Method for improving stability of fluorescent microsphere labeled antibody freeze-dried product

Abstract

The invention discloses a method for improving the stability of a fluorescent microsphere labeled antibody freeze-dried product, which comprises the following steps: S1, mixing a polystyrene microsphere solution containing a fluorescent dye, an EDC solution and an NHS solution, and carrying out an activation reaction to obtain an activated microsphere solution; s2, an antibody solution is added into the activated microsphere solution for a coupling reaction, and a coupling reaction solution is obtained; s3, adding a microsphere confining liquid into the coupling reaction liquid to carry out confining reaction, then carrying out centrifugation, removing a supernatant, then adding a microsphere washing liquid to carry out cleaning, and then carrying out centrifugation to remove the supernatant so as to obtain confined microspheres; and S4, adding a microsphere storage solution into the closed microspheres, ultrasonically mixing uniformly to obtain a microsphere storage solution, and storing the microsphere storage solution in a dark place at 2-8 DEG C for later use. The complex formula of the microsphere preserving fluid can effectively prevent the antibody structure from being damaged in the freeze-drying process, meanwhile, the stability of fluorescein is kept, the activity retention rate and the fluorescence intensity of the antibody are remarkably improved, and the shelf life of the product is prolonged.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay technology, and in particular to a method for improving the stability of a freeze-dried product of a fluorescent microsphere-labeled antibody. Background Art

[0002] Antigen-antibody atopy testing is of great significance in medical and biological research, and can be applied in disease diagnosis, immune status assessment, allergic reaction detection, autoimmune disease diagnosis, and other fields. Currently, methods for antigen-antibody atopy testing include fluorescence immunochromatography (FICA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), and digital fluorescence immunosorbent assay systems. The digital fluorescence immunosorbent assay system is an immunoassay technology based on digital fluorescent signals, combining the advantages of immunoadsorption and fluorescence detection. It generally includes a plate-based platform, fluorescent particles, a fluorescence microscopy system, and image recognition and data processing software. During the detection process, the capture substance corresponding to the bioactive substance is first adsorbed onto the plate platform. Then, the fluorescent particles are combined with the detection substance corresponding to the bioactive substance to form a fluorescently labeled complex. The fluorescently labeled bioactive substance is then combined with the capture substance on the plate platform to form a stable adsorption complex. Finally, the fluorescence signal is enhanced through a specific fluorescence amplification technology and digitally encoded for subsequent imaging and analysis. The fluorescence signal is imaged using a fluorescence microscopy system, and then the image is analyzed using image recognition and data processing software to generate the final detection result.

[0003] The digital fluorescent immunosorbent assay system has the advantages of high sensitivity, high specificity and digital analysis. Fluorescent particles are its core components. Fluorescent particles generally include core materials, surface modification layers and stabilizing components. The core materials are fluorescent dyes, quantum dots, fluorescent microspheres, etc. The surface modification layer is used to connect the fluorescent particles and the ligands of the target biological molecules, such as antibody coupling, nucleic acid probe coupling, etc. The stabilizing components are surfactants, buffers, antioxidants, etc. However, the liquid storage stability of fluorescent microsphere-labeled antibodies is poor, and they are also very unstable during the preparation of fluorescent microsphere-labeled antibodies into freeze-dried products. Specifically, on the one hand, in the traditional freeze-drying process, fluorescent microsphere-labeled antibodies are easily deactivated due to ice crystal formation, protein denaturation or aggregation during freezing and sublimation. Especially in the absence of an effective protective agent, the structural integrity of the antibody is difficult to maintain, resulting in a significant decrease in the activity of the freeze-dried product. On the other hand, the fluorescent dyes (such as FITC, Alexa Fluor, etc.) in fluorescent microsphere-labeled antibodies are sensitive to light, oxygen and temperature, and are easily degraded or the fluorescence intensity is weakened during the freeze-drying process. In addition, the binding of fluorescent dyes to antibodies in fluorescent microsphere-labeled antibodies may also be destroyed during the freeze-drying process, affecting the sensitivity and accuracy of the detection.

[0004] Therefore, there is an urgent need for a fluorescent-labeled antibody freeze-dried product with high stability. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the stability of a freeze-dried product of a fluorescent microsphere-labeled antibody, so as to solve the problem of poor stability of the freeze-dried product of the fluorescent microsphere-labeled antibody.

[0006] To achieve the above objectives, the present invention provides a method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody, comprising the following steps:

[0007] S1: Activation of polystyrene microspheres containing fluorescent dyes

[0008] A polystyrene microsphere solution containing a fluorescent dye, an EDC solution and an NHS solution are mixed to perform an activation reaction to obtain an activated microsphere solution;

[0009] S2: Coupling reaction

[0010] adding the antibody solution to the activated microsphere solution to perform a coupling reaction to obtain a coupling reaction solution;

[0011] S3: Sealing of microspheres

[0012] Adding microsphere blocking solution to the coupling reaction solution to perform blocking reaction, then centrifuging, removing the supernatant, then adding microsphere washing solution to wash, and then centrifuging to remove the supernatant to obtain blocked microspheres;

[0013] S4: Storage of Microspheres

[0014] Add microsphere storage solution to the sealed microspheres, mix thoroughly by ultrasonication to obtain microsphere storage solution, and store in the dark at 2-8°C until use;

[0015] S5: Preparation of lyophilized products

[0016] The microsphere storage solution is freeze-dried to obtain a fluorescent microsphere-labeled antibody freeze-dried product.

[0017] Preferably, in step S1, the preparation method of the polystyrene microsphere solution containing fluorescent dye is:

[0018] S11: A 1% solid content polystyrene microsphere suspension containing fluorescent dye was added to the coupling buffer, ultrasonically mixed, then centrifuged and washed twice with coupling buffer;

[0019] S12: adding the solid material obtained in step S11 into a coupling buffer solution, and mixing the mixture evenly by ultrasonication to obtain a polystyrene microsphere solution containing a fluorescent dye.

[0020] Preferably, in step S1, the coupling buffer is 10 mM pH = 6.2 ± 0.05 MES and 0.05% ProClin 300;

[0021] The EDC solution was prepared by adding EDC to the coupling buffer solution and mixing evenly to form an EDC solution with a concentration of 10 mg / mL;

[0022] The NHS solution was prepared by adding NHS to the coupling buffer solution and mixing uniformly to form an NHS solution with a concentration of 10 mg / mL.

[0023] Preferably, in step S1, the activation reaction is carried out on a turntable at 37° C. and 40 r / min in the dark for 15 to 30 min.

[0024] Preferably, in step S1, after the activation reaction, centrifugation is performed, and then washing is performed with a coupling buffer. After washing, a coupling buffer is added and mixed evenly to obtain an activated microsphere solution.

[0025] Preferably, in step S2, the antibody solution is prepared by adding the antibody to be labeled into a coupling buffer and mixing uniformly to obtain an antibody solution; the coupling reaction is carried out on a turntable at 37° C. and 40 r / min in the dark for 15 to 30 minutes.

[0026] Preferably, in step S3, the microsphere blocking solution is a borate buffer solution composed of 5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, and 0.05% Tween-20, pH = 9.0 ± 0.05, 1% BSA, and 0.24% ethanolamine; the blocking reaction is carried out on a turntable at 37° C. and 40 rpm in the dark for 1 hour; the microsphere washing solution is 50 mM Tris, pH = 8.0 ± 0.05, 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300.

[0027] Preferably, in step S4, the microsphere storage solution is 25 mM pH=7.2±0.05 Tris, 150 mM NaCl, 2‰ Proclin 300, 1% BSA, 1‰ ADP and 5% trehalose; the concentration of the microsphere storage solution is 1 mg / mL.

[0028] The second aspect of the present invention provides a lyophilized product of fluorescent microsphere-labeled antibodies prepared by the above method.

[0029] The third aspect of the present invention provides the use of a lyophilized product of fluorescent microsphere-labeled antibodies in a digital fluorescent immunosorbent assay system.

[0030] Therefore, the present invention adopts the above-mentioned method for improving the stability of fluorescent microsphere-labeled antibody freeze-dried products, which has the following beneficial effects:

[0031] (1) The present invention uses fluorescent microsphere-labeled antibodies as detection reagent components in a digital fluorescent immunosorbent assay system. Based on the basic principle of the double antibody sandwich method, the antibodies are first solid-phased on the detection plate. When the antigen in the sample binds to the solid-phased antibody, the fluorescein-labeled antibody binds to another antigenic determinant on the antigen, thereby forming a "sandwich" complex between the solid-phased antibody and the fluorescein-labeled antibody. In this way, the present invention can achieve high-sensitivity and high-specificity detection of antigens and quantify the antigen content by counting fluorescent particles.

[0032] (2) The present invention proposes a method for preparing a fluorescently labeled antibody freeze-dried product, aiming to address the existing difficulty in simultaneously maintaining antibody activity and fluorescence properties. By developing a new lyoprotectant and optimizing freeze-drying process parameters, the present invention can improve the storage stability of the product. This technology is expected to significantly enhance the quality and performance of fluorescently labeled antibody freeze-dried products, meeting the needs of biomedical and life science research.

[0033] (3) The composite formula of the microsphere preservation solution developed in the present invention can effectively prevent the destruction of the antibody structure during the freeze-drying process while maintaining the stability of the fluorescein, significantly improving the activity retention rate and fluorescence intensity of the antibody and extending the shelf life of the product.

[0034] (4) The freeze-dried product prepared by the present invention also has good rehydration properties. The freeze-dried product can quickly recover to its original properties when rehydrated, and the rehydration is faster and more complete.

[0035] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the fluorescence of the analyte detected by fluorescent microsphere-labeled antibodies at an excitation wavelength of 350-550nm / emission wavelength of 530-650nm. DETAILED DESCRIPTION

[0037] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and specific operation process, but the present invention is not limited to this embodiment.

[0038] Example 1

[0039] A method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody comprises the following steps:

[0040] S1: Raw material preparation and formulation

[0041] The fluorescent microspheres used in this example are polystyrene microspheres containing fluorescent dye (hereinafter referred to as fluorescent microspheres), purchased from Suzhou Weidu Biotechnology Co., Ltd. (item number FG0300CA, particle size 300 nm, green fluorescence, excitation wavelength 488 nm, emission wavelength 520 nm, surface modified with carboxyl groups, solid content 1.0%, named green fluorescent microspheres, fluorescent dye embedded in the microspheres).

[0042] The solution preparation includes coupling buffer, EDC solution, NHS solution, microsphere blocking solution, microsphere washing solution and microsphere storage solution. The specific preparation process is as follows:

[0043] S11: Coupling buffer: 10 mM MES (pH 6.2 ± 0.05), containing 0.05% ProClin 300;

[0044] S12: EDC solution: 10 mg / mL, prepared with coupling buffer, ready for use;

[0045] S13: NHS solution: 10 mg / mL, prepared with coupling buffer, ready for use;

[0046] S14: Microsphere blocking solution: borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0 ± 0.05), 1% BSA, 0.24% ethanolamine;

[0047] S15: microsphere washing solution: 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300;

[0048] S16: Microsphere storage solution: 25 mM Tris (pH 7.2±0.05), 150 mM NaCl, 2‰ Proclin300, 1% BSA, 1‰ ADP, 5% trehalose.

[0049] S2: Activation of microspheres

[0050] S21: 0.05 mL of fluorescent microsphere suspension (1% solid content) was added to a 2 mL centrifuge tube containing 1 mL of coupling buffer, mixed by sonication, and centrifuged at 15°C and 20,000 rpm for 10 min. The supernatant was removed.

[0051] S22: Add 1 mL of microsphere coupling buffer, mix thoroughly by ultrasonication, centrifuge at 15°C and 20,000 rpm for 10 min, and remove the supernatant;

[0052] S23: Add 1 mL of microsphere coupling buffer and mix thoroughly by ultrasonication;

[0053] S24: Add 3.5 μL of EDC solution and vortex mix, then add 33 μL of NHS solution and sonicate to mix;

[0054] S25: Place the centrifuge tube on a turntable and activate it in the dark for 25 min at 37°C and 40 rpm.

[0055] S26: Centrifuge at 15°C, 20,000 rpm for 10 min and remove the supernatant;

[0056] S27: Add 1.5 mL of coupling buffer, mix thoroughly by ultrasonication, centrifuge at 15°C, 20,000 rpm for 10 min, and remove the supernatant;

[0057] S28: Add 1.5 mL of coupling buffer and repeat washing once;

[0058] S29: Add 0.75 mL of coupling buffer and mix thoroughly by ultrasonication to obtain an activated microsphere solution;

[0059] S3: Coupling reaction

[0060] S31: Add 50 μg of the antibody to be labeled to 0.25 mL of coupling buffer to obtain an antibody solution;

[0061] S32: adding the antibody solution to the mixed activated microsphere solution and vortexing to mix;

[0062] S33: Place the centrifuge tube on a turntable and incubate at 37°C, 40 rpm, in the dark for 2 h.

[0063] S4: Sealing and storage of microspheres

[0064] S41: Add 0.5 mL of microsphere blocking solution to the centrifuge tube and vortex to mix;

[0065] S42: Place the centrifuge tube on a turntable and incubate at 37°C, 40 rpm, in the dark for 1 h.

[0066] S43: Centrifuge at 15°C, 20,000 rpm for 10 min and remove the supernatant;

[0067] S44: Add 1.5 mL of microsphere washing solution, mix thoroughly by ultrasonication, centrifuge at 15°C and 20,000 rpm for 10 min, and remove the supernatant;

[0068] S45: Add 1.5 mL of microsphere washing solution and repeat the washing process once;

[0069] S46: Finally, add 0.5 mL of microsphere storage solution (microsphere final concentration 1 mg / mL) to the centrifuge tube, mix thoroughly by ultrasonication, and store in the dark at 2-8°C until use.

[0070] Example 2

[0071] This example studies the effect of the composition of the microsphere storage solution on the lyophilized product. The other steps are the same as in Example 1. Commonly used PBS and Tris-NaCl are selected for comparison. In addition, considering the stability of the lyophilized product, a certain amount of protein BSA and protein protective agent ADP are added. Considering the appearance of the lyophilized product, sucrose or trehalose is added.

[0072] Specifically, the types and concentrations of buffer, protein, and sugar added to the microsphere storage solution are as follows:

[0073] Formula A: PBS + 2‰ Proclin 300 + 1% BSA + 1‰ ADP + 5% trehalose;

[0074] Formula B: PBS + 2‰ Proclin 300 + 2% BSA + 1‰ ADP + 5% sucrose;

[0075] Formula C: Tris-NaCl + 2‰ Proclin 300 + 1% BSA + 1‰ ADP + 5% trehalose;

[0076] Formula D: Tris-NaCl + 2‰ Proclin 300 + 2% BSA + 1‰ ADP + 5% sucrose;

[0077] It should be noted that: Formula C is Example 1.

[0078] The microsphere storage solution prepared with the above formula was diluted 20-fold and then dispensed into brown glass bottles for lyophilization. The lyophilization process included pre-freezing and vacuum freeze-drying. The pre-freezing was performed in a -80°C refrigerator overnight, and then the solution was transferred to a vacuum freeze dryer for vacuum drying. The lyophilization temperature and time are detailed in Table 1. The lyophilized products were stored at 37°C for 10 days and at 2-8°C, respectively. The stability and appearance of the lyophilized products were compared by measuring the number of fluorescent particles in the lyophilized products.

[0079] The fluorescent particle count test procedure involved adding samples of varying concentrations to an ELISA plate. Samples P1 to P5 had concentrations of 100, 33, 11, 3, and 1 pg / mL, respectively. The lyophilized products prepared using Recipes A to D were then added to the plate. The samples and lyophilized products reacted on the plate for 30 minutes. After the reaction, the plate was washed five times with microsphere wash buffer and then placed in a digital fluorescence immunosorbent assay system for imaging and signal counting. The test results are shown in Tables 2 and 3. Figure 1 This is the imaging result of formula C. It can be seen from the figure that the microspheres have obvious green fluorescence signals.

[0080] Table 1 Detailed vacuum drying process parameter settings

[0081]

[0082] Table 2 Comparison results of freeze-dried product stability

[0083]

[0084]

[0085] Table 3 Comparison results of freeze-dried product appearance

[0086] Freeze-dried product form Recipe A groove Recipe B round cake shape Recipe C round cake shape Recipe D honeycomb

[0087] As shown in Table 2, freeze-dried products prepared using different preservative solutions showed no significant differences when stored at 2-8°C. However, after 10 days at 37°C, the luminescence values of Formulations A, B, and D varied significantly, with Formulation C showing the greatest variation. Therefore, Formulation C was selected as the optimal formulation. As shown in Table 3, the freeze-dried appearance of Formulation C was also satisfactory. In summary, Formulation C is the preferred preservative solution for fluorescently labeled antibodies.

[0088] The above freeze-dried products were subjected to rehydration tests as follows:

[0089] Ten sets of freeze-dried fluorescent microspheres were randomly selected and numbered 1 to 10 for each concentration, and measured three times in the following order.

[0090] Measurement order: 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9.

[0091] Record the measurement results and calculate the F value and CV according to the following formula 瓶内 and CV 瓶间 When the statistical result F value is less than or equal to 10, it is considered that the uniformity between fluorescent microsphere bottles is good. And calculate the average value of all test results, and then perform CV 瓶内 and CV 瓶间 The calculation of CV is required to be less than 10%. When the statistical result F value is greater than 10, it is considered that the uniformity in the fluorescent microsphere bottle is poor and CV is no longer calculated. 瓶内 and CV 瓶间。

[0092] When the statistical result F≤1, the standard deviation within the bottle is used instead of the standard deviation between bottles, that is, S bb =Sr.

[0093] Formula 1

[0094] Formula 2 Formula 3 SS 瓶内 =SS 总和 -SS 瓶间 Formula 4 Formula 5 Formula 6 Formula 7 Formula 8 Formula 9 Formula 10

[0095] Where:

[0096] SS—variance;

[0097] ν—degrees of freedom;

[0098] MS—mean square;

[0099] F—F test value;

[0100] n i —Number of repeated measurements of sample i;

[0101] n0—effective measurement times;

[0102] s bb —Inter-bottle standard deviation;

[0103] sr —Repeatability standard deviation (i.e., within-bottle standard deviation); x i —Measurement or calculation result of each sample; x—measurement or calculation result — overall mean value;

[0104] The test results are as follows (unit: mAU / mL):

[0105] Record measurement results and calculate F value and CV 瓶内 and CV 瓶间 :

[0106] Table 4 Rehydration uniformity test results

[0107]

[0108] The above data show that the F value of the calibrator is less than 10, indicating good inter-bottle uniformity of the fluorescent microspheres and their suitability for use as calibrators. Furthermore, both the intra-bottle and inter-bottle CVs are less than 10%, meeting the requirements for the use of lyophilized fluorescent microspheres and demonstrating good rehydration properties.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody, characterized by: The following steps are involved: S1: Activation of polystyrene microspheres containing fluorescent dyes A polystyrene microsphere solution containing a fluorescent dye, an EDC solution and an NHS solution are mixed to perform an activation reaction to obtain an activated microsphere solution; S2: Coupling reaction adding the antibody solution to the activated microsphere solution to perform a coupling reaction to obtain a coupling reaction solution; S3: Sealing of microspheres Adding microsphere blocking solution to the coupling reaction solution to perform blocking reaction, then centrifuging, removing the supernatant, then adding microsphere washing solution to wash, and then centrifuging to remove the supernatant to obtain blocked microspheres; S4: Storage of Microspheres Add microsphere storage solution to the sealed microspheres, mix thoroughly by ultrasonication to obtain microsphere storage solution, and store in the dark at 2-8°C until use; S5: Preparation of lyophilized product The microsphere storage solution is freeze-dried to obtain a fluorescent microsphere-labeled antibody freeze-dried product.

2. A method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody according to claim 1, characterized in that: In step S1, the preparation method of the polystyrene microsphere solution containing fluorescent dye is as follows: S11: Add a 1% solid content polystyrene microsphere suspension containing fluorescent dye to the coupling buffer, mix thoroughly by ultrasonication, then centrifuge and wash twice with coupling buffer; S12: adding the solid material obtained in step S11 into a coupling buffer solution, and mixing the mixture evenly by ultrasonication to obtain a polystyrene microsphere solution containing a fluorescent dye.

3. A method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody according to claim 2, characterized in that: In step S1, the coupling buffer was 10 mM pH = 6.2 ± 0.05 MES and 0.05% ProClin 300; The EDC solution was prepared by adding EDC to the coupling buffer solution and mixing evenly to form an EDC solution with a concentration of 10 mg / mL; The NHS solution was prepared by adding NHS to the coupling buffer solution and mixing uniformly to form an NHS solution with a concentration of 10 mg / mL.

4. The method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody according to claim 1, characterized in that: In step S1, the activation reaction is carried out on a turntable at 37° C. and 40 r / min in the dark for 15 to 30 minutes.

5. The method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody according to claim 3, characterized in that: In step S1, after the activation reaction, centrifugation is performed, and then washing is performed with a coupling buffer. After washing, a coupling buffer is added and mixed evenly to obtain an activated microsphere solution.

6. The method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody according to claim 3, characterized in that: In step S2, the antibody solution is prepared by adding the antibody to be labeled into the coupling buffer and mixing uniformly to obtain the antibody solution; the coupling reaction is carried out on a turntable at 37° C. and 40 rpm in the dark for 15 to 30 minutes.

7. The method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody according to claim 1, characterized in that: In step S3, the microsphere blocking solution is a pH = 9.0 ± 0.05 borate buffer composed of 5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, and 0.05% Tween-20, 1% BSA, and 0.24% ethanolamine. The blocking reaction is carried out on a turntable at 37°C and 40 rpm for 1 hour in the dark. The microsphere washing solution is 50 mM pH = 8.0 ± 0.05 Tris, 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300.

8. The method for improving the stability of a lyophilized product of a fluorescent microsphere-labeled antibody according to claim 1, characterized in that: In step S4, the microsphere storage solution is 25 mM pH = 7.2 ± 0.05 Tris, 150 mM NaCl, 2‰ Proclin 300, 1% BSA, 1‰ ADP and 5% trehalose; the concentration of the microsphere storage solution is 1 mg / mL.

9. A lyophilized product of fluorescent microsphere-labeled antibody prepared by the method according to any one of claims 1 to 8.

10. Use of the lyophilized product of fluorescent microsphere-labeled antibody according to claim 9 in a digital fluorescent immunosorbent assay system.

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