A preservation solution for antibody-microsphere conjugates, its preparation method, and its application.

Abstract

This invention discloses a preservation solution for antibody-microsphere conjugates, its preparation method, and its applications, relating to the field of immunoassay. The preservation solution comprises a buffer solution, 0.05%–1% protein protectant, 0.01%–0.5% surfactant, 0.5%–1.5% sugar, and 0.02%–0.1% preservative. The buffer solution includes Good's buffer, with a pH 2–3 higher than the isoelectric point of the antibody. This buffer environment, 2–3 points higher than the isoelectric point of the labeled antibody, allows the colloidal particles to acquire a higher negative charge. The Good's buffer system, combined with a low salt ion concentration, ensures the colloidal solution is at a low ion concentration, providing good protection for the electric double layer on the colloidal particle surface. The above components, in a specific ratio, synergistically improve the colloidal and biostability of the antibody-microsphere conjugates, providing a pathway for their storage and application.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and more specifically, to a preservation solution for an antibody-microsphere conjugate, its preparation method, and its application. Background Technology

[0002] Polystyrene (PS) latex microspheres are spherical colloidal particles whose surfaces are often modified with bioactive groups such as carboxyl or amino groups to facilitate the formation of conjugates with antibodies. They are commonly used in the field of in vitro diagnostics.

[0003] Microspheres not conjugated with antibodies possess excellent monodispersity due to the presence of numerous charged groups such as carboxyl or amino groups on their surface, allowing them to remain stable for extended periods and resist aggregation. However, when microspheres are conjugated with antibodies, these charged groups are replaced by the antibody, leading to decreased latex stability. In unsuitable preservation solutions, they become susceptible to aggregation and sedimentation due to external environmental influences, severely impacting the performance of immunoassay reagents. Therefore, prepared microsphere-antibody conjugates must be stored in specific preservation solutions to maintain good dispersibility, protect the antibody activity on the microsphere surface, and ensure the microsphere-antibody conjugates are in optimal condition. Currently reported preservation solutions are often only applicable to certain specific antibody-microsphere conjugates and mainly include buffers, proteins, surfactants, and preservatives; a universal preservation solution preparation method is lacking.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a preservation solution for antibody-microsphere conjugates, its preparation method, and its application.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide a preservation solution for an antibody-microsphere conjugate, wherein the preservation solution comprises, by mass fraction, the following components: buffer solution, 0.05%–1% protein protectant, 0.01%–0.5% surfactant, 0.5%–1.5% sugar, and 0.02%–0.1% preservative;

[0008] The buffer solution includes Good's buffer, with a pH 2-3 higher than the isoelectric point of the antibody.

[0009] Secondly, embodiments of the present invention provide a product for preserving antibody-microsphere conjugates, the active ingredient of which includes a preservation solution for antibody-microsphere conjugates as described in the foregoing embodiments.

[0010] Thirdly, embodiments of the present invention provide a method for preparing a preservation solution for antibody-microsphere conjugates as described in the foregoing embodiments, comprising: mixing the components of the preservation solution according to mass fraction and / or effective concentration.

[0011] Fourthly, embodiments of the present invention provide the use of a preservation solution for antibody-microsphere conjugates as described in the foregoing embodiments, or a product for preserving antibody-microsphere conjugates as described in the foregoing embodiments, in the preparation of immunoassay reagents or kits.

[0012] Fifthly, embodiments of the present invention provide an immunoassay reagent or kit, which includes a preservation solution for antibody-microsphere conjugates as described in the foregoing embodiments or a product for preserving antibody-microsphere conjugates as described in the foregoing embodiments.

[0013] The present invention has the following beneficial effects:

[0014] The preservation solution of this invention provides a buffer environment 2-3 points above the isoelectric point of the labeled antibody, resulting in a higher negative charge on the surface of the colloidal particles. Simultaneously, the surfactant forms a protective layer around the colloidal particles, further improving the dispersibility of the conjugate particles. Furthermore, the Good's buffer system, combined with a low salt ion concentration, ensures the colloidal solution is at a low ion concentration, providing good protection for the electrical double layer on the colloidal particle surface. The sugars and protein protectants provide good protection for the antibody conjugated to the particle surface, enabling it to maintain its biological activity for a longer period. The preservatives inhibit the growth and reproduction of bacteria and other microorganisms in the solution, preventing solution deterioration. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The signal changes over time of the D-Dimer antibody-microsphere conjugates stored in the preservation solutions of Examples 1-2 and Comparative Examples 1-3;

[0017] Figure 2 The particle size of D-Dimer antibody-microsphere conjugates stored in the preservation solutions of Examples 1-2 and Comparative Examples 1-3 changes over time;

[0018] Figure 3 The signal changes over time of the IL-6 antibody-microsphere conjugates stored in the preservation solutions of Example 3 and Comparative Example 4;

[0019] Figure 4 The reaction curves of the IL-6 antibody-microsphere conjugate stored in the preservation solution of Example 3 and Comparative Example 4 on the chromatography reagent;

[0020] Figure 5 The change in particle size of IL-6 antibody-microsphere conjugates stored in the preservation solution of Example 3 and Comparative Example 4 over time. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] First, this embodiment of the invention provides a preservation solution for an antibody-microsphere conjugate, which, by mass fraction, comprises the following components: buffer solution, 0.05%–1% protein protectant, 0.01%–0.5% surfactant, 0.5%–1.5% sugar, and 0.02%–0.1% preservative; wherein the buffer solution includes Good's buffer, and the pH is 2–3 higher than the isoelectric point of the antibody.

[0023] This invention uses the Good's buffer system instead of inorganic salts to avoid introducing excessive inorganic ions and increasing the ionic strength of the solution. Furthermore, controlling the inorganic salt content to a low level, or even omitting it altogether, also helps maintain a low ionic strength, providing better protection for the electric double layer on the colloidal particle surface. In addition, the pH of the buffer solution is determined based on the isoelectric point of the antibody; the solution pH needs to be 2-3 points higher than the antibody's isoelectric point to ensure sufficient negative charge on the microsphere surface. Simultaneously, the surfactant forms a protective layer around the colloidal particles, further improving the dispersibility of the conjugate particles. Sugars and protein protectants provide good protection for the antibody conjugated to the particle surface, allowing it to maintain its biological activity for a longer period. Preservatives inhibit the growth and reproduction of bacteria and other microorganisms in the solution, preventing solution deterioration.

[0024] In this document, the value "pH is 2-3 points higher than the isoelectric point of the antibody" refers to the pH value. In some embodiments, the pH of the buffer solution can be any one of the values ​​of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3 points higher than the isoelectric point of the antibody, or a range between any two values.

[0025] In some embodiments, the Good's buffer includes at least one of HEPES, DIPSO, Tris, Gly, TAPS, and Bis-Tris propane.

[0026] Preferably, the effective concentration of the buffer solution is 10 to 100 mM, specifically any one or any two of the following: 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM.

[0027] In some embodiments, the preservation solution may contain inorganic salts or may not contain inorganic salts.

[0028] In some embodiments, when an inorganic salt is included, the effective concentration of the inorganic salt is 0.01 mM to 50 mM, and the effective concentration can specifically be any one or any two of 0.01 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM and 50 mM.

[0029] Optionally, the inorganic salt is selected from at least one of sodium chloride and potassium chloride.

[0030] In some embodiments, the mass fraction of the protein protectant in the preservation solution can be any one or any two of 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%.

[0031] In some embodiments, the protein protectant includes at least one of bovine serum albumin, casein, and gelatin.

[0032] In some embodiments, the mass fraction of the surfactant in the preservation solution can be any one or any two of 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%.

[0033] In some embodiments, the surfactant includes anionic surfactants and / or nonionic surfactants.

[0034] Preferably, the surfactant includes at least one of Tween-20, Tween-80, and Triton X-100.

[0035] In some embodiments, the mass fraction of the sugars in the preservation solution can be any one or a range between any two of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%.

[0036] In some embodiments, the sugars include at least one of sucrose, glucose, dextran, and trehalose.

[0037] In some embodiments, the mass fraction of the preservative in the preservation solution can be any one or any two of 0.02%, 0.04%, 0.06%, 0.08%, and 0.1%.

[0038] Optionally, the preservative is selected from at least one of sodium azide, thimerosal, and proclin 300.

[0039] This invention also provides a product for preserving antibody-microsphere conjugates, the active ingredient of which includes a preservation solution for antibody-microsphere conjugates as described in any of the foregoing embodiments.

[0040] Optionally, the product may be a reagent or a kit.

[0041] The present invention also provides a method for preparing a preservation solution for antibody-microsphere conjugates as described in any of the foregoing embodiments, comprising: mixing the components of the preservation solution according to mass fraction and / or effective concentration.

[0042] In some embodiments, the preparation method further includes the preparation of a buffer solution: based on the isoelectric point of the antibody, the pH of the buffer solution is prepared to be 2 to 3 higher than the isoelectric point of the antibody.

[0043] The present invention also provides the use of the preservation solution for antibody-microsphere conjugates as described in any of the foregoing embodiments, or the product for preserving antibody-microsphere conjugates as described in the foregoing embodiments, in the preparation of immunoassay reagents or kits.

[0044] Preferably, the immunoassay includes, but is not limited to, chemiluminescence immunoassay, immunoturbidimetry, and fluorescence immunochromatography.

[0045] The present invention also provides an immunoassay reagent or kit, which includes a preservation solution for antibody-microsphere conjugates as described in any of the foregoing embodiments or a product for preserving antibody-microsphere conjugates as described in any of the foregoing embodiments.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] Example 1: Preservation solution for anti-D-Dimer antibody-microsphere conjugate

[0048] The anti-D-Dimer antibody in this embodiment is a mouse monoclonal antibody with an isoelectric point of approximately 6.0. The microspheres are white polystyrene microspheres with a diameter of 220 nm. The microspheres in this embodiment are used in an immunoturbidimetric assay kit.

[0049] The preservation solution for the antibody-microsphere conjugate in this embodiment is formulated as follows: the buffer system is a 50mM Tris system with pH=8.0; BSA is used as a protein protectant with a concentration of 0.1%; no inorganic salts are added; Tween 20 is used as the surfactant with a concentration of 0.04%; sucrose is selected as the sugar with a concentration of 1%; and sodium azide is selected as the preservative with a concentration of 0.1%.

[0050] Example 2: Preservation solution for anti-D-Dimer antibody-microsphere conjugate

[0051] The anti-D-Dimer antibody in this embodiment is a mouse monoclonal antibody with an isoelectric point of approximately 6.0. The microspheres are white polystyrene microspheres with a diameter of 220 nm. The microspheres in this embodiment are used in an immunoturbidimetric assay kit.

[0052] In this embodiment, a preferred preservation solution of the present invention is used, the formulation of which is as follows: the buffer system is a 50mM HEPES system with pH=8.0; BSA is used as a protein protectant with a concentration of 0.1%; no inorganic salts are added; Tween 20 is used as a surfactant with a concentration of 0.04%; sucrose is selected as the sugar with a concentration of 1%; and sodium azide is selected as the preservative with a concentration of 0.1%.

[0053] Example 3: Preservation solution for anti-IL-6 antibody-fluorescent microsphere conjugate

[0054] The anti-IL-6 antibody in this embodiment is a mouse monoclonal antibody with an isoelectric point of approximately 6.0. The fluorescent microspheres are polystyrene microspheres with a diameter of 300 nm. The microspheres in this embodiment are used as a reagent in fluorescent immunochromatography.

[0055] In this embodiment, a preferred preservation solution of the present invention is used, the formulation of which is as follows: the buffer system is a 50mM Tris system with pH=9.0; BSA is used as a protein protectant with a concentration of 0.1%; no inorganic salts are added; Tween 20 is used as a surfactant with a concentration of 0.04%; trehalose is selected as the sugar with a concentration of 1%; and sodium azide is selected as the preservative with a concentration of 0.1%.

[0056] Comparative Example 1: Preservative solution for anti-D-Dimer antibody-microsphere conjugate

[0057] The anti-D-Dimer antibody in this comparative example is a mouse monoclonal antibody with an isoelectric point of approximately 6.0. The microspheres are white polystyrene microspheres with a diameter of 220 nm. These microspheres are used in an immunoturbidimetric assay kit.

[0058] The preservation solution used in this comparative example has the following formulation: a 50 mM Tris buffer system with pH = 8.0; BSA as a protein protectant with a concentration of 0.1%; NaCl as an inorganic salt with a concentration of 150 mM; Tween 20 as a surfactant with a concentration of 0.04%; sucrose as a sugar with a concentration of 1%; and sodium azide as a preservative with a concentration of 0.1%.

[0059] Comparative Example 2: Preservative solution for anti-D-Dimer antibody-microsphere conjugate

[0060] The anti-D-Dimer antibody in this comparative example is a mouse monoclonal antibody with an isoelectric point of approximately 6.0. The microspheres are white polystyrene microspheres with a diameter of 220 nm. The microspheres in this comparative example are used in an immunoturbidimetric assay kit.

[0061] The preservation solution used in this comparative example has the following formulation: the buffer system is a 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate (hereinafter referred to as PB) system, pH=8.0; BSA is used as the protein protectant, with a BSA concentration of 0.1%; no inorganic salts are added; the surfactant used is Tween 20, with a concentration of 0.04%; the sugar used is sucrose, with a concentration of 1%; and the preservative used is sodium azide, with a concentration of 0.1%.

[0062] Comparative Example 3: Preservative solution for anti-D-Dimer antibody-microsphere conjugate

[0063] The anti-D-Dimer antibody in this comparative example is a mouse monoclonal antibody with an isoelectric point of approximately 6.0. The microspheres are white polystyrene microspheres with a diameter of 220 nm. The microspheres in this comparative example are used in an immunoturbidimetric assay kit.

[0064] The preservation solution used in this comparative example has the following formulation: the buffer system is a 50mM HEPES system with pH=7.0; BSA is used as the protein protectant with a concentration of 0.1%; no inorganic salts are added; the surfactant used is Tween 20 with a concentration of 0.04%; the sugar used is sucrose with a concentration of 1%; and the preservative used is sodium azide with a concentration of 0.1%.

[0065] Comparative Example 4: Preservative solution for anti-IL-6 antibody-fluorescent microsphere conjugate

[0066] The anti-IL-6 antibody in this comparative example is a mouse monoclonal antibody with an isoelectric point of approximately 6.0. The fluorescent microspheres are polystyrene microspheres with a diameter of 300 nm. The microspheres in this comparative example are used as reagents in fluorescent immunochromatography.

[0067] The formulation used in this comparative example is as follows: the buffer system is a 50mM PB system with pH=7.3; BSA is used as a protein protectant with a concentration of 0.1%; NaCl is used as the inorganic salt with a concentration of 150mM; Tween 20 is used as the surfactant with a concentration of 0.04%; trehalose is used as the sugar with a concentration of 1%; and sodium azide is used as the preservative with a concentration of 0.1%.

[0068] Verification Example 1

[0069] To verify the performance of the preservation solutions described in Examples 1-2 and Comparative Examples 1-3, a batch of anti-D-Dimer antibody-microsphere conjugates was labeled, and the conjugates were preserved using the preservation solutions described in Examples 1-2 and Comparative Examples 1-3, respectively. The conjugates were stored at 37°C, their particle size was tracked, and a D-Dimer detection kit (immunoturbidimetric assay) was prepared to test the change in their signal over time.

[0070] Figure 1 The changes in the signals of the conjugates stored in the preservation solutions of Examples 1-2 and Comparative Examples 1-3 over time were shown. It can be observed that the conjugate signals in Examples 1-2 remained basically stable over 31 days, but the conjugate signals in Comparative Examples 1-3 continued to rise, with the largest increase observed in Comparative Example 3.

[0071] Figure 2 The changes in particle size of the coupling compounds stored in the preservation solutions of Examples 1-2 and Comparative Examples 1-3 over time are shown. Similar to the signal change trend, the particle size in Examples 1-2 remained basically stable, but the particle size of the coupling compounds in Comparative Examples 1-3 continuously increased, with the largest increase observed in Comparative Example 3. Simultaneously, visual observation of the coupling compound sedimentation revealed that the coupling compounds in Examples 1-2 showed almost no sedimentation, while the coupling compounds in Comparative Examples 1-3 exhibited significant sedimentation, with the most severe sedimentation observed in Comparative Example 3.

[0072] Comparing Example 1 and Comparative Example 1, it can be seen that increasing the salt concentration of the preservation solution leads to instability of the conjugate when the buffer system and pH are the same. Comparing Example 1 and Comparative Example 2, it can be seen that, without adding NaCl, changing the buffer system from a Tris system to a PB system also makes the conjugate unstable. This is because both NaCl and PB increase the ionic strength of the preservation solution. Excessively high ionic strength weakens the electrostatic repulsion caused by the surface charge of the antibody-microsphere conjugate, making the nanoparticles prone to aggregation and sedimentation.

[0073] Comparing Example 2 and Comparative Example 3, it can be seen that after the pH of the preservation solution was lowered from 8.0 to 7.0, the conjugates in the preservation solution became unstable and easily aggregated and precipitated. This is because the isoelectric point of the antibodies conjugated to the nanospheres is 6.0. When the pH of the preservation solution is 8.0, the antibodies will carry a sufficient negative charge, thus generating enough electrostatic repulsion between the microspheres to resist the van der Waals forces and hydrophobic forces between them. The latter two tend to cause the microspheres to aggregate. However, when the pH of the preservation solution is lowered, the negative charge carried by the antibodies decreases, and the electrostatic repulsion between the microspheres is insufficient to resist the attraction, therefore the microspheres will aggregate and precipitate.

[0074] As can be seen from Examples 1 and 2, the conjugate can be kept stable in the preservation solution using either Tris or HEPES buffer systems. In fact, other Good's buffers can also be used, as long as the ionic strength is kept low (Good's buffers have a lower ionic strength than inorganic salt buffers at the same concentration).

[0075] Verification Example 2

[0076] To verify the performance of the preservation solutions described in Example 3 and Comparative Example 4, a batch of IL-6 antibody-fluorescent microsphere conjugates were labeled and preserved using the preservation solutions described in Example 3 and Comparative Example 4, respectively. The conjugates were stored at 2-8°C, their particle size was tracked, and they were used to prepare an IL-6 detection kit (fluorescent immunochromatography) to test the reaction curves and signal changes over time.

[0077] Figure 3 and Figure 4 The changes in signal and reaction curves of the conjugates stored in the preservation solutions of Example 3 and Comparative Example 4 over time are shown. It can be seen that the signal of the conjugate in Comparative Example 4 decreases significantly with prolonged storage time, and the baseline of the reaction curve becomes significantly warped. In contrast, the signal of the conjugate in Example 3 is stable, and the reaction curve is relatively flat.

[0078] Figure 5 The changes in particle size of the couplings stored in the preservation solutions of Example 3 and Comparative Example 4 are shown. It can be seen that the particle size of the couplings stored in Example 3 is relatively stable, while the particle size of the couplings stored in Comparative Example 4 increases significantly over time. Furthermore, visual observation of the sedimentation of the couplings reveals that the couplings in Example 3 show virtually no sedimentation after 30 days of storage, while significant sedimentation is observed in the couplings in Comparative Example 4 after 30 days of storage.

[0079] As can be seen from Example 3 and Comparative Example 4, the composition of the preservation solution described in this invention is applicable to different methodological platforms and has a certain degree of versatility.

[0080] In summary, as can be seen from the examples provided in this invention, the preservation solution of this invention has a good preservation effect on microsphere-antibody conjugates and can be widely used as a reagent in different methodologies such as immunochromatography and immunoturbidimetry, demonstrating good versatility. The preservation solution of this invention can enhance the stability of the stored conjugates, significantly improve reagent performance, and has good commercial value.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A preservation solution for an antibody-microsphere conjugate, characterized in that, The preservation solution comprises, by mass fraction, the following components: buffer solution, 0.05%–1% protein protectant, 0.01%–0.5% surfactant, 0.5%–1.5% sugars, and 0.02%–0.1% preservative; The buffer solution is Good's buffer solution, with a pH 2-3 higher than the isoelectric point of the antibody; the surfactant is a nonionic surfactant.

2. The preservation solution for the antibody-microsphere conjugate according to claim 1, characterized in that, The Good's buffer comprises at least one of HEPES, DIPSO, Tris, Gly, TAPS, and Bis-Tris propane.

3. The preservation solution for the antibody-microsphere conjugate according to claim 1, characterized in that, The effective concentration of the buffer solution is 10~100mM.

4. The preservation solution for the antibody-microsphere conjugate according to any one of claims 1 to 3, characterized in that, The protein protectant includes at least one of bovine serum albumin, casein, and gelatin.

5. The preservation solution for the antibody-microsphere conjugate according to claim 1, characterized in that, The surfactant includes at least one of Tween-20, Tween-80, and Triton X-100.

6. The preservation solution for the antibody-microsphere conjugate according to any one of claims 1 to 3, characterized in that, The sugars include at least one of sucrose, glucose, dextran, and trehalose.

7. The preservation solution for the antibody-microsphere conjugate according to any one of claims 1 to 3, characterized in that, The preservative is selected from at least one of sodium azide, thimerosal, and proclin 300.

8. A product for preserving conjugates of antibodies and microspheres, characterized in that, Its active ingredient includes a preservation solution for the antibody-microsphere conjugate as described in any one of claims 1 to 7.

9. The method for preparing the preservation solution of the antibody-microsphere conjugate according to any one of claims 1 to 7, characterized in that, It includes: The components of the preservation solution are mixed according to their mass fraction and / or effective concentration.

10. The use of the preservation solution for the antibody-microsphere conjugate as described in any one of claims 1 to 7, or the product for preserving the antibody-microsphere conjugate as described in claim 8, in the preparation of immunoassay reagents or kits.

11. The application according to claim 10, characterized in that, The immunoassay includes at least one of chemiluminescence immunoassay, immunoturbidimetry, and fluorescence immunochromatography.

12. An immunoassay reagent or kit, characterized in that, It includes a preservation solution for antibody-microsphere conjugates as described in any one of claims 1 to 7, or a product for preserving antibody-microsphere conjugates as described in claim 8.

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