A freeze-dried protective agent composition for AlphaLISA detection and preparation method thereof

Abstract

The present invention discloses a lyophilized protective agent composition for AlphaLISA detection and a preparation method thereof. The method comprises: diluting a biotinylated antibody and an antibody-labeled receptor bead with a diluent to a desired concentration, mixing the two solutions to obtain a detection reagent A, and diluting a streptavidin-coupled donor microsphere with a diluent to a desired concentration to obtain a detection reagent B; adding a lyophilized protective agent to A and B respectively, and mixing them evenly; transferring the reagents to a bead dropper, quick-freezing the reagents in liquid nitrogen to form ice balls, and vacuum-drying the quick-frozen reagent balls with liquid nitrogen to obtain freeze-dried beads of detection reagents A and B. After adding the lyophilized protective agent and using liquid nitrogen for quick freeze drying, the freeze-dried reagents are in the form of small balls with a good round effect, uniform shape and volume, consistent size, low moisture content, and can be stored stably for a long time without affecting the detection signal value, and the time required is shorter than that of conventional freeze drying.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a freeze-dried protective agent composition for AlphaLISA detection and a preparation method thereof. Background Art

[0002] The AlphaLISA (amplified luminescent proximity homogeneous assay linked immunosorbent assay) assay requires no washing, is simple to use, offers high sensitivity, a wide dynamic range (3-4 orders of magnitude), a wide affinity range (both high- and low-affinity antibodies can be used), and exhibits strong anti-interference capabilities, making it a viable alternative to traditional ELISA assays. AlphaLISA assays have been successfully applied in a variety of areas, including enzyme assays, interaction assays (including receptor / ligand, protein / protein, and protein / DNA assays), immunoassays, and GPCR functional assays (cAMP, IP3). Currently, commercially available kits are liquid reagents. The AlphaLISA assay system, containing protein components such as antibodies, biotin, and streptavidin, requires storage at low temperatures and then returns to room temperature before use. This is not only cumbersome but also temperature-sensitive. Temperature variations can lead to signal fluctuations, impacting reagent reproducibility. Furthermore, low-temperature storage increases storage and transportation costs, as well as energy consumption.

[0003] Freeze-drying is the most commonly used technique for preserving proteins in the research and production of biological products. It not only preserves the biological properties of the product but also extends its shelf life at room temperature. Freeze-drying utilizes the principle of ice crystal sublimation. Under a high vacuum, the water in the frozen biological product is sublimated directly from solid ice to vapor, without melting the ice. Freeze-drying offers numerous advantages. The dried biological product maintains its biological activity and volume, while removing over 95% to 99% of its water content, enabling long-term storage without deterioration. The porous structure formed by freeze-drying readily absorbs water and returns to its original shape. Therefore, it is highly suitable for the development of immunoassay reagents, facilitating their storage and transportation without compromising their detection. However, conventional freeze-drying techniques require a relatively long pre-freezing time, and the stability of the donor and acceptor spheres in the AlphaLISA assay system is easily compromised by prolonged freezing, making conventional freeze-drying unsuitable for the AlphaLISA system.

[0004] Compared with conventional freeze-drying, liquid nitrogen quick freezing has the advantage of fast pre-freezing time, which reduces the time that the detection reagent is exposed to the room temperature environment. In addition, liquid nitrogen freeze-drying can also form freeze-dried beads, which has the advantages of saving freeze-drying space (no need to put the reagent strips and other packaging into the freeze dryer for freeze drying, which improves the utilization rate of the freeze dryer), convenient transfer (one small bead for each person's reagent, in the early research and development and small-batch production, the beads can be manually packaged; after the production capacity is increased, they can be automatically packaged by automated equipment), and rapid re-dissolution (the freeze-dried beads have no contact surface with the tube wall, and the entire sphere is a loose mesh structure). However, the freeze-dried beads also have the characteristics of being easy to break, so it is necessary to find a freeze-drying protection system required for the AlphaLISA detection reagent to prepare complete, smooth, and non-breakable freeze-dried beads that are easier to store for a long time. Summary of the Invention

[0005] The purpose of the present invention is to provide a freeze-dried protective agent composition for AlphaLISA detection and a preparation method thereof.

[0006] The lyophilization protectant composition for AlphaLISA detection provided by the present invention comprises an AlphaLISA detection reagent and a lyophilization protectant.

[0007] The AlphaLISA detection reagent includes detection reagent A and detection reagent B for AlphaLISA reaction, wherein detection reagent A includes biotinylated antibody, antibody-labeled acceptor beads and diluent, and detection reagent B includes streptavidin-coupled donor microspheres and diluent;

[0008] Specifically, the detection reagent A is composed of: 0.05-1.0 μg / mL biotinylated antibody, 5-50 μg / mL antibody-labeled receptor beads and diluent, wherein the diluent is composed of: 50 mM phosphate buffer, pH 7.4, 50 mM sodium chloride, 2 mM diethylenetriaminepentaacetic acid, 1 mg / mL dextran 500, 0.5% bovine serum albumin, 0.1% Tween-20, 0.01% proclin300;

[0009] The detection reagent B is composed of: 5-50 μg / mL streptavidin-coupled donor microspheres and diluent, wherein the diluent is composed of: 50 mM phosphate buffer, pH 7.4, 50 mM sodium chloride, 2 mM diethylenetriaminepentaacetic acid, 1 mg / mL dextran 500, 0.5% bovine serum albumin, 0.1% Tween-20, and 0.01% proclin 300.

[0010] The freeze-drying protective agent is a complex of one or more of the following reagents: dextran, trehalose, sucrose, bovine serum albumin, and casein.

[0011] The weight-to-volume ratio of the lyophilized protective agent to the AlphaLISA detection reagent is 1%-10%, that is, every 100 ml of the AlphaLISA detection reagent contains 1-10 g of lyophilized protective agent.

[0012] Specifically, each 100 ml of detection reagent A contains 1-10 g of lyoprotectant, and each 100 ml of detection reagent B also contains 1-10 g of lyoprotectant;

[0013] More specifically, every 100 ml of the detection reagent A or B contains 1-10 g of at least one of sucrose, dextran, and trehalose and 1-5 g of bovine serum albumin and / or casein.

[0014] In an embodiment of the present invention, each 100 ml of detection reagent A contains 5 g of sucrose and 2 g of bovine serum albumin (BSA); each 100 ml of detection reagent B contains 5 g of sucrose and 2 g of bovine serum albumin (BSA).

[0015] Through a large number of experimental studies, it was found that the freeze-dried reagent can have a better morphology, better reagent performance and can be stored stably for a long time.

[0016] The AlphaLISA detection reagent is a freeze-dried bead pellet with good stability.

[0017] The present invention also provides a method for preparing the above-mentioned freeze-dried protectant composition for AlphaLISA detection.

[0018] The preparation method of the lyoprotectant composition for AlphaLISA detection provided by the present invention comprises the following steps:

[0019] 1) Diluting the biotinylated antibody and the antibody-labeled receptor beads to the desired concentrations with a diluent, respectively, and mixing the two solutions to obtain detection reagent A; adding a lyoprotectant and mixing thoroughly to obtain detection reagent A containing a lyoprotectant; transferring the solution to a bead dropper, and rapidly freezing the reagent in liquid nitrogen to form ice balls; vacuum drying the rapidly frozen reagent balls with liquid nitrogen to obtain lyophilized beads of detection reagent A;

[0020] 2) Diluting the streptavidin-coupled donor microspheres with a diluent to the desired concentration to obtain detection reagent B; adding a lyoprotectant and mixing to obtain detection reagent B containing a lyoprotectant; transferring the microspheres to a bead dropper, quick-freezing the reagent in liquid nitrogen to form ice balls, and vacuum drying the quick-frozen reagent balls with liquid nitrogen to obtain lyophilized beads of detection reagent B.

[0021] In steps 1) and 2) of the above method, the drop volume is set to 10-20 μL / drop, specifically 20 μL / drop;

[0022] The conditions for vacuum drying after quick freezing with liquid nitrogen are as follows:

[0023] 1) Pre-freezing stage: liquid nitrogen for 1-10 minutes, pressure of 1 atm;

[0024] 2) Sublimation stage: control the temperature at -50℃~-40℃, the pressure at 0.1-2.0Pa, and maintain for 1-2h;

[0025] 3) Sublimation stage: control the temperature at -40℃~-30℃, the pressure at 0.1-2.0Pa, and maintain for 3-5h;

[0026] 4) Sublimation stage: control the temperature at -30℃~-20℃, the pressure at 0.1-2.0Pa, and maintain for 1-2h;

[0027] 5) Sublimation stage: control the temperature at -20℃~-10℃, the pressure at 0.1-2.0Pa, and maintain for 1-2h;

[0028] 6) Desorption stage: control the temperature at 0°C to 10°C and the pressure at 0.1-2.0 Pa for 2-4 hours;

[0029] 7) Analysis stage: control the temperature at 10℃~20℃ and the pressure at 0.1-2.0Pa for 2-4h.

[0030] In an embodiment of the present invention, the conditions for vacuum drying after liquid nitrogen quick freezing are as follows:

[0031] 1) Pre-freezing stage: liquid nitrogen for 1-10 minutes, pressure of 1 atm;

[0032] 2) Sublimation stage: control the temperature at -50℃~-40℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0033] 3) Sublimation stage: control the temperature at -40℃~-30℃, the pressure at 0.1-2.0Pa, and maintain for 4 hours;

[0034] 4) Sublimation stage: control the temperature at -30℃~-20℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0035] 5) Sublimation stage: control the temperature at -20℃~-10℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0036] 6) Analysis stage: control the temperature at 0°C to 10°C and the pressure at 0.1-2.0 Pa for 3 hours;

[0037] 7) Analysis stage: control the temperature at 10℃~20℃ and the pressure at 0.1-2.0Pa for 3h.

[0038] Through a large number of experimental studies, it was found that the freeze-dried reagent was in the form of small pellets using the above-mentioned liquid nitrogen quick freeze-drying treatment, with a good round effect, uniform shape and volume, consistent size, low moisture content, and could be stored stably for a long time without affecting the detection signal value, and took less time than conventional freeze-drying treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The following are photos of the AlphaLISA detection reagent prepared by liquid nitrogen quick freeze-drying of the present invention and the AlphaLISA detection reagent prepared by conventional pre-freeze-drying (both are detection reagent A).

[0040] Figure 2 The stability of the reagents obtained by liquid nitrogen quick freeze drying of the present invention and conventional pre-freeze drying is compared.

[0041] Figure 3 The effect of adding or not adding a lyoprotectant on the appearance of the reagent in Example 2 of the present invention (both are detection reagent A).

[0042] Figure 4 This is a repeatability experiment of the freeze-dried bead reagent prepared with or without the addition of a freeze-dried protective agent in Example 2 of the present invention.

[0043] Figure 5 The effects of a single sugar protective agent, a single protein protective agent, and a combined protective agent on the appearance of the reagent (all detection reagent A) in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0045] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0046] Example 1

[0047] 1) Preparation of AlphaLISA Detection Reagent A and Quick Freeze-Drying with Liquid Nitrogen

[0048] Mice were immunized with recombinantly expressed Staphylococcus aureus enterotoxin B antigen, and spleen cells were harvested for cell fusion to generate hybridomas. High-titer cell lines were screened and purified to obtain SEB1# and SEB6#, Staphylococcus aureus enterotoxin B-specific antibodies. Antibody biotinylation was performed according to the instructions for the Thermo Fisher Scientific biotinylation kit. Acceptor microspheres and streptavidin-conjugated donor microspheres were purchased from Perkin Elmer. Antibody conjugation procedures were described in the Perkin Elmer microsphere labeling instructions.

[0049] Biotinylated Staphylococcus aureus enterotoxin B antibody (SEB6#) and Staphylococcus aureus enterotoxin B antibody (SEB1#) labeled receptor spheres were diluted to 0.25 μg / mL and 25 μg / mL, respectively, using diluent (50 mM phosphate buffer, pH 7.4, 50 mM sodium chloride, 2 mM diethylenetriaminepentaacetic acid, 1 mg / mL dextran 500, 0.5% bovine serum albumin, 0.1% Tween-20, 0.01% proclin300). The two solutions were mixed and the reagents were mixed evenly by vortexing or inverting to obtain detection reagent A. Then, add the lyoprotectant (5% sucrose and 2% BSA) to the above-mentioned detection reagent A according to the weight-to-volume ratio and mix thoroughly. Then, transfer the lyoprotectant-added reagent to a bead dropper, adjust the drop volume to 20 μL / drop, and quickly freeze the reagent in liquid nitrogen to form uniform ice balls. Pour off the excess liquid nitrogen, transfer the quick-frozen reagent balls with a small amount of liquid nitrogen to a penicillin bottle, and vacuum dry them in a freeze dryer. The specific procedure is as follows:

[0050] 1) Pre-freezing stage: liquid nitrogen for 1-10 minutes, pressure of 1 atm;

[0051] 2) Sublimation stage: control the temperature at -50℃~-40℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0052] 3) Sublimation stage: control the temperature at -40℃~-30℃, the pressure at 0.1-2.0Pa, and maintain for 4 hours;

[0053] 4) Sublimation stage: control the temperature at -30℃~-20℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0054] 5) Sublimation stage: control the temperature at -20℃~-10℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0055] 6) Analysis stage: control the temperature at 0°C to 10°C and the pressure at 0.1-2.0 Pa for 3 hours;

[0056] 7) Analysis stage: control the temperature at 10℃~20℃ and the pressure at 0.1-2.0Pa for 3h.

[0057] Remove the vial containing the freeze-dried beads from the freeze dryer and seal it for later use.

[0058] 2) Preparation of AlphaLISA Detection Reagent B and Liquid Nitrogen Freeze-Drying

[0059] Dilute the streptavidin-conjugated donor microspheres to 10 μg / mL using diluent (50 mM phosphate buffer, pH 7.4, 50 mM sodium chloride, 2 mM diethyltriamine pentaacetic acid, 1 mg / mL dextran 500, 0.5% bovine serum albumin, 0.1% Tween-20, 0.01% proclin 300). Mix thoroughly by vortexing or inverting the solution. Add lyoprotectant (5% sucrose and 2% BSA) to the above-mentioned detection reagent B according to a weight-to-volume ratio and mix thoroughly. Transfer the lyoprotectant-added reagent to a bead dryer, adjusting the drop volume to 20 μL / drop. Quick-freeze the reagent in liquid nitrogen to form uniform ice balls. Pour off excess liquid nitrogen, transfer the quick-frozen reagent balls with a small amount of liquid nitrogen to a vial, and vacuum dry in a freeze dryer. The specific procedure is as follows:

[0060] 1) Pre-freezing stage (liquid nitrogen quick freezing): liquid nitrogen 1-10 min, pressure 1atm;

[0061] 2) Sublimation stage: control the temperature at -50℃~-40℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0062] 3) Sublimation stage: control the temperature at -40℃~-30℃, the pressure at 0.1-2.0Pa, and maintain for 4 hours;

[0063] 4) Sublimation stage: control the temperature at -30℃~-20℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0064] 5) Sublimation stage: control the temperature at -20℃~-10℃, the pressure at 0.1-2.0Pa, and maintain for 1h;

[0065] 6) Analysis stage: control the temperature at 0°C to 10°C and the pressure at 0.1-2.0 Pa for 3 hours;

[0066] 7) Analysis stage: control the temperature at 10℃~20℃ and the pressure at 0.1-2.0Pa for 3h.

[0067] Remove the vial containing the freeze-dried beads from the freeze dryer and seal it for later use.

[0068] Comparative Example 1

[0069] 1) Preparation and routine lyophilization of AlphaLISA detection reagent A

[0070] Prepare AlphaLISA Assay Reagent A as described above. Aliquot 20 μL of the reagent, adding a lyoprotectant, into 0.2 mL Eppendorf tubes and place in a freeze dryer for conventional freeze-vacuum drying. Replace the liquid nitrogen quick freeze with a pre-freeze in the freeze dryer at -50°C and 1 atm for 2 hours. After the pre-freeze, proceed to the sublimation and desorption stages, following the same procedures as described above. After lyophilization, cap the 0.2 mL Eppendorf tubes and store until ready for use.

[0071] 2) Preparation and routine lyophilization of AlphaLISA detection reagent B

[0072] Prepare AlphaLISA Assay Reagent B as described above. Aliquot 20 μL of the lyoprotectant-added reagent into 0.2 mL Eppendorf tubes and place in a freeze dryer for freeze-vacuum drying. Replace the liquid nitrogen quick freeze with a pre-freeze in the freeze dryer at -50°C and 1 atm for 2 hours. After the pre-freeze, proceed to the sublimation and desorption stages, following the same procedures as described above. After lyophilization, cap the 0.2 mL Eppendorf tubes and store until ready for use.

[0073] Depend on Figure 1 Comparing the two freeze-drying methods, it can be seen that AlphaLISA detection reagent A produced using the liquid nitrogen quick-freeze drying method of the present invention is in the form of small spheres, with a good rounded effect, uniform shape and volume, and consistent size. In contrast, AlphaLISA detection reagent A produced using conventional pre-freeze drying has a rough, uneven surface, and its appearance is affected by the freeze-drying container. Meanwhile, AlphaLISA detection reagent B produced using the liquid nitrogen quick-freeze drying method of the present invention has the same morphology as detection reagent A, also in the form of small spheres, with a good rounded effect, uniform shape and volume, and consistent size. In contrast, AlphaLISA detection reagent B produced using conventional pre-freeze drying has a rough, uneven surface, and its appearance is also affected by the freeze-drying container.

[0074] Reconstitution and testing of AlphaLISA assay reagents A and B

[0075] Detection reagents A and B, prepared by quick-freeze vacuum drying with liquid nitrogen in Example 1 or conventional freeze drying in Comparative Example 1, were removed and reconstituted with 20 μL of reconstitution solution (0.01 M phosphate buffer, pH 7.4). Staphylococcus aureus enterotoxin B antigen was diluted to 100 ng / mL using the reconstitution solution. 10 μL of the antigen was added to 20 μL of the reconstituted AlphaLISA detection reagent A. The mixture was reacted in a 37°C incubator for 15 minutes. The antigen and detection reagent A mixture was then transferred to detection reagent B and reacted in a 37°C incubator for 10 minutes. AlphaLISA detection was performed on a SPECTRAMAXi3 multifunctional microplate reader (Molecular Devices).

[0076] Accelerated stability testing of liquid nitrogen freeze-dried or conventional pre-freeze-dried test reagents

[0077] The detection reagents A and B that were freeze-dried with liquid nitrogen or pre-freeze-dried with conventional methods were placed in a 37°C incubator and taken out at 0 days, 1 day, 7 days and 14 days, respectively, for reconstitution and testing to compare the stability of the freeze-dried reagents obtained by the two freeze-drying methods. The signal value of the liquid nitrogen freeze-dried reagents detected at 100 ng / mL of Staphylococcus aureus enterotoxin B antigen remained basically unchanged after being stored in a 37°C incubator for 0 days, 1 day, 7 days and 14 days, while the signal value of the reagent pre-freeze-dried at room temperature decreased significantly after being stored in a 37°C incubator for 7 days, and the signal value decreased by more than 50% after 14 days ( Figure 2 ). This shows that compared with the conventional pre-freezing method, liquid nitrogen quick freeze drying can better ensure the stability of the reagent.

[0078] Example 2: Effect of Lyoprotectants on the Appearance of Liquid Nitrogen Lyophilized Beads

[0079] Referring to the procedure of Example 1, AlphaLISA detection reagent A and detection reagent B were prepared, and lyophilization protective agent was added according to the weight-to-volume ratio (5% sucrose and 2% BSA), and the bead volume was adjusted to 20 μL / drop, and liquid nitrogen was quick-freeze-dried. Alternatively, no lyophilization protective agent was added, and the bead volume was adjusted to 20 μL / drop, and liquid nitrogen was quick-freeze-dried. The appearance difference of the liquid nitrogen freeze-dried beads with and without lyophilization protective agent was compared. After adding lyophilization protective agent, the prepared freeze-dried beads were more complete, smooth, and not easy to break (see Figure 3 , Detection Reagent A. Detection Reagent B with and without lyoprotectant had the same appearance as Detection Reagent A). After reconstitution of the two lyophilized beads, 0.1 ng / mL Staphylococcus aureus enterotoxin B antigen was detected. The coefficient of variation (CV) of the results of six repeated tests was calculated. Compared with the test without lyoprotectant, the CV was smaller (<5%) after adding lyoprotectant, the lyophilized appearance was more uniform, the reagent concentration was uniform, and more consistent results were obtained ( Figure 4 ).

[0080] Example 3: Effect of Lyoprotectant Composition on the Appearance of Liquid Nitrogen Freeze-Dried Beads

[0081] AlphaLISA Detection Reagents A and B were prepared by following the procedures in Example 1. 5% sucrose and 2% BSA were added as lyoprotectants in a weight-to-volume ratio. The drop volume was adjusted to 20 μL / drop, and the beads were freeze-dried using liquid nitrogen. Alternatively, only 5% sucrose was added, the drop volume was adjusted to 20 μL / drop, and the beads were freeze-dried using liquid nitrogen. Alternatively, only 2% BSA was added, the drop volume was adjusted to 20 μL / drop, and the beads were freeze-dried using liquid nitrogen.

[0082] Compare the differences in the appearance of the liquid nitrogen freeze-dried beads by the three methods. Sugar or protein alone will affect the appearance of the liquid nitrogen freeze-dried beads. Adding only sucrose will cause the freeze-dried beads to absorb water severely and become smaller, while adding only BSA will result in poor spherical effect of the freeze-dried beads ( Figure 5 , detection reagent A). A lyoprotectant of a combination of at least one sugar and at least one protein is also most suitable for detection reagent B. Therefore, the lyoprotectant is preferably a combination of at least one sugar and at least one protein.

[0083] Based on experimental tests, we found that sugar and protein play different roles. No single component can achieve a better appearance. Even increasing the concentration cannot replace the role of another component. Increasing the concentration of protein too high will affect the detection signal.

[0084] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for preparing a composition for AlphaLISA detection, comprising the following steps: 1) diluting a biotinylated antibody and an antibody-labeled receptor bead to a desired concentration with a diluent, mixing the two solutions to obtain a detection reagent A; adding a lyoprotectant and mixing thoroughly to obtain a detection reagent A containing the lyoprotectant; transferring the mixture to a bead dropper, rapidly freezing the reagent in liquid nitrogen to form ice balls, and vacuum drying the rapidly frozen reagent balls with liquid nitrogen to obtain lyophilized beads of the detection reagent A; 2) Diluting the streptavidin-coupled donor microspheres with diluent to the desired concentration to obtain detection reagent B; adding a lyoprotectant and mixing to obtain detection reagent B containing a lyoprotectant; transferring the microspheres to a bead dropper, rapidly freezing the microspheres in liquid nitrogen to form ice balls; and vacuum drying the rapidly frozen microspheres containing liquid nitrogen to obtain lyophilized beads of detection reagent B; The lyoprotectants were 5% w / v sucrose and 2% w / v BSA.

2. The method according to claim 1, wherein: In steps 1) and 2), set the bead volume to 10-20 μL / drop.

3. The method according to claim 1, wherein: The conditions for vacuum drying after quick freezing with liquid nitrogen are as follows: 1) Pre-freezing stage: liquid nitrogen for 1-10 minutes, pressure of 1 atm; 2) Sublimation stage: control the temperature at -50℃~-40℃, the pressure at 0.1-2.0Pa, and maintain for 1-2h; 3) Sublimation stage: control the temperature at -40℃~-30℃, the pressure at 0.1-2.0Pa, and maintain for 3-5h; 4) Sublimation stage: control the temperature at -30℃~-20℃, the pressure at 0.1-2.0Pa, and maintain for 1-2h; 5) Sublimation stage: control the temperature at -20℃~-10℃, the pressure at 0.1-2.0Pa, and maintain for 1-2h; 6) Analysis stage: control the temperature at 0℃~10℃ and the pressure at 0.1-2.0Pa for 2-4h; 7) Analysis stage: control the temperature at 10℃~20℃ and the pressure at 0.1-2.0Pa for 2-4h.

4. A composition for AlphaLISA detection, wherein the composition for AlphaLISA detection is prepared according to the method according to any one of claims 1 to 3. The composition for AlphaLISA detection comprises an AlphaLISA detection reagent and a lyophilization protectant, The AlphaLISA detection reagents include detection reagent A and detection reagent B for AlphaLISA reaction, wherein detection reagent A includes biotinylated antibody, antibody-labeled receptor ball and diluent, Detection Reagent B includes streptavidin-coupled donor microspheres and diluent.

5. The composition for AlphaLISA detection according to claim 4, characterized in that: The detection reagent A is composed of: 0.05-1.0 μg / mL biotinylated antibody, 5-50 μg / mL antibody-labeled receptor beads and diluent, wherein the diluent is composed of: 50 mM phosphate buffer, pH 7.4, 50 mM sodium chloride, 2 mM diethylenetriaminepentaacetic acid, 1 mg / mL dextran 500, 0.5% bovine serum albumin, 0.1% Tween-20, and 0.01% proclin300.

6. The composition for AlphaLISA detection according to claim 4, characterized in that: The detection reagent B is composed of: 5-50 μg / mL streptavidin-coupled donor microspheres and diluent, wherein the diluent is composed of: 50 mM phosphate buffer, pH 7.4, 50 mM sodium chloride, 2 mM diethylenetriaminepentaacetic acid, 1 mg / mL dextran 500, 0.5% bovine serum albumin, 0.1% Tween-20, and 0.01% proclin300.

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