Homogeneous freeze-dried microspheres as well as preparation method and application thereof

Abstract

The invention relates to immunoassay, in particular to homogeneous freeze-dried microspheres as well as a preparation method and application thereof, and the homogeneous freeze-dried microspheres comprise an oxygen-receiving microsphere coating raw material, an oxygen-supplying microsphere coating streptavidin and a coupling antigen-antibody; the storage agent comprises a freeze-drying protective agent, PEG6000 (Polyethylene Glycol 6000) and an antioxidant. The kit is applied to immunoassay, and has the advantages of good stability, easy carrying and the like.

Description

Technical Field

[0001] The invention relates to immunoassay, and in particular to homogeneous freeze-dried microspheres and a preparation method and application thereof. Background Art

[0002] The lyophilization process of homogeneous microsphere reagents and its application in immunoassays is an important development in the fields of biotechnology and clinical diagnosis. This technology combines the high efficiency of microsphere technology with the stability advantages of lyophilization technology, aiming to improve the sensitivity, specificity and convenience of immunoassay detection.

[0003] In immunoassay, homogeneous microspheres are used as carriers to immobilize antigens or antibodies, and through chemical modification of the microsphere surface, high-density and uniform distribution of biomolecules can be achieved. This design simplifies the detection steps because there is no need to separate bound and unbound components, and the reaction is completed directly in the solution, which improves the detection efficiency.

[0004] Freeze drying is a process of pre-freezing a water-containing sample to below freezing point and then sublimating the water under vacuum conditions to preserve the structural integrity and activity of the sample. For biological reagents, freeze drying can significantly extend their shelf life, facilitate transportation and storage, while maintaining their biological activity.

[0005] Currently, homogeneous lyophilized microspheres have the following deficiencies in immunoassays: 1. Homogeneous microspheres are prone to aggregation and deformation during the freeze-drying process, resulting in reduced uniformity and stability of the microspheres, affecting their performance in immunoassays, such as reducing binding efficiency and detection sensitivity.

[0006] 2. It is impossible to effectively prevent the denaturation of proteins or other bioactive molecules during freeze-drying and subsequent storage, which affects the long-term stability and accuracy of the reagents.

[0007] 3. The freeze-drying process parameters (such as freezing rate, vacuum degree, sublimation time, etc.) lack fine control, resulting in damage to the microsphere structure or inability to quickly restore to its original state during rehydration, affecting its rapid response and efficient binding ability in practical applications.

[0008] 4. It is difficult to completely and evenly rehydrate the microspheres in a short period of time, which prolongs the test preparation time and reduces the experimental efficiency. Summary of the invention

[0009] In order to solve the deficiencies in the above-mentioned prior art solutions, the present invention provides a homogeneous freeze-dried microsphere.

[0010] The objective of the present invention is achieved through the following technical solutions: A homogeneous freeze-dried microsphere, comprising oxygen-accepting microsphere coating raw materials, oxygen-supplying microsphere coating streptavidin and coupled antigen-antibody; the homogeneous freeze-dried microsphere also includes: The storage agent comprises a lyophilization protectant, PEG6000 and an antioxidant.

[0011] Another object of the present invention is to provide a method for preparing the above-mentioned homogeneous freeze-dried microspheres, which is achieved by the following technical solutions: The preparation method of homogeneous freeze-dried microspheres comprises the following steps: (A1) preparing reagents using oxygen accepting microspheres coated with raw materials, oxygen supplying microspheres coated with streptavidin and coupled antigen-antibody; (A2) adding the storage agent to the reagent; (A3) Using liquid nitrogen to freeze-dry, the sample is frozen in aliquots to form frozen microspheres.

[0012] Another object of the present invention is to provide an application of the above-mentioned homogeneous freeze-dried microspheres, and the object of the invention is achieved by the following technical solutions: The invention provides an application of freeze-dried microspheres in immunoassay.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. Improve the stability and uniformity of microspheres; By optimizing the formulation and addition strategy of lyoprotectants and improving the lyophilization process, the aggregation and deformation of microspheres during processing can be reduced, and the stability of their physical and chemical properties can be ensured, thereby improving the accuracy and repeatability of immunoassays; 2. Enhance the protection of bioactive molecules; Develop new or improved lyoprotectant systems, especially for antibodies, antigens and other biomolecules commonly used in immunoassays, to ensure their structural integrity and functional activity during lyophilization and long-term storage, and to improve the sensitivity and specificity of detection; 3. Optimize freeze-drying process parameters; By precisely controlling key parameters in the freeze-drying process, such as optimizing the freezing rate, adjusting the vacuum conditions, and managing the sublimation phase, the damage to the microsphere structure and embedded biomolecules can be minimized, thus ensuring high-quality recovery of microspheres after freeze-drying; 4. Design efficient rehydration scheme; Research and implement rapid and uniform rehydration technology to shorten reagent preparation time, simplify operating procedures, and improve the convenience and efficiency of laboratory and clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The disclosure of the present invention will become easier to understand with reference to the accompanying drawings. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. In the drawings: Figure 1It is a schematic diagram of a preparation method according to an embodiment of the present invention. DETAILED DESCRIPTION

[0015] Figure 1 The following description describes the optional embodiments of the present invention to teach those skilled in the art how to implement and reproduce the present invention. In order to teach the technical solution of the present invention, some conventional aspects have been simplified or omitted. It should be understood by those skilled in the art that variations or substitutions derived from these embodiments will be within the scope of the present invention. It should be understood by those skilled in the art that the following features can be combined in various ways to form multiple variations of the present invention. Thus, the present invention is not limited to the following optional embodiments, but is only limited by the claims and their equivalents.

[0016] Example 1.

[0017] The homogeneous freeze-dried microspheres of the embodiment of the present invention include: Oxygen accepting microspheres coated with raw materials, oxygen supplying microspheres coated with streptavidin and coupled antigen antibodies; The storage agent comprises a lyophilization protectant, PEG6000 and an antioxidant.

[0018] In order to improve the structural integrity and functional activity during freeze-drying and long-term storage, the freeze-drying protective agent further includes trehalose, mannitol, a surfactant, sucrose, BSA, a preservative, PVP-K30 and a buffer.

[0019] In order to prevent the reagent from oxidative deterioration during the freeze-drying process and storage process, further, the antioxidants are sodium thiosulfate and EDTA-2Na.

[0020] In order to reduce ice-water interfacial tension during the freeze-drying process, the surfactant is further Tween 20, Tween 80 or triton×100.

[0021] In order to achieve extensive antibacterial activity, the preservative is preferably Proclin 300 with good biocompatibility.

[0022] According to the preparation method of homogeneous freeze-dried microspheres in this embodiment, Figure 1 As shown, the following steps are included: (A1) preparing reagents using oxygen accepting microspheres coated with raw materials, oxygen supplying microspheres coated with streptavidin and coupled antigen-antibody; (A2) adding the storage agent to the reagent; (A3) Using liquid nitrogen to freeze-dry, the sample is frozen in aliquots to form frozen microspheres.

[0023] In order to solve the problem that the product cannot be stored at room temperature during transportation, the preparation method further comprises the following steps: (A4) The frozen microspheres are transferred to a freeze dryer, and after freeze drying, they are filled with protective gas and packaged for storage.

[0024] Example 2.

[0025] An example of using the homogeneous freeze-dried microspheres and method according to Example 1 of the present invention in a chemiluminescent immunoassay reagent.

[0026] In this application example, Figure 1 As shown, the preparation method comprises the following steps: (A1) preparing reagents using oxygen accepting microspheres coated with raw materials, oxygen supplying microspheres coated with streptavidin and coupled antigen-antibody; (A2) adding the storage agent to the reagent, wherein the storage agent comprises a lyoprotectant, PEG6000 and an antioxidant, and the lyoprotectant comprises trehalose, mannitol, a surfactant, sucrose, BSA, a preservative, PVP-K30 and a buffer.

[0027] Mannitol: As a freeze-drying filler, it is non-hygroscopic and freeze-dries quickly. The freeze-drying process can prevent the effective components from sublimating with water vapor and make the effective components take shape.

[0028] Trehalose: It plays a role of low-temperature protection during the freezing process and acts as a dehydration protectant during the drying and dehydration process.

[0029] BSA: It plays a role in protecting proteins and acts as a filler in the dehydration and drying process. It can also control the background and eliminate false positives in immunological reagents.

[0030] Surfactants, such as Tween 20, Tween 80 or Triton × 100, can reduce freezing and dehydration deformation caused by ice-water interfacial tension during the freeze-drying process, and act as a wetting agent and re-wrinkling agent for the active components during the rehydration process. At the same time, it can reduce non-specific reactions during the immune response process.

[0031] The preservative used is Proclin 300: it has broad antibacterial properties and good biocompatibility.

[0032] PEG6000: It acts as a low-temperature protectant and a dehydration protectant. It can also accelerate immune responses and increase reagent sensitivity.

[0033] Antioxidants include sodium thiosulfate and EDTA-2Na to prevent the reagents from oxidative deterioration during freeze-drying and storage.

[0034] (A3) Using liquid nitrogen to freeze-dry, forming freeze-dried beads, splitting and freezing the sample to form frozen microspheres, and realizing a single-serving packaging test mode; (A4) The frozen microspheres are transferred to a freeze dryer, and after freeze drying, they are filled with protective gas and packaged for storage.

[0035] The freeze-drying process parameters are shown in the following table.

[0036] .

[0037] The components of the lyoprotectant are shown in the table below.

[0038] .

[0039] Homogeneous chemiluminescent immunoassay reagent is a new type of chemiluminescent detection reagent. This reagent contains two types of microspheres. One is the oxygen supply microsphere, which contains compounds such as phthalocyanine molecules that can produce singlet oxygen; the other is the oxygen acceptor microsphere, which contains fluorescent substances that can accept singlet oxygen. The oxygen supply microsphere can produce a large number of singlet oxygen molecules under the excitation of light with a wavelength of about 680nm. The half-life of singlet oxygen is only 4us, and it decays quickly in water medium, and can be transmitted as far as 200nm. If the oxygen acceptor microsphere can approach the oxygen supply microsphere through specific recognition methods such as immune recognition, the fluorescent group in the oxygen acceptor microsphere can quickly absorb the singlet oxygen generated under the excitation of 680nm light, and at the same time generate 520-620nm light signals. Therefore, when the oxygen supplying microspheres and the oxygen accepting microspheres are coated with appropriately matched antibodies, antigens or other specific molecules, and can form bridges and aggregations through the corresponding analytes in the test sample, a 520-620nm light signal will be generated under 680nm light excitation, and its intensity is linearly related to the amount of the analyte in the test sample within a certain range. By detecting the luminescence intensity and then comparing it with the calibration curve, the concentration of the analyte (here, the antigen) in the sample can be quantitatively detected.

Claims

1. Homogeneous freeze-dried microspheres, comprising oxygen-accepting microspheres coated with raw materials, oxygen-supplying microspheres coated with streptavidin and coupled antigen-antibody; characterized in that: The homogeneous freeze-dried microspheres also include: The storage agent comprises a lyophilization protectant, PEG6000 and an antioxidant.

2. The homogeneous freeze-dried microspheres according to claim 1, characterized in that The lyoprotectant comprises trehalose, mannitol, a surfactant, sucrose, BSA, a preservative, PVP-K30 and a buffer.

3. The homogeneous freeze-dried microspheres according to claim 1, characterized in that The antioxidants are sodium thiosulfate and EDTA-2Na.

4. The homogeneous freeze-dried microspheres according to claim 2, characterized in that: The surfactant is Tween 20, Tween 80 or Triton×100.

5. The homogeneous freeze-dried microspheres according to claim 2, characterized in that The preservative used was Proclin 300.

6. The method for preparing the homogeneous freeze-dried microspheres according to any one of claims 1 to 5, comprising the following steps: (A1) preparing reagents using oxygen accepting microspheres coated with raw materials, oxygen supplying microspheres coated with streptavidin and coupled antigen-antibody; (A2) adding the storage agent to the reagent; (A3) Using liquid nitrogen to freeze-dry, the sample is frozen in aliquots to form frozen microspheres.

7. The preparation method according to claim 1, characterized in that: The following steps are involved: (A4) The frozen microspheres are transferred to a freeze dryer, and after freeze drying, they are filled with protective gas and packaged for storage.

8. The preparation method according to claim 1, characterized in that: The freeze-dried microspheres are chemiluminescent immunoassay reagent freeze-dried microspheres.

9. Use of the freeze-dried microspheres according to any one of claims 1 to 5 in immunoassay.

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