A preservation solution, an adiponectin detection kit, its preparation method and application

Abstract

This invention relates to the field of biodetection technology, disclosing a preservation solution, an adiponectin detection kit, its preparation method, and its application. The preservation solution comprises Tris, trehalose, Tween 20, sodium caseinate, PVP-10, Proclin-300, TX-100, surfactant S13, and glycine. This specific formulation effectively prevents quantum dot aggregation and maintains protein activity for a long time. The adiponectin detection kit is based on quantum dot immunofluorescence chromatography technology and includes components such as a sample pad, a release pad, and a nitrocellulose membrane. The release pad is simultaneously coated with quantum dot-labeled antibodies and unlabeled detection line antibodies, and the quantum dot-labeled antibodies are coupled in a cascade manner of "quantum dot-streptavidin-biotinylated antibody". This invention, through liquid dialysis linking technology and a unique release pad antibody premixing process, utilizes the signal amplification effect of the biotin system and the principle of homogeneous reaction, significantly improving the sensitivity, accuracy, and reagent stability of the detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a preservation solution, an adiponectin detection kit, its preparation method, and its application. Background Technology

[0002] Adiponectin (APN) is an endogenous bioactive polypeptide or protein secreted by adipocytes. Initially, adiponectin was discovered in human subcutaneous adipose tissue, plasma, and adipocytes of mice. Human adiponectin consists of 244 amino acids with a molecular weight of 30 kDa, encoded by the apM1 gene on chromosome 3q27. The main sources of adiponectin secretion include adipocytes, skeletal muscle cells, endothelial cells, and cardiomyocytes. Structurally, adiponectin is composed of monomers that link together to form trimers, which further combine to form a macromolecular structure of 4-6 trimers. Notably, adiponectin monomers are only present in adipocytes; they are only secreted into the bloodstream after forming polymers. As an insulin-sensitizing hormone, adiponectin promotes fatty acid oxidation and glucose uptake in skeletal muscle cells, significantly enhances the inhibitory effect of insulin on gluconeogenesis, and inhibits hepatic glucose production. It is an important regulator in the body's lipid metabolism and glucose homeostasis regulation network.

[0003] In recent years, with changes in lifestyle, the number of people with diabetes has increased exponentially. Diabetes, as a lifelong disease, is not only incurable, requiring lifelong medication to delay the onset of complications, but its complications have also become a leading cause of death. Clinical studies have shown that adiponectin differs significantly from glycated hemoglobin and blood glucose levels: serum or plasma adiponectin levels can predict the risk of developing type 2 diabetes 4-7 years in advance. For individuals with metabolic abnormalities, adiponectin testing can enable early detection, prevention, and treatment. Furthermore, adiponectin levels are negatively correlated with insulin resistance, which can not only aid in clinical classification and diagnosis, reflecting the degree of insulin resistance, but also be used to assess the reversal effect of drug treatment, providing guidance for the clinical application of insulin.

[0004] Currently, the main methods for detecting adiponectin on the market are latex immunoturbidimetry and chemiluminescence. Although these methods are relatively mature in large laboratories, they have obvious limitations: (1) Long detection cycle and low efficiency: The above traditional methods usually require a long reaction and analysis time, and medical staff and patients have to wait for the results for a long time, which is not conducive to rapid diagnosis; (2) Complex operation and high threshold: The professional skills of the operators are required to a high degree, and a long period of training and learning is usually required; (3) Expensive equipment and large size: It relies on large automated analytical instruments, and the equipment cost and maintenance cost are high, making it difficult to popularize in primary hospitals, community clinics or point-of-care testing (POCT) scenarios; (4) Difficult waste disposal: A lot of medical waste is generated, and the disposal cost is high.

[0005] While immunochromatographic techniques (such as colloidal gold methods) offer advantages in speed and convenience, there are currently few mature adiponectin chromatography products on the market. This is mainly because adiponectin exists in blood in a polymeric form with a complex structure, requiring high detection sensitivity and reagent stability. Ordinary chromatography kits often suffer from technical challenges such as insufficient sensitivity (difficulty in accurately detecting low-concentration samples), poor storage stability (easily leading to false negatives due to antibody inactivation), and significant background interference. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a preservation solution, an adiponectin detection kit, its preparation method and application. By constructing a unique quantum dot-streptavidin-biotin signal amplification system, combined with a specially formulated antibody preservation solution and liquid formulation, and employing a unique antibody release pad treatment process, a rapid adiponectin detection solution with high sensitivity, high stability, simple operation and low cost is achieved.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] In a first aspect, the present invention provides a preservation solution comprising, by mass fraction, the following components: 9-11 mM Tris with a pH of 6.0-6.5, 9-11% trehalose, 4-6% Tween 20, 4-6% sodium caseinate, 1.1-1.3% PVP-10, 0.8-1.2% proclin-300, 1-3% TX-100, 0.04-0.06% surfactant S13, and 0.4-0.6% glycine, with the remaining components made up to 100% with water, thereby obtaining a preservation solution with a pH finely adjusted to 6.0-6.5.

[0009] Preferably, the preservation solution comprises the following components by mass fraction: 10mM Tris with a pH of 6.0-6.5, 10% trehalose, 5% Tween 20, 5% sodium caseinate, 1.2% PVP-10, 5% sodium caseinate, 1% proclin-300, 2% TX-100, 0.05% surfactant S13, and 0.5% glycine. The remaining content is made up to 100% with water, and the pH of the preservation solution is finely adjusted to 6.0-6.5.

[0010] Secondly, the present invention provides an adiponectin detection kit, comprising, in sequence, a sample pad, a quantum dot binding and release pad, a nitrocellulose membrane, absorbent paper, and a PVC base plate. The quantum dot binding and release pad is coated with quantum dot-labeled adiponectin monoclonal antibody and unlabeled adiponectin mouse monoclonal antibody. The quantum dot-labeled adiponectin monoclonal antibody is dispersed and stored in the above-mentioned preservation solution before being coated onto the release pad. The nitrocellulose membrane is coated with adiponectin monoclonal antibody as a detection line and anti-mouse IgG antibody as a quality control line.

[0011] Preferably, the kit also includes a sample processing solution, which comprises the following components by mass fraction: 0.03M-0.06M PBS with a pH of 6.0-9.0, 0.1-0.3% Tween 20, 0.5-1% casein, 0.01-0.1% Proclin-300, and the remaining amount is made up to 100% with water.

[0012] Thirdly, the present invention provides a method for preparing an adiponectin detection kit, for use in the adiponectin detection kit of claim above, comprising the following steps:

[0013] S1: Preparation of labeled antibody: activate quantum dots, couple streptavidin, block and biotinylated adiponectin monoclonal antibody, and finally link QD-COOH-SA with APN-Ab-B and store in the preservation solution as described in claim 1;

[0014] S2: Preparation of labeling solution: Dilute the labeled antibody prepared in step S1, and add unlabeled adiponectin monoclonal antibody, and mix well;

[0015] S3: Prepare the coating solution: dilute the detection line antibody and the control line antibody;

[0016] S4: Spraying and coating: Spray the labeling solution from step S2 onto the quantum dot binding and release pad, and coat the coating solution from step S3 onto the nitrocellulose membrane.

[0017] S5: Drying, lamination assembly and cutting.

[0018] Preferably, the preparation of the labeled antibody in S1 specifically involves:

[0019] A1. Activation: Add EDC and NHS to QD-COOH at a concentration of 0.2-0.8 μM for activation;

[0020] A2. SA coupling: Activated QD-COOH is coupled with SA at a volume ratio of 1:30-1:60;

[0021] A3. Blocking and Biotinylation: First, block with BSA; then link APN-Ab and biotin at a volume ratio of 1:50-1:100 and dialyze in 0.3-0.9M phosphate buffer;

[0022] A4. Final linking: QD-COOH-SA and APN-Ab-B are linked at a ratio of 1:10 to 1:20 and stored in the preservation solution as described above.

[0023] Preferably, in step S2, the labeling solution is prepared using a labeled antibody diluent, which comprises the following components by mass fraction: 80-120 mM PBS, 0.5-1.5% casein, 0.1-0.6% Tween 20, 100-200 mM EDTA.2NA, 0.09-0.11% Proclin 300, with the remaining content made up to 100% with water. The pH of the labeled antibody diluent is adjusted to 6.0-10.0. The concentration of the additionally added unlabeled adiponectin monoclonal antibody in the labeling solution is 0.1-1 mg / mL.

[0024] Preferably, in step S3, the coating solution comprises the following components by mass fraction: 0.01-0.05M disodium hydrogen phosphate, 0.05-0.1M sodium dihydrogen phosphate, 1-5% sodium chloride, 0.5-1.5% trehalose, 0.1-0.5% casein, 0.5-2% sucrose, 0.09-0.11% Proclin 300, with the remaining content made up to 100% with water, and pH = 7.0-9.0; the detection line antibody concentration is 0.5-1 mg / mL, the quality control line antibody concentration is 0.5-1 mg / mL, and the dilution ratio is 1:10-1:30.

[0025] Preferably, the drying process in step S5 is as follows: the quantum dot-bound release pad is dried at 35-45℃ for 25-35 min; the nitrocellulose membrane is dried at 50-70℃ for 10-14 h.

[0026] Fourthly, the present invention provides the application of the above-mentioned adiponectin detection kit in the preparation of products for detecting the adiponectin content in human serum, plasma or whole blood.

[0027] The beneficial effects of this invention are:

[0028] (1) This invention employs a cascade coupling method of “QD-COOH-SA-APN-Ab-B”. Utilizing the extremely high affinity and multi-site binding characteristics of streptavidin (SA) and biotin (one SA molecule can bind four biotin molecules), significant signal amplification is achieved. Compared to direct coupling, this method reduces steric hindrance, maximizes the preservation of antibody immunoreactivity, thereby significantly enhancing the measurable fluorescence signal of the reagent card, greatly improving detection sensitivity, and enabling accurate detection of low-concentration adiponectin samples.

[0029] (2) This invention innovatively pre-adds detection line antibodies to the quantum dot binding release pad. When the sample passes through the release pad during chromatography, the adiponectin antigen in the sample will first come into contact with the "labeled antibody" and "detection line antibody" on the release pad simultaneously and undergo a partial liquid-phase reaction. This design improves reaction kinetics and, compared with the traditional method of immobilizing detection antibodies only on the NC membrane, can more effectively capture antigens, further improving the sensitivity and speed of detection.

[0030] (3) This invention has developed a special preservation solution containing Tris, trehalose, sodium caseinate, PVP-10, and a special surfactant S13. This formulation can effectively prevent the aggregation of quantum dot microspheres, protect the protein structure from damage, and maintain the activity of the labeled complex even during long-term storage. In addition, the protective film formed by trehalose and PVP enhances the heat and moisture resistance of the kit.

[0031] (4) The present invention uses liquid dialysis linking technology in the preparation process. This step can not only effectively remove unreacted small molecule impurities and by-products in the raw materials, but also provide a stable buffer environment for the coupling reaction, thereby significantly improving the coupling efficiency and batch-to-batch consistency, and ensuring the uniformity of product quality.

[0032] (5) The kit provided by the present invention is easy to operate and does not require complicated professional training; the detection speed is fast, and the results can be obtained in just 10 minutes, which greatly reduces the waiting time for medical staff and patients; only a small fluorescence reader is needed, the equipment is small in size and low in cost, which is very suitable for primary hospitals and point-of-care testing (POCT), and helps to detect and prevent adiponectin-related diseases (such as type II diabetes risk assessment). Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of the present invention;

[0034] 1 is the sample pad, 2 is the PVC board, 3 is the quantum dot binding and release pad, 4 is the nitrocellulose membrane (NC membrane), 5 is the absorbent paper, 6 is the detection line, and 7 is the quality control line. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] like Figure 1 As shown, the adiponectin detection kit provided by the present invention includes, in sequence along the detection direction: a sample pad 1, a quantum dot binding and release pad 3, a nitrocellulose membrane (NC membrane) 4, and absorbent paper 5. The above components are fixedly mounted on a PVC base plate 2.

[0037] On the nitrocellulose membrane 4, a detection line 6 and a quality control line 7 are sequentially arranged along the detection direction.

[0038] The test line 6 is coated with adiponectin monoclonal antibody for specific capture of adiponectin; the control line 7 is coated with anti-mouse IgG antibody to indicate whether the test process is effective.

[0039] Example 1

[0040] A preservation solution is prepared in 500 mL portions, with the formulation and dosage shown in Table 1. The remaining content is made up to 100% with water.

[0041] Prepare a Tris solution by adding 70% of the total water volume to the Tris solution, adjust the pH to 6.0±0.2 with 6mol / L hydrochloric acid, then add the other reagents. Finally, after all the reagents have dissolved, add the remaining water to make up to 500mL, and then finely adjust the pH to 6.0±0.2 with 2mol / L sodium hydroxide.

[0042] Table 1

[0043]

[0044] Prepare 500 mL of sample processing solution as shown in Table 2. First, dissolve the PBS formula (sodium chloride, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate) in 80% of the total water volume to prepare PBS buffer. Then, add Tween 20, casein, and Proclin-300 to dissolve. Finally, after all the drugs are dissolved, add the remaining water to make up to 500 mL.

[0045] Table 2

[0046]

[0047] The preparation of an adiponectin detection kit includes the following steps:

[0048] S1: Preparation of labeled antibody: activate quantum dots, conjugate streptavidin, biotinylate adiponectin monoclonal antibody, and finally link QD-COOH-SA with APN-Ab-B and store in preservation solution;

[0049] Specifically, S1 is:

[0050] A1. Activation: Add EDC and NHS to 0.2 μM QD-COOH (the ratio is 10 μL of 8 mM EDC and 10 μL of 8 mM NHS per mL of QD-COOH) for activation (the EDC and NHS are prepared with water and used immediately; prepare 100 μL of EDC (0.1534 g + 100 μL of water) and 100 μL of NHS (0.092 g + 100 μL of water).

[0051] A2. SA Coupling: Activated QD-COOH is coupled with SA (concentration of 10 mg / mL) at a volume ratio of 1:30;

[0052] (To couple 1 mL of QD-COOH, you need to add 30 mL of SA at a concentration of 10 mg / mL. The SA is prepared using 20 mM PBS.)

[0053] A3. Blocking and Biotinylation: Blocked with bovine serum albumin (BSA) (5% concentration); APN-Ab was linked with biotin at a volume ratio of 1:50 and dialyzed in 0.3M phosphate buffer;

[0054] A4. Final linking: QD-COOH-SA and APN-Ab-B are linked at a ratio of 1:10 and stored in a preservation solution.

[0055] S2: Preparation of labeling solution: Dilute the labeled antibody prepared in step S1, and add unlabeled adiponectin monoclonal antibody, and mix well;

[0056] The labeled antibody diluent, by mass fraction, comprises the following components: 80 mM PBS, 0.5% casein, 0.1% Tween 20, 100 mM EDTA.2NA, 0.09% Proclin 300, with the remainder made up to 100% with water, pH = 6.0; the additional unlabeled adiponectin monoclonal antibody is added at a concentration of 0.1 mg / mL in the labeled solution.

[0057] S3: Prepare the coating solution: dilute the detection line antibody and the control line antibody;

[0058] The detection line uses adiponectin mouse / rabbit monoclonal antibody at a concentration of 0.5 mg / mL; the control line uses goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody at a concentration of 0.5 mg / mL; the coating solution comprises the following components by mass fraction: 0.01 M disodium hydrogen phosphate, 0.05 M sodium dihydrogen phosphate, 1% sodium chloride, 0.5% trehalose, 0.1% casein, 0.5% sucrose, 0.09% Proclin 300, with the remaining content made up to 100% with water, pH = 7.0-9.0; the dilution ratio is 1:10-1:30.

[0059] S4: Spraying and coating: Spray the marking solution from step S2 onto the quantum dot binding and release pad, and coat the coating solution from step S3 onto the nitrocellulose membrane. The quantum dot binding and release pad is required to be made of glass fiber.

[0060] S5: Drying, lamination assembly and cutting.

[0061] The drying process is as follows: the quantum dot-bound release pad is dried at 35℃ for 25 minutes; the nitrocellulose membrane is dried at 50℃ for 14 hours. The components are then prepared according to... Figure 1 The components are assembled in a manner that ensures a 3mm overlap between each other. The sample processing pad is made of cotton fiber, and the absorbent paper is 1mm thick. After the test strip is prepared, the absorbent paper is held and inserted into a special card holder to form a test reagent card. The test line and control line on the nitrocellulose membrane should be at 1 / 3 and 2 / 3 of the viewing window, respectively; the lower 1 / 3 of the sample processing pad should be at the center of the sample well.

[0062] Example 2

[0063] A preservation solution is prepared in 500 mL portions, with the formulation and dosage shown in Table 3. The remaining content is made up to 100% with water.

[0064] First, add water to prepare Tris to a volume of 70%, then adjust the pH to 6.0±0.2 with 6 mol / L hydrochloric acid. Then add other reagents, and finally add the remaining water to make up to 500 mL after all the reagents have dissolved. Finally, use 2 mol / L sodium hydroxide to finely adjust the pH to 6.0±0.2.

[0065] Table 3

[0066]

[0067] Prepare 500 mL of sample processing solution as shown in Table 4. Dissolve the PBS formula (sodium chloride, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate) in 80% of the total water volume to prepare PBS buffer. Then add Tween 20, casein, and Proclin-300 to dissolve. Finally, after all the drugs are dissolved, add the remaining water to make up to 500 mL.

[0068] Table 4

[0069]

[0070] The preparation of an adiponectin detection kit includes the following steps:

[0071] S1: Preparation of labeled antibody: activate quantum dots, conjugate streptavidin, biotinylate adiponectin monoclonal antibody, and finally link QD-COOH-SA with APN-Ab-B and store in preservation solution;

[0072] Specifically, S1 is:

[0073] A1. Activation: Add EDC and NHS to 0.5 μM QD-COOH (the ratio is 20 μL of 10 mM EDC and 20 μL of 10 mM NHS per mL of QD-COOH) for activation;

[0074] A2. SA Coupling: Activated QD-COOH is coupled with SA (concentration of 15 mg / mL) at a volume ratio of 1:45;

[0075] A3. Blocking and Biotinylation: Blocked with bovine serum albumin (BSA) (8% concentration); APN-Ab was linked with biotin at a volume ratio of 1:75 and dialyzed in 0.6M phosphate buffer;

[0076] A4. Final linking: QD-COOH-SA and APN-Ab-B are linked at a ratio of 1:15 and stored in a preservation solution.

[0077] S2: Preparation of labeling solution: Dilute the labeled antibody prepared in step S1, and add unlabeled adiponectin monoclonal antibody, and mix well;

[0078] The labeled antibody dilution solution was formulated as follows: 100mM PBS, 0.5-1.5% casein, 0.35% Tween 20, 150mM EDTA.2NA, 0.10% Proclin 300, with the remaining amount made up to 100% with water, pH=8.0; the additional unlabeled adiponectin monoclonal antibody was added at a concentration of 0.55mg / mL in the labeled solution.

[0079] S3: Prepare the coating solution: dilute the detection line antibody and the control line antibody;

[0080] The coating solution comprises the following components by mass fraction: 0.03M disodium hydrogen phosphate, 0.075M sodium dihydrogen phosphate, 3% sodium chloride, 1% trehalose, 0.3% casein, 1.25% sucrose, and 0.1% Proclin 300. The remaining content is made up to 100% with water, and the pH is 8.0. The detection line antibody concentration is 0.75 mg / mL, the control line antibody concentration is 0.75 mg / mL, and the dilution ratio is 1:20.

[0081] S4: Spraying and coating: Spray the marking solution from step S2 onto the quantum dot binding and release pad, and coat the coating solution from step S3 onto the nitrocellulose membrane. The quantum dot binding and release pad is required to be made of glass fiber.

[0082] S5: Drying, lamination assembly and cutting.

[0083] The drying process is as follows: the quantum dot-bound release pad is dried at 40℃ for 30 min; the nitrocellulose membrane is dried at 60℃ for 12 h. The components are then prepared according to... Figure 1 The components are assembled in a manner that ensures a 3mm overlap between each other. The sample processing pad is made of cotton fiber, and the absorbent paper is 1mm thick. After the test strip is prepared, the absorbent paper is held and inserted into a special card holder to form a test reagent card. The test line and control line on the nitrocellulose membrane should be at 1 / 3 and 2 / 3 of the viewing window, respectively; the lower 1 / 3 of the sample processing pad should be at the center of the sample well.

[0084] Example 3

[0085] A preservation solution is prepared in 500 mL portions, with the formulation and dosage shown in Table 5. The remaining content is made up to 100% with water.

[0086] First, add water to prepare Tris to a volume of 70%, then adjust the pH to 6.0±0.2 with 6 mol / L hydrochloric acid. Then add other reagents, and finally add the remaining water to make up to 500 mL after all the reagents have dissolved. Finally, use 2 mol / L sodium hydroxide to finely adjust the pH to 6.0±0.2.

[0087] Table 5

[0088]

[0089] Prepare 500 mL of sample processing solution as shown in Table 6. First, dissolve the PBS formula (sodium chloride, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate) in 80% of the total water volume to prepare PBS buffer. Then, add Tween 20, casein, and Proclin-300 to dissolve. Finally, after all the drugs are dissolved, add the remaining water to make up to 500 mL.

[0090] Table 6

[0091]

[0092] The preparation of an adiponectin detection kit includes the following steps:

[0093] S1: Preparation of labeled antibody: activate quantum dots, conjugate streptavidin, biotinylate adiponectin monoclonal antibody, and finally link QD-COOH-SA with APN-Ab-B and store in preservation solution;

[0094] Specifically, S1 is:

[0095] A1. Activation: Add EDC and NHS to 0.8 μM QD-COOH (the ratio is 30 μL of 12 mM EDC and 30 μL of 12 mM NHS per mL of QD-COOH) for activation;

[0096] A2. SA Coupling: Activated QD-COOH is coupled with SA (concentration of 20 mg / mL) at a volume ratio of 1:60;

[0097] A3. Blocking and Biotinylation: Blocked with bovine serum albumin (BSA) (10% concentration); APN-Ab was linked with biotin at a volume ratio of 1:100 and dialyzed in 0.9M phosphate buffer;

[0098] A4. Final linking: QD-COOH-SA and APN-Ab-B are linked at a ratio of 1:20 and stored in a preservation solution.

[0099] S2: Preparation of labeling solution: Dilute the labeled antibody prepared in step S1, and add unlabeled adiponectin monoclonal antibody, and mix well;

[0100] The labeled antibody diluent was formulated as follows: 120 mM PBS, 1.5% casein, 0.6% Tween 20, 200 mM EDTA.2NA, 0.11% Proclin 300, with the remainder made up to 100% with water, pH = 10.0; the additional unlabeled adiponectin monoclonal antibody was added at a concentration of 1 mg / mL in the labeled solution.

[0101] S3: Prepare the coating solution: dilute the detection line antibody and the control line antibody;

[0102] The coating solution comprises the following components by mass fraction: 0.05M disodium hydrogen phosphate, 0.1M sodium dihydrogen phosphate, 5% sodium chloride, 1.5% trehalose, 0.5% casein, 2% sucrose, and 0.11% Proclin 300. The remaining content is made up to 100% with water, and the pH is 9.0. The detection line antibody concentration is 1 mg / mL, the control line antibody concentration is 1 mg / mL, and the dilution ratio is 1:30.

[0103] S4: Spraying and coating: Spray the marking solution from step S2 onto the quantum dot binding and release pad, and coat the coating solution from step S3 onto the nitrocellulose membrane. The quantum dot binding and release pad is required to be made of glass fiber.

[0104] S5: Drying, lamination assembly and cutting.

[0105] The drying process is as follows: the quantum dot-bound release pad is dried at 45℃ for 35 minutes; the nitrocellulose membrane is dried at 70℃ for 10 hours. The components are then prepared according to... Figure 1 The components are assembled in a manner that ensures a 3mm overlap between each other. The sample processing pad is made of cotton fiber, and the absorbent paper is 1mm thick. After the test strip is prepared, the absorbent paper is held and inserted into a special card holder to form a test reagent card. The test line and control line on the nitrocellulose membrane should be at 1 / 3 and 2 / 3 of the viewing window, respectively; the lower 1 / 3 of the sample processing pad should be at the center of the sample well.

[0106] Comparative Example 1

[0107] The difference from Example 2 is that the pH of Tris was adjusted to 8.3±0.2, the pH of the preservation solution was adjusted to 8.3±0.2, and the preservation solution contained 2% less TX-100, 0.05% less surfactant S13, and 0.5% less glycine.

[0108] Comparative Example 2

[0109] The difference from Example 2 is that the pH of Tris was adjusted to 7.3±0.2, the pH of the preservation solution was adjusted to 7.3±0.2, and the preservation solution contained 2% less TX-100, 0.05% less surfactant S13, and 0.5% less glycine.

[0110] Comparative Example 3

[0111] The difference from Example 2 is that the preservation solution contains 2% less TX-100, 0.05% less surfactant S13, and 0.5% less glycine.

[0112] Comparative Example 4

[0113] The difference from Example 2 is that no detection line antibody is added to the quantum dot binding release pad.

[0114] Comparative Example 5

[0115] The difference from Example 2 is that the sample processing solution includes the following components by mass fraction: 0.1 mol / L Tris, 1% sucrose, 1% mannitol, 1% trehalose, 0.5% Tween 20, and 1% PC.

[0116] Comparative Example 6

[0117] The difference from Example 2 is that biotin is not added in step S1.

[0118] Adiponectin Detection Kit Detection Method

[0119] 1. Take the test card and the serum sample to be tested, and allow them to equilibrate to room temperature for 10 minutes to allow each component to return to room temperature.

[0120] 2. Use an SD card to calibrate the instrument.

[0121] 3. Take 20 μL of sample and add it to the sample diluent. Mix well and then add 80 μL of sample vertically into the well.

[0122] 4. Read the results using the instrument after 10 minutes. For males, a result >3.4 μg / mL is considered negative, and for females, a result >3 μg / mL is considered positive.

[0123] First, the adiponectin content of comparative examples 1-3 was measured to verify the most suitable pH.

[0124] Table 7

[0125]

[0126] As shown in Table 7, the accuracy and specificity of the adiponectin monoclonal antibody preserved using Comparative Example 3 are higher than those of Comparative Examples 1-2. This is because the pH of the preservation solution in Comparative Example 3 is 6.3±0.2, while the preservation solution in Comparative Example 2 allows QD-COOH-SA-APN-Ab-B to be released more cleanly at the quantum dot binding release pad, resulting in higher sensitivity and accuracy.

[0127] Repeatability tests were conducted on 20 groups of Comparative Example 3 and Example 2, with adiponectin content of 1.2 μg / mL.

[0128] Table 8

[0129]

[0130] The results in Table 8 confirm that the reproducibility of the adiponectin monoclonal antibody preserved in Example 2 is better than that of Comparative Example 3. This is because of the pH value of the preservation solution. The addition of various surfactants in Example 2 improved the binding and release efficiency of biotin antibody and quantum dots, resulting in better reproducibility.

[0131] The adiponectin content of Examples 1-3 and Comparative Example 4 was detected.

[0132] Table 9

[0133]

[0134] The results in Table 9 confirm that adding a detection line antibody to the quantum binding release pad in Example 2 can improve the sensitivity of the detection kit.

[0135] The adiponectin content of Example 2 and Comparative Example 5 was detected.

[0136] Table 10

[0137]

[0138] The results in Table 10 confirm that the sample processing solution of the adiponectin monoclonal antibody labeled using the labeling method of Example 2 has higher accuracy and specificity than that of the sample processing solution of Comparative Example 5.

[0139] The adiponectin content of Example 2 and Comparative Example 6 was detected.

[0140] Table 11

[0141]

[0142] The results in Table 11 confirm that the adiponectin monoclonal antibody kit labeled using the labeling method of Example 2 has higher accuracy and specificity than the kit without biotin in Comparative Example 6.

[0143] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A preservation solution, characterized in that, The preservation solution comprises the following components by mass fraction: 9-11 mM Tris with a pH of 6.0-6.5, 9-11% trehalose, 4-6% Tween 20, 4-6% sodium caseinate, 1.1-1.3% PVP-10, 0.8-1.2% proclin-300, 1-3% TX-100, 0.04-0.06% surfactant S13, and 0.4-0.6% glycine. The remaining components are made up to 100% with water, and the pH of the preservation solution is adjusted to 6.0-6.

5.

2. The preservation solution according to claim 1, characterized in that, The preservation solution comprises the following components by mass fraction: 10mM Tris with a pH of 6.0-6.5, 10% trehalose, 5% Tween 20, 5% sodium caseinate, 1.2% PVP-10, 5% sodium caseinate, 1% proclin-300, 2% TX-100, 0.05% surfactant S13, and 0.5% glycine. The remaining components are made up to 100% with water, and the pH of the preservation solution is finely adjusted to 6.0-6.

5.

3. An adiponectin detection kit, characterized in that: The device comprises, in sequence, a sample pad, a quantum dot binding and release pad, a nitrocellulose membrane, absorbent paper, and a PVC base plate. The quantum dot binding and release pad is coated with quantum dot-labeled adiponectin monoclonal antibody and unlabeled adiponectin monoclonal antibody. The quantum dot-labeled adiponectin monoclonal antibody is dispersed and stored in the preservation solution as described in any one of claims 1-2 before being coated onto the release pad. The nitrocellulose membrane is coated with adiponectin monoclonal antibody as a detection line and anti-mouse IgG antibody as a quality control line.

4. The adiponectin detection kit according to claim 3, characterized in that, The kit also includes a sample processing solution, which comprises the following components by mass fraction: 0.03M-0.06M PBS buffer with a pH of 6.0-9.0, 0.1-0.3% Tween 20, 0.5-1% casein, and 0.01-0.1% Proclin-300, with the remaining amount made up to 100% with water.

5. A method for preparing an adiponectin detection kit, used to prepare the adiponectin detection kit according to claim 3 or 4, characterized in that, Includes the following steps: S1: Preparation of labeled antibody: Activate quantum dots, conjugate streptavidin, biotinylate adiponectin monoclonal antibody, link QD-COOH-SA with APN-Ab-B, and store in the preservation solution as described in claim 1; S2: Preparation of labeling solution: Dilute the labeled antibody prepared in step S1, and add unlabeled adiponectin mouse monoclonal antibody, then mix well; S3: Prepare the coating solution: dilute the detection line antibody and the control line antibody; S4: Spraying and coating: Spray the labeling solution from step S2 onto the quantum dot binding and release pad, and coat the coating solution from step S3 onto the nitrocellulose membrane. S5: Drying, lamination assembly and cutting.

6. The preparation method according to claim 5, characterized in that, The preparation of the labeled antibody in step S1 is specifically as follows: A1. Activation: Add EDC and NHS to QD-COOH at a concentration of 0.2-0.8 μM for activation; A2. SA coupling: Activated QD-COOH is coupled with SA at a volume ratio of 1:30-1:60; A3. Blocking and Biotinylation: First, block with BSA, then link APN-Ab and biotin at a volume ratio of 1:50-1:100, and dialyze in 0.3-0.9M phosphate buffer; A4. Final linking: QD-COOH-SA and APN-Ab-B are linked at a ratio of 1:10 to 1:20 and stored in the preservation solution as described in claim 1 or 2.

7. The preparation method according to claim 5, characterized in that, In step S2, the labeling solution is prepared using a labeled antibody diluent, which comprises the following components by mass fraction: 80-120 mM PBS, 0.5-1.5% casein, 0.1-0.6% Tween 20, 100-200 mM EDTA·2Na, 0.09-0.11% Proclin 300, with the remaining amount made up to 100% with water. The pH of the labeled antibody diluent is adjusted to 6.0-10.

0. The concentration of the additionally added unlabeled adiponectin monoclonal antibody in the labeling solution is 0.1-1 mg / mL.

8. The preparation method according to claim 5, characterized in that, In step S3, the coating solution comprises the following components by mass fraction: 0.01-0.05M disodium hydrogen phosphate, 0.05-0.1M sodium dihydrogen phosphate, 1-5% sodium chloride, 0.5-1.5% trehalose, 0.1-0.5% casein, 0.5-2% sucrose, 0.09-0.11% Proclin 300, with the remaining content made up to 100% with water, and pH = 7.0-9.0; the detection line antibody concentration is 0.5-1 mg / mL, the quality control line antibody concentration is 0.5-1 mg / mL, and the dilution ratio is 1:10-1:

30.

9. The preparation method according to claim 5, characterized in that, The drying process in step S5 is as follows: the quantum dot-bound release pad is dried at 35-45℃ for 25-35 minutes; the nitrocellulose membrane is dried at 50-70℃ for 10-14 hours.

10. The use of the adiponectin detection kit according to any one of claims 2-4 in the preparation of products for detecting the adiponectin content in human serum, plasma or whole blood.

Images