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

Abstract

The application discloses a method for improving the stability of freeze-dried fluorescent microsphere-labeled antibody, which comprises the following steps: S1, mixing polystyrene microsphere solution containing fluorescent dye, EDC solution and NHS solution to perform activation reaction, and obtaining activated microsphere solution; S2, adding antibody solution into the activated microsphere solution to perform coupling reaction, and obtaining coupling reaction liquid; S3, adding microsphere blocking solution into the coupling reaction liquid to perform blocking reaction, then performing centrifugation to remove supernatant, then adding microsphere washing solution to perform cleaning, then performing centrifugation to remove supernatant, and obtaining blocked microspheres; and S4, adding microsphere storage solution into the blocked microspheres, and uniformly mixing by ultrasonic to obtain microsphere storage solution, which is stored at 2-8 DEG C in dark for standby use. The microsphere storage solution composite formula developed by the application can effectively prevent the destruction of antibody structure in the freeze-drying process, meanwhile, the stability of fluorescein is maintained, the activity retention rate of the antibody and the fluorescence intensity are significantly improved, and the shelf life of the product is prolonged.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a method for improving the stability of lyophilized products containing fluorescent microsphere-labeled antibodies. Background Technology

[0002] Antigen-antibody atopy detection plays a crucial role in medical and biological research, with applications in disease diagnosis, immune status assessment, allergy detection, and autoimmune disease assessment. Currently, methods for antigen-antibody atopy detection include fluorescence immunochromatography (FICA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), and digital fluorescence immunoassay systems. Among these, the digital fluorescence immunoassay system is an immunoassay technique based on digital fluorescence signals, combining the advantages of immunoadsorption and fluorescence detection. It typically includes a plate platform, fluorescent particles, a fluorescence microscopy imaging system, and image recognition and data processing software. In 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. Next, the fluorescently labeled bioactive substance is combined with the capture substance on the plate platform to form a stable adsorption complex. Finally, the fluorescence signal is enhanced by a specific fluorescence amplification technique and digitally encoded for subsequent imaging and analysis. The fluorescence signal is imaged using a fluorescence microscopy imaging system, and then the image is analyzed by image recognition and data processing software to generate the final detection result.

[0003] Digital fluorescence immunoassay systems offer advantages such as high sensitivity, high specificity, and digital analysis. Fluorescent particles are their core component, typically comprising a core material, a surface modification layer, and stabilizing agents. The core material includes fluorescent dyes, quantum dots, and fluorescent microspheres. The surface modification layer connects the fluorescent particles to ligands of the target biomolecules, such as antibody conjugation or nucleic acid probe conjugation. Stabilizing agents include surfactants, buffers, and antioxidants. However, fluorescent microsphere-labeled antibodies exhibit poor stability in liquid storage and are highly unstable during the lyophilization process. Specifically, in traditional lyophilization processes, fluorescent microsphere-labeled antibodies are prone to losing activity due to ice crystal formation, protein denaturation, or aggregation during freezing and sublimation, especially in the absence of effective protective agents. Maintaining the structural integrity of the antibody is difficult, leading to a significant reduction in the activity of the lyophilized product. Furthermore, the fluorescent dyes in fluorescent microsphere-labeled antibodies (such as FITC and Alexa Fluor) are sensitive to light, oxygen, and temperature, and are prone to degradation or weakening of fluorescence intensity during lyophilization. In addition, the binding of fluorescent dyes to antibodies in fluorescent microsphere-labeled antibodies may be disrupted during the freeze-drying process, affecting the sensitivity and accuracy of detection.

[0004] Therefore, there is an urgent need for a highly stable lyophilized fluorescently labeled antibody. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the stability of lyophilized fluorescent microsphere-labeled antibodies, so as to solve the problem of poor stability of lyophilized fluorescent microsphere-labeled antibodies.

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

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

[0008] An activated microsphere solution was obtained by mixing a polystyrene microsphere solution containing fluorescent dye, an EDC solution, and an NHS solution.

[0009] S2: Coupling reaction

[0010] The antibody solution was added to the activated microsphere solution to carry out the coupling reaction, and the coupling reaction solution was obtained.

[0011] S3: Closure of microspheres

[0012] Microsphere blocking solution was added to the coupling reaction solution to carry out the blocking reaction, followed by centrifugation to remove the supernatant. Then, microsphere washing solution was added for washing, followed by centrifugation to remove the supernatant, and the blocked microspheres were obtained.

[0013] S4: Preservation of Microspheres

[0014] Add microsphere preservation solution to the sealed microspheres, mix evenly by ultrasonication to obtain microsphere storage solution, and store at 2-8℃ in the dark for later use;

[0015] S5: Preparation of freeze-dried products

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

[0017] Preferably, in step S1, the method for preparing the polystyrene microsphere solution containing fluorescent dye is as follows:

[0018] S11: Take a 1% solid content polystyrene microsphere suspension containing fluorescent dye and add it to the coupling buffer. Mix it evenly by sonication, then centrifuge and wash it twice with the coupling buffer.

[0019] S12: Add the solid material obtained in step S11 to the coupling buffer, and mix it evenly by sonication to obtain a polystyrene microsphere solution containing 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 is prepared by adding EDC to the coupling buffer solution and mixing thoroughly to form an EDC solution with a concentration of 10 mg / mL.

[0022] The NHS solution is prepared by adding NHS to a coupling buffer solution and mixing thoroughly 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 rotating disk, and activated in the dark for 15 to 30 minutes at 37°C and 40 r / min.

[0024] Preferably, in step S1, after the activation reaction, centrifugation is performed, followed by washing with coupling buffer. After washing, coupling buffer is added again 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 the coupling buffer and mixing it evenly to obtain the antibody solution; the coupling reaction is carried out on a turntable and coupled in the dark for 15-30 minutes at 37°C and 40 r / min.

[0026] Preferably, in step S3, the microsphere blocking solution is a borate buffer solution with pH = 9.0 ± 0.05 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 r / min, in the dark for 1 h; 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.

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

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

[0029] The third aspect of this invention provides the application of lyophilized fluorescent microsphere-labeled antibodies in a digital fluorescence immunoassay detection system.

[0030] Therefore, the method described above for improving the stability of lyophilized fluorescent microsphere-labeled antibodies has the following beneficial effects:

[0031] (1) This invention uses fluorescent microsphere-labeled antibodies as a detection reagent component in a digital fluorescence immunoassay detection system. Based on the fundamental principle of the double-antibody sandwich method, the antibody is first immobilized on the detection plate. When the antigen in the sample binds to the immobilized antibody, the fluorescently labeled antibody binds to another antigenic determinant on the antigen, thus forming a sandwich-like complex between the immobilized antibody and the fluorescently labeled antibody. This invention achieves high sensitivity and high specificity detection of antigens and quantifies the antigen content by the number of fluorescent particles.

[0032] (2) This invention proposes a method for preparing lyophilized fluorescently labeled antibodies, aiming to solve the problem of simultaneously maintaining antibody activity and fluorescence performance in existing technologies. By developing novel lyophilization protectants and optimizing lyophilization process parameters, this invention can improve the storage stability of the product. This technology is expected to significantly improve the quality and performance of lyophilized fluorescently labeled antibodies, meeting the needs of biomedical and life science research.

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

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

[0035] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0036] Figure 1 It is a fluorescence detection method using fluorescent microsphere-labeled antibodies to detect the fluorescence of the analyte at an excitation wavelength of 350-550 nm and an emission wavelength of 530-650 nm. Detailed Implementation

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

[0038] Example 1

[0039] A method for improving the stability of lyophilized products containing fluorescent microsphere-labeled antibodies includes the following steps:

[0040] S1: Raw material preparation and formulation

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

[0042] The solution preparation includes coupling buffer, EDC solution, NHS solution, microsphere blocking solution, microsphere washing solution, and microsphere preservation 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, freshly prepared and used immediately;

[0045] S13: NHS solution: 10 mg / mL, prepared with coupling buffer, and used immediately;

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

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

[0048] S16: Microsphere preservation solution: 25mM Tris (pH 7.2±0.05), 150mM NaCl, 2‰ Proclin 300, 1% BSA, 1‰ ADP, 5% Trehalose.

[0049] S2: Activation of microspheres

[0050] S21: Take 0.05 mL of fluorescent microsphere suspension (1% solid content) and add it to a 2 mL centrifuge tube containing 1 mL of coupling buffer. Mix well by sonication and centrifuge at 15℃ and 20000 rpm for 10 min. Remove the supernatant.

[0051] S22: Add 1 mL of microsphere coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000 rpm for 10 min, and remove the supernatant;

[0052] S23: Add 1 mL of microsphere coupling buffer and sonicate to mix well;

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

[0054] S25: Place the centrifuge tubes on a turntable and activate them for 25 minutes in the dark at 37℃ and 40r / min.

[0055] S26: Centrifuge at 15℃ and 20000rpm for 10min and remove the supernatant;

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

[0057] S28: Add 1.5 mL of coupling buffer and wash once more;

[0058] S29: Add 0.75 mL of coupling buffer, sonicate to mix, and obtain the 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 the antibody solution;

[0061] S32: Add the antibody solution to the well-mixed activated microsphere solution and vortex to mix.

[0062] S33: Place the centrifuge tubes on a turntable and couple them in the dark for 2 hours at 37℃ and 40r / min.

[0063] S4: Sealing and Preservation 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 tubes on a turntable and seal them in the dark at 37℃ and 40r / min for 1 hour.

[0066] S43: Centrifuge at 15℃ and 20000rpm for 10min and remove the supernatant;

[0067] S44: Add 1.5 mL of microsphere washing solution, sonicate to mix, centrifuge at 15℃ and 20000 rpm for 10 min, and remove the supernatant;

[0068] S45: Add 1.5 mL of microsphere washing solution and wash once more;

[0069] S46: Finally, add 0.5 mL of microsphere preservation solution (final microsphere concentration 1 mg / mL) to the centrifuge tube, sonicate to mix well, and store at 2–8 °C in the dark for later use.

[0070] Example 2

[0071] This embodiment studied the effect of the composition of the microsphere preservation solution on the lyophilized product. Other steps were the same as in Example 1. Commonly used PBS and Tris-NaCl were selected for comparison. In addition, considering the stability of the lyophilized product, a certain amount of protein BSA and protein protectant ADP were added. Considering the appearance of the lyophilized product, sucrose or trehalose was added.

[0072] Specifically, the types and concentrations of buffer, protein, and sugar added to the microsphere preservation 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 the same as Example 1.

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

[0079] The procedure for detecting fluorescent particles was as follows: Samples of different concentration gradients were added to ELISA plates, with concentrations of samples P1 to P5 being 100, 33, 11, 3, and 1 pg / mL, respectively. The lyophilized products prepared according to formulations A to D were added to the ELISA plates, allowing the samples and lyophilized products to react on the plates for 30 minutes. After the reaction, the ELISA plates were washed five times with microsphere washing buffer. The plates were then placed in a digital fluorescence immunoassay system for imaging and signal counting. The test results are shown in Tables 2 and 3. Figure 1 The image shows the imaging results of formulation C. As can be seen from the image, the microspheres have a clear green fluorescence signal.

[0080] Table 1 Detailed Vacuum Drying Process Parameter Settings

[0081]

[0082] Table 2. Results of Stability Comparison of Freeze-Dried Products

[0083]

[0084]

[0085] Table 3. Comparison Results of Appearance of Freeze-Dried Products

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

[0087] Table 2 shows that the lyophilized products prepared under different preservation solutions showed no significant difference when stored at 2–8°C. However, after 10 days at 37°C, the luminescence values ​​of formulations A, B, and D showed the largest variations, while formulation C exhibited the best variation. Therefore, formulation C is the preferred formulation. Table 3 shows that the lyophilized appearance of formulation C is also satisfactory. In conclusion, formulation C is the preferred preservation solution for fluorescently labeled antibodies.

[0088] The rehydration properties of the above-mentioned freeze-dried products were tested, as follows:

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

[0090] Measurement sequence: 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 using the formulas below. 瓶内 and CV 瓶间 When the statistical result F-value is less than or equal to 10, the uniformity between fluorescent microspheres is considered to be good. The average value of all test results is calculated, and then CV is performed. 瓶内 and CV 瓶间 The calculation requires that the CV be less than 10%; when the statistical result F value is greater than 10, the uniformity inside the fluorescent microsphere bottle is considered poor, and the CV is no longer calculated. 瓶内 and CV 瓶间。

[0092] When the statistical result F≤1, the within-bottle standard deviation is used instead of the between-bottle standard deviation, i.e., 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] In the formula:

[0096] SS—Variance;

[0097] ν—degree of freedom;

[0098] MS—mean square;

[0099] F-F test value;

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

[0101] n0—Number of valid measurements;

[0102] s bb —Standard deviation between bottles;

[0103] sr —Repeatability standard deviation (i.e., within-bottle standard deviation); x i —The measurement or calculation result for each sample; x—The measurement or calculation result —Overall average;

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

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

[0106] Table 4 Results of Rehydration Uniformity Test

[0107]

[0108] The data above shows that the F-value of the calibrator is less than 10, indicating good uniformity between the fluorescent microsphere vials, making it suitable as a calibrator. Furthermore, the CV values ​​within and between vials are both less than 10%, meeting the requirements for use of lyophilized fluorescent microspheres, indicating good rehydration properties of the lyophilized product.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions 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 lyophilized products containing fluorescent microsphere-labeled antibodies, characterized in that: Includes the following steps: S1: Activation treatment of polystyrene microspheres containing fluorescent dyes An activated microsphere solution was obtained by mixing a polystyrene microsphere solution containing fluorescent dye, an EDC solution, and an NHS solution. S2: Coupling reaction The antibody solution was added to the activated microsphere solution to carry out the coupling reaction, and the coupling reaction solution was obtained. S3: Closure of microspheres Microsphere blocking solution was added to the coupling reaction solution to carry out the blocking reaction, followed by centrifugation to remove the supernatant. Then, microsphere washing solution was added for washing, followed by centrifugation to remove the supernatant, and the blocked microspheres were obtained. S4: Preservation of Microspheres Add microsphere preservation solution to the sealed microspheres, mix thoroughly by ultrasonication to obtain microsphere storage solution, and store at 2~8℃ in the dark for later use. S5: Preparation of freeze-dried products The microsphere storage solution was freeze-dried to obtain a lyophilized product of fluorescent microsphere-labeled antibody. In step S3, the microsphere blocking solution was a borate buffer (pH 9.0 ± 0.05) consisting 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 was carried out on a turntable at 37°C and 40 r / min, in the dark for 1 h. The microsphere washing solution was 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300. In step S4, the microsphere preservation solution consisted of 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 was 1 mg / mL.

2. The method for improving the stability of lyophilized fluorescent microsphere-labeled antibody products 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: Take a 1% solid content polystyrene microsphere suspension containing fluorescent dye and add it to the coupling buffer. Mix it evenly by sonication, then centrifuge and wash it twice with the coupling buffer. S12: Add the solid material obtained in step S11 to the coupling buffer, and mix it evenly by sonication to obtain a polystyrene microsphere solution containing fluorescent dye.

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

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

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

6. The method for improving the stability of lyophilized fluorescent microsphere-labeled antibody products 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 it evenly to obtain the antibody solution; the coupling reaction is carried out on a turntable and coupled for 15-30 min in the dark at 37℃ and 40r / min.

7. A lyophilized product of fluorescent microsphere-labeled antibody prepared by the method of any one of claims 1 to 6.

8. The application of the fluorescent microsphere-labeled antibody lyophilized product according to claim 7 in a digital fluorescence immunoassay detection system.

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