Composition and application thereof in improving stability of immunological detection reagent

Abstract

The invention relates to the technical field of immunological detection, in particular to a composition and application thereof in improving the stability of an immunological detection reagent. The invention develops a compound stabilizer composition, and realizes multi-dimensional protection on an immunological detection reagent (saccharides: maintaining a hydrated layer on the surface of an antibody molecule; the amino acid is used for regulating the ionic strength and pH buffer of the solution; the zwitterionic surfactant is used for inhibiting non-specific adsorption of protein; and a stable formula proportion model is established, so that a synergistic stable system is constructed, the activity of the reagent is kept for more than or equal to 1 month at the ambient temperature of 37 DEG C, the stability of reagent detection is greatly improved, and the method is applied to wider regions.

Description

Technical Field

[0001] This invention relates to the field of immunological detection technology, and more particularly to compositions and their application in improving the stability of immunological detection reagents. Background Technology

[0002] Von Willebrand factor (vWF) is a glycoprotein secreted into the plasma by endothelial cells and megakaryocytes, participating in the initial hemostasis and coagulation processes. It plays a central role in primary hemostasis, mediating platelet adhesion to the damaged vascular endothelium, leading to platelet aggregation. Plasma vWF:Ag levels in healthy individuals vary considerably, depending on many factors. Type O blood is associated with a 25% reduction in vWF:Ag levels. vWF:Ag levels increase with age. Other factors contributing to fluctuations in vWF:Ag levels include the menstrual cycle, stress, pregnancy, and immune responses. VWF deficiency causes von Willebrand disease (vWD), a common autosomal dominant inherited disorder characterized by impaired hemostasis due to reduced vWF antigen (vWF Ag) and vWF activity. A decrease in vWF:Ag indicates a reduction in the amount of vWF. On the other hand, chronic or acute inflammatory diseases or processes involving vascular endothelial damage can produce abnormally high concentrations of vWF. Patients with myocardial infarction may also have elevated vWF levels. Therefore, the detection of vWF:Ag is often used as an important tool for clinical diagnosis.

[0003] There are various methods for determining vWF content. Compared to other methods, latex immunoturbidimetry is simpler, and its accuracy and sensitivity meet clinical requirements. Latex immunoturbidimetry utilizes the agglutination reaction formed when latex particles bind to vWF antigens to detect vWF content. This method is simple to operate, provides intuitive results, and is suitable for rapid screening and testing of large numbers of samples. It has high accuracy and sensitivity, and can accurately reflect the content and functional activity of vWF. Latex immunoturbidimetry has the advantage of simplicity and fast detection speed, which can improve work efficiency. Traditional immunoturbidimetric reagents often use a single stabilizer (such as BSA or sucrose), which has poor heat resistance. Commercially available products (such as the Siemens von Willebrand factor antigen assay kit) require cold chain transportation, limiting their application in resource-scarce areas; therefore, there is an urgent market need to develop a von Willebrand factor antigen assay kit to help solve existing problems. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a composition and its application in improving the stability of vWF latex detection reagents.

[0005] The composition provided by the present invention consists of trehalose, glycine, cocamidopropyl hydroxysulfonate betaine (CAHSB) and sodium lauroyl amphoteric acetate.

[0006] The composition provided by this invention is based on a synergistic mechanism of a ternary stabilizing system of "sugar + amino acid + zwitterionic surfactant". CAHSB forms an electrical double layer with the surface of latex microparticles, and glycine stabilizes the conformation of the antibody Fab region through ion interactions. This composition achieves antibody activity protection in immunoturbidimetric reaction solution and can be stably stored at 37°C for 1 month.

[0007] Experiments show that the triple-stabilized reagent formulation of "sugar + amino acid + zwitterionic surfactant," when mixed in a certain proportion, significantly improves stability while meeting sensitivity and linearity requirements. Preferably, the mass ratio of trehalose, glycine, cocamidopropyl hydroxysulfonate, and sodium lauroyl amphoteric acetate is (20–60):(20–30):2:1. As a feasible example, the mass ratio of trehalose, glycine, cocamidopropyl hydroxysulfonate, and sodium lauroyl amphoteric acetate is 60:30:2:1, 40:30:2:1, 20:30:2:1, 60:20:2:1, 40:20:2:1, or 20:20:2:1.

[0008] More preferably, the mass ratio of trehalose, glycine, cocamidopropyl hydroxysulfonate, and sodium lauroyl amphotericate is 60:30:2:1.

[0009] The composition provided by this invention, when applied to immunological detection reagents, enables the reagents to maintain detection stability over a wide temperature range of 25-37°C, with stability extended to 30 days at 37°C, and an intra-assay coefficient of variation (CV) ≤2%. It is particularly suitable for rapid vWF detection in primary healthcare institutions and tropical regions. Therefore, this invention provides the application of the aforementioned composition in improving the stability of immunological detection reagents.

[0010] Furthermore, the present invention also provides a detection reagent based on latex immunoturbidimetry, comprising reagent 1 and reagent 2; wherein reagent 1 and / or reagent 2 contain the composition as described above.

[0011] In this invention, reagent 1 comprises: 2.15wt% to 4.65wt% of the composition, 0.1wt% to 5wt% of the salt, 10 to 500 μg / mL of the blocking agent, and 0.1 to 1 mL / L of the preservative;

[0012] In this invention, reagent 2 comprises: 2.15wt% to 4.65wt% of the composition, 0.1 to 10 mg / mL of polystyrene latex particles, 0.1wt% to 5wt% of salt, 0.1 to 10 mL / L of liquid preservative, 0.5 to 5 g / L of solid preservative, and 0.1 to 1 mL / L of preservative.

[0013] In this invention,

[0014] The salt is NaCl;

[0015] The preservative is proclin300.

[0016] The liquid preservative is glycerol, and the solid preservative is BSA.

[0017] In a specific embodiment,

[0018] The reagent 1 comprises: trehalose 3wt%, glycine 1.5wt%, cocamidopropyl hydroxysulfonate betaine (CAHSB) 0.1wt%, sodium lauroyl amphoteric acetate 0.05wt%, NaCl 2.5wt%, MAB 3310 1μg / mL, and proclin 300 1mL / L.

[0019] The reagent 2 comprises: trehalose 3wt%, glycine 1.5wt%, cocamidopropyl hydroxysulfonate betaine (CAHSB) 0.1wt%, sodium lauroyl amphoteric acid 0.05wt%, 2.5wt% NaCl, polystyrene latex particles 1mg / mL, glycerol 5mL / L, BSA 2g / L, and proclin 300 1mL / L.

[0020] Taking the latex immunoturbidimetric assay reagent for vWF detection as an example: the polystyrene latex particles in reagent 2 are coated with vWF antibodies.

[0021] This invention significantly improves the stability of immunological detection reagents by adding the aforementioned composition to them. Accelerated stability at 37°C can reach 30 days, effectively extending the product's shelf life. Furthermore, the pH values ​​of reagents R1 and R2 are adjusted. The preparation of reagent 2 depends on the selection of the activator and the type, concentration, and pH of the buffer solution.

[0022] In some embodiments, the method for preparing the antibody-coated polystyrene latex particles includes:

[0023] After activation, the latex microspheres undergo a coupling reaction in the presence of EDC and Sulfo-NHS, followed by conjugation with an antibody. After blocking, they are resuspended in a preservation solution. In this embodiment of the invention, the antibody is a mouse monoclonal vWF antibody.

[0024] In a specific embodiment, the sealing agent for the sealing treatment is ethanolamine, and the preservation solution contains water, trehalose 3wt%, glycine 1.5wt%, cocamidopropyl hydroxysulfonate betaine (CAHSB) 0.1wt%, sodium lauroylamphoacetate 0.05wt%, NaCl 2.5wt%, glycerol 5mL / L, BSA 2g / L, and proclin 300 at a concentration of 1mL / L.

[0025] This invention develops a composite stabilizer composition that achieves multi-dimensional protection of immunological detection reagents (carbohydrates: maintain the hydration layer on the surface of antibody molecules; amino acids: regulate the ionic strength and pH buffer of the solution; zwitterionic surfactants: inhibit non-specific protein adsorption); and establishes a formulation ratio model with a thermodynamic stability range (ΔG<0), thereby constructing a synergistic stabilizing system that allows the reagents to maintain their activity for ≥1 month at an ambient temperature of 37°C, greatly improving the stability of reagent detection and thus enabling its application in a wider range of regions. Attached Figure Description

[0026] Figure 1 The graph shows the trend of thermal stability of the reagents in the example and commercially available reagents over days. Detailed Implementation

[0027] This invention provides compositions and their application in improving the stability of immunological detection reagents. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0028] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0029] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0030] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0031] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0032] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0033] The reagents and consumables used in this invention are all commercially available products and can be purchased on the market. Specifically:

[0034] The fully automated coagulation analyzer is the SunBio UP1500.

[0035] The preparation of antibody microspheres includes:

[0036] (1) Wash the latex microspheres once with morpholine ethanesulfonic acid activation buffer, centrifuge at 18000 r / min, 4℃ for 30 min to obtain latex microsphere precipitate;

[0037] (2) The latex microsphere precipitate prepared in step (1) is suspended in an activation buffer solution and mixed to obtain a latex suspension.

[0038] (3) The latex suspension prepared in step (2) was coupled. A mixed morpholine ethanesulfonic acid buffer containing EDC and Sulfo-NHS (mass ratio of 5:3) was added to the latex suspension. The mixture was gently stirred at room temperature for 15 min, centrifuged at 18000 r / min for 30 min, and the supernatant was removed to obtain latex particle precipitate.

[0039] (4) The latex particle precipitate prepared in step (3) is suspended in 40 mM borate buffer solution and mixed well;

[0040] (5) The mouse monoclonal antibody against von Willebrand factor was diluted with 40mM borate buffer solution. When coating the antibody, the antibody solution was coated on latex particles with a diameter of 200nm. The latex particle concentration was 5mg / ml. The amount of vWF antibody was 1 times the saturated adsorption capacity of the microspheres. The mixture was incubated at room temperature for 2h.

[0041] (6) Add the blocking agent ethanolamine to the reaction solution of step (5) to make the final concentration 1%, and vortex incubate at room temperature for 30 min;

[0042] (7) Centrifuge the solution prepared in step (6) at 18000 r / min for 30 min, remove the supernatant, add a small amount of reagent 2 (without antibody microspheres) to treat the precipitate, mix the antibody latex evenly, sonicate and dilute to a latex suspension of 1 mg / mL, age at 37℃ for 12 h, and then store at 4℃.

[0043] It should be understood that in the various embodiments of this application, the sequence number of the above processes does not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased through legitimate channels.

[0044] Example 1: Preparation of Reagent Kit 1

[0045] The concentrations of each component in the kit (where % is a mass percentage) are as follows:

[0046] Reagent 1: Trehalose 3%, glycine 1.5%, cocamidopropyl hydroxysulfonate betaine (CAHSB) 0.1%, sodium lauroyl amphoteric acid 0.05%, NaCl 2.5%, MAB33 concentration 101 μg / mL, proclin 300 concentration 1 mL / L.

[0047] Reagent 2: Polystyrene latex particles (containing specific anti-human vWF antibody) 1 mg / mL, trehalose 3%, glycine 1.5%, cocamidopropyl hydroxysulfobetaine (CAHSB) 0.1%, sodium lauroylamphoacetate 0.05%, NaCl 2.5%, glycerol 5 mL / L, BSA 2 g / L, and proclin 300 at a concentration of 1 mL / L.

[0048] Preparation methods include:

[0049] Step 1: Prepare reagent 1

[0050] Add a certain amount of purified water to the container, then add glycine according to the concentration ratio, stir until it is completely dissolved, and then add NaCl, MAB33, cocamidopropyl hydroxysulfonate betaine (CAHSB), sodium lauroyl amphoteric acetate, trehalose, and proclin300 in sequence.

[0051] Stir until completely dissolved, adjust the pH of the reagent to 7, add purified water to the container to make up the total volume, fill into reagent bottles to obtain reagent 1;

[0052] Step 2: Prepare reagent 2

[0053] 0.3 mL of 200 nm diameter polystyrene latex microspheres (10% concentration) were added to 1.2 mL of activation buffer MES (pH 5.6). Then, 1.5 mL of a 1:1 mixture of EDC·HCl solution (8 mg / mL) and sulfo-NHS solution (4.8 mg / mL) was added. The mixture was stirred at room temperature for 15 min, followed by centrifugation at 18000 rpm and 4 °C for 30 min. The supernatant was removed, and 40 mM coupling buffer (borate buffer, pH 7.4) was added. 3 mL of coupling buffer was used to dilute 2 mg of the styrene latex microspheres. vWF antibody was added to the above conjugation buffer and mixed at room temperature for 2 h. Then, 60 μL of ethanolamine solution was added and mixed at room temperature for 30 min. The mixture was then centrifuged at 18000 rpm and 4 °C for 30 min. The supernatant was discarded, and the antibody microsphere precipitate was mixed with 6 ml of Reagent 2 (without antibody microspheres). The microspheres were then dispersed by sonication and diluted with Reagent 2 (without antibody microspheres) to a latex microsphere solution with a concentration of 1 mg / ml. The solution was aged at 37 °C for 12 h and then stored at 4 °C to obtain Reagent 2.

[0054] Example 2: Detection of reagent repeatability

[0055] The detection-related kit in Example 1 and the commercially available vWF antigen detection kit (immunoturbidimetric assay) were used to perform repeatability tests on normal and abnormal quality control plasmas. The test results are shown in Table 1. The vWF detection kit in Example 1 has a smaller CV and better repeatability compared with the commercially available vWF antigen detection kit (immunoturbidimetric assay).

[0056] Table 1

[0057]

[0058]

[0059] As can be seen from the results in Table 1, the vWF detection reagent of the present invention has a smaller repeatability CV, and its detection repeatability is better than that of commercially available reagents.

[0060] Example 3: Determination of the thermal stability of the reagent at 37°C

[0061] The detection-related reagent kit from Example 1 and a vWF antigen detection kit (immunoturbidimetric assay) from a certain company were each aliquoted into five equal-volume aliquots and placed in a 37°C incubator. On days 0, 4, 7, 14, and 30, the abnormal and normal quality control plasma samples from the same batch were measured. The measurements were repeated three times, and the average value was taken. The test results are shown in Tables 2 and 3. Figure 1 As shown.

[0062] Table 2

[0063]

[0064] Table 3

[0065]

[0066] As can be seen from the results in Tables 2 and 3, the vWF detection reagent of the present invention has stable test results within 30 days at 37°C, while the measured values ​​of commercially available reagents continue to decrease from the 7th day. This indicates that the present kit has good heat resistance stability at 37°C, which is superior to commercially available reagents and is suitable for use in high-temperature areas.

[0067] Comparative Example 1 (Reagent Kit 2)

[0068] The difference between Comparative Example 1 and Example 1 is that the concentration of the overall complex (trehalose, glycine, cocamidopropyl hydroxysulfonate and sodium lauroyl amphotericate) in Example 1 was increased to twice that of Example 1, while the other components and processes were the same as in Example 1.

[0069] Comparative Example 2 (Reagent Kit 3)

[0070] The difference between Comparative Example 2 and Example 1 is that the 0.05% sodium lauroyl amphotericate in Example 1 was replaced with 0.05% Tween-20, while the other components and processes were the same as in Example 1.

[0071] Comparative Example 3 (Reagent Kit 4)

[0072] The difference between Comparative Example 3 and Example 1 is that 3% trehalose was removed, while the other components and processes were the same as in Example 1.

[0073] Performance Verification: Accelerated Stability Test at 37℃

[0074] The reagent kits prepared in Example 1 and Comparative Examples 1-3 were placed at a constant temperature of 37°C to investigate the stability of the calibration test results. Each kit was aliquoted into three equal small volumes and placed in a 37°C incubator. Calibration tests were performed on the same batch of calibrators on days 0, 7, and 30, with three repeated measurements taken as the average. The relative deviations of the measurements on days 7 and 30 compared to day 0 are shown in Table 4. The data in Table 4 represent the relative deviations of the absorbance of the calibrators on days 7 and 30 compared to before incubation (day 0).

[0075] Table 4. Thermal stability on days 7 and 30

[0076]

[0077]

[0078] As shown in Table 4, the latex reagent kit prepared by the method of this application has significantly better thermal stability at 37℃ than the comparative examples (reagents 2-4). After being placed at 37℃ for 7 days, the relative deviation of the absorbance value is less than 5%. It can be seen from the comparison that the reagent kit prepared by the method of this application has significantly better thermal stability than the control reagent.

[0079] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition comprising trehalose, glycine, cocamidopropyl hydroxysulfonate betaine and sodium lauroyl amphoteric acetate, wherein the mass ratio of trehalose, glycine, cocamidopropyl hydroxysulfonate betaine and sodium lauroyl amphoteric acetate is (20-60):(20-30):2:

1.

2. The composition according to claim 1, characterized in that, The mass ratio of trehalose, glycine, cocamidopropyl hydroxysulfonate, and sodium lauroyl amphoteric acetate is 60:30:2:

1.

3. The use of the composition according to any one of claims 1 to 3 in improving the stability of immunological detection reagents.

4. A detection reagent based on latex immunoturbidimetry, comprising reagent 1 and reagent 2; wherein reagent 1 and / or reagent 2 contain the composition according to any one of claims 1 to 3.

5. The detection reagent according to claim 4, characterized in that, The reagent 1 comprises: 2 wt% to 5 wt% of the composition according to any one of claims 1 to 3, 0.1 wt% to 5 wt% of a salt, 10 to 500 μg / mL of an inhibitor, and 0.1 to 1 mL / L of a preservative; The reagent 2 comprises: 2wt% to 5wt% of the composition according to any one of claims 1 to 3, 0.1 to 10 mg / mL of polystyrene latex particles, 0.1wt% to 5wt% of salt, 0.1 to 10 mL / L of liquid preservative, 0.5 to 5 g / L of solid preservative, and 0.1 to 1 mL / L of preservative.

6. The detection reagent according to claim 5, characterized in that, The salt is NaCl; The preservative is proclin300. The liquid preservative is glycerol, and the solid preservative is BSA.

7. The detection reagent according to claim 5 or 6, characterized in that: The reagent 1 comprises: 2 wt% to 5 wt% of the composition according to any one of claims 1 to 3, 2.5 wt% of NaCl, 101 μg / mL of MAB33 and 1 mL / L of proclin 300; The reagent 2 comprises: 2 wt% to 5 wt% of the composition according to any one of claims 1 to 3, 0.1 wt% to 5 wt% of NaCl, 1 mg / mL of polystyrene latex particles, 5 mL / L of glycerol, 2 g / L of BSA, and 1 mL / L of proclin 300.

8. The detection reagent according to any one of claims 5 to 7, characterized in that: The method for preparing the antibody-coated polystyrene latex particles includes: After activation, the latex microspheres undergo a coupling reaction in the presence of EDC and Sulfo-NHS, then are coupled with antibodies, blocked, and resuspended in a preservation solution.

9. The detection reagent according to claim 8, characterized in that, The preservation solution contains water, 1.5 wt% glycine, 0.1 wt% cocamidopropyl hydroxysulfonate betaine, 0.05 wt% sodium lauroylamphoacetate, 3 wt% trehalose, 2.5 wt% NaCl, 5 mL / L glycerol, 2 g / L BSA, and 1 mL / L proclin 300.

10. The detection reagent according to claim 8 or 9, characterized in that, The antibody in the antibody-coated polystyrene latex particles is a vWF antibody.

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