Freeze-drying protective solution, freeze-drying process, freeze-drying standard and detection kit for CD89-poly-IgA detection system

Abstract

This invention provides a lyophilization protectant, lyophilization process, lyophilized standards, and detection kit for a CD89-Poly-IgA detection system. The lyophilization protectant, by mass-volume ratio, comprises the following components: 1%–10% disaccharide protectant, 0.5%–5% polysaccharide / polymer stabilizer, 1%–10.0% sugar alcohol auxiliary stabilizer, 0.05%–0.5% disulfide bond protectant, 0.01%–0.1% surfactant, with the balance being a buffer system. The lyophilization protectant proposed in this invention provides synergistic protection of disaccharides and proteins, maintenance of ligand-receptor activity, long-term stability, and process compatibility.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a lyophilization protective solution, lyophilization process, lyophilized standard, and detection kit for a CD89-Poly-IgA detection system. Background Technology

[0002] Poly-IgA is an immunoglobulin complex formed by two or more IgA monomers covalently linked by a J chain. Recent studies have shown that serum poly-IgA levels are closely related to the development of IgA nephropathy (IgAN), mucosal inflammatory diseases, and certain autoimmune diseases, and it has become an emerging biomarker in the field of nephrology. Poly-IgA contains a complex glycosylation structure, characterized by: O-glycosylation in the hinge region, N-glycosylation in the Fc region, and J-chain glycosylation.

[0003] CD89 (FcαRI) is a specific receptor for the Fc segment of IgA. CD89 has a low affinity for monomeric IgA (mIgA) (Ka ~ 10⁻⁶ M, micromolar level), but it achieves high-affinity and sustained binding with poly-IgA due to the affinity effect of multivalent binding. This characteristic makes it an ideal probe for detecting serum poly-IgA levels. The extracellular region of CD89 contains six potential N-glycosylation sites (Asn-X-Ser / Thr motifs) and may also contain O-glycosylation modifications. Glycosylation not only participates in intramolecular interactions of CD89 (maintaining the correct folding of the D1 / D2 domains) but may also affect the oligomerization state of the receptor through glycan-glycan interactions, thereby regulating the affinity binding efficiency with poly-IgA.

[0004] The binding of CD89 to monomeric IgA is a typical low-affinity, rapidly dissociating acceptor-ligand interaction. Poly-IgA generates an affinity effect by simultaneously binding multiple IgA monomers to multiple CD89 molecules, significantly improving the overall binding stability. This mechanism requires both CD89 and Poly-IgA polymers to maintain structural integrity; after lyophilization and rehydration, both CD89 and Poly-IgA must simultaneously restore their functional conformations. Damage to the structure of either leads to the loss of the affinity effect.

[0005] Freeze-drying is a common technique for the long-term preservation of biological products. It removes moisture through low-temperature vacuum sublimation, allowing proteins to remain stable in a solid state for extended periods. However, existing freeze-drying preservation technologies have the following main shortcomings: Designed for non-glycosylated or low-glycosylated proteins: Conventional lyophilization protection solutions (such as PBS containing 5%-10% trehalose or sucrose) are mainly designed for antibody drugs (IgG, with only one N-glycosylation site in the Fc segment) or simple proteins, without considering the special protection requirements of glycan chains of highly glycosylated proteins during the lyophilization-rehydration process. Single protein protection mode: Existing technologies typically protect a single active component (such as a monoclonal antibody drug or a single standard), and do not address the complex scenario where the same formulation needs to simultaneously protect two proteins with different glycosylation modes and functional interactions (receptor + ligand); Neglecting the preservation of ligand-receptor activity: Current evaluation indicators for lyophilization protection are mostly protein structural integrity (SDS-PAGE, circular dichroism) or single activity recovery rate, without taking the ligand-receptor interaction activity after rehydration as the core evaluation criterion. Insufficient glycan protection: The glycans of highly glycosylated proteins are prone to breakage, dehydration, or conformational changes during freeze-drying. Although conventional protective agents can protect the protein backbone through water substitution mechanisms, their specific protection of glycans is limited. Therefore, there is an urgent need for a lyophilization protection solution and a matching lyophilization process specifically designed for the CD89-Poly-IgA ligand-receptor detection system, which can simultaneously protect the structural integrity and interaction activity of the two highly glycosylated proteins, and achieve long-term stable preservation of the kit components (CD89 protein, Poly-IgA standard / quality control). Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a lyophilization protective solution, lyophilization process, lyophilized standard and detection kit for the CD89-Poly-IgA detection system, so as to at least solve the deficiencies in the above-mentioned technologies.

[0007] This invention proposes a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: The composition consists of 1%~10% disaccharide protectant, 0.5%~5% polysaccharide / polymer stabilizer, 1%~10.0% sugar alcohol auxiliary stabilizer, 0.05%~0.5% disulfide bond protectant, 0.01%~0.1% surfactant, and the remainder is a buffer system.

[0008] Furthermore, the disaccharide protectant is selected from at least one of trehalose, sucrose, lactose, and maltose.

[0009] Furthermore, the polysaccharide / polymer stabilizer is selected from at least one of dextran, hydroxyethyl starch, polyethylene glycol, and polyvinylpyrrolidone.

[0010] Furthermore, the sugar alcohol auxiliary stabilizer is selected from at least one of mannitol, sorbitol, inositol, and xylitol.

[0011] Furthermore, the disulfide bond protectant is selected from at least one of reduced glutathione, cysteine, dithiothreitol, and tris(2-carboxyethyl)phosphine.

[0012] Furthermore, the surfactant is selected from at least one of Tween-20, Tween-80, and Triton-X100.

[0013] This invention also proposes a lyophilization process using the above-mentioned CD89-Poly-IgA detection system lyophilization protectant, comprising the following steps: S1: Sample preparation: Mix the CD89 protein solution or Poly-IgA standard / quality control solution with the lyophilization protection solution in a certain proportion and dispense them into lyophilization containers; S2: Pre-freezing: Cool the sample to -40°C to -50°C at a rate of 1-3°C / min, without vacuum, and hold for 2-4 hours; S3: First stage freeze-drying: Under vacuum conditions ≤15Pa, the temperature is increased to -25℃ to -20℃ in a stepwise manner at a rate of 0.3-0.6℃ / min, and held for 1-2 hours after each 5℃ increase; S4: Second stage freeze-drying: Increase the temperature by 25-30℃ at a rate of 0.3-0.6℃ / min, holding the temperature for 2-3 hours after each 10℃ increase, while maintaining a vacuum of ≤15Pa. S5: Store in a sealed container. After drying, seal under vacuum or nitrogen-filled conditions and store at 2-8°C away from light.

[0014] Furthermore, in step S1, the final concentration of CD89 protein is 0.5-2 mg / mL, the final concentration of Poly-IgA protein is 2-10 μg / mL, and the lyophilization container is a vial or lyophilization tube, with each vial / tube containing 25-100 μL. In step S2, the cooling rate is controlled at 2℃ / min to avoid rapid cooling forming too many small ice crystals that could cause mechanical damage.

[0015] The present invention also proposes a freeze-dried Poly-IgA standard, which is prepared by the above-mentioned freeze-drying process.

[0016] This invention also proposes a CD89-Poly-IgA complex enzyme-linked immunosorbent assay kit, characterized in that it comprises: lyophilized CD89 coated protein, lyophilized Poly-IgA standard, lyophilized Poly-IgA quality control, enzyme-labeled detection antibody, coating buffer, sample dilution buffer, washing buffer, blocking buffer, chromogenic solution (TMB substrate A and B), stop solution, ELISA plate, and sealing film, wherein the lyophilized CD89 coated protein, the lyophilized Poly-IgA standard, and the lyophilized Poly-IgA quality control are prepared using the lyophilization process described in any one of claims 8-9.

[0017] Compared with the prior art, the CD89-Poly-IgA detection system lyophilization protective solution, lyophilization process, lyophilized standards and detection kit of the present invention have the following beneficial effects: 1. Synergistic protection of disaccharide proteins: The same lyophilization protection solution formulation simultaneously protects CD89 receptor protein and Poly-IgA ligand / standard, maintaining the functional integrity of both after lyophilization and rehydration; 2. Ligand-receptor activity retention: After lyophilization and rehydration, the binding activity of CD89-Poly-IgA was restored, with a relative deviation of less than 10% compared to before lyophilization; 3. Long-term stability: After being stored at 2-8℃ for 12 months or accelerated aging at 25℃ for 1 month, the core analytical performance of the freeze-dried product does not deteriorate. 4. Process compatibility: The lyophilization protective solution is compatible with other components of the CD89-Poly-IgA detection system and does not affect the subsequent ELISA detection process. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the CD89-Poly-IgA ligand-receptor interaction pattern in the eighth embodiment of the present invention; Figure 2 This is a comparison chart of the standard curves of the CD89 pre-coated plate before freeze-drying and after freeze-drying and rehydration in the eighth embodiment of the present invention. Figure 3 This is a comparison of the appearance of the two freeze-drying processes in the eighth embodiment of the present invention.

[0019] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Several embodiments of the invention are illustrated in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0022] Example 1 The first embodiment of the present invention provides a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: The composition consists of 1%~10% disaccharide protectant, 0.5%~5% polysaccharide / polymer stabilizer, 1%~10.0% sugar alcohol auxiliary stabilizer, 0.05%~0.5% disulfide bond protectant, 0.01%~0.1% surfactant, and the remainder is a buffer system.

[0023] In this embodiment, the function of the disaccharide protectant is to maintain the protein backbone and glycan hydrogen bond network through a water substitution mechanism. It is selected from at least one of trehalose, sucrose, lactose, and maltose. Preferably, the disaccharide protectant is trehalose, with a content of 4% to 6%. Trehalose is a non-reducing disaccharide with a high glass transition temperature (Tg' about -30°C) and is not prone to Maillard reaction during rehydration. The polysaccharide / polymer stabilizer is formed through a glassy matrix, which increases the Tg' of the system and prevents collapse. It is selected from at least one of dextran (molecular weight 10-70 kDa), hydroxyethyl starch (HES), polyethylene glycol (PEG, molecular weight 3.35-6 kDa), and polyvinylpyrrolidone (PVP, molecular weight 10-40 kDa). Preferably, the polysaccharide / polymer stabilizer is polyvinylpyrrolidone (PVP K30) with a content of 1% to 2%. The high molecular weight polymer improves the viscosity and glassy stability of the system and prevents freeze-drying collapse. The sugar alcohol auxiliary stabilizer can reduce the ice crystal growth rate and protect the fine structure of the sugar chain. It is selected from at least one of mannitol, sorbitol, inositol, and xylitol. Preferably, the sugar alcohol auxiliary stabilizer is mannitol, with a content of 4% to 6%. Mannitol acts as a filler to form a good cake structure. The disulfide bond protectant is capable of maintaining the stability of the J-chain disulfide bond and the CD89 D1 domain disulfide bond, and is selected from at least one of reduced glutathione (GSH), cysteine, dithiothreitol (DTT, low concentration ≤0.1%), and tris(2-carboxyethyl)phosphine (TCEP, low concentration ≤0.05%). Preferably, the disulfide bond protectant is reduced glutathione (GSH) with a content of 0.1%~0.2%. GSH maintains a moderately reducing environment in the liquid phase before freeze-drying, and stabilizes the disulfide bond conformation in the solid state after freeze-drying through an intermolecular hydrogen bond network. The surfactant is capable of preventing protein aggregation and is selected from at least one of Tween-20, Tween-80, and Triton-X100. Preferably, the surfactant is Tween-80, and its content is 0.01% to 0.05%. The buffer system can maintain a pH value between 6.5 and 7.5 and is selected from at least one of phosphate buffer (PBS, pH 6.8-7.4), histidine buffer (pH 6.5-7.0), and Tris-HCl buffer (pH 7.0-7.5). Preferably, the buffer system is PBS, and the weakly acidic to neutral environment is beneficial to reducing the protein deamidation reaction during the freeze-drying process.

[0024] In this embodiment, the lyophilization protective solution of the CD89-Poly-IgA detection system comprises the following components by mass-volume ratio: 5.0% trehalose, 0.5% PVP K30, 5.0% mannitol, 0.15% reduced glutathione (GSH), 0.01% Tween-80, balance PBS (pH 7.0).

[0025] Example 2 The second embodiment of the present invention provides a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: 5% sucrose, 0.5% PVP K30, 5% mannitol, 0.15% GSH, 0.01% Tween-80, balance PBS.

[0026] Example 3 The third embodiment of the present invention provides a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: 5% trehalose, 0.5% dextran, 5% mannitol, 0.15% GSH, 0.01% Tween-80, balance PBS.

[0027] Example 4 The fourth embodiment of the present invention provides a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: 5% Trehalose + Sucrose, 0.5% PVP K30, 5% Inositol, 0.15% GSH, 0.01% Tween-80, balance PBS.

[0028] Example 5 The fifth embodiment of the present invention provides a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: 5% trehalose, 0.5% dextran, 5% mannitol, 0.15% DTT, 0.01% Tween-80, balance PBS.

[0029] Example 6 The sixth embodiment of the present invention provides a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: 5% Trehalose, 0.5% PVP K30 + 1% dextran 40, 5% mannitol, 0.15% GSH, 0.01% Tween-80, balance PBS.

[0030] Example 7 The seventh embodiment of the present invention provides a lyophilization protective solution for a CD89-Poly-IgA detection system, which comprises the following components by mass-volume ratio: 5% trehalose + 5% sucrose, 0.5% PVP K30 + 1% dextran 40, 5% inositol, 0.15% DTT, 0.01% triton X100, balance PBS.

[0031] Furthermore, in each embodiment, the freeze-dried CD89, high-concentration calibrator, medium-concentration standard, low-concentration calibrator, and quality control sample were stored in two batches. One batch was stored at 4°C, and the other at 25°C. The same batch of calibrators was then used to perform tests on days 1, 6, 15, 20, and 30. The relative deviation from the calibrator was calculated, and the average value was taken based on the storage temperature. Relative deviation = (Formula - Calibrator) / Calibrator * 100.

[0032] At a given storage temperature, the relative deviations of the above embodiments were calculated. Please refer to Table 1 below for details:

[0033] As shown in Table 1 above, the average relative deviation of the formulations in Example 1 and Example 6 at a given storage temperature is relatively good, with the formulation in Example 1 being better than that in Example 6.

[0034] The detection concentrations of different concentrations of lyophilization protective solutions in the above embodiments at different incubation temperatures and different storage days are compared in Table 2 below: Table 2

[0035] Example 8 The eighth embodiment of the present invention provides a lyophilization process for the lyophilization protective solution of the above-mentioned CD89-Poly-IgA detection system, including the following steps: S1: Sample preparation: Mix the CD89 protein solution or Poly-IgA standard / quality control solution with the lyophilization protection solution in a certain proportion and dispense them into lyophilization containers; S2: Pre-freezing: Cool the sample to -40°C to -50°C at a rate of 1-3°C / min, without vacuum, and hold for 2-4 hours; S3: First stage freeze-drying: Under vacuum conditions ≤15Pa, the temperature is increased to -25℃ to -20℃ in a stepwise manner at a rate of 0.3-0.6℃ / min, and held for 1-2 hours after each 5℃ increase; S4: Second stage freeze-drying: Increase the temperature by 25-30℃ at a rate of 0.3-0.6℃ / min, holding the temperature for 2-3 hours after each 10℃ increase, while maintaining a vacuum of ≤15Pa. S5: Store in a sealed container. After drying, seal under vacuum or nitrogen-filled conditions and store at 2-8°C away from light.

[0036] Furthermore, in step S1, the final concentration of the CD89 protein is 0.5-2 mg / mL, the final concentration of the Poly-IgA protein is 2-10 μg / mL, and the lyophilization container is a vial or lyophilization tube, with each vial / tube containing 25-100 μL.

[0037] Furthermore, in step S2, the cooling rate is controlled at 2℃ / min to avoid rapid cooling forming too many small ice crystals that could cause mechanical damage.

[0038] For a detailed diagram of the CD89-Poly-IgA ligand-receptor interaction, please refer to [link / reference needed]. Figure 1 Please refer to the comparison chart of the standard curves of CD89 pre-coated plates before freeze-drying and after freeze-drying and rehydration. Figure 2 ; In this embodiment, a second freeze-drying process is also provided, which differs from the freeze-drying process described above in that: In step S3, under vacuum conditions ≤15Pa, the temperature is increased stepwise to -25℃ to -20℃ at a rate of 0.3-0.6℃ / min and maintained for 6-10 hours.

[0039] In step S4, the temperature is increased by a gradient of 25-30℃ at a rate of 0.3-0.6℃ / min and maintained for 8-12 hours, while keeping the vacuum level ≤15Pa.

[0040] In this embodiment, the appearance of the two freeze-drying processes described above is examined (e.g., Figure 3 (As shown in the image) and the relative concentration deviations before and after freeze-drying are listed below. Please refer to Tables 3 and 4 for details: Table 3: Appearance after freeze-drying process

[0041] Table 4: Relative Concentration Deviation Before and After Freeze-drying

[0042] Understandably, the appearance and performance deviation of the freeze-drying process 1 are better, so freeze-drying process 1 is selected as the best freeze-drying process.

[0043] Furthermore, the following comparison examines the relative deviations in luminescence before and after freeze-drying in different batches: Using the freeze-drying process 1 described above, three batches of CD89, standard products, and quality control products were freeze-dried respectively. The relative deviation of the luminescence value before and after freeze-drying was detected to verify the stability between batches. For details, please refer to Table 5.

[0044] Table 5

[0045] Furthermore, the key process parameters of the above-mentioned freeze-drying process 1 are adapted to the thermodynamic properties of the CD89-Poly-IgA system:

[0046] Example 9 This embodiment also proposes a CD89-Poly-IgA complex enzyme-linked immunosorbent assay kit, comprising: lyophilized CD89 coated protein, lyophilized Poly-IgA standard, lyophilized Poly-IgA quality control, enzyme-labeled detection antibody (such as HRP-labeled anti-IgA antibody), coating buffer, sample dilution buffer, washing buffer, blocking buffer, chromogenic solution (TMB substrate A and B), stop solution, ELISA plate, and sealing film. The lyophilized CD89 coated protein, lyophilized Poly-IgA standard, and lyophilized Poly-IgA quality control are all lyophilized using the lyophilization process 1 described in Example 8.

[0047] Example 10 This embodiment also proposes a lyophilization preparation method for Poly-IgA standards / quality control products. The Poly-IgA complex (concentration set according to the detection range, such as 10 μg / mL) is dissolved in the lyophilization protection solution in Example 1 above, dispensed, lyophilized according to the lyophilization process 1 in Example 8, and sealed for storage.

[0048] Furthermore, please refer to Table 6 below for the comparison results of the prepared Poly-IgA standards / controls before and after lyophilization: Table 6

[0049] Understandably, the relative deviation of the OD (luminosity) of the calibrator raw material Poly-IgA before and after lyophilization is within ±10%, which meets the performance requirements.

[0050] Furthermore, the long-term stability of the prepared Poly-IgA standard / control sample after lyophilization was tested, specifically: The samples were stored at 2℃~8℃ for 12 months, and tested at 0, 3, 6, 9, and 12 months respectively. The prepared Poly-IgA standard / quality control was lyophilized to prepare the detection kit in Example 9. The kit components are as follows:

[0051] The performance of the test kit is monitored, including accuracy, repeatability, batch-to-batch variation, calibrator homogeneity, calibrator accuracy, intra-vial homogeneity of calibrators, inter-vial homogeneity of calibrators, and quality control accuracy, intra-vial homogeneity of quality control materials, and inter-vial homogeneity of quality control materials. The standards are as follows: (1) Accuracy The recovery rate should be within the range of 85% to 115%. (2) Linear Within the range of [15.63~500] U / mL, the linear correlation coefficient (r) should not be less than 0.9900; (3) Repeatability The coefficient of variation (CV) should not exceed 10.0%; (4) Batch-to-batch variation The coefficient of variation (CV) should not exceed 15.0%; (5) Accuracy of calibrators The relative deviation shall not exceed ±10%.

[0052] Please refer to Table 7 for detailed results: Table 7

[0053] Understandably, all stability indicators meet the standards, and the freeze-drying protective liquid and products made using the freeze-drying process in Example 1 can maintain stability for 12 months.

[0054] Example 11 This embodiment also proposes a method for detecting the Poly-IgA content in a sample using a kit containing the above-mentioned lyophilized reagent, including the following steps: Take out the CD89 protein lyophilized powder, add coating buffer, and let stand at room temperature for 10-15 minutes to reconstitute; Lyophilized standards / quality control samples are used after being rehydrated with sample diluent. Perform the standard ELISA test according to the usual operating procedures.

[0055] In summary, the lyophilization protective solution for the CD89-Poly-IgA detection system in the above embodiments of the present invention has the following beneficial effects: 1. Synergistic protection of disaccharide proteins: The same lyophilization protection solution formulation simultaneously protects CD89 receptor protein and Poly-IgA ligand / standard, maintaining the functional integrity of both after lyophilization and rehydration; 2. Ligand-receptor activity retention: After lyophilization and rehydration, the binding activity of CD89-Poly-IgA was restored, with a relative deviation of less than 10% compared to before lyophilization; 3. Long-term stability: After being stored at 2-8℃ for 12 months or accelerated aging at 25℃ for 1 month, the core analytical performance of the freeze-dried product does not deteriorate. 4. Process compatibility: The lyophilization protective solution is compatible with other components of the CD89-Poly-IgA detection system and does not affect the subsequent ELISA detection process.

[0056] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively 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 this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A lyophilization protective solution for a CD89-Poly-IgA detection system, characterized in that, Based on mass-volume ratio, it includes the following components: The composition consists of 1%~10% disaccharide protectant, 0.5%~5% polysaccharide / polymer stabilizer, 1%~10.0% sugar alcohol auxiliary stabilizer, 0.05%~0.5% disulfide bond protectant, 0.01%~0.1% surfactant, and the remainder is a buffer system.

2. The lyophilization protective solution for the CD89-Poly-IgA detection system according to claim 1, characterized in that, The disaccharide protectant is selected from at least one of trehalose, sucrose, lactose, and maltose.

3. The lyophilization protective solution for the CD89-Poly-IgA detection system according to claim 1, characterized in that, The polysaccharide / polymer stabilizer is selected from at least one of dextran, hydroxyethyl starch, polyethylene glycol, and polyvinylpyrrolidone.

4. The lyophilization protective solution for the CD89-Poly-IgA detection system according to claim 1, characterized in that, The sugar alcohol auxiliary stabilizer is selected from at least one of mannitol, sorbitol, inositol, and xylitol.

5. The lyophilization protective solution for the CD89-Poly-IgA detection system according to claim 1, characterized in that, The disulfide bond protectant is selected from at least one of reduced glutathione, cysteine, dithiothreitol, and tris(2-carboxyethyl)phosphine.

6. The lyophilization protective solution for the CD89-Poly-IgA detection system according to claim 1, characterized in that, The surfactant is selected from at least one of Tween-20, Tween-80, and Triton-X100.

7. A lyophilization process using the CD89-Poly-IgA detection system lyophilization protective solution according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Sample preparation: Mix the CD89 protein solution or Poly-IgA standard / quality control solution with the lyophilization protection solution in a certain proportion and dispense them into lyophilization containers; S2: Pre-freezing: Cool the sample to -40°C to -50°C at a rate of 1-3°C / min and hold for 2-4 hours; S3: First stage freeze-drying: Under vacuum conditions ≤15Pa, the temperature is increased to -25℃ to -20℃ in a stepwise manner at a rate of 0.3-0.6℃ / min, and held for 1-2 hours after each 5℃ increase; S4: Second stage freeze-drying: Increase the temperature by 25-30℃ at a rate of 0.3-0.6℃ / min, holding the temperature for 2-3 hours after each 10℃ increase, while maintaining a vacuum of ≤15Pa. S5: Store in a sealed container. After drying, seal under vacuum or nitrogen-filled conditions and store at 2-8°C away from light.

8. The freeze-drying process according to claim 7, characterized in that, In step S1, the final concentration of the CD89 protein solution is 0.5-2 mg / mL, the final concentration of the Poly-IgA standard / quality control solution is 2-10 μg / mL, and the lyophilization container is a vial or lyophilization tube, with each vial / tube containing 25-100 μL. In step S2, the cooling rate is controlled at 2℃ / min to avoid rapid cooling forming too many small ice crystals that could cause mechanical damage.

9. A freeze-dried Poly-IgA standard, characterized in that, It is made using the freeze-drying process described in any one of claims 7-8.

10. A CD89-Poly-IgA complex enzyme-linked immunosorbent assay kit, characterized in that, include: The freeze-dried CD89 coated protein, the freeze-dried Poly-IgA standard, and the freeze-dried Poly-IgA quality control product are prepared using the freeze-drying process described in any one of claims 7-8.

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