High stability calibrators, antigen detection kit

Abstract

This invention discloses a highly stable calibrator and an antigen detection kit, relating to the field of in vitro diagnostic reagent technology. The highly stable calibrator of this invention is prepared by adding a free antibody with antigen specificity to the antigen, mixing and reacting at room temperature for 2-5 hours to form an immune complex with the antigen, and then adding calibrator buffer to obtain a highly stable calibrator product. This invention, by adding a free antibody with overlapping antigen binding sites but weaker affinity to the immunoassay system, preserves the antigen in the calibrator as a more stable immune complex. The calibrator provided by this invention effectively protects the antigen structure without affecting the quantitative detection of the antigen by the kit, significantly extending the antigen inactivation time and solving the problem of poor antigen stability in the medical testing field.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, and in particular to a highly stable calibrator and antigen detection kit. Background Technology

[0002] In the field of clinical medical testing, reagent stability is a crucial parameter for the quality of in vitro diagnostic reagents, especially chemiluminescence reagents. The stability of chemiluminescence reagents typically includes the stability of the reagent itself, as well as the stability of calibrators and quality control materials. Reagent stability requires accurate measurements after calibration with reliable calibrators. When calibrators exhibit poor stability, especially for some diagnostic gold standards such as high-sensitivity troponin I, it can directly affect the objectivity and accuracy of test results, leading clinicians to make incorrect diagnoses or treatments based on inaccurate test results.

[0003] Factors affecting protein stability can be roughly divided into two parts: (1) the stability of the protein itself, which is determined by the molecular structure of the protein itself, including its physical stability (stability directly related to structure and conformation) and chemical stability (whether it is prone to chemical reactions such as deamidation, oxidation, isomerization, and hydrolysis). These factors may lead to protein inactivation, aggregation, etc., making it unusable for clinical use (Trends in Biotechnology, 2014, 32(7), 372-380); (2) the storage environment of the protein. Most protein reagents are stored in the liquid phase in a buffer solution with water as the solvent to maintain the stability of antibodies (reagents) and antigens (calibrators, quality control products) in the solution. The purpose is to maintain the structural integrity and activity of the protein during storage. In other words, the stability of a protein is not only related to its own structure, but also to the storage environment (pH, temperature, salt ion concentration, surfactant, etc.). The interaction between the protein and the solute and solvent in the solution can also affect the protein structure. The stability of the same protein in different buffer solutions may vary greatly (Proteinstability and storage, ThermoSCIENTIFIC). Analyzing the factors that affect protein stability and regulating the protein storage environment are of great significance for improving protein stability.

[0004] In the field of in vitro diagnostics, antigens in test kits often need to be diluted to very low concentrations, which can affect the stability of many antigens. Single-component buffers are often insufficient to maintain the stability of low-concentration antigens. Typically, optimization of calibrator stability focuses on optimizing the buffer solution, including the matrix (serum, diluent), buffer system (MES, PB, Tris, etc.), solution pH, ionic strength, metal ion concentration, protease inhibitors, and protective proteins (serum albumin, casein, collagen peptides, etc.). The goal is to identify key factors that improve stability and determine the optimal combination concentration (US8071723B2, Stable D-Dimer Liquid Preparation). For proteins with good inherent stability, the optimized addition of these components can be highly beneficial. However, for proteins with poor inherent stability, such optimization efforts are often ineffective, yielding minimal results despite significant time, manpower, and resources invested, and failing to effectively improve protein structural stability. For this type of protein, there is also a lyophilized calibrator production process on the market. However, the existing lyophilization preparation process involves large investment, complex process, many control points, and extremely high requirements for time and personnel. Although it has greatly extended the product shelf life and facilitated storage and transportation, there are still certain risks to its stability after reconstitution and reproducibility in clinical use. It has not fundamentally solved the problem of poor antigen stability. Summary of the Invention

[0005] The technical problem to be solved by this invention is that existing calibrators have poor stability, complex production processes, and high production costs.

[0006] To address the above problems, the present invention proposes the following technical solution:

[0007] A highly stable calibrator includes an antigen, an antigen-specific free antibody, and a calibrator buffer, wherein the free antibody binds to the antigen to form an immune complex.

[0008] Furthermore, the concentration of the free antibody is 2-4 times that of the antigen.

[0009] The present invention provides a highly stable calibrator, which, by adding an excess of free antibody to the calibrator, promotes the equilibrium reaction of antigen-free antibody binding in the calibrator towards the formation of immune complexes, reduces the proportion of antigen without bound free antibody, and thus achieves a better protective effect against antigen.

[0010] Furthermore, the calibration buffer comprises a buffer solution, a surfactant, inorganic salt ions, a stable protein, and a preservative.

[0011] Further, the buffer solution is selected from one of TRIS buffer, phosphate buffer, and MES buffer, with a concentration of 50-100 mmol / L; the surfactant is selected from one or more of Tween 20, Tween 80, Brij 35, Triton 100, and NP40, with a concentration of 0.5-2 g / L.

[0012] The inorganic salt ions are selected from sodium chloride or potassium chloride at a concentration of 50-100 mmol / L; the stable protein is selected from casein or BSA at a concentration of 5-15 g / L; the preservative is selected from NaN3, procline300 or mercuric sulfide at a concentration of 0.4-1.2 g / L.

[0013] On the other hand, the present invention discloses an antigen detection kit containing the aforementioned calibrator.

[0014] Furthermore, the kit also includes a labeled antibody labeled with a detectable marker.

[0015] Furthermore, the detectable markers include fluorescent markers, luminescent markers, or solid-phase carrier markers.

[0016] Furthermore, the fluorescent label includes, but is not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin;

[0017] The luminescent label includes, but is not limited to, luminol, acridinium ester, and ruthenium tripyridine; bioluminescent substances include, but are not limited to, luciferase, luciferin, and jellyfish luminescent protein.

[0018] The solid-phase carrier labeling includes time-resolved fluorescent microspheres, magnetic nanoparticles, immunolatex particles, quantum dots, or upconversion luminescent particles.

[0019] Furthermore, the affinity of the labeled antibody for the antigen is greater than that of the free antibody for the antigen.

[0020] This invention preserves the antigen in the calibrator as a more stable immune complex by adding a free antibody that has overlapping antigen binding sites with the immunoassay system but has a weaker affinity.

[0021] Furthermore, the labeled antibody and the free antibody have overlapping antigen-binding sites.

[0022] The term "overlap" as used in this invention refers to the presence of some identical antigen binding sites.

[0023] Furthermore, the antigen detection kit is a chemiluminescent detection kit.

[0024] When using the chemiluminescence detection kit, the antigen in the sample reacts fully with the detection reagent to form a sandwich immune complex of streptavidin magnetic beads-biotin-labeled antibody-antigen-alkaline phosphatase-labeled antibody. The magnetic particles are then washed to remove substances that did not participate in the immune reaction and other substances. The chemiluminescence substrate solution is then added, and an enzymatic reaction with alkaline phosphatase is carried out to generate photons. The relative luminescence intensity (RLU) is measured to obtain an accurate measurement result.

[0025] The free antibodies described in this invention can be obtained by screening using an antigen detection kit.

[0026] Further explanation regarding the aforementioned free antibody:

[0027] (1) The free antibody and the labeled antibody in the detection system have overlapping antigen-binding sites, but the free antibody has weaker affinity: In existing immunoassay systems, biotin-labeled antibodies or enzyme-labeled antibodies are usually used to recognize antigens. Because the free antibody and the biotin-labeled antibody or enzyme-labeled antibody have overlapping antigen-binding sites, the free antibody and the labeled antibody in the detection system compete for antigen binding. When screening free antibodies using an antigen detection kit, this is visually apparent: the addition of the free antibody to the calibrator causes a significant drop in signal value, meaning the addition of the free antibody has an inhibitory effect on the signal value. Due to the weak affinity of the free antibody, the detection system still retains approximately 40%-80% of the signal. This characteristic helps to screen for free antibodies that offer both antigen protection and cost savings.

[0028] (2) The free antibody has good protection against the antigen: after adding the free antibody to the calibrator, the accelerated degradation of the reagent within 1 year was simulated by accelerating it at 37°C for 7 days, and then the stability performance was evaluated, and the signal value drop could be controlled within 10%.

[0029] This invention adds free antibodies with overlapping antigen-binding sites but weaker affinity to the immunoassay system to the calibrators. While considering cost, this ensures that the antigen-binding sites recognized by the labeled antibodies in the assay system are not destroyed during storage. The antibodies are preserved in the calibrators in the form of more stable immune complexes, effectively protecting the antigen structure without affecting quantitative detection and significantly extending the antigen inactivation time.

[0030] This invention does not limit the types of antigens and antigen-specific free antibodies. Those skilled in the art can select different antigens and antigen-specific free antibodies according to different detection items.

[0031] Beneficial effects:

[0032] The high-stability calibrator provided by this invention forms an immune complex by adding antigen-specific free antibodies to the calibrator. This allows the antigen to be preserved in the calibrator buffer as an immune complex, thereby protecting the antigen sites recognized by the antibodies in the immunoassay system. This reduces protein damage caused by exposure of recognition sites during storage, thus improving the stability of the calibrator. The calibrator provided by this invention effectively protects the antigen structure without affecting the quantitative detection of antigens in the kit, significantly extending the antigen inactivation time and solving the problem of poor antigen stability in the field of medical testing.

[0033] Compared to conventional methods for optimizing the matrix, buffer system, solution pH, ionic strength, metal ion concentration, protease inhibitors, and types and concentrations of protective proteins in calibrator buffers, the method for preparing highly stable calibrators provided by this invention has high specificity, high feasibility, and can effectively protect the protein structure of the antigen from damage, thus having promotional and application value.

[0034] Compared to the production process of directly freeze-drying calibrators, the high-stability calibrators provided by this invention have a simple preparation method. Once the raw materials are determined, the production process is simple and does not require additional equipment or manpower. There is no risk of instability after reconstitution in clinical use, which better meets clinical needs.

[0035] The assay kit calibrator provided by this invention contains specific free antibodies to protect the antigen protein, effectively stabilizing the protein structure of the antigen and significantly improving the stability of the calibrator, thereby meeting the needs of clinical testing. Attached Figure Description

[0036] Figure 1 The data are real-time stability monitoring data of calibrators with an antigen concentration of 0.05 ng / mL in Example 1 and Comparative Example 1 added within 1 year; Example 1 is the antibody-added group; Comparative Example 1 is the antibody-free group.

[0037] Figure 2 The data are real-time stability monitoring data of calibrators with an antigen concentration of 48 ng / mL in Example 2 and Comparative Example 2 over one year; Example 2 is the antibody-added group; Comparative Example 2 is the antibody-free group. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. 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.

[0039] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0040] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0041] Examples 1-2

[0042] In this embodiment, an existing troponin I (cTnI) detection kit (magnetic microparticle chemiluminescence method) was used to screen for free troponin I antibodies to be added to the troponin I calibrator.

[0043] Table 1. Reagent components and concentration ranges of existing troponin I (cTnI) detection kits

[0044]

[0045]

[0046] The specific reagents selected for the existing troponin I (cTnI) detection kit are as follows:

[0047] Table 2. Reagent Components of Existing Troponin I (cTnI) Detection Kits

[0048]

[0049] The procedure for screening free antibodies in this embodiment is as follows:

[0050] (1) Based on the concentrations of calibrators and quality control materials used in existing troponin I (cTnI) detection kits (magnetic microparticle chemiluminescence method), dilute the cTnI antigen concentrate using two methods to prepare cTnI antigen samples:

[0051] A. Dilute the cTnI antigen concentrate directly to 0.05 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 2.0 μg / mL using diluent.

[0052] B. Dilute the cTnI antigen concentrate containing 2 times the concentration of cTnI antigen to be screened to 0.05 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 2.0 μg / mL using diluent.

[0053] (2) The signals of cTnI antigen samples prepared in the two ways were detected using troponin I (cTnI) detection reagents, and the differences in their optical values ​​were compared. The results are shown in Table 3 below:

[0054] Table 3 shows the detection of signals from cTnI antigen samples.

[0055]

[0056] The results in Table 3 show that the addition of screening antibody 2# and screening antibody 3# had no significant effect on the detection light value, while the addition of screening antibody 1# led to a decrease of about 40% of the signal value in the detection system. This indicates that the binding site of screening antibody 1# partially overlaps with the antigen recognition site of the labeled antibody in the detection system, resulting in a certain degree of competition among the three. At the same time, the binding ability of screening antibody 1# to the antigen is weaker than that of the antibody in the detection system, and the detection system retains about 60% of the light value.

[0057] (3) The cTnI antigens prepared using the two methods described above were placed at 37℃ and 2-8℃ for 7 days, respectively. The cTnI antigens were then detected using a troponin I (cTnI) detection kit, and the rate of change of light intensity over 7 days was calculated for each group (with and without antibody addition). The results are shown in Tables 4-1 to 4-4 below:

[0058] Table 4-1 Accelerated 7-day decrease in light intensity in the antibody-free group

[0059]

[0060] Table 4-2 Accelerated Light Value Decline in Group 1 (with Added Antibodies for Screening) over 7 Days

[0061]

[0062]

[0063] Table 4-3 shows the accelerated 7-day light intensity drop in group #2 with added antibodies to be screened.

[0064]

[0065] Table 4-4 shows the accelerated 7-day light intensity drop in group #3 after adding antibodies to be screened.

[0066]

[0067] The above results indicate that the stability of cTnI protein alone in the unantibody group is poor, with a maximum decrease of 38% after 7 days of accelerated testing at 37℃. However, after adding antibody #1, the free antibody binds to the cTnI protein to form an immune complex, which significantly improves the stability of the calibrator and has a significant effect on protecting the antigen. The decrease at each point after 7 days of accelerated testing at 37℃ can be controlled within 10%, confirming the effectiveness of antibody #1 in protecting the antigen.

[0068] In summary, antibody #1 was selected as a free troponin I antibody, specifically a mouse anti-human troponin I antibody, which meets the following characteristics:

[0069] (1) The free antibody and the labeled antibody in the detection system have overlapping antigen binding sites, but the binding ability is weaker: the addition of the free antibody in the calibrator will cause the signal value to drop significantly, while the detection system still retains about 40%-80% of the signal.

[0070] (2) The free antibody has good protection against the antigen: as shown by the addition of the free antibody during calibration, the signal value drop can be controlled within 10% after 7 days of accelerated calibration at 37℃.

[0071] The screened mouse anti-human troponin I antibody 1# was added to the calibrator as a free antibody to prepare the calibrator concentrate: the concentration of human troponin I antigen was selected as 2000 ng / mL, and the concentration of the added free antibody was 10-20 ug / mL. After adding the free antibody to the human troponin I antigen, it was mixed at room temperature for 2-5 hours to allow the human troponin I antigen to fully react with the free antibody to form an immune complex. Then, it was diluted with calibrator buffer to further prepare calibrators of different concentrations.

[0072] Specifically, the calibrators for the troponin I assay kits provided in Examples 1 and 2 of this invention, with their components and concentrations as shown in Table 5. In this example, the antigen concentration was selected from two low-to-high values ​​within the detectable range of 0.03 ng / mL to 50 ng / mL of the troponin I (cTnI) assay kit (magnetic microparticle chemiluminescence method). In practice, those skilled in the art can select appropriate concentrations according to their needs; concentrations may vary for different items, therefore, this invention does not limit the concentration.

[0073] Table 5. Calibration composition of Examples 1 and 2

[0074] Calibrator components Example 1 Example 2 Human troponin I antigen 0.05 ng / mL 50 ng / mL Mouse anti-human troponin I antibody 0.1 ng / mL 100ng / mL Tris buffer 50mmol / L 100mmol / L surfactants 0.5g / L 2g / L Inorganic salt ions 50mmol / L 100mmol / L Stable proteins 5g / L 15g / L preservative 0.4g / L 1.2g / L The solvent is purified water. The solvent is purified water. The solvent is purified water.

[0075] Comparative Example 1:

[0076] A calibrator for a troponin I assay kit, which differs from Example 1 in that Comparative Example 1 did not contain mouse anti-human troponin I antibody.

[0077] Comparative Example 2:

[0078] The calibrator for the troponin I assay kit differs from that in Example 2 in that Comparative Example 2 did not contain mouse anti-human troponin I antibody.

[0079] Stability tests were performed on the calibrators of Example 1 and Comparative Example 1, and Example 2 and Comparative Example 2. Their storage conditions at 4°C for one year were monitored in real time. Samples were taken and tested at months 2, 4, 6, 8, and 12 to obtain the real-time stability test results. (See attached figures.) Figures 1-2 .

[0080] according to Figure 1 , Figure 2 The results showed that, under storage conditions at 4°C, the stability of the calibrators with antibodies added in Examples 1 and 2 was significantly better than that of Comparative Examples 1 and 2. The calibrators of Examples 1 and 2 showed a degradation of less than 10% over 12 months, confirming that the method can effectively protect the antigen, improve the stability of the calibrators, and meet the needs of clinical testing.

[0081] Example 3:

[0082] This embodiment compares the protective effects of adding different concentrations of free troponin I antibody. The procedure is as follows:

[0083] (1) Based on the concentrations of calibrators and quality control samples used in the existing troponin I (cTnI) detection kit (magnetic microparticle chemiluminescence method) in Examples 1-2, dilute the cTnI antigen concentrate using the following three methods to prepare cTnI antigen samples:

[0084] A. Dilute the cTnI antigen concentrate directly to 0.05 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 2.0 μg / mL using diluent.

[0085] B. Dilute the cTnI antigen concentrate containing 2 times the concentration of cTnI antigen to be screened to 0.05 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 2.0 μg / mL using diluent.

[0086] C. Dilute the cTnI antigen concentrate containing 4 times the concentration of cTnI antigen to be screened to 0.05 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 2.0 μg / mL using diluent.

[0087] (2) The cTnI antigens prepared using the three methods described above were placed at 37℃ and 2-8℃ for 7 days, respectively. The cTnI antigens were then detected using a troponin I (cTnI) detection kit. The rate of change of light intensity over 7 days was calculated to simulate the protective effect of free cTnI antibodies against the antigen at different concentrations. The results are as follows:

[0088] Table 6-1 Protective effect of free troponin I antibody against antigens.

[0089]

[0090] Table 6-2 Protective effect of troponin I free antibody with 2 times the antigen concentration against the antigen.

[0091]

[0092]

[0093] Table 6-3 Protective effect of troponin I free antibody with 4 times the antigen concentration against the antigen.

[0094]

[0095] The above results indicate that the signal drop of antigens with 2-4 times the concentration of free troponin I antibody was within 10% after 7 days of accelerated treatment at 37°C, confirming the effectiveness of free antibody 1# in protecting the antigen at a concentration 2-4 times the antigen concentration.

[0096] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0097] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this invention, and these modifications or substitutions should all be covered within the scope of protection of this invention. For example, the selection of other detection items is not limited to troponin I; or the calibrator is not limited to use in antigen detection, but theoretically can also be used in antibody detection based on the balance reaction between antigen and antibody. Therefore, the scope of protection of this invention should be determined by the scope of the claims.

Claims

1. A highly stable calibrator, characterized in that, The mixture includes an antigen, an antigen-specific free antibody, an immune complex, and a calibrator buffer, wherein the immune complex is formed by the binding of the free antibody to the antigen; wherein the antigen includes troponin I, and the free antibody is a mouse anti-human troponin I antibody; the concentration of the free antibody is 2-4 times that of the antigen.

2. The high-stability calibrator as described in claim 1, characterized in that, The calibration buffer solution includes a buffer solution, a surfactant, inorganic salt ions, a stable protein, and a preservative.

3. The high-stability calibrator as described in claim 2, characterized in that, The buffer solution is selected from one of the following: TRIS buffer, phosphate buffer, and MES buffer, with a concentration of 50-100 mmol / L; The surfactant is selected from one or more of Tween 20, Tween 80, Brij 35, Triton 100, and NP40, with a concentration of 0.5-2 g / L; The inorganic salt ions are selected from sodium chloride or potassium chloride, with a concentration of 50-100 mmol / L. The stable protein is selected from casein or BSA, with a concentration of 5-15 g / L; The preservative is selected from NaN3, procline300 or mercuric sulfide, with a concentration of 0.4~1.2 g / L.

4. A kit for antigen detection, characterized in that, It includes the calibrator as described in any one of claims 1-3.

5. The kit according to claim 4, characterized in that, The kit also includes labeled antibodies labeled with detectable markers.

6. The reagent kit as described in claim 5, characterized in that, The detectable markers include fluorescent markers, luminescent markers, or solid-phase carrier markers; The fluorescent label includes umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin. The luminescent markers include luminol, acridinium ester, ruthenium tripyridine, luciferase, luciferin, and jellyfish luminescent protein; The solid-phase carrier labeling includes time-resolved fluorescent microspheres, magnetic nanoparticles, immunolatex particles, quantum dots, or upconversion luminescent particles.

7. The kit according to any one of claims 4-6, characterized in that, The labeled antibody has a greater affinity for the antigen than the free antibody has for the antigen.

8. The kit according to claim 7, characterized in that, The labeled antibody and the free antibody have overlapping antigen-binding sites.

9. The kit according to any one of claims 4-6, characterized in that, The kit is a chemiluminescence detection kit.

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