proteins or antibody molecules with interchain disulfide bonds, the antigen proteins or antibodies can be effectively stabilized, storage stability can be improved, the reliability of immunoassay can be maintained within the shelf life, and the accuracy and precision can be improved to meet the needs of clinical testing. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0037] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0038] In this article, "N or more" means at least N, such as "one or more" which includes one, two, three, etc.

[0039] Unless otherwise specified, the content of a reagent component in this article is in mass percentage or per thousand.

[0040] Disulfide bonds (SS bonds), formed by the oxidation of two cysteine ​​residues, are important covalent bonds in proteins. They maintain the tertiary and higher-order structures of proteins by connecting different regions of the same polypeptide chain or different polypeptide chains, thereby enhancing molecular stability. The more disulfide bonds there are, the stronger the protein's stability against external factors such as temperature, pH, and reducing gases. The presence of disulfide bonds is directly related to the biological activity of proteins. Disulfide bonds are mainly divided into two types: intrachain disulfide bonds and interchain disulfide bonds. Intrachain disulfide bonds connect two cysteine ​​residues within the same polypeptide chain, while interchain disulfide bonds connect cysteine ​​residues between different polypeptide chains. Interchain disulfide bonds can promote the assembly of multi-subunit complexes, and their dynamic characteristics allow for breakage and reformation, affecting the adjustability of protein function. Furthermore, as the number of disulfide bonds increases, the protein molecule's ability to resist external influences increases. The presence of multiple disulfide bonds helps maintain the protein's spatial conformation, thereby enhancing its overall stability. The protective agent of this invention mainly targets interchain disulfide bonds in polyproteins and polyantibodies, exhibiting a significant protective effect.

[0041] For proteins with large molecular weights and complex structures, the more disulfide bonds they contain, the more stable their structure, and the more effectively they can perform their immune function. The essential reason for disulfide bond reduction involves the redox reaction of the thioredoxin system. This system is composed of thioredoxin (Trx), thioredoxin reductase (TrxR), and reduced coenzyme II (NADPH). The thioredoxin system is a known enzyme system that promotes disulfide bond reduction and maintains redox balance. Therefore, all factors related to the catalytic reaction of the thioredoxin system will affect disulfide bond reduction. However, for in vitro detection reagents, firstly, the addition of a protective agent should not affect the normal progress of the immunoassay reaction or the signal detection while providing protection; secondly, the protective agent should be universal, and also as simple and readily available as possible.

[0042] This invention significantly improves reagent stability by adding any two or more of the following types of protective agents.

[0043] (1) Inhibition of enzymes involved in reduction reactions: Metal ion chelating agents can inhibit enzyme-catalyzed redox reactions. Commonly used metal ion chelating agents can be selected, such as ethylenediaminetetraacetic acid (EDTA) or its sodium salt.

[0044] (2) Competitive consumption of enzymes involved in the reduction reaction: Small molecule oxidants react with NADPH in the disulfide bond reduction reaction, competitively consuming the enzyme and thus preventing it from reducing the disulfide bond. Small molecule oxidants refer to oxidizing substances with a molecular weight not exceeding 1000 Da. In this invention, hydrogen peroxide or L-cysteine ​​are preferred.

[0045] (3) Thioredoxin (Trx): a protein protectant that can reduce disulfide bonds through thiodisulfide bond exchange, improve protein solubility, and can act as a fusion tag to assist folding, thereby indirectly protecting disulfide bonds.

[0046] (4) Protein disulfide isomerase (PDI): It can catalyze the correct formation of disulfide bonds and has a variety of activities, including disulfide bond isomerization, reduction and oxidation. It plays an important role in the protein folding process and helps to protect the correct conformation and stability of disulfide bonds.

[0047] Among them, the protective agents of types (1) and (2) mainly play the role of inhibiting the reduction of disulfide bonds, while those of types (3) and (4) play a protective role from the perspective of the reduction and recovery after the disulfide bond is broken. The recovery of disulfide bonds involves the thiol-disulfide bond exchange reaction between the antibody and the redox agent, which includes two steps: First, the nucleophilic thioester group (S-) formed by the deprotonation of the free thiol group attacks a sulfur atom of the redox agent, and a disulfide bond is formed between the redox agent and the antibody; Second, other thiol groups in the antibody attack the newly formed disulfide bond, release the redox agent and rearrange the disulfide bond.

[0048] Based on this, the present invention provides a highly stable immunoassay reagent, comprising: An antibody or antigen protein having one or more interchain disulfide bonds in its molecule; The protective agent combination includes at least two of the following: metal ion chelators, small molecule oxidants of NADPH, thioredoxins, and protein disulfide isomerases.

[0049] For example, the protective agent combination can be a combination of a metal ion chelating agent and a small molecule oxidant of NADPH, or a combination of a metal ion chelating agent and thioredoxin, or a combination of a metal ion chelating agent and protein disulfide isomerase, or a combination of a small molecule oxidant of NADPH and thioredoxin, or a combination of thioredoxin and protein disulfide isomerase, or a combination of three or four of these combinations. However, if two combinations can meet the requirements, then there is no need for more combinations.

[0050] In one embodiment, the metal ion chelating agent is ethylenediaminetetraacetic acid or its sodium salt, and the small molecule NADPH oxidant is H2O2 or L-cysteine.

[0051] Generally, the dosage of various protective agents in immunoassay reagents is as follows: ethylenediaminetetraacetic acid or its sodium salt content is 0.01-3‰, hydrogen peroxide content is 0.01-0.5‰, L-cysteine ​​content is 0.5-20‰, thioredoxin content is 0.5-5‰, and protein disulfide isomerase content is 1-5 KU / L. Adjustments should be made according to actual needs when using them in combination.

[0052] The protective agent of the present invention is particularly useful in immunoassay reagents where the antibody or antigen protein is a protein molecule containing multiple subunits, especially where there are interchain disulfide bonds between the subunits. For example, common examples include: the antigen protein may be complement C1q, complement C3, complement C4, or D-dimer protein, and the antibody may be IgM or IgA.

[0053] In one embodiment, the immunoassay reagent is used in immunoturbidimetry, particularly in conventional immunoturbidimetry. Immunoturbidimetry is divided into conventional immunoturbidimetry and latex microsphere-enhanced immunoturbidimetry. The difference lies in that the former does not contain latex microspheres in its detection system, meaning the detection antibody or antigen is not linked to latex microspheres, while the latter's detection system contains latex microspheres coupled to the detection antibody or antigen. If the molecular weight of the antibody or antigen protein is above 100 kDa, its structure is generally relatively complex, containing multiple subunits and interchain disulfide bonds. The more disulfide bonds, the more stable the overall molecular structure. Adding the protective agent combination of the present invention can effectively maintain its stability.

[0054] For immunoassay reagents based on conventional immunoturbidimetry, coagulants such as PEG (polyethylene glycol) can be added.

[0055] In another embodiment, the immunoassay reagent is used in a luminescent immunoassay, such as a magnetic microparticle chemiluminescence assay. In this case, the antibody or antigen protein molecule is an enzyme-labeled antibody or enzyme-labeled antigen, or an antibody or antigen coated with magnetic beads.

[0056] Whether using immunoturbidimetric or chemiluminescent immunoassay, immunoassay reagents may contain buffer solutions, surfactants, or preservatives. However, it is important to note that the added protective agents should not react with other components in the reagent besides the antigen protein or antibody under room temperature conditions. For example, sodium azide, used as a preservative, should not be added to the reagent simultaneously with the protective agent H₂O₂.

[0057] Reagent kits, such as immunoturbidimetric kits or magnetic microparticle chemiluminescence assay kits, can be prepared using the above-mentioned immunoassay reagents. Magnetic microparticle chemiluminescence assay kits generally include reagent R1 and reagent R2, optionally as calibrators. R1 contains magnetic bead-coated antibodies or magnetic bead-coated antigens capable of capturing the analyte, forming a magnetic bead-antigen-antibody complex after mixing with the sample; R2 contains enzyme-labeled antibodies or enzyme-labeled antigens capable of binding to the magnetic bead-antigen-antibody complex.

[0058] In one embodiment, the R2 reagent or calibrator in the kit is an immunoassay reagent as described above, or both the R2 reagent and the calibrator in the kit are immunoassay reagents as described above, but the composition of the R2 reagent and the calibrator differs. The R2 reagent contains an antibody or antigen protein capable of immunobinding to the analyte.

[0059] The immunoturbidimetric assay is used as an example. An immunoturbidimetric assay kit includes R1, which provides the reaction environment; R2, which contains a protective agent; and a calibrator, which also contains a protective agent. The reagent formulation is shown in Table 1, wherein either or both of reagent R2 and the calibrator contain a protective agent combination.

[0060] Table 1. Composition of a detection kit (immunoturbidimetric assay)

[0061] The reagent kit is prepared as follows: Preparation of reagent R1: Weigh an appropriate amount of ultrapure water, add the buffer substance, and stir until completely dissolved. After all substances have been dissolved, adjust the pH to the desired level.

[0062] Preparation of reagent R2: Weigh an appropriate amount of ultrapure water, add the buffer substance, and stir until completely dissolved. After all substances have been dissolved, adjust the pH to the desired level.

[0063] Preparation of calibrators: Weigh an appropriate amount of ultrapure water, add a buffer solution, and stir until completely dissolved. After all substances have been dissolved, adjust the pH to the desired level and then freeze-dry.

[0064] The present invention will be further described with reference to specific embodiments.

[0065] (a) Complement C1q detection Example 1: Complement C1q Detection Kit (Immunoturbidimetric Assay) A complement C1q detection kit, the calibrator contains a protective agent, and the reagent formulation is shown in Table 2.

[0066] Table 2 Composition of the complement C1q detection kit

[0067] Compare with Example 1 The calibrator consists of 100 mM MOPS, 10‰ sodium chloride, 1‰ PC300, 2% sucrose, 8% mannitol, and complement C1q protein, and does not contain disulfide bond protectants. Reagents R1 and R2 are the same as in Example 1.

[0068] Compare with Example 2 The calibrator consisted of 100 mM MOPS, 10‰ sodium chloride, 1‰ PC300, 2% sucrose, 8% mannitol, complement C1q protein, 2‰ EDTA, and 30 μM potassium ferricyanide. Reagents R1 and R2 were the same as in Example 1.

[0069] Detection method The kit uses immunoturbidimetry to determine the content of biomarkers in the human body. The biomarker in the sample binds to the polyclonal antibody in the reagent, causing adjacent antibodies to cross-link and resulting in an agglutination reaction that produces a change in turbidity. This turbidity is directly proportional to the biomarker content in the sample. The biomarker content in the sample can be calculated by measuring the absorbance at a specific wavelength, and the biomarker content in the sample can be detected based on the working curve.

[0070] The samples were tested using standard methods on a fully automated biochemical analyzer (Chemistry Analyzer AU5800, Beckman Coulter). The complement C1q parameters were set as follows: Method: Endpoint method; Reaction direction: upward; Sample: Reagent R1: Reagent R2: 4:240:60; Wavelength: 340 nm; Subwavelength: 700nm; Reaction temperature: 37℃; Reaction time: 10 min; Freshly prepared calibrators were used as samples for calibration, while other test samples were derived from clinical sources. The sample processing procedure was as follows: R1 was mixed with the sample, incubated at 37°C for 3-5 minutes, and the absorbance A1 was measured. Then, R2 was added, mixed, incubated at 37°C for 5 minutes, and the absorbance A2 was measured. The result was ΔA = A2 - A1. A multi-point nonlinear / semi-logarithmic calibration mode was used, with spline functions as the calculation method. A dose / response curve was plotted based on the calibrator values ​​and absorbance changes. The content of the target analyte in the sample could be calculated from its absorbance change on the dose / response curve.

[0071] Calibration response As shown in Table 3, the reactivity of each group, from highest to lowest, is Example 1, Control Example 1, and Control Example 2, all of which meet the detection requirement of S2 test value > 0.01. However, a comparison revealed that Control Example 2, despite the addition of EDTA and potassium ferricyanide as protective agents, showed a lower reactivity than Control Example 1, which did not contain any protective agents. Although potassium ferricyanide is a commonly used oxidizing additive in detection reagents, capable of consuming reductases such as NAPDH and inhibiting the reduction of disulfide bonds in protein molecules, its combination with EDTA did not improve the test results compared to the blank Control Example 1. Previous research by the inventors found that adding a single protective agent, EDTA, did not significantly improve the test results. In Example 1, the addition of a protective agent combination of 2‰ EDTA and 10‰ L-cysteine ​​significantly improved the reactivity compared to Control Example 1.

[0072] Table 3 Calibration test results for different calibrators

[0073] Accuracy As shown in Table 4, the experiment found that the accuracy of Control Example 1 and Example 1 was comparable and better than that of Control Example 2. However, the detection accuracy of Control Example 2 did not meet the requirement of relative deviation of accuracy <5%.

[0074] Table 4 Comparison of detection accuracy of different calibration groups

[0075] Precision Ten tests were performed at each concentration level for different calibrator groups, and the mean, standard deviation (SD), and systematic deviation (CV) were calculated. As shown in Table 5, the experiment revealed that after adding the protective agent, the detection precision of Example 1, especially the low-to-median precision, was significantly improved compared to Control Example 1. Both groups met the requirement of CV < 5% for precision at different levels. Control Example 2 did not meet this requirement for precision in low-value tests.

[0076] Table 5 Comparison of detection precision of different calibration groups

[0077] 37°c accelerated heat stability As shown in Table 6, after the calibrators of Example 1 were treated at 37°C, the changes in reagent reactivity were within 5% for 3 days and 7 days. However, the reagent reactivity of both the control group without protective agent (Example 1) and the control group with added EDTA and potassium ferricyanide (Example 2) decreased significantly, failing to meet the requirement of accelerated deviation <10% after 7 days.

[0078] Table 6. Comparison of accelerated thermal stability at 37℃ for different calibration groups

[0079] 2-8°c storage stability As shown in Tables 7-9, the test values ​​of the kit in Example 1 remained within 10% after storage for up to 14 months, indicating that the addition of the protective agent combination maintained the stability of the antibody protein while the specific reaction of the antigen and antibody was not affected, and the stability of the reagent was significantly improved during storage. However, the test values ​​of both the control kits without protective agents (Example 1) and the control kits with EDTA and potassium ferricyanide (Example 2) decreased significantly with prolonged storage time, failing to meet the requirement of a test value deviation of <10% after 12 months of storage at 2-8℃.

[0080] Table 7. Storage stability of the kit for Control Example 1 at 2-8°C

[0081] Table 8. Storage stability of the kit for Control Example 2 at 2-8°C Table 9. Storage stability of the kit in Example 1 at 2-8°C

[0082] Clinical sample measurement study Based on the above data, it is evident that the combination of EDTA and L-cysteine ​​as protective agents significantly improves the stability of calibrators. To investigate whether the addition of protective agents affects clinical measurements, 20 randomized, fresh, freeze-thaw-free clinical samples were selected for comparison. The results are shown in Table 10. The clinical measurements of Example 1 and the control Example 1 without protective agents showed high consistency, indicating that the addition of protective agents improves the stability of calibrators without affecting clinical testing.

[0083] Table 10 Comparison of the effects of adding and not adding protective agents on clinical measurements

[0084] (ii) IgM detection Immunoglobulin M (IgM) is an important antibody in the human immune system, primarily produced during the primary immune response. Detecting serum IgM levels allows for the assessment of an individual's immune status and function. IgM is the largest immunoglobulin in the blood, secreted by plasma cells in the spleen and lymph nodes. It consists of five Y-type monomers linked by J chains and disulfide bonds to form a cyclic pentamer, with a total molecular weight of approximately 970 kDa. Each monomer contains one μ heavy chain and one light chain. Disulfide bonds play a crucial role in the structure of IgM.

[0085] The antibodies used in IgM detection kits mainly include IgM and IgG types. IgM antibodies consist of two identical heavy chains (H chains) and two identical light chains (L chains). The variable region of the antibody is responsible for specifically binding to IgM antigen epitopes. Cysteine ​​is an easily reduced amino acid in the variable region of IgM antibodies and mainly participates in the formation of disulfide bonds. IgM monomers are linked by disulfide bonds formed by the penultimate cysteine ​​residue of the C-terminal tail peptide of the heavy chain, promoting their polymerization into pentamers. In the heavy chain region, the disulfide bonds between Cys414 residues further stabilize the cyclic structure of IgM. However, during reagent storage, the disulfide bonds in the CDR region are susceptible to the effects of reducing gases in the air or reagent chamber (hydrogen sulfide (H2S), carbon monoxide (CO), ammonia (NH3), methane (CH4), alcohols (such as ethanol), leading to changes or disintegration of the protein's three-dimensional structure, thereby disrupting the spatial conformation of the CDR region. Once the conformation of the CDR region is disrupted, the antibody's ability to bind to the antigen will significantly decrease or even be completely lost, leading to the depolymerization of the IgM antibody structure and a decrease in titer.

[0086] Therefore, both antibody reagents and calibrators in IgM detection kits face the challenge of maintaining stability.

[0087] Example 2: IgM Detection Kit (Immunoturbidimetric Assay) --- Adding Protectant to Calibrator An IgM detection kit, the calibrator contains a protective agent, and the reagent formulation is shown in Table 11 below.

[0088] Table 11 Composition of the IgM detection kit in Example 2

[0089] Compare with Example 3 The calibrator consists of 100 mM Tris, 8‰ sodium chloride, 1.2‰ PC-300, 3% sucrose, 10% mannitol, and IgM protein, and does not contain disulfide bond protectants. Reagents R1 and R2 are the same as in Example 2.

[0090] Compare with Example 4 The calibrator consisted of Tris 100 mM, sodium chloride 8‰, PC-300 1.2‰, sucrose 3%, mannitol 10%, and IgM protein, along with EDTA 1.5‰ and potassium permanganate 10 mM as a protective agent combination. Reagents R1 and R2 were the same as in Example 2.

[0091] Detection method The samples were tested using conventional methods on a fully automated biochemical analyzer (Chemistry Analyzer AU5800, Beckman Coulter).

[0092] Immunoglobulin M parameter settings: Method: Endpoint method Reaction direction: upward Sample:Reagent R1:Reagent R2: 3:250:50 Wavelength: 340 nm Subwavelength: 700nm Reaction temperature: 37℃ Reaction time: 10 min The sample processing procedure is as follows: R1 is mixed with the sample, kept at 37℃ for 5 minutes, and then the absorbance A1 is measured. The mixture is then mixed again, kept at 37℃ for 5 minutes, and then the absorbance A2 is measured. The result is ΔA = A2 - A1. A multi-point nonlinear / semi-logarithmic calibration mode is used, with a spline function as the calculation model. A dose / response curve is plotted based on the calibrator values ​​and absorbance changes. The content of the target analyte in the sample can be calculated from its absorbance change value on the dose / response curve.

[0093] Its accelerated thermal stability and low-temperature storage stability were tested using test calibrators, and the effect of adding protective agents on clinical measurements was confirmed by testing clinical samples.

[0094] 37°c accelerated stability As shown in Table 12, in Example 2 with added protective agent, the reagent reactivity of the calibrator after treatment at 37°C for 3 and 7 days was within 5%. However, the reagent reactivity of the two groups, Control Example 3 without added protective agent and Control Example 4 with added EDTA and potassium permanganate, decreased significantly and did not meet the requirement of accelerated deviation <10% after 7 days.

[0095] Table 12 Comparison of accelerated thermal stability at 37℃ for experimental groups with different calibrators

[0096] 2-8°c storage stability As shown in Tables 13-15, the test values ​​of the kit in Example 2 remained within 10% after storage for up to 14 months, indicating that the addition of the protective agent combination maintained the stability of the reagent during storage. However, the test values ​​of both the control kits (without protective agents, Example 3) and the control kits (with EDTA and potassium permanganate, Example 4) decreased significantly with prolonged storage time, failing to meet the requirement of a test value deviation of <10% after 12 months of storage at 2-8℃.

[0097] Table 13. Storage stability of the kit for Control Example 3 at 2-8°C

[0098] Table 14. Storage stability of the kit for Control Example 4 at 2-8°C

[0099] Table 15. Storage stability of the kit for Example 2 at 2-8°C Clinical sample measurement study Based on the above data, it is evident that the combination of EDTA and L-cysteine ​​as protective agents significantly improves the stability of the calibrators. To investigate whether the addition of protective agents affects clinical measurements, 20 random clinical samples were compared, and the results are shown in Table 16. The clinical measurements of Example 2 and the control Example 3 (without protective agents) showed high consistency, indicating that the addition of protective agents improves the stability of the calibrators without affecting clinical testing.

[0100] Table 16 Comparison of the effects of adding and not adding protective agents on clinical measurements in calibrators.

[0101] (III) IgM detection: The effect of adding a protective agent to reagent R2 Examples 3-4: IgM Detection Kit (Immunoturbidimetric Assay) The IgM detection kit in Example 3 uses the same reagents R1 and calibrators as in Example 2; the difference is that a protective agent is added to reagent R2. Reagent R2 consists of: 100 mM MOPS, 8‰ sodium chloride, 1‰ PC300, 1.5% sucrose, 6% mannitol, antibody, and a protective agent combination of 2.5‰ EDTA and 0.2‰ hydrogen peroxide.

[0102] The kit for Example 4 is basically the same as that for Example 3, except that the protective agent combination in reagent R2 is: 10‰ L-cysteine ​​and 3KU / L protein disulfide isomerase antibody.

[0103] Compare with Example 5 The kit used in Control Example 5 is the same as that used in Example 2.

[0104] The kits for Examples 3, 4, and Control Example 5 were subjected to an accelerated thermal stability test at 37°C, and the differences in clinical sample measurements were compared. The detection method was the same as in Part (II).

[0105] 37°c accelerated stability As shown in Table 17, after treatment at 37°C for 3 and 7 days, the reactivity of the reagent kits in Examples 3 and 4 remained within 10%. Examples 3 and 4 showed comparable reactivity, both superior to Control Example 5. The reactivity of Control Example 5 did not meet the requirement of a deviation <10% after 7 days of accelerated treatment. This indicates that the addition of a protective agent to reagent R2 effectively improved thermal stability.

[0106] Table 17 Comparison of accelerated thermal stability at 37℃ for experimental groups with different R2 reagents

[0107] Clinical sample measurement study Based on the above data, the addition of the protective agent combination significantly affects the stability of reagent R2. To investigate whether the addition of the protective agent affects clinical measurements, 20 random fresh clinical samples were compared, and the results are shown in Table 18. It can be seen that the clinical measurements of the kits with and without the protective agent combination are highly consistent. Combined with the results of the accelerated thermal stability test, it can be concluded that the addition of the protective agent improves the stability of reagent R2 without affecting clinical testing.

[0108] Table 18 Comparison of the effects of adding and not adding a protective agent to reagent R2 on clinical measurements.

[0109] (iv) D-dimer detection Example 5: D-dimer Detection Kit (Magnetic Particle Chemiluminescence Method) --- Addition of Protective Agent to Calibrator A D-dimer detection kit, reagents R1 and R2 are the same as those in commercially available kits (Zhongyuan Huiji Biotechnology Co., Ltd.), and the calibrator contains a protective agent. The formula is as follows: HEPES 150mM, sodium chloride 10‰, PC300 1‰, sucrose 2%, mannitol 5%, disulfide bond protectant—1‰ thioredoxin (Trx) + 2KU / L protein disulfide isomerase (PDI) and D-dimer natural protein.

[0110] Compare with Example 6 The calibrator consists of 150 mM HEPES, 10‰ sodium chloride, 1‰ PC300, 2% sucrose, 5% mannitol, and D-dimer natural protein, without disulfide bond protectants. Reagents R1 and R2 are the same as in Example 5.

[0111] Compare with Example 7 The calibrator consists of 150 mM HEPES, 10‰ sodium chloride, 1‰ PC300, 2% sucrose, 5% mannitol, 1‰ thioredoxin (Trx), and D-dimer native protein. Reagents R1 and R2 are the same as in Example 5.

[0112] Compare with Example 8 The calibrator consists of 150 mM HEPES, 10‰ sodium chloride, 1‰ PC300, 2% sucrose, 5% mannitol, 2 KU / L protein disulfide isomerase (PDI), and D-dimer native protein. Reagents R1 and R2 are the same as in Example 5.

[0113] Detection method The samples were tested using conventional methods on the Zhongyuan Huiji EXI1800 fully automated chemiluminescence immunoassay analyzer.

[0114] 1. Place the refrigerated reagents directly into the reagent tray of the analyzer to avoid the formation of air bubbles.

[0115] 2. The analyzer automatically remixes the magnetic particles before use.

[0116] 3. The analyzer reads specific parameters by scanning the reagent barcode.

[0117] 4. How to use calibrators: 4.1 Before use, slowly mix the calibrators and quality control samples to avoid the formation of air bubbles; 4.2 Hold the reagent bottle vertically and add the recommended amount to the corresponding sample cup: Calibration material for each level: minimum 6 drops; calibration: 1. The master calibration curve card for each kit contains specific calibration information for each batch of reagents.

[0118] 2. The analyzer scans the reagents and the main calibration curve card, reads the main calibration curve information, and calibrates the main calibration curve by measuring the calibrator.

[0119] Sample testing: 1. The D-dimer in the sample reacts with biotin-labeled D-Dimer specific antibody and alkaline phosphatase-labeled D-Dimer antibody to form immune complexes. Streptavidin-coated magnetic microparticles bind the immune complexes to the surface of the magnetic microparticles. 2. Unreacted and other substances are removed by washing the magnetic microparticles. The substrate solution for the automated immunoassay system is added, and an enzymatic reaction with alkaline phosphatase occurs to generate photons. The relative luminescence intensity (RLU) is measured. 3. The final test results are obtained by calculating the calibration curve of the analyzer.

[0120] The effects of adding a protective agent on clinical measurements were confirmed by testing the accelerated thermal stability and low-temperature storage stability of the calibrators, as well as by testing clinical samples.

[0121] 37°c accelerated stability In Example 5, the calibrator with added protective agent showed a reaction rate change of less than 5% after 3 and 7 days of treatment at 37°C. In contrast, the reaction rates of the calibrators in Control Examples 6-8 all decreased to varying degrees. In Control Examples 6 and 7, the reaction rate deviation no longer met the requirement of <10% after 7 days of accelerated reaction. Although Control Example 8 met this requirement, the deviation was significantly larger than that of Example 5.

[0122] Table 19 Comparison of accelerated thermal stability at 37℃ for experimental groups with different calibrators

[0123] 2-8°c storage stability As shown in Tables 20-23, the calibrators of Example 5 maintained a measurement deviation of less than 10% for up to 14 months of storage, indicating that the addition of the protective agent combination maintained the stability of the reagents during storage. However, the calibrators of Control Example 6 without the addition of a protective agent, and Control Examples 7 and 8 with the addition of one protective agent, showed a significant decrease in measurement values ​​as the storage time increased, failing to meet the requirement of a measurement deviation of <10% after 12 months of storage at 2-8℃.

[0124] Table 20. Storage stability of the kit for Control Example 6 at 2-8°C

[0125] Table 21. Storage stability of the kit for Control Example 7 at 2-8°C

[0126] Table 22. Storage stability of the kit for control example 8 at 2-8°C

[0127] Table 23. Storage stability of the kit in Example 5 at 2-8°C Clinical sample measurement study Based on the above data, it is evident that the combination of thioredoxin (Trx) and protein disulfide isomerase as protective agents significantly improves the stability of the calibrators. To investigate whether the addition of protective agents affects clinical measurements, 20 randomized clinical samples were compared, and the results are shown in Table 24. The clinical measurements of Example 5 and the control Example 6 (without protective agents) showed high consistency, indicating that the addition of protective agents improves the stability of the calibrators without affecting clinical testing.

[0128] Table 24 Comparison of the effects of adding and not adding protective agents on clinical measurements in calibrators

[0129] Based on the above research, adding a protective agent combination to detection reagents containing antigen proteins / antibodies or to analyte calibrators can improve the accelerated thermal stability and low-temperature storage stability of the reagents, keeping the measurement deviation of the kit within the specified range during its shelf life without affecting clinical measurements. Furthermore, the protective agent combination of the present invention can also improve reagent reactivity and enhance detection precision. The protective agent combination of the present invention is based on its protective effect on protein molecules with interchain disulfide bonds, and is universally applicable to detection antigen reagents, detection antibody reagents, and calibrators containing such protein molecules.

[0130] All data, reagents, and procedures described herein should be understood as illustrative rather than restrictive. Although the invention has been described in conjunction with the specific embodiments described above, many modifications and other variations will be apparent to those skilled in the art. All such modifications and other variations also fall within the scope of the invention.

Claims

1. An immunoassay reagent, characterized in that, include: An antibody or antigen protein, wherein the antibody or antigen protein molecule has one or more interchain disulfide bonds; A protective agent combination comprising at least two of the following: a metal ion chelating agent, a small molecule oxidant of NADPH, thioredoxin, and a protein disulfide isomerase.

2. The immunoassay reagent as described in claim 1, characterized in that: The protective agent combination may be a combination of a metal ion chelating agent and a small molecule oxidant of NADPH, or a combination of a metal ion chelating agent and thioredoxin, or a combination of a metal ion chelating agent and protein disulfide isomerase, or a combination of a small molecule oxidant of NADPH and thioredoxin, or a combination of a small molecule oxidant of NADPH and protein disulfide isomerase, or a combination of thioredoxin and protein disulfide isomerase.

3. The immunoassay reagent as described in claim 1 or 2, characterized in that: The metal ion chelating agent is ethylenediaminetetraacetic acid or its sodium salt, and the small molecule NADPH oxidant is H2O2 or L-cysteine.

4. The immunoassay reagent as described in claim 3, characterized in that: The immunoassay reagent contains 0.01-3‰ ethylenediaminetetraacetic acid or its sodium salt; and / or, The hydrogen peroxide content in the immunoassay reagent is 0.01-0.5‰; and / or, The L-cysteine ​​content in the immunoassay reagent is 0.5-20‰; and / or, The immunoassay reagent contains 0.5-5‰ thioredoxin; and / or, The content of protein disulfide isomerase in the immunoassay reagent is 1-5 KU / L.

5. The immunoassay reagent as described in claim 1 or 2, characterized in that: The antibody or antigen protein is a protein molecule containing multiple subunits; preferably, the subunits of the antibody or antigen protein molecule have interchain disulfide bonds.

6. The immunoassay reagent as described in claim 1 or 2, characterized in that: The immunoassay reagent is used for immunoturbidimetry; preferably, it is used for conventional immunoturbidimetry.

7. The immunoassay reagent as described in claim 1 or 2, characterized in that: The immunoassay reagent is used in chemiluminescent immunoassay; preferably, the antibody or antigen protein molecule is an enzyme-labeled antibody or enzyme-labeled antigen, or an antibody or antigen coated with magnetic beads.

8. A kit comprising the immunoassay reagent as described in any one of claims 1 to 7.

9. The application of a protective agent combination in the preparation of an immunoassay reagent, wherein the protective agent combination comprises at least two of a metal ion chelating agent, a small molecule oxidant of NADPH, a thioredoxin, and a protein disulfide isomerase; wherein the immunoassay reagent contains an antibody or an antigen protein, wherein the antibody or antigen protein molecule has one or more interchain disulfide bonds.

10. The application as described in claim 9, wherein the protective agent combination is a combination of a metal ion chelating agent and a small molecule oxidant of NADPH, or a combination of a metal ion chelating agent and thioredoxin, or a combination of a metal ion chelating agent and protein disulfide isomerase, or a combination of a small molecule oxidant of NADPH and thioredoxin, or a combination of a small molecule oxidant of NADPH and protein disulfide isomerase, or a combination of thioredoxin and protein disulfide isomerase.

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