Test strip and kit for detecting interleukin 6

Abstract

The invention relates to the technical field of biomolecule detection, in particular to a test strip and a kit for detecting interleukin 6. The invention discloses a test strip for detecting interleukin-6, the test strip comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and pasted on the bottom plate according to a chromatography direction, and the nitrocellulose membrane is coated with a detection line and a quality control line; the detection line is coated with an anti-IL-6 monoclonal antibody; the quality control line is coated with rabbit anti-DNP-BSA or goat anti-chicken IgY (Immunoglobulin Y); the combination pad is coated with a biotinylated ProteinA / G-IL-6 monoclonal antibody marked by an AIE polystyrene fluorescent microsphere, and a biotinylated antibody or antigen marked by the AIE polystyrene fluorescent microsphere; the test strip and the kit for detecting the interleukin 6, disclosed by the invention, are good in stability, higher in sensitivity, wider in detection range and simple and convenient to operate compared with similar products, are suitable for rapid detection, and have low professional requirements on personnel.

Description

Technical Field

[0001] The present application relates to the technical field of biomolecule detection, and in particular to a test strip and a kit for detecting interleukin 6. Background Art

[0002] Interleukin 6 is a small molecule polypeptide, composed of four α-helices, with a molecular weight of 19-28KD and 184 amino acid residues. It usually exists in the form of a monomer. IL-6 is an important member of the cytokine network and plays a central role in acute inflammatory reactions. It was first discovered by Weissenbach in 1980. It is a multifunctional cytokine that plays an important role in human metabolism, autoimmune cell differentiation, disease treatment, etc. Its synthesis pathway can be produced in almost all matrix cells and immune system cells, such as B lymphocytes, T lymphocytes, macrophages, monocytes, dendritic cells, mast cells and other non-lymphocytes, such as fibroblasts, endothelial cells and tumor cells. IL-6 can promote T cell population expansion and activation and B cell differentiation, regulate acute phase reactions, and can affect vascular diseases, lipid metabolism, insulin resistance, mitochondrial activity, neuroendocrine system and neuropsychological behavior hormone-like properties. IL-6 is involved in the occurrence and development of many diseases, and its blood level is closely related to inflammation, viral infection, and autoimmune diseases. Therefore, the clinical significance and application prospects of interleukin-6 are mainly reflected in the following aspects: (1) Testing for interleukins can determine whether the patient has inflammation or other infections and severe systemic inflammatory responses; (2) Guide the rational use of antibiotics and reduce their abuse; (3) IL-6 can be used as a more sensitive early identification and monitoring indicator for postoperative infection; (4) IL-6 can accurately diagnose focal bacterial infections such as pneumonia and reduce missed diagnoses; (5) Studies have shown that PCT and IL-6 have a high accuracy in diagnosing sepsis, and the combined diagnostic value of the two is slightly better than that of a single indicator; (6) Interleukin-6 is significantly overexpressed in the stroma of many malignant tumors and is a key factor involved in the connection between inflammation and tumors; (7) Many studies have shown that IL-6 is highly expressed in a variety of malignant tumors, including breast cancer, lung adenocarcinoma, gastric cancer, liver cancer, and ovarian cancer. High expression of IL-6 is correlated with poor prognosis in patients and has been proposed as a diagnostic marker for detecting inflammation and malignant tumors.

[0003] The changes of IL-6 are earlier than those of CRP and PCT. Compared with the three, IL-6 has higher sensitivity than PCT and CRP, but lower specificity than PCT. PCT and CRP can make up for its shortcomings. The combination of the three can continuously monitor the high-risk population for sepsis, which is of great significance for early detection and early treatment.

[0004] At present, the determination of interleukin-6 mainly adopts chemiluminescence immunoassay and fluorescence immunochromatography. The chemiluminescence immunoassay has high sensitivity, but the stability of the reagent is difficult to control, the instrument failure rate is high, and professional personnel are required to perform the test, and the cost is relatively high. The immunochromatographic reagent has problems such as insufficient sensitivity and narrow detection range.

[0005] Therefore, it is necessary to develop an IL-6 assay kit with higher sensitivity and simpler operation. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present application provides a test strip and a kit for detecting interleukin-6.

[0007] In a first aspect, the present application provides a test strip for detecting interleukin-6, which adopts the following technical solution: A test strip for detecting interleukin-6, the test strip comprising a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction; The nitrocellulose membrane is coated with a detection line and a quality control line; the detection line is coated with anti-IL-6 monoclonal antibody; the quality control line is coated with rabbit anti-DNP-BSA or goat anti-chicken IgY; The conjugate pad is coated with biotinylated ProteinA / G-antibody labeled with AIE polystyrene fluorescent microspheres, and biotinylated antibody or antigen labeled with AIE polystyrene fluorescent microspheres.

[0008] In a specific embodiment, the conjugate pad is coated with biotinylated ProteinA / G-IL-6 antibody labeled with AIE polystyrene fluorescent microspheres and biotinylated antibody labeled with AIE polystyrene fluorescent microspheres.

[0009] In a specific embodiment, the conjugate pad is coated with biotinylated ProteinA / G-antibody labeled with AIE polystyrene fluorescent microspheres and biotinylated antigen labeled with AIE polystyrene fluorescent microspheres.

[0010] Preferably, the biotinylated ProteinA / G-antibody labeled with AIE polystyrene fluorescent microspheres is biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres.

[0011] By adopting the above technical solution, the labeling model of the probe is improved in the present application, a biotin-avidin system is introduced, and Protein A / G is labeled on biotin, and then the IL-6 antibody is indirectly labeled on Protein A / G. Since Protein A / G has the common characteristics of Protein A and Protein G, it has a larger specific surface area and more antigen binding sites, and thus can provide a high number of antigen bindings. In addition, the biotin-avidin system is used to increase the spacer arm and reduce the steric hindrance, thereby improving the sensitivity of the reagent.

[0012] Preferably, the biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres comprises the following steps: (1) Mixing Protein A / G with biotin to obtain biotinylated Protein A / G; (2) Mixing the streptavidin-modified AIE microspheres with biotinylated Protein A / G to obtain AIE microsphere-SA-biotin-Protein A / G conjugates; (3) mixing the IL-6 monoclonal antibody with the AIE microsphere-SA-biotin-ProteinA / G conjugate to obtain the IL-6 monoclonal antibody microsphere marker; (4) Add a blocking agent to the IL-6 monoclonal antibody microsphere marker to block it, and obtain biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres.

[0013] Preferably, in step (1), the molar ratio of ProteinA / G to biotin is 1:(20-40).

[0014] Preferably, in step (2), the mass ratio of streptavidin-modified AIE microspheres to biotinylated ProteinA / G is 1:(0.001-0.02).

[0015] In the step (3), the mass ratio of the AIE microsphere-SA-biotin-ProteinA / G conjugate to the IL-6 monoclonal antibody is 1:(0.015-0.05).

[0016] In the step (4), the mass ratio of the IL-6 monoclonal antibody microsphere marker to the blocker is 1:(0.015-0.03).

[0017] Preferably, the biotin is NHS-PEG12-biotin.

[0018] By adopting the above technical scheme, due to the small molecular weight of biotin, the formed biotinylated label is easily affected by the steric hindrance effect of large molecular proteins. This kit adopts NHS-PEG12-biotin to make the labeled conjugate have a longer spacer arm to reduce the problem of low sensitivity due to limited binding caused by spatial steric hindrance when the antigen-antibody and biotin and SA are combined. At the same time, AIE polystyrene fluorescent microspheres (aggregation-induced emission microspheres) have higher fluorescence efficiency than rare earth element time-resolved fluorescent microspheres. After the amplification of the biotin-avidin system and the simultaneous extension of the distance between the biotin parent structure and the antibody, it is more conducive to the binding of biotin and avidin. In addition, biotin and ProteinA / G are coupled and then the antibody and ProteinA / G are blocked by a blocking agent. Since ProteinA / G has the common characteristics of ProteinA and ProteinG, it has a larger specific surface area and more antigen binding sites. Therefore, it can provide a high number of antigen binding, thereby helping to improve the sensitivity of the reagent.

[0019] In the present application, aggregation-induced luminescence microspheres are used and the biotin-avidin system is amplified while Protein A / G is introduced to improve the sensitivity of the reagent and broaden the detection range of the detection reagent.

[0020] Preferably, the biotinylated antibody or antigen labeled with AIE polystyrene fluorescent microspheres is biotinylated chicken IgY labeled with AIE polystyrene fluorescent microspheres or biotinylated DNP-BSA labeled with AIE polystyrene fluorescent microspheres.

[0021] Preferably, the preparation method of the biotinylated chicken IgY labeled with AIE polystyrene fluorescent microspheres comprises the following steps: adding chicken IgY to biotin and mixing them to obtain biotin-chicken IgY; The streptavidin-modified AIE microspheres were mixed with biotin-chicken IgY to obtain biotinylated chicken IgY labeled with AIE polystyrene fluorescent microspheres.

[0022] Preferably, the molar ratio of biotin to chicken IgY is (10-40):1.

[0023] Preferably, the mass ratio of the streptavidin-modified AIE microspheres to biotin-chicken IgY is 1:(0.02-0.05).

[0024] Preferably, the preparation method of the biotinylated DNP-BSA labeled with AIE polystyrene fluorescent microspheres comprises the following steps: Adding DNP-BSA to biotin and mixing them to obtain biotin-DNP-BSA; The streptavidin-modified AIE microspheres were mixed with biotin-DNP-BSA to obtain biotinylated DNP-BSA labeled with AIE polystyrene fluorescent microspheres.

[0025] Preferably, the molar ratio of biotin to DNP-BSA is (10-40):1.

[0026] Preferably, the mass ratio of the streptavidin-modified AIE microspheres to biotin-DNP-BSA is 1:(0.02-0.05).

[0027] Preferably, the biotin is NHS-PEG12-biotin.

[0028] Preferably, the preparation method of the streptavidin-modified AIE microspheres comprises the following steps: labeling streptavidin onto the AIE microspheres using the carbodiimide method to obtain AIE microspheres-SA; Among them, the mass ratio of AIE microspheres and streptavidin is 1:(0.01-0.3).

[0029] AIE microspheres are AIE microspheres with carboxyl groups modified on the surface.

[0030] In a second aspect, the present application provides a kit for detecting interleukin-6, which adopts the following technical solution: A kit for detecting interleukin-6 comprises a diluent and a cartridge, wherein a test strip is arranged in the cartridge.

[0031] In summary, the present application includes at least one of the following beneficial technical effects: The present application discloses a test strip and a test kit for detecting interleukin-6, which have good stability, higher sensitivity than similar products, wider detection range, simple operation, and are suitable for rapid detection, and do not require high professional skills of personnel. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram of methodological comparison results. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present invention; some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0034] The raw materials involved in this application are all commercially available products, among which: Blocker: The manufacturer is Roche MAK33.

[0035] NHS-PEG12-biotin: Manufacturer: Thermo Fisher Scientific.

[0036] The present application is further described in detail below in conjunction with embodiments and comparative examples.

[0037] Preparation of raw materials: The preparation method of AIE microsphere-SA, the specific steps are as follows: Take 1 mg AIE fluorescent microspheres (200 nm), add 10 μL of 20 mg / mL EDC and 15 μL of 20 mg / mL NHS, mix and react at room temperature for 20 min, centrifuge at 12000 rpm and 4°C for 10 min, remove the supernatant, add 1 mL of coupling buffer and ultrasonically resuspend, centrifuge at 12000 rpm and 4°C for 10 min, remove the supernatant, add 1 mL of coupling buffer and ultrasonically resuspend, add 30 μg of streptavidin and shake for 2 hours, add 20 μL of 10% BSA and mix for half an hour; centrifuge at 12000 rpm and 4°C for 10 min, remove the supernatant, add 1 mL of cleaning solution and ultrasonically resuspend, centrifuge at 12000 rpm and 4°C for 10 min, remove the supernatant, add 100 μL of conjugate preservation solution and store at 2-8°C to obtain AIE microspheres-SA.

[0038] Among them, the AIE fluorescent microspheres are AIE microspheres with carboxyl groups modified on the surface.

[0039] The solution used in this example is configured as follows: Example 1: A test kit for detecting interleukin-6 1. Preparation of conjugate pad: 1.1. Preparation of biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres: (1) 1 mL of 2 mg / mL Protein A / G was mixed with 4.8 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotinylated Protein A / G. The biotin marker was desalted using a desalting column, and the recovered solution was added with 50% glycerol and stored at 2-8°C for later use; (2) 1 mg of AIE microsphere-SA was mixed with 15 μg of biotinylated Protein A / G to obtain AIE microsphere-SA-biotin-Protein A / G conjugate; (3) 30 μg of IL-6 monoclonal antibody was mixed with 1 mg of AIE microsphere-SA-biotin-ProteinA / G conjugate to obtain IL-6 monoclonal antibody microsphere marker; (4) 1 mg of IL-6 monoclonal antibody microsphere marker was blocked by adding 30 μg of blocking agent (Roche MAK33) to obtain AIE carboxyl microsphere-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate.

[0040] 1.2. Preparation of biotinylated chicken IgY labeled with AIE polystyrene fluorescent microspheres: (1) Mix 1 mL of 2 mg / mL chicken IgY with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-chicken IgY; (2) 1 mg of AIE microsphere-SA was mixed with 30 μg of biotin-chicken IgY to obtain an AIE microsphere-SA-biotin-chicken IgY conjugate.

[0041] 1.3. Preparation of conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) AIE carboxyl microsphere-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate and AIE microsphere-SA-biotin-chicken IgY conjugate were diluted 110 times with fluorescent microsphere marker storage solution to obtain a fluorescent marker solution; (3) Spray the fluorescent marker solution at 5 μL / cm2 onto the dried and cut conjugate pad, and dry it for later use to obtain a conjugate pad coated with AIE carboxyl microspheres-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate and AIE microspheres-SA-biotin-chicken IgY conjugate.

[0042] 2. Preparation of nitrocellulose membrane: (1) Before scratching, the nitrocellulose membrane needs to be equilibrated in a humidity environment of 45%-60% for 3-4 hours; (2) Preparation of test line: The immunologically prepared anti-IL-6 monoclonal antibody was prepared with coating buffer to a concentration of 1 mg / mL, and the membrane was streaked to the T-line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; the streaked membrane was dried at 55°C for 2-3 days.

[0043] (3) Preparation of quality control line: The goat anti-chicken IgY antibody was prepared into a concentration of 1 mg / mL using a coating buffer, and the membrane was streaked onto the C line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm, and then dried at 55°C for 2-3 days.

[0044] 3. Preparation of sample pad: The sample pad was evenly coated with the sample pad blocking solution to completely wet it, and then dried at 55°C for 1 day and cut.

[0045] 4. Preparation of test strips: The sample pad, the conjugate pad, the NC membrane and the absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction to obtain a test strip.

[0046] 5. Preparation of the kit: (1) Test card: Put the prepared test strip into the card housing to obtain a test card; (2) Sample diluent.

[0047] Example 2: A test kit for detecting interleukin-6 1. Preparation of conjugate pad 1.1. Preparation of biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres (1) 1 mL of 2 mg / mL Protein A / G and 3.2 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) were mixed and reacted for 30 minutes to obtain biotinylated Protein A / G; the biotin marker was desalted using a desalting column, and the recovered solution was added with 50% glycerol and stored at 2-8°C for later use; (2) 1 mg of AIE microsphere-SA was mixed with 10 μg of biotinylated Protein A / G to obtain AIE microsphere-SA-biotin-Protein A / G conjugate; (3) 15 μg of IL-6 monoclonal antibody was mixed with 1 mg of AIE microsphere-SA-biotin-ProteinA / G conjugate to obtain IL-6 monoclonal antibody microsphere marker; (4) 15 μg of blocking agent (Roche MAK33) was added to 1 mg of IL-6 monoclonal antibody microsphere marker to obtain AIE carboxyl microsphere-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate.

[0048] 1.2. Preparation of biotinylated chicken IgY labeled with AIE polystyrene fluorescent microspheres: (1) Mix 1 mL of 2 mg / mL chicken IgY with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotinylated chicken IgY; (2) 1 mg of AIE microsphere-SA was mixed with 30 μg of biotin-chicken IgY to obtain AIE microsphere-SA-biotin-chicken IgY conjugate; 1.3. Preparation of conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) AIE carboxyl microsphere-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate and AIE microsphere-SA-biotin-chicken IgY conjugate were diluted 110 times with fluorescent microsphere marker storage solution to obtain a fluorescent marker solution; (3) Spray the fluorescent marker solution at 5 μL / cm2 onto the dried and cut conjugate pad, and dry it for later use to obtain a conjugate pad coated with AIE carboxyl microspheres-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate and AIE microspheres-SA-biotin-chicken IgY conjugate.

[0049] 2. Preparation of nitrocellulose membrane: (1) Before scratching, the nitrocellulose membrane needs to be equilibrated in a humidity environment of 45%-60% for 3-4 hours; (2) Preparation of test line: The immunologically prepared anti-IL-6 monoclonal antibody was prepared with coating buffer to a concentration of 1 mg / mL, and the membrane was streaked to the T-line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; the streaked membrane was dried at 55°C for 2-3 days.

[0050] (3) Preparation of quality control line: The goat anti-chicken IgY antibody was prepared into a concentration of 1 mg / mL using a coating buffer, and the membrane was streaked onto the C line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm, and then dried at 55°C for 2-3 days.

[0051] 3. Preparation of sample pad: The sample pad was evenly coated with the sample pad blocking solution to completely wet it, and then dried at 55°C for 1 day and cut.

[0052] 4. Preparation of test strips: The sample pad, the conjugate pad, the NC membrane and the absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction to obtain a test strip.

[0053] 5. Preparation of the kit: (1) Test card: Put the prepared test strip into the card housing to obtain a test card; (2) Sample diluent.

[0054] Example 3: A test kit for detecting interleukin-6 1. Preparation of conjugate pad: 1.1. Preparation of biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres (1) 1 mL of 2 mg / mL Protein A / G and 6.4 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) were mixed and reacted for 30 min to obtain biotinylated Protein A / G; the biotin marker was desalted using a desalting column, and the recovered solution was added with 50% glycerol and stored at 2-8°C for later use; (2) 1 mg of AIE microsphere-SA was mixed with 20 μg of biotinylated Protein A / G to obtain AIE microsphere-SA-biotin-Protein A / G conjugate; (3) Mix 30 μg of IL-6 monoclonal antibody with 1 mg of AIE microsphere-SA-biotin-ProteinA / G conjugate to obtain IL-6 monoclonal antibody microsphere marker; (4) 15 μg of blocking agent (Roche MAK33) was added to 1 mg of IL-6 monoclonal antibody microsphere marker to obtain AIE carboxyl microsphere-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate.

[0055] 1.2. Preparation of biotinylated chicken IgY labeled with AIE polystyrene fluorescent microspheres: (1) Mix 1 mL of 2 mg / mL chicken IgY with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-chicken IgY; (2) 1 mg of AIE microsphere-SA was mixed with 30 μg of biotin-chicken IgY to obtain AIE microsphere-SA-biotin-chicken IgY conjugate; 1.3. Preparation of conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) AIE carboxyl microsphere-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate and AIE microsphere-SA-biotin-chicken IgY conjugate were diluted 110 times with fluorescent microsphere marker storage solution to obtain a fluorescent marker solution; (3) Spray the fluorescent marker solution at 5 μL / cm2 onto the dried and cut conjugate pad, and dry it for later use to obtain a conjugate pad coated with AIE carboxyl microspheres-SA-biotin-ProteinA / G-IL-6 monoclonal antibody conjugate and AIE microspheres-SA-biotin-chicken IgY conjugate.

[0056] 2. Preparation of nitrocellulose membrane: (1) Before scratching, the nitrocellulose membrane needs to be equilibrated in a humidity environment of 45%-60% for 3-4 hours; (2) Preparation of test line: The immunologically prepared anti-IL-6 monoclonal antibody was prepared with coating buffer to a concentration of 1 mg / mL, and the membrane was streaked to the T-line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm; the streaked membrane was dried at 55°C for 2-3 days.

[0057] (3) Preparation of quality control line: The goat anti-chicken IgY antibody was prepared into a concentration of 1 mg / mL using a coating buffer, and the membrane was streaked onto the C line area of ​​the NC membrane using a gold spray streaking instrument with a streaking volume of 1 μL / cm, and then dried at 55°C for 2-3 days.

[0058] 3. Preparation of sample pad: The sample pad was evenly coated with the sample pad blocking solution to completely wet it, and then dried at 55°C for 1 day and cut.

[0059] 4. Preparation of test strips: The sample pad, the conjugate pad, the NC membrane and the absorbent pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction to obtain a test strip.

[0060] 5. Preparation of the kit: (1) Test card: Put the prepared test strip into the card housing to obtain a test card; (2) Sample diluent.

[0061] Comparative Example 1: The difference between this comparative example and Example 1 is that the detection line markers of the conjugate pad are different.

[0062] The preparation process of the conjugate pad is as follows: 1. Preparation of carboxyl microsphere labeled IL-6 monoclonal antibody: 30 μg of IL-6 monoclonal antibody was mixed with 1 mg of carboxyl microspheres to obtain carboxyl microsphere-labeled IL-6 monoclonal antibody; 2. Preparation of AIE microsphere-SA-biotin-chicken IgY conjugate: 1 mL of 2 mg / mL chicken IgY was mixed with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-chicken IgY; 1 mg of AIE microsphere-SA was mixed with 30 μg of biotin-chicken IgY to obtain AIE microsphere-SA-biotin-chicken IgY conjugate; 3. Prepare the conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) diluting the carboxyl microsphere labeled IL-6 monoclonal antibody and the AIE microsphere-SA-biotin-chicken IgY conjugate 110 times with the fluorescent microsphere marker storage solution to obtain a fluorescent marker solution; (3) Spray the fluorescent marker solution at 5 μL / cm2 onto the dried and cut conjugate pad, dry it for later use, and obtain a conjugate pad.

[0063] Comparative Example 2: The difference between this comparative example and Example 1 is that the detection line markers of the conjugate pad are different.

[0064] The preparation process of the conjugate pad is as follows: 1. Preparation of SA microsphere-labeled biotinylated IL-6 monoclonal antibody: (1) 1 mL of 2 mg / mL IL-6 monoclonal antibody was mixed with 3.2 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-IL-6 monoclonal antibody; (2) 1 mg of SA microspheres was mixed with 30 μg of biotin-IL-6 monoclonal antibody to obtain SA microspheres labeled with biotinylated IL-6 monoclonal antibody.

[0065] 2. Preparation of AIE microsphere-SA-biotin-chicken IgY conjugate: 1 mL of 2 mg / mL chicken IgY was mixed with 0.9 μL of 0.25 mol / L biotin (NHS-PEG12-biotin) for 30 min to obtain biotin-chicken IgY; 1 mg of AIE microsphere-SA was mixed with 30 μg of biotin-chicken IgY to obtain AIE microsphere-SA-biotin-chicken IgY conjugate.

[0066] 3. Prepare the conjugate pad (1) Evenly apply the conjugate pad with the conjugate pad blocking solution to completely wet it, dry it, and cut it; (2) mixing the SA microsphere-labeled biotinylated IL-6 monoclonal antibody and the AIE microsphere-SA-biotin-chicken IgY conjugate with a fluorescent microsphere marker storage solution 110 times diluted to obtain a fluorescent marker solution; (3) Spray the fluorescent marker solution at 5 μL / cm onto the dried and cut conjugate pad, dry it for later use, and obtain a conjugate pad coated with SA microspheres labeled with IL-6 monoclonal antibody.

[0067] Performance testing: 1. Linear detection: The kits prepared in Examples 1-3 and Comparative Examples 1-2 were used to test Roche fixed-value serum samples, respectively. Five test strips were tested with a fluorescence analyzer for each concentration test, and the average value was calculated. The linear results are shown in the table.

[0068] Table 1 Test strip T line, C line signal values ​​and T / C values ​​of Example 1 Concentration (pg / mL) T C T / C 0 156 102235 0.0015 2.4 692 108562 0.0064 14.1 3098 84620 0.0366 31.3 7690 106563 0.0722 121.5 17120 94870 0.1805 506.8 28121 83494 0.3368 1331.2 103598 67074 1.5445 2450.8 111139 54035 2.0568 4578.6 189343 48899 3.8721 5006.4 195436 47689 4.0981 5976.2 166751 40937 4.0734 Table 2 Test strip T line, C line signal values ​​and T / C values ​​of Example 2 Concentration (pg / mL) T C T / C 0 232 98146 0.0024 2.4 247 104220 0.0023 14.1 800 81235 0.0098 31.3 2746 102300 0.0268 121.5 6114 91075 0.0671 506.8 10043 80154 0.1253 1331.2 36999 64391 0.5746 2450.8 39693 51874 0.7652 4578.6 67623 46943 1.4405 5006.4 69799 45781 1.5246 5976.2 59554 39300 1.5154 Table 3 Test strip T line, C line signal values ​​and T / C values ​​of Example 3 Concentration (pg / mL) T C T / C 0 2340 100190 0.0284 2.4 3114 106391 0.0293 14.1 3253 82928 0.0392 31.3 8075 104432 0.0773 121.5 17976 92973 0.1933 506.8 29527 81824 0.3609 1331.2 108778 65733 1.6548 2450.8 116696 52954 2.2037 4578.6 198810 47921 4.1487 5006.4 205208 46785 4.3909 5976.2 175089 40118 4.3643 Table 4 Test strip T line, C line signal value and T / C value of comparative example 1 Concentration (pg / mL) T C T / C 0 188 104252 0.0018 2.4 214 110704 0.0019 14.1 1589 86290 0.0184 31.3 3944 104512 0.0377 121.5 11190 96742 0.1157 506.8 18880 85141 0.2159 1831.2 61711 71397 0.8643 2450.8 72640 55101 1.3183 4578.6 117871 49864 2.3639 5006.4 117932 48630 2.4251 5976.2 105988 42745 2.4795 Table 5 Test strip T line, C line signal value and T / C value of comparative example 2 Concentration (pg / mL) T C T / C 0 168 100395 0.0017 2.4 254 106608 0.0024 14.1 2065 83097 0.0249 31.3 5127 100645 0.0509 121.5 11413 93162 0.1225 506.8 18747 81991 0.2287 1331.2 69065 65867 1.0486 2450.8 74093 53062 1.3963 4578.6 120229 48019 2.5038 5006.4 120291 46831 2.5686 5976.2 111167 40200 2.7653 Combining the detection results of Example 1, Comparative Example 1, and Comparative Example 2, it can be seen that the kit prepared by Example 1 has a wider low-end signal, a better signal-to-noise ratio, and a better sensitivity, indicating that the labeling model of the probe is improved in the present application, the biotin-avidin system is introduced, and Protein A / G is labeled on biotin, and then the IL-6 antibody is indirectly labeled on Protein A / G. Since Protein A / G has the common characteristics of Protein A and Protein G, it has a larger specific surface area and more binding sites, and thus can provide a high number of antigen binding, and the biotin-avidin system is used to increase the spacer arm and reduce the steric hindrance, thereby improving the sensitivity of the reagent.

[0069] Combining the detection results of Example 1 and Example 3, it can be seen that the background of Example 3 is higher and the signal-to-noise ratio is not as good as that of Example 1. This is because insufficient blocking agent may cause excessive Protein A / G to not be blocked, but directly bind to the NC membrane-coated antibody, resulting in a higher background.

[0070] Combining the linear results of Examples 1-3, it can be seen that Example 1 has the best effect, and the kit prepared in Example 1 is used for subsequent testing.

[0071] 2. Sensitivity detection The assay reagent was prepared by the method of Example 1, Comparative Example 1 and Comparative Example 2, and 5 low-value samples with concentrations close to the detection limit were tested. Each sample was tested 5 times, and the test results were sorted according to size. The number of test results that were lower than the blank limit value (the blank limit was 1.0 mg / mL) should be less than or equal to 3, and the detection limit LOD was not greater than 2.0 pg / mL.

[0072] Table 6 Test strip sensitivity detection of Example 1 Table 7 Test strip sensitivity detection of comparative example 1 Table 8 Test strip sensitivity detection of comparative example 2 According to the test results in Tables 6-8, it is shown that the kit prepared in Example 1 has good sensitivity.

[0073] 3. Stability test Three kits of the same batch were prepared using Example 1 and Comparative Examples 1-2. One kit was placed at 2-8°C, one was placed at a constant temperature at 37°C for 7 days, and one was placed at a constant temperature at 37°C for 14 days. Two samples with concentrations of 15 pg / mL and 250 pg / mL were tested. Two samples were tested respectively using the kits that were kept at a constant temperature at 37°C for 7 days, at a constant temperature at 37°C for 14 days, and at 2-8°C. Each test was performed in parallel 3 times, and the relative deviation of the accelerated result from 2-8°C was calculated.

[0074] Table 9 Test results of high-value samples of test strips in Example 1 Table 10 Test results of low-value samples of test strips of Example 1 Table 11 Test results of high-value samples of test strips of comparative example 1 Table 12 Test results of low-value samples of test strips of comparative example 1: Table 13 Test results of high-value samples of test strips of comparative example 2 Table 14 Test results of low-value samples of test strips of comparative example 2 According to the test results, the relative deviation of the kit prepared in Example 1 is less than 10%, indicating that the kit prepared by the method of the present application has good stability.

[0075] 4. Methodological comparison The detection kit was prepared by the method of Example 1 of the present application, and compared with Roche's interleukin 6 detection kit (electrochemiluminescence method) Elecsys IL-6 National Medical Device Injection No. 20162404369 to verify the accuracy of the kit of this embodiment: Table 15 Accuracy test results of the kit of Example 1 and the commercially available kit The sample test results are shown in Table 15. A scatter plot is made of the results and a fitting curve is shown in Table 15. Figure 1 The linear correlation equation is: y = 1.0415x + 4.8505, R 2=0.9976, correlation coefficient R=0.9987 (≥0.975), slope K=1.0415 (between 0.9-1.1), the above results show that the detection of this kit and Roche electrochemiluminescence kit has good consistency.

[0076] The IL-6 content of normal healthy people is less than 7pg / mL. The detection kit prepared by the above Example 1 has an LOD of 2.0pg / mL, a linear range comparable to that of Roche electrochemiluminescence, and good stability.

Claims

1. A test strip for detecting interleukin-6, characterized in that: The test strip comprises a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing pad are overlapped and pasted on the bottom plate in sequence according to the chromatography direction; The nitrocellulose membrane is coated with a detection line and a quality control line; the detection line is coated with anti-IL-6 monoclonal antibody; the quality control line is coated with rabbit anti-DNP-BSA or goat anti-chicken IgY; The conjugate pad is coated with biotinylated ProteinA / G-antibody labeled with AIE polystyrene fluorescent microspheres, and biotinylated antibody or antigen labeled with AIE polystyrene fluorescent microspheres.

2. A test strip for detecting interleukin-6 according to claim 1, characterized in that: The biotinylated ProteinA / G-antibody labeled with AIE polystyrene fluorescent microspheres is biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres.

3. A test strip for detecting interleukin-6 according to claim 2, characterized in that: The biotinylated ProteinA / G-IL-6 labeled with the AIE polystyrene fluorescent microspheres is prepared as follows: (1) Mixing Protein A / G with biotin to obtain biotinylated Protein A / G; (2) Mixing the streptavidin-modified AIE microspheres with biotinylated Protein A / G to obtain AIE microsphere-SA-biotin-Protein A / G conjugates; (3) Mixing the IL-6 monoclonal antibody with the AIE microsphere-SA-biotin-ProteinA / G conjugate to obtain the IL-6 monoclonal antibody microsphere marker; (4) Add a blocking agent to the IL-6 monoclonal antibody microsphere marker to block it, and obtain biotinylated ProteinA / G-IL-6 labeled with AIE polystyrene fluorescent microspheres.

4. A test strip for detecting interleukin-6 according to claim 3, characterized in that: In the step (1), the molar ratio of ProteinA / G to biotin is 1:(20-40); In step (2), the mass ratio of streptavidin-modified AIE microspheres to biotinylated ProteinA / G is 1:(0.001-0.02); In step (3), the mass ratio of the AIE microsphere-SA-biotin-ProteinA / G conjugate to the IL-6 monoclonal antibody is 1:(0.015-0.05); In the step (4), the mass ratio of the IL-6 monoclonal antibody microsphere marker to the blocker is 1:(0.015-0.03).

5. A test strip for detecting interleukin-6 according to claim 1, characterized in that: The biotinylated antibody or antigen labeled with AIE polystyrene fluorescent microspheres is biotinylated chicken IgY labeled with AIE polystyrene fluorescent microspheres or biotinylated DNP-BSA labeled with AIE polystyrene fluorescent microspheres.

6. A test strip for detecting interleukin-6 according to claim 5, characterized in that: The preparation method of the biotinylated antibody or antigen labeled with the AIE polystyrene fluorescent microspheres comprises the following steps: adding the antibody or antigen to biotin and mixing them to obtain biotin-antibody or antigen; The streptavidin-modified AIE microspheres are mixed with biotin-antibody or antigen to obtain biotinylated antibody or antigen labeled with AIE polystyrene fluorescent microspheres; The antibody is chicken IgY; the antigen is DNP-BSA.

7. A test strip for detecting interleukin-6 according to claim 6, characterized in that: The molar ratio of biotin to antibody or antigen is (10-40):1; The mass ratio of the streptavidin-modified AIE microspheres to the biotin-antibody or antigen is 1:(0.02-0.05).

8. A test strip for detecting interleukin-6 according to claim 3 or 6, characterized in that: The biotin is NHS-PEG12-biotin.

9. A test paper kit for detecting interleukin-6, characterized in that: The test kit comprises a test card, which comprises a card housing, in which the test strip according to any one of claims 1 to 8 is arranged.

10. A test strip for detecting interleukin-6 according to claim 1, characterized in that: The kit also includes a diluent.

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