A lyophilized diluent and methods of making and using the same

Abstract

The application discloses a freeze-dried diluent as well as a preparation method and application thereof, and belongs to the technical field of biological detection. The freeze-dried diluent is added with a simple buffer system of a low-concentration inorganic acid (such as 1-5 mM hydrochloric acid), so that the stability problem of a recombinant protein (GDF-15) caused by high pI and strong hydrophobicity can be overcome in a targeted manner. The system provides a strong electrostatic repulsive force for protein molecules by providing an acidic environment, and fundamentally eliminates non-specific adsorption and salt-induced aggregation, so that high-activity recovery and long-term stability of a freeze-dried recombinant protein standard are realized. In addition, through synergistic action between the inorganic acid and other components (protein stabilizers, sugar compounds, metal ion chelating agents and preservatives) in the freeze-dried diluent, the attenuation of the recombinant protein standard can be effectively reduced, and the stability of the recombinant protein standard can be significantly improved, and then the long-term effectiveness of an ELISA kit and the detection accuracy can be significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a lyophilized diluent, its preparation method, and its application. Background Technology

[0002] Enzyme-linked immunosorbent assay (ELISA) is an enzyme immunoassay technique developed after immunofluorescence and radioimmunoassay. Due to its sensitivity, specificity, cost-effectiveness, simplicity, and safety, ELISA is currently the most commonly used detection method in immunology laboratories and has been widely applied in biomedical research and clinical disease diagnosis and treatment. Based on the two main methodologies of competitive assays and double-antibody sandwich assays, ELISA kits covering more than ten fields, including oncology, hormones, autoimmunity, cardiovascular, and metabolism, have been developed in the industry. Among them, the commonly used double-antibody sandwich assay involves binding specific antibodies to a solid-phase carrier to form a solid-phase antibody, which then binds to the corresponding antigen in the sample to form an immune complex. Biotin-labeled antibodies are then added, binding to the immune complex to form a biotin-labeled antibody-antigen-solid-phase antibody complex. HRP-labeled avidin is then added, followed by TMB substrate for color development, allowing for quantitative analysis of the antigen content in the sample.

[0003] Growth differentiation factor 15 (GDF-15) belongs to the TGF-β superfamily, and its mature form is a disulfide-linked dimer protein. In mice, GDF-15 participates in metabolic regulation, neuroprotection, and disease progression by binding to its receptor GFRAL and activating the downstream RET signaling pathway. Under normal physiological conditions, GDF-15 is expressed at low levels in the placenta, prostate, and central nervous system; its expression is significantly upregulated under stress conditions (such as tissue damage, metabolic imbalance, or inflammation), for example, liver injury or a high-fat diet can induce the liver to secrete GDF-15. Recombinant GDF-15 protein and gene-edited mouse models (such as systemic or tissue-specific knockout) are widely used in research on metabolic syndrome, neurodegenerative diseases, and cancer mechanisms.

[0004] In the development of ELISA kits, recombinant proteins are frequently used as standards for various ELISA kits. Currently, commonly used lyophilization diluents for recombinant GDF-15 protein standards typically contain one of PBS buffer, TRIS buffer, or HEPES buffer, with the addition of 0.5%-2% BSA and 0.05%-0.1% Proclin 300. These diluents offer advantages such as ease of preparation, low cost, good batch stability, and ease of storage. However, due to the unique properties of mouse recombinant GDF-15 protein (isoelectric point pI 9.41, strong hydrophobicity), lyophilization with conventional lyophilization buffers (PBS buffer, TRIS buffer, or HEPES buffer) leads to significant attenuation of the recombinant GDF-15 protein standard after lyophilization, and its long-term stability cannot meet the performance requirements of ELISA kits. Furthermore, the insufficient attenuation and stability of lyophilized standards severely affect the shelf life of enzyme-linked immunosorbent assay (ELISA) kits and the accuracy of experimental results. Summary of the Invention

[0005] The purpose of this invention is to provide a lyophilized diluent, its preparation method, and its application. This addresses the problems of significant attenuation and poor stability of existing lyophilized diluents after lyophilizing recombinant GDF-15 protein standards.

[0006] In a first aspect, the present invention provides a lyophilized diluent comprising the following components: 1-5 mM basal buffer, 0.5-2% by mass of a protein stabilizer, 1-5% by mass of a carbohydrate compound, 0.1-0.4% by mass of a metal ion chelating agent, and 0.05-0.1% by volume of a preservative; wherein the basal buffer comprises a hydrochloric acid solution.

[0007] In this invention, the inventors discovered that by adding a simple buffer system of low-concentration inorganic acid (such as 1-5 mM hydrochloric acid) to the lyophilization diluent, the stability problem of recombinant protein (GDF-15) caused by its high pI and strong hydrophobicity can be specifically overcome. This system provides an acidic environment that imparts strong electrostatic repulsion to the protein molecules, fundamentally eliminating non-specific adsorption and salt-induced aggregation, thereby achieving high-activity recovery and long-term stability of the lyophilized recombinant protein standard. In addition, the inorganic acid and other components in the lyophilization diluent work synergistically to effectively reduce the attenuation of the recombinant protein standard and significantly improve its stability, thereby significantly improving the long-term effectiveness and detection accuracy of the ELISA kit.

[0008] In some implementations, the protein stabilizer includes at least one of bovine serum albumin and goat serum albumin.

[0009] In some embodiments, the carbohydrate compound includes at least one of arabinose, allulose, and isomaltitol.

[0010] In some embodiments, the metal ion chelating agent includes at least one of trisodium methylglycine diacetate and tetrasodium glutamate diacetate.

[0011] In some implementations, the preservative includes Proclin 300.

[0012] In some implementations, the pH of the lyophilized diluent is 2-3.

[0013] In a second aspect, the present invention provides a method for preparing a lyophilized diluent as described in any of the above claims, comprising the following steps: providing a base buffer; dissolving a protein stabilizer, a carbohydrate compound, and a metal ion chelating agent in the base buffer to obtain a first diluent; and adding a preservative to the first diluent to obtain a lyophilized diluent.

[0014] In a third aspect, the present invention provides the use of the lyophilized diluent as described above or the lyophilized diluent prepared by the above preparation method in the preparation of recombinant protein lyophilized standards.

[0015] In some implementations, recombinant protein lyophilized standards include recombinant GDF-15 protein lyophilized standards.

[0016] In a fourth aspect, the present invention provides an ELISA kit for the detection of recombinant GDF-15 protein, comprising any of the above-described lyophilized diluents or lyophilized diluents prepared by the above-described preparation methods or any of the above-described recombinant protein lyophilized standards.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention, by adding a simple buffer system of low-concentration inorganic acid (such as 1-5 mM hydrochloric acid) to the lyophilization diluent, can specifically overcome the stability problem of recombinant protein (GDF-15) caused by its high pI and strong hydrophobicity. This system provides an acidic environment that imparts strong electrostatic repulsion to the protein molecules, fundamentally eliminating non-specific adsorption and salt-induced aggregation, thereby achieving high-activity recovery and long-term stability of the lyophilized recombinant protein standard. In addition, the inorganic acid, through synergistic effects with other components in the lyophilization diluent, can effectively reduce the attenuation of the recombinant protein standard and significantly improve its stability, thereby significantly improving the long-term effectiveness and detection accuracy of the ELISA kit. Attached Figure Description

[0018] Figure 1 This is a standard curve diagram of the liquid protein standard of the present invention; Figure 2A standard curve diagram of lyophilized standards lyophilized using the lyophilization diluent in Example 1 of this invention; Figure 3 This is a standard curve diagram of lyophilized standards using the lyophilization diluent in Example 2 of the present invention. Figure 4 This is a standard curve diagram of lyophilized standards using the lyophilization diluent in Example 3 of the present invention. Figure 5 A standard curve diagram of the lyophilized standard sample lyophilized using the lyophilized diluent in Comparative Example 1 of this invention; Figure 6 This is a standard curve diagram of the lyophilized standard sample lyophilized using the lyophilization diluent in Comparative Example 2 of the present invention. Figure 7 A standard curve diagram of the lyophilized standard sample lyophilized using the lyophilization diluent in Comparative Example 3 of this invention. Figure 8 A standard curve diagram of the lyophilized standard sample lyophilized using the lyophilization diluent in Comparative Example 4 of this invention. Figure 9 A standard curve diagram of the lyophilized standard sample lyophilized using the lyophilized diluent in Comparative Example 5 of this invention; Figure 10 This is a standard curve diagram of the lyophilized standard sample lyophilized using the lyophilized diluent in Comparative Example 6 of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0021] Currently, existing lyophilized diluents exhibit significant degradation and poor stability after lyophilizing recombinant GDF-15 protein standards.

[0022] To address the issues of significant attenuation and poor stability of existing lyophilized diluents after lyophilizing recombinant GDF-15 protein standards, this invention provides a lyophilized diluent, its preparation method, and its application.

[0023] In a first aspect, the present invention provides a lyophilized diluent comprising the following components: 1-5 mM basal buffer, 0.5-2% by mass of a protein stabilizer, 1-5% by mass of a carbohydrate compound, 0.1-0.4% by mass of a metal ion chelating agent, and 0.05-0.1% by volume of a preservative; wherein the basal buffer comprises a hydrochloric acid solution.

[0024] In this invention, the inventors discovered that by adding a simple buffer system of low-concentration inorganic acid (such as 1-5 mM hydrochloric acid) to the lyophilization diluent, the stability problem of recombinant protein (GDF-15) caused by its high pI and strong hydrophobicity can be specifically overcome. This system provides an acidic environment that imparts strong electrostatic repulsion to the protein molecules, fundamentally eliminating non-specific adsorption and salt-induced aggregation, thereby achieving high-activity recovery and long-term stability of the lyophilized recombinant protein standard. The protein stabilizer in the lyophilization diluent mainly plays a competitive protection and physical support role. Its core function is to act as an interfacial active protein, preferentially adsorbing onto the gas-liquid interface and container surface, effectively preventing interfacial denaturation and non-specific adsorption loss of the target protein. Simultaneously, it inhibits intermolecular aggregation of the target protein in solution and forms a porous framework after lyophilization, maintaining the physical structural integrity of the lyophilized cake and facilitating reconstitution. Sugar alcohols play a crucial role in maintaining protein conformation and long-term stability in lyophilization diluents. Their core mechanism involves a "water substitution" effect, where hydroxyl groups form hydrogen bond networks with proteins during dehydration, maintaining their native spatial structure. After lyophilization, they form a high-viscosity glassy matrix, isolating and immobilizing protein molecules, thus significantly inhibiting chemical degradation and physical aggregation during storage and ensuring the long-term stability of recombinant protein standards. Metal ion chelators in lyophilization diluents primarily function by selectively complexing transition metal ions in the solution, effectively inhibiting protein oxidation reactions catalyzed by these ions and eliminating metal ion-induced protein misfolding, aggregation, or precipitation. This maintains the chemical integrity and conformational stability of proteins during lyophilization and subsequent storage, making them key components for ensuring the activity and shelf life of recombinant protein standards. Through synergistic effects, these components effectively reduce the degradation of recombinant protein standards and significantly improve their stability.

[0025] In some implementations, the protein stabilizer includes at least one of bovine serum albumin and goat serum albumin.

[0026] It is understandable that the type of protein stabilizer can be conventionally selected according to actual needs, as long as it can stabilize the structure of the target protein. For example, in this invention, the protein stabilizer preferably includes at least one of bovine serum albumin and goat serum albumin.

[0027] In some embodiments, the carbohydrate compound includes at least one of arabinose, allulose, and isomaltitol.

[0028] It is understandable that the types of carbohydrate compounds can be conventionally selected according to actual usage needs, as long as they can maintain protein conformation and long-term stability. For example, in this invention, the carbohydrate compounds preferably include at least one of arabinose, allulose, and isomaltitol.

[0029] In some embodiments, the metal ion chelating agent includes at least one of trisodium methylglycine diacetate (MGDA) and tetrasodium glutamate diacetate (GLDA).

[0030] It is understandable that the type of metal ion chelating agent can be conventionally selected according to actual application needs, as long as it can maintain protein conformation and long-term stability. For example, in this invention, the metal ion chelating agent preferably includes at least one of trisodium methylglycine diacetate (MGDA) and tetrasodium glutamate diacetate (GLDA).

[0031] In some implementations, the preservative includes Proclin 300.

[0032] It is understandable that the type of preservative can be conventionally selected according to actual usage needs, as long as it can maintain protein conformation and long-term stability. For example, in this invention, the preservative preferably includes Proclin 300.

[0033] In some implementations, the pH of the lyophilized diluent is 2-3, for example, it can be 2, 2.2, 2.4, 2.6, 2.8, 3 or other values ​​within this range.

[0034] In this invention, by controlling the pH value of the lyophilization diluent within a specific range, the conformation and long-term stability of proteins during the lyophilization process can be further maintained.

[0035] In a second aspect, the present invention provides a method for preparing a lyophilized diluent as described in any of the above claims, comprising the following steps: providing a base buffer; dissolving a protein stabilizer, a carbohydrate compound, and a metal ion chelating agent in the base buffer to obtain a first diluent; and adding a preservative to the first diluent to obtain a lyophilized diluent.

[0036] The preparation method provided by this invention is simple, and the raw materials used are cheap and readily available, making it suitable for large-scale industrial production applications.

[0037] In a third aspect, the present invention provides the use of the lyophilized diluent as described above or the lyophilized diluent prepared by the above preparation method in the preparation of recombinant protein lyophilized standards.

[0038] In some implementations, recombinant protein lyophilized standards include recombinant GDF-15 protein lyophilized standards.

[0039] It is understandable that the types of recombinant protein lyophilized standards can be conventionally selected according to actual needs, as long as they are common proteins. For example, in this invention, the recombinant protein lyophilized standards preferably include recombinant GDF-15 protein lyophilized standards.

[0040] In a fourth aspect, the present invention provides an ELISA kit for the detection of recombinant GDF-15 protein, comprising any of the above-described lyophilized diluents or lyophilized diluents prepared by the above-described preparation methods or any of the above-described recombinant protein lyophilized standards.

[0041] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] Example 1 A lyophilized diluent comprising the following components: 3.96 mM hydrochloric acid solution, 1% bovine serum albumin, 2% allulose, 0.4% trisodium methylglycine diacetate, and 0.1% Proclin 300.

[0043] The preparation method of this lyophilized diluent includes the following steps: S1. Add about 700-800mL of pure water to a clean 1L volumetric flask, and use a pipette to add 0.33mL of 12M concentrated hydrochloric acid slowly while stirring until well mixed to obtain a hydrochloric acid solution. S2. Weigh 10g bovine serum albumin, 20g allulose and 4g trisodium methylglycine diacetate, and add them sequentially to the hydrochloric acid solution obtained in step S1. Stir until completely dissolved to obtain the first dilution. S3. Measure 1 ml of Proclin 300 and add it to the first diluent obtained in step S2. Stir and mix well. Add pure water to the 1 L mark of the volumetric flask. Invert the volumetric flask 10-15 times, take a sample and test the pH with a pH meter. Adjust the pH to 2.3-3.0 with hydrochloric acid or sodium hydroxide to obtain the lyophilized diluent.

[0044] Example 2 A lyophilized diluent comprising the following components: 2.04 mM hydrochloric acid solution, 2% goat serum albumin (w / w), 1% allulose (w / w), 0.2% tetrasodium glutamate diacetate (w / w), and 0.05% Proclin 300 (v / v).

[0045] The preparation method of this lyophilized diluent includes the following steps: S1. Add about 700-800mL of pure water to a clean 1L volumetric flask, and use a pipette to add 0.17mL of 12M concentrated hydrochloric acid slowly while stirring until well mixed to obtain a hydrochloric acid solution. S2. Weigh 20g of goat serum albumin, 10g of allulose and 2g of tetrasodium diacetate of glutamate, and add them sequentially to the hydrochloric acid solution obtained in step S1. Stir until completely dissolved to obtain the first dilution. S3. Measure 500 μL of Proclin 300 and add it to the first diluent obtained in step S2. Stir and mix well. Add pure water to the 1L mark of the volumetric flask. Invert the volumetric flask 10-15 times, take a sample and test the pH with a pH meter. Adjust the pH to 2.3-3.0 with hydrochloric acid or sodium hydroxide to obtain the lyophilized diluent.

[0046] Example 3 A lyophilized diluent comprising the following components: 4.8 mM hydrochloric acid solution, 0.6% bovine serum albumin, 5% allulose, 0.1% tetrasodium glutamate diacetate, and 0.08% Proclin 300.

[0047] The preparation method of this lyophilized diluent includes the following steps: S1. Add about 700-800mL of pure water to a clean 1L volumetric flask, and use a pipette to add 0.4mL of 12M concentrated hydrochloric acid slowly and stir well to obtain a hydrochloric acid solution. S2. Weigh 6g of bovine serum albumin, 50g of allulose and 1g of tetrasodium diacetate of glutamate, and add them sequentially to the hydrochloric acid solution obtained in step S1. Stir until completely dissolved to obtain the first dilution. S3. Measure 800 μL of Proclin 300 and add it to the first diluent obtained in step S2. Stir and mix well. Add pure water to the 1L mark of the volumetric flask. Invert the volumetric flask 10-15 times, take a sample and test the pH with a pH meter. Adjust the pH to 2.3-3.0 with hydrochloric acid or sodium hydroxide to obtain the lyophilized diluent.

[0048] Comparative Example 1 The lyophilization diluent in this comparative example is prepared by replacing the hydrochloric acid solution in the lyophilization diluent of Example 1 with phosphate buffer. The specific preparation method includes the following steps: S1. First, weigh 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of anhydrous disodium hydrogen phosphate and 0.24g of anhydrous potassium dihydrogen phosphate and dissolve them in a 1L volumetric flask containing 800mL of purified water. Stir until completely dissolved to obtain 0.01M phosphate buffer. S2. Weigh 10g bovine serum albumin, 20g allulose and 4g trisodium methylglycine diacetate, and add them sequentially to the phosphate buffer obtained in step S1. Stir until completely dissolved to obtain the first dilution. S3. Measure 1 ml of Proclin 300 and add it to the first diluent obtained in step S2. Stir and mix well. Add pure water to the 1 L mark of the volumetric flask. Invert the volumetric flask 10-15 times, take a sample and test the pH with a pH meter. Adjust the pH to about 7.4 with hydrochloric acid or sodium hydroxide to obtain the lyophilized diluent.

[0049] Comparative Example 2 The lyophilization diluent in this comparative example is prepared by replacing the hydrochloric acid solution in the lyophilization diluent of Example 1 with Tris-HCl buffer. The specific preparation method includes the following steps: S1. First, weigh 3.15g Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride and 5.84g sodium chloride and dissolve them in a 1L volumetric flask containing 800mL purified water. Stir until completely dissolved to obtain Tris-HCl buffer solution. S2. Weigh 10g bovine serum albumin, 20g allulose and 4g trisodium methylglycine diacetate, and add them sequentially to the Tris-HCl buffer solution obtained in step S1. Stir until completely dissolved to obtain the first dilution solution. S3. Measure 1 ml of Proclin 300 and add it to the first diluent obtained in step S2. Stir and mix well. Add pure water to the 1 L mark of the volumetric flask. Invert the volumetric flask 10-15 times. Take a sample and test the pH with a pH meter. Adjust the pH to about 8.0 with hydrochloric acid or sodium hydroxide to obtain the lyophilized diluent.

[0050] Comparative Example 3 The lyophilization diluent in this comparative example is prepared by replacing the hydrochloric acid solution in the lyophilization diluent of Example 1 with HEPES buffer. The specific preparation method includes the following steps: S1. First, weigh 5.84g of sodium chloride and 4.77g of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) and dissolve them in a 1L volumetric flask containing 800mL of purified water. Stir until completely dissolved to obtain HEPES buffer. S2. Weigh 10g bovine serum albumin, 20g allulose and 4g trisodium methylglycine diacetate, and add them sequentially to the HEPES buffer obtained in step S1. Stir until completely dissolved to obtain the first dilution. S3. Measure 1 ml of Proclin 300 and add it to the first diluent obtained in step S2. Stir and mix well. Add pure water to the 1 L mark of the volumetric flask. Invert the volumetric flask 10-15 times, take a sample and test the pH with a pH meter. Adjust the pH to about 7.4 with hydrochloric acid or sodium hydroxide to obtain the lyophilized diluent.

[0051] Comparative Example 4 In this comparative example, the composition and preparation method of the lyophilized diluent are basically the same as in Example 1, except that allulose and trisodium methylglycine diacetate are not added.

[0052] Comparative Example 5 In this comparative example, the composition and preparation method of the lyophilized diluent are basically the same as in Example 1, except that allulose is not added.

[0053] Comparative Example 6 In this comparative example, the composition and preparation method of the lyophilized diluent are basically the same as in Example 1, except that trisodium methylglycine diacetate is not added.

[0054] Performance testing 1. Preparation of ELISA kit, sample processing, and kit operation procedures 1) Preparation of standards: Take one vial of recombinant GDF-15 liquid protein from mice and dilute it with the lyophilization diluents prepared in Examples 1-3 and Comparative Examples 1-6 to obtain a lyophilized working solution with a concentration of 5000 pg / ml. Then, take 100 μL from each mouse in the above lyophilized working solution and add it to a cryovial and lyophilize it. This will be used as a lyophilized standard in the ELISA kit. Each group needs to lyophilize an appropriate amount to ensure that there is enough for the experiment.

[0055] The preparation of liquid standards is as follows: a) Take the liquid protein from the same branch used for lyophilization as a liquid standard, and dilute it to 500 pg / ml with sample diluent (50 mM Tris-HCl, 150 mM NaCl, 1% BSA, 0.05% Tween-20, 0.1% Proclin 300). Mix thoroughly with a pipette to obtain standard S7, and set aside for later use.

[0056] (b) Arrange seven 1.5 mL centrifuge tubes (S0-S6) sequentially, and add 250 μL of sample diluent to each. Pipette 250 μL of standard S7 into the first centrifuge tube (S6) and gently mix by pipetting. Pipette 250 μL from S6 into the second EP tube (S5) and gently mix by pipetting. Continue this serial dilution process for the standard. S0 is the sample diluent.

[0057] The freeze-dried standards are prepared as follows: a) Take out the above-mentioned lyophilized standards and centrifuge at 6000-10000 rpm for 30 seconds. Dissolve them with 1 mL of sample diluent (50 mM Tris-HCl, 150 mM NaCl, 1% BSA, 0.05% Tween-20, 0.1% Proclin 300), and repeatedly pipette the solution 5 times from the bottom of the cryovial to aid dissolution. Mix thoroughly to obtain standard S7, and set aside for later use.

[0058] (b) Take seven 1.5 mL centrifuge tubes (S0-S6) from each group and arrange them sequentially. Add 250 μL of sample diluent to each tube. Transfer 250 μL of standard S7 to the first centrifuge tube (S6) and gently mix. Transfer 250 μL from S6 to the second EP tube (S5) and gently mix. Continue this serial dilution process for the standards. S0 is the sample diluent.

[0059] 2) Preparation of the working solution: Dilute the concentrated washing solution with deionized water at a ratio of 1:25. For example, measure 240 mL of deionized water using a graduated cylinder, pour it into a beaker or other clean container, then measure 10 mL of the concentrated washing solution, add it evenly, stir and mix well, and prepare the solution just before use. Salt precipitation may occur if the concentrated washing solution is stored at low temperatures; heating in a water bath can aid dissolution during dilution.

[0060] 3) Preparation of biotin-labeled antibody working solution: Dilute the biotin-labeled antibody solution according to the usage ratio. For example, if diluting at a ratio of 1:100 with antibody diluent (20mM Tris-HCl, 150mM NaCl, 1% BSA, 0.05% Tween-20, 0.05% Proclin 300), add 10μL of biotin-labeled antibody to 990μL of antibody diluent, mix gently, and prepare within 10 minutes before use.

[0061] 4) Preparation of horseradish peroxidase-labeled avidin working solution: Dilute the 1:40 horseradish peroxidase-labeled avidin intermediate solution 1:100 with enzyme diluent (20 mM Tris-HCl, 150 mM NaCl, 1% BSA, 0.05% Tween-20, 0.05% Proclin 300). For example, add 990 μL of enzyme diluent to 10 μL of horseradish peroxidase-labeled avidin, mix gently, and prepare within 10 minutes before use.

[0062] 5) Sample Processing: a) Serum: Whole blood samples should be incubated at room temperature for 2 hours or overnight at 4°C, then centrifuged at 1000xg for 15 minutes at 2-8°C. The supernatant is ready for immediate testing; alternatively, aliquots can be used, and the samples can be stored at -20°C or -80°C, but repeated freeze-thaw cycles should be avoided. Thawed samples should be centrifuged again before testing. b) Plasma: EDTA or heparin can be used as anticoagulants. Within 30 minutes of collection, samples should be centrifuged at 1000xg for 15 minutes at 2-8°C. The supernatant is ready for immediate testing; alternatively, aliquots can be used, and the samples can be stored at -20°C or -80°C, but repeated freeze-thaw cycles should be avoided. Thawed samples should be centrifuged again before testing.

[0063] 6) The reagent kit operation steps are as follows: a) Move all reagents to room temperature (18-25℃) and equilibrate for at least 30 minutes. Prepare the reagents according to the above method and set aside.

[0064] b) Sample addition: Set up separate wells for standard and test samples. Add 100 μL of standard or test sample to each well, cover with a plate label, and incubate at 37°C for 120 min.

[0065] c) Discard the liquid in the wells, spin dry, and wash the plate twice. Soak for 1 minute each time, 200 μL / well, then spin dry.

[0066] d) Add biotin-labeled antibody: Add 100 μL of biotin-labeled antibody working solution to each well, cover with a plate, and incubate at 37°C for 60 min.

[0067] e) Discard the liquid in the wells, spin dry, and wash the plate 3 times. Soak for 1 minute each time, 200 μL / well, then spin dry.

[0068] f) Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a plate, and incubate at 37°C for 60 min.

[0069] g) Discard the liquid in the wells, spin dry, and wash the plate 5 times. Soak for 1 minute each time, 200 μL / well, then spin dry.

[0070] h) Add 90 μL of substrate solution to each well in sequence and incubate at 37 °C for 20 min for color development.

[0071] i) Add 50 μL of the stop solution to each well sequentially to terminate the reaction.

[0072] j) Within 5 minutes after the reaction is terminated, measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm using an ELISA reader.

[0073] 2. Parameter validation of ELISA kit The ELISA kits prepared above were used to conduct comparative experiments on parameters (standard curve testing, standard stability testing, and sample recovery rate testing). The lyophilized standards used in the experiments were obtained by lyophilizing the prepared lyophilized diluents from Examples 1-3 and Comparative Examples 1-6. Specific data are as follows: 1) The results of the standard curve test comparison are shown in Table 1 below.

[0074] Table 1. Standard curve test results of lyophilized standards using the lyophilization diluents from Examples 1-3 and Comparative Examples 1-6.

[0075] The standard curves of the above lyophilized standards prepared from the data in Table 1 are as follows: Figure 1-10 As shown. The standard curve formula for liquid protein standards is as follows: y = d + (ad) / (1 + (x / c)) b In the formula, a = 17.5157, b = -0.8530, c = 2653.8695, d = 0.0438; the standard curve formula for the lyophilized standard using the lyophilized diluent in Example 1 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a = 10.6266, b = -0.9657, c = 1044.3797, d = 0.0522; the standard curve formula for the lyophilized standard using the lyophilized diluent in Example 2 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a = 120.0692, b = -0.7831, c = 47122.0325, d = 0.0452; the standard curve formula for the lyophilized standard using the lyophilized diluent in Example 3 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a = 11.9570, b = -0.9363, c = 1219.1259, d = 0.0483; the standard curve formula for the lyophilized standard using the lyophilized diluent from Comparative Example 1 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a = 426292.1715, b = -0.8847, c = 1415804149.6755, d = 0.0848; the standard curve formula for the lyophilized standard using the lyophilized diluent from Comparative Example 2 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a = 597523.5727, b = -0.8793, c = 1592878307.9307, d = 0.0865; the standard curve formula for the lyophilized standard using the lyophilized diluent from Comparative Example 3 is as follows: y = d + (ad) / (1 + (x / c)).b In the formula, a = 973631.7912, b = -0.9415, c = 1752683467.7774, d = 0.0770; the standard curve formula for the lyophilized standard using the lyophilized diluent from Comparative Example 4 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a = 26.5887, b = -0.8953, c = 8200.7306, d = 0.0549; the standard curve formula for the lyophilized standard using the lyophilized diluent from Comparative Example 5 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a = 6.5624, b = -1.0265, c = 491.8440, d = 0.0644; the standard curve formula for the lyophilized standard using the lyophilized diluent from Comparative Example 6 is as follows: y = d + (ad) / (1 + (x / c)). b In the formula, a=7.2890, b=-0.8969, c=628.8725, d=0.0781.

[0076] From the above formula, it can be seen that the relevant parameters of the standard curve after the liquid standard is lyophilized with the lyophilized diluent in Examples 1-3 are: R 2 >0.99, P / N>45, and the average OD value decay rate of the standard curves compared to the liquid standards is <6%, indicating that the standard curves of the three lyophilized standards in Examples 1-3 all meet industry requirements. The relevant parameters of the standard curves after the liquid standards were lyophilized with the lyophilized diluents in Comparative Examples 1-4 and then reconstituted are: R 2 >0.99, P / N<35, and the average OD value attenuation rate of the standard curves compared to the liquid standards are all >15%. The results indicate that the standard curves of the lyophilized diluents in Comparative Examples 1-4 do not meet the requirements and no further verification is needed. The relevant parameters of the standard curves after the liquid standards were lyophilized with the lyophilized diluents in Comparative Examples 5-6 and then reconstituted are: R 2 With values ​​>0.99 and P / N >45, the average OD value decay rate of the standard curves is <10% compared to the liquid form, indicating that the three standard curves of Comparative Examples 5 and 6 meet industry standards. Therefore, the linearity of the standard curve of the kit prepared by the lyophilized diluent provided in the embodiments of this application is not affected.

[0077] 2) Stability test of freeze-dried standards To assess the stability of ELISA standards and scientifically estimate their shelf life, an accelerated degradation assay is typically used: the standards are subjected to accelerated degradation at 37°C for 7-10 days. Subsequently, this 37°C-stored standard is simultaneously subjected to parallel ELISA assays with fresh standards stored in a chromatography cabinet at 2°C–8°C (i.e., the initial standards that have not undergone accelerated degradation). The key parameters of the standard curves (such as OD value and linear correlation coefficient R) of the two standards are compared. 2 The experiment aimed to verify whether the performance of the standards after accelerated treatment at 37℃ remained within the preset acceptable range. The core objective of this experiment was to simulate the protein degradation trend during long-term storage, thereby verifying the stability of the standards under recommended storage conditions (2℃~8℃) in advance, and thus ensuring the quantitative accuracy and reliability of the results throughout the ELISA detection process. The results are shown in Table 2 below.

[0078] Table 2. Stability test results of lyophilized standards using the lyophilization diluents from Examples 1-3 and Comparative Examples 5-6

[0079] As shown in Table 2, the OD value deviation rate of the standard curves determined using the lyophilized diluents in Examples 1-3 of this invention is less than 10%, which meets the quality control standards of the reagent kit industry. However, the OD value deviation rate of the standard curves determined using the lyophilized diluents in Comparative Examples 5-6 of this invention is mostly greater than 20%, which does not meet the quality control standards of the reagent kit industry, and no further related verification is required. Therefore, the lyophilized standards prepared using the lyophilized diluents provided in the examples of this invention meet industry requirements.

[0080] 3) Sample recovery rate test To verify that the lyophilized standards using the lyophilization diluents from Examples 1-3 do not interfere with ELISA detection results, and to evaluate the recovery rate of mouse GDF-15 in different matrix samples, the experiment was designed as follows: Liquid control standards and lyophilized standards using the lyophilization diluents from Examples 1-3 were added to the target matrix samples. The recovery rates of these standards were detected by ELISA and calculated. By comparing the differences in the recovery rates of these standards, the consistency of detection between the lyophilized standards and the liquid control standards in different matrices was verified. If there was no significant difference in the recovery rates and both met the preset acceptable range (e.g., 80%-120%), it was proven that the lyophilization process did not affect the detection performance of the standards, thus ensuring that the ELISA detection results based on these lyophilized standards could truly reflect the actual concentration of endogenous mouse GDF-15 in the samples, providing core data support for the reliability of subsequent sample detection. The results are shown in Table 3 below.

[0081] Table 3. Recovery rate test results of lyophilized standards using the lyophilization diluents from Examples 1-3.

[0082] As shown in Table 3, the recovery rates of serum and plasma tested using the lyophilized standards prepared with the lyophilized diluents in Examples 1-3 ranged from 80% to 120%, which meets the quality control standards of the reagent kit industry. Therefore, the lyophilized standards prepared with the lyophilized diluents provided in the embodiments of this invention did not affect the detection performance of the reagent kit and met the industry quality control standards.

[0083] Furthermore, the results of the parameters (standard curve test, standard stability test and sample recovery rate test) of the above ELISA kits are summarized in Table 4 below.

[0084] Table 4. Parameter validation results of the ELISA kit

[0085] As shown in Table 4, the lyophilized standards prepared using the lyophilized diluents in Examples 1-3 of this invention fully comply with the industry quality control standards for ELISA kits.

[0086] In summary, the lyophilized diluent provided by this invention can significantly and effectively reduce the attenuation of standards and improve their stability through the interaction between protein stabilizers, metal ion chelators, and carbohydrate compounds. This helps to extend the shelf life of enzyme-linked immunosorbent assay kits and improve the accuracy of experimental results, thereby obtaining more reliable and accurate experimental results.

[0087] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0088] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A lyophilized diluent, characterized in that, The lyophilized diluent comprises the following components: 1-5 mM basal buffer, 0.5-2% protein stabilizer, 1-5% carbohydrate compound, 0.1-0.4% metal ion chelating agent, and 0.05-0.1% preservative; The basic buffer solution includes a hydrochloric acid solution.

2. The lyophilized diluent according to claim 1, characterized in that, The protein stabilizer includes at least one of bovine serum albumin and goat serum albumin.

3. The lyophilized diluent according to claim 1, characterized in that, The carbohydrate compound includes at least one of arabinose, allulose, and isomaltitol.

4. The lyophilized diluent according to claim 1, characterized in that, The metal ion chelating agent includes at least one of trisodium methylglycine diacetate and tetrasodium glutamate diacetate.

5. The lyophilized diluent according to claim 1, characterized in that, The preservatives include Proclin 300.

6. The lyophilized diluent according to claim 1, characterized in that, The pH value of the lyophilized diluent is 2-3.

7. A method for preparing a lyophilized diluent as described in any one of claims 1-6, characterized in that, Includes the following steps: Provide basic buffer solution; Protein stabilizers, carbohydrate compounds, and metal ion chelating agents are added to the base buffer and dissolved to obtain the first dilution. After adding a preservative to the first diluent, the lyophilized diluent is obtained.

8. The use of the lyophilized diluent as described in any one of claims 1-6 or the lyophilized diluent prepared by the preparation method described in claim 7 in the preparation of recombinant protein lyophilized standards.

9. The application according to claim 8, characterized in that, The recombinant protein lyophilized standard includes the recombinant GDF-15 protein lyophilized standard.

10. An ELISA kit for detecting recombinant GDF-15 protein, characterized in that, Includes the lyophilized diluent of any one of claims 1-6, the lyophilized diluent prepared by the preparation method of claim 7, or the lyophilized recombinant protein standard of any one of claims 8-9.

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