Single-molecule immunodetection kit for interleukin 6

Abstract

The invention provides a monomolecular immunodetection kit for interleukin 6, and relates to the technical field of immunodetection, the monomolecular immunodetection kit comprises magnetic beads coated with an IL-6 antibody, a biotin-labeled IL-6 antibody and SAPE-PE, and the lowest detection limit of the kit can reach the fg / mL level, which means that extremely trace interleukin 6 (IL-6) can be detected. In the early stage of the disease, the concentration of the IL-6 may be very low, the level change of the IL-6 can be found earlier due to high sensitivity, and a key basis is provided for early diagnosis of the disease. For example, in the early stage of infection or the early stage of some chronic diseases, the kit can sensitively capture tiny changes which can not be detected by a traditional detection method, so that intervention measures can be taken in time, the treatment success rate is increased, the prognosis of a patient is improved, the detection range covers 0.1-2000pg / mL, the common concentration range of IL-6 in a clinical sample can be met, and the clinical application prospect is wide. A double-antibody sandwich fluorescence immunoassay method is adopted, specific epitopes of the IL-6 protein are detected, and cross reaction with other similar proteins is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and more specifically, particularly relates to a single molecule immunoassay kit for interleukin-6. Background Art

[0002] In the field of modern medicine, accurate detection of cytokines is of vital importance for disease diagnosis, treatment monitoring and prognosis assessment. Interleukin-6 (IL-6) is a key cytokine, and its level changes are closely related to the occurrence and development of various diseases.

[0003] With the continuous deepening of medical research, people have gradually realized that IL-6 plays an important role in many fields such as immune regulation, inflammatory response, cardiovascular disease, autoimmune disease and tumor. For example, in infectious diseases, the rapid increase of IL-6 often indicates the body's inflammatory response; in cardiovascular diseases, IL-6 levels are related to the severity and prognosis of the disease; in autoimmune diseases, IL-6 can be used as an indicator of disease activity. Therefore, accurate determination of IL-6 levels has important guiding value for clinical diagnosis and treatment decisions.

[0004] However, traditional IL-6 detection methods have many limitations. Early detection kits often have low sensitivity and are difficult to detect low concentrations of IL-6, which can easily lead to missed diagnosis in the early stages of the disease or when there are trace changes in IL-6. Moreover, these methods have a narrow detection range and cannot cover the various IL-6 concentration levels that may occur clinically, limiting their application in patients at different stages of the disease and with large individual differences.

[0005] In terms of specificity, some detection methods may be interfered by other similar cytokines or biological molecules, which may affect the accuracy of the test results and bring confusion and risk of misdiagnosis to clinical diagnosis.

[0006] At the same time, traditional detection methods also have shortcomings in terms of accuracy and repeatability. There may be a large deviation between the measured value and the true value, and the consistency of multiple test results is poor, which makes it difficult for doctors to make reliable diagnosis and treatment decisions based on the test results.

[0007] In addition, the complexity of the testing process is also a problem. Some testing methods require cumbersome operating steps, professional technicians and expensive instruments and equipment, which not only increases the cost of testing, but also reduces the efficiency of testing, and cannot meet the needs of clinical rapid and accurate diagnosis.

[0008] In this context, there is an urgent need for a new IL-6 detection kit that can overcome the limitations of traditional methods. This kit should have high sensitivity and be able to detect extremely low concentrations of IL-6; have a wide detection range to adapt to changes in IL-6 levels under different disease states; have good specificity and accurately distinguish IL-6 from other similar molecules; ensure the accuracy and repeatability of the test results, and provide a reliable basis for clinical diagnosis; at the same time, the operation process should be simple and fast, and the test results can be obtained in a relatively short time, thereby improving clinical work efficiency. The single molecule fluorescence detection kit for interleukin 6 (IL-6) of the present invention came into being just under such a demand. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a single molecule immunoassay kit for interleukin-6 to solve the above problems.

[0010] A single molecule immunoassay kit for interleukin-6 comprises magnetic beads coated with an IL-6 antibody, an IL-6 antibody labeled with biotin and SAPE-PE.

[0011] Preferably, the magnetic beads are MS300 / Carboxy l carboxyl magnetic beads with a particle size of 3 um.

[0012] Preferably, in the IL-6 antibody-coated magnetic beads, the mass ratio of the antibody to the magnetic beads is 1:50, and the concentration used is 40 μg / mL.

[0013] Preferably, in the biotin-labeled IL-6 antibody, the molar ratio of biotin to antibody is 20:1, and the concentration used is 1 μg / mL.

[0014] Preferably, the preparation method of the SAPE-PE is:

[0015] Step 1: Prepare BIO-PE by mixing biotin and PE in a molar ratio of 20:1;

[0016] Step 2: Dilute SAPE and BIO-PE to 0.1 mg / mL, mix them in a volume ratio of 4:1, and react at 37°C for 1 h.

[0017] Preferably, the reagent reaction steps are as follows:

[0018] Step 1: Take a 96-well ELISA plate, add 50 μL IL-6 sample, 50 μL reagent 1 and 50 μL reagent 2 to each well, and incubate in a 37°C incubator for 30 min;

[0019] Step 2: Magnetic aspiration, discard the supernatant, wash three times with 200 μL of PBST washing solution, and then add 50 μL of reagent 3;

[0020] Step 3: Magnetic suction, discard the supernatant, wash three times with 200μL PBST cleaning solution, add 60μL PBS to each well to resuspend the magnetic beads, and load the sample on the single molecule detection liquid path for detection.

[0021] Preferably, the minimum detection limit of the kit is at the fg / mL level.

[0022] Preferably, the detection range of the kit is 0.1-2000 pg / mL.

[0023] Preferably, the precision of the kit is less than 8%.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The minimum detection limit of this kit can reach the fg / mL level, which means that it can detect extremely small amounts of interleukin 6 (IL-6). In the early stages of the disease, the concentration of IL-6 may be very low. High sensitivity can detect changes in its level earlier, providing a key basis for early diagnosis of the disease. For example, in the early stages of infection or certain chronic diseases, traditional detection methods may not be able to detect small changes, but this kit can capture them sharply, so that timely intervention measures can be taken to increase the success rate of treatment and improve patient prognosis.

[0026] 2. The detection range covers 0.1-2000pg / mL, which can meet the common IL-6 concentration range in clinical samples. Whether it is a mild inflammatory response with less IL-6 secretion or a severe condition with a large amount of IL-6 release, it can be accurately detected. This makes the kit suitable for different courses of various diseases and differences between different patients, providing strong support for comprehensive assessment of the patient's condition.

[0027] 3. The double antibody sandwich fluorescent immunoassay is used to detect the specific epitope of IL-6 protein, reducing cross-reactions with other similar proteins. This means that the test results are more accurate and reliable, and will not be interfered by other cytokines or similar substances. It can provide clinicians with accurate diagnostic basis, avoid misdiagnosis and mistreatment, and ensure that patients receive targeted treatment plans.

[0028] 4. By testing the company's reference products and samples of known concentrations, the deviation between the measured value and the true value is within an acceptable range. Accurate test results help doctors make an objective assessment of the patient's condition and develop a reasonable treatment strategy. During the treatment process, accurate monitoring of changes in IL-6 levels can also adjust the treatment plan in a timely manner, improve treatment effects, and reduce unnecessary drug use and waste of medical resources.

[0029] 5. The coefficient of variation (CV) of the same concentration of samples was less than 8% after repeated testing, indicating that the kit has good repeatability. This means that the results can maintain high consistency when tested at different times, by different operators, and under different experimental conditions. Stable and reliable repeatability helps to ensure the reliability and comparability of test results, providing a solid foundation for clinical diagnosis and research.

[0030] 6. The test steps of the kit are relatively simple, the operation process is easy to master, and no complicated instruments and equipment or professional technical training are required. This allows the kit to be widely used in medical institutions and laboratories at all levels, shortens the time for sample testing, improves testing efficiency, and can provide a basis for clinical diagnosis and treatment in a timely manner, helping patients to get accurate diagnosis and effective treatment as soon as possible.

[0031] 7. It plays an important role in the diagnosis, treatment monitoring and prognosis evaluation of cardiovascular diseases, autoimmune diseases, infectious diseases and other diseases. It can assist doctors in judging the severity of the disease, the treatment effect and the prognosis of the patient, and provide strong support for the formulation of personalized medical plans, thereby improving the treatment effect and quality of life of patients.

[0032] 8. Through strict quality control and optimized production process, the stability and reliability of the test kit are guaranteed. At the same time, compared with other similar detection methods, under the premise of ensuring detection performance, it has certain cost advantages, reduces the cost of medical testing, reduces the economic burden of patients, and improves the utilization efficiency of medical resources.

[0033] 9. The high sensitivity, wide detection range and good repeatability of this kit provide a powerful tool for medical research. Researchers can more accurately explore the role of IL-6 in the pathogenesis of diseases and gain a deeper understanding of its relationship with other cytokines and biomolecules. It is helpful to carry out large-scale clinical studies, reveal the distribution pattern and change trend of IL-6 in different disease populations, provide data support for new therapeutic targets and drug development, and accelerate the innovation process of medical research.

[0034] 10. This test kit is not only suitable for clinical laboratories in hospitals, but can also be used in primary medical institutions, mobile testing vehicles and other scenarios. Its portability and ease of operation enable rapid testing of IL-6 in special circumstances such as emergency medical rescue and medical services in remote areas. It provides the possibility of accurate diagnosis of diseases in areas with relatively scarce medical resources, helps to achieve fairness and accessibility of medical services, and promotes the improvement of overall medical standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the composition of the kit of the present invention;

[0036] Figure 2 It is the standard curve drawn according to the test results in the present invention Figure 1 ;

[0037] Figure 3 It is the standard curve drawn according to the test results in the present invention Figure 2 . DETAILED DESCRIPTION

[0038] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0039] Interleukin 6 (IL-6), as an important cytokine, plays a key role in physiological and pathological processes such as human immune regulation and inflammatory response. Accurate detection of IL-6 levels is of great significance for disease diagnosis, treatment monitoring and prognosis assessment. The present invention provides a single-molecule fluorescence detection kit for interleukin 6 (IL-6), aiming to achieve more sensitive, accurate and convenient detection of IL-6.

[0040] The kit includes IL-6 antibody-coated magnetic beads, biotin-labeled IL-6 antibody, and SAPE-PE;

[0041] In the magnetic beads coated with IL-6 antibody, the magnetic beads used are MS300 / Carboxy l carboxyl magnetic beads from JSR Company with a particle size of 3um, and the antibody used is IL-6 monoclonal antibody C00504 from Dongkang Biological. The coating method is as follows: the magnetic beads are collected with 0.1MMES (pH5.0) and washed twice to remove impurities and unbound substances. EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) is added and activated on a rotator for 30 minutes. After collecting the magnet and washing twice, the antibody is added at a mass ratio of 1:50 between the antibody and the magnetic beads, and reacted on a rotator for 2 hours. Then, a blocking solution is added and the beads are blocked on a rotator for 30 minutes. Finally, the beads are washed with a cleaning solution to obtain IL-6 antibody-coated magnetic beads. The concentration used is 40μg / mL;

[0042] In the biotin-labeled IL-6 antibody, the biotin is sulfo-NHS-LC-LC-biotin from Thermo Company, and the antibody is IL-6 monoclonal antibody C00503 from Dongkang Biological. The labeling method is: biotin and antibody are mixed in a 20:1 molar ratio and reacted at room temperature for 40 minutes. The reactant is purified by a desalting column to obtain a biotin-labeled IL-6 antibody.

[0043] The concentration used was 1 μg / mL;

[0044] In the materials of SAPE-PE, PE comes from Langya Biotechnology and SAPE comes from Langya Biotechnology. The preparation method is as follows: first, biotin and PE are mixed in a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and BIO-PE are diluted to 0.1 mg / mL, mixed in a volume ratio of 4:1, and reacted at 37°C for 1 hour to obtain SAPE-PE. The concentration used is 1 μg / mL.

[0045] Experimental preparation:

[0046] (1) Experimental consumables include: 96-well ELISA plate, 96-well plate magnetic stand, pipette and matching tips (different specifications), centrifuge tubes (different specifications), micropipette, disposable gloves, disposable masks, and laboratory coats;

[0047] (2) Experimental instruments include: 37°C incubator, single molecule detection liquid system, centrifuge, vortex oscillator, refrigerator (4°C, -20°C), timer, electronic balance (accuracy 0.01 mg);

[0048] (3) Reagent preparation includes:

[0049] Phosphate buffered saline (PBS): Weigh a certain amount of sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate, dissolve in deionized water, adjust the pH to 7.4, and dilute to the required volume;

[0050] PBST washing solution: add 0.05% Tween-20 to PBS;

[0051] Blocking solution: PBS solution containing 1% bovine serum albumin (BSA).

[0052] Sample collection and processing:

[0053] (1) Serum sample collection: Use a sterile vacuum blood collection tube to collect 3-5 mL of fasting venous blood from the patient, let it stand at room temperature for 30 minutes to allow the blood to coagulate, centrifuge it at 3000 rpm for 10 minutes to separate the serum, transfer the serum to a new centrifuge tube, mark the patient information and collection time, and if it cannot be tested immediately, store the serum sample in a -20°C refrigerator to avoid repeated freezing and thawing;

[0054] (2) Collection of plasma samples: Use a vacuum blood collection tube containing an anticoagulant (such as heparin or EDTA) to collect 3-5 mL of venous blood from the patient. Gently invert the tube to allow the anticoagulant and blood to mix thoroughly. Centrifuge at 3000 rpm for 10 minutes to separate the plasma. Transfer the plasma to a new centrifuge tube and mark the patient information and collection time. If the plasma sample cannot be tested immediately, store it in a -20°C refrigerator to avoid repeated freezing and thawing.

[0055] During the test, take out the kit from the refrigerator, balance it to room temperature, gently shake reagent 1 (IL-6 antibody-coated magnetic beads), reagent 2 (biotin-labeled IL-6 antibody) and reagent 3 (SAPE-PE), take a 96-well ELISA plate, add 50 μL IL-6 sample (serum or plasma) to each well, add 50 μL reagent 1 and 50 μL reagent 2 to each well, mix well, and place the ELISA plate in a 37°C incubator for 30 minutes. During the incubation process, avoid shaking and moving the ELISA plate. After the incubation, use a 96-well plate magnetic stand for magnetic absorption, discard the supernatant, add 200 μL PBST cleaning solution to each well, gently shake, then magnetically absorb, and discard the supernatant. Repeat the washing three times, add 50 μL of reagent 3 to the washed wells, mix gently, magnetically aspirate, discard the supernatant, wash three times with 200 μL of PBST washing solution, add 60 μL of PBS to each well to resuspend the magnetic beads, and load the resuspended samples on the single molecule detection liquid path for detection.

[0056] Preparation of standard curve using international standards:

[0057] The IL-6 second generation international standard (No. 21 / 308, specification 1 μg / tube) was diluted to different concentrations such as 2000pg / mL, 1000pg / mL, 500pg / mL, 200pg / mL, 100pg / mL, 50pg / mL, 20pg / mL, 10pg / mL, 8pg / mL, 4pg / mL, 2pg / mL, 1pg / mL, 0.5pg / mL with phosphate buffer containing 1% BSA. Step: Detect the standard of each concentration, analyze the ratio of r2 / r1 (i.e., the positive percentage) and the PE average value (MeanPE). In the low concentration group (0-10pg / mL), as the concentration increases, the positive percentage gradually increases, and MeanPE does not change significantly; in the high concentration group (10-2000pg / mL), the positive percentage gradually saturates and no longer increases significantly, and MeanPE gradually increases. Draw a standard curve based on the test results. The standard curve is shown in the figure. Figure 2 and Figure 3 .

[0058] Blank limit:

[0059] Use blank PBS buffer as the sample and perform the test according to the test steps. Repeat the measurement 20 times to obtain the corresponding percentage coefficients. Calculate the mean (MEAN) and standard deviation (SD) of the percentage coefficients. Use the concentration value of the mean percentage coefficient plus twice the standard deviation as the detection limit of the kit.

[0060]

[0061] Through calculation, the blank limit of this test is 0.0191pg / mL, and the sensitivity can reach the fg / mL level, which is far higher than the pg / mL sensitivity performance of the test kits on the market.

[0062] Linear:

[0063] The high-value samples (close to 2000 pg / mL) were mixed with diluent and diluted into 8 dilution concentrations: 2442 pg / mL, 5947 pg / mL, 225031 pg / mL, 684485 pg / mL, 6396 pg / mL, 11717 pg / mL, 36790 pg / mL, and 74448 pg / mL. Each concentration was tested, and the linear regression equation was obtained with the dilution concentration as the independent variable and the test result value as the dependent variable, and the correlation coefficient r of the linear regression was calculated;

[0064]

[0065] Through calculation, within the range of (0.5-2000.0) pg / mL, the correlation coefficient r should be no less than 0.9900.

[0066] Repeatability Assessment:

[0067] The measurement was repeated 10 times with samples containing IL-6 (12.0 ± 6.0) pg / mL and (250.0 ± 100.0) pg / mL, and the mean and standard deviation of the 10 measurement results were calculated to calculate the coefficient of variation CV;

[0068]

[0069] The samples with the concentration of (12.0±6.0) pg / mL and (250.0±100.0) pg / mL were tested 10 times each, and the coefficient of variation (CV) was no more than 8%, indicating good repeatability.

[0070] Accuracy Assessment:

[0071] The company reference product with a concentration of (8.0±1.2) pg / mL and (200.0±30.0) pg / mL was tested as a regular sample, repeated 3 times, and the relative deviation was calculated;

[0072]

[0073] When measuring enterprise reference products, the relative deviation of the measurement results should be within the range of ±15%, with good accuracy.

[0074] Negative and positive controls should be set up for each experiment. The negative control uses serum or plasma samples without IL-6, and the positive control uses serum or plasma samples with known high concentrations of IL-6. The test results of the negative control should have no obvious signal, and the test results of the positive control should be within the expected range. If the negative control has a positive result or the positive control result is abnormal, the experiment should be repeated.

[0075] According to the standard curve, the signal value of the test sample is converted into IL-6 concentration. If the test result is lower than the minimum detection limit of the kit (0.0191pg / mL), it will be reported as "below the detection limit". The test result is valid within the range of 0.1-2000pg / mL. If it exceeds this range, the sample should be diluted and retested.

[0076] The experimental process should strictly adhere to the principle of aseptic operation to prevent contamination of samples and reagents. Reagents should be shaken thoroughly before use to avoid stratification and precipitation affecting the test results. Bubbles should be avoided during the operation to avoid affecting the accuracy of sample addition. The incubation time and temperature should be strictly controlled to ensure the full progress of the reaction. The cleaning steps should be thorough to remove unbound substances and impurities. Instruments and equipment should be calibrated and maintained regularly to ensure the accuracy and reliability of the test results.

[0077] The minimum detection limit of this kit can reach fg / mL level, and it can detect extremely low concentrations of IL-6, which is of great significance for early disease diagnosis and trace IL-6 detection. Compared with existing detection kits on the market, the sensitivity is significantly improved, and the potential risk of the disease can be discovered earlier. The kit uses double antibody sandwich fluorescent immunoassay to detect specific epitopes of IL-6 protein, reducing cross-reactions with other similar proteins. By detecting a variety of cytokines that may have cross-reactions, it is verified that the kit has good specificity and can accurately detect IL-6 without interference from other factors. By testing corporate reference products and samples of known concentrations, the results show that the deviation between the measured value of the kit and the true value is within an acceptable range, which is consistent with other established The results showed that the accuracy of the kit was comparable to or better than other methods. The same concentration of samples was tested repeatedly, and the results showed that the coefficient of variation (CV) was less than 8%, indicating that the kit had good repeatability. The test results of the same sample from different batches of the kit were highly consistent, proving that the production process of the kit was stable and the repeatability was reliable. The detection range of the kit was 0.1-2000pg / mL, which could cover the common IL-6 concentration range in clinical samples. Within the linear range, there was a good linear relationship between the test result and the sample concentration, and the correlation coefficient r was not less than 0.9900, ensuring the accuracy and reliability of the test result.

[0078] The test kit should be stored in a refrigerator at 2-8℃, avoiding freezing and high temperature. During storage, the test kit should be protected from direct sunlight and humid environment. During transportation, a refrigerator or ice pack should be used to maintain low temperature to ensure that the quality of the test kit is not affected.

[0079] The single molecule fluorescence detection kit for interleukin 6 (IL-6) of the present invention has the advantages of high sensitivity, strong specificity, good accuracy, good repeatability, wide linear range, etc., and can meet the clinical demand for IL-6 detection. Through strict quality control and standardized operating procedures, reliable test results can be provided for disease diagnosis, treatment monitoring and prognosis evaluation. In future clinical applications, it is expected to provide strong support for the diagnosis and treatment of related diseases and bring positive effects to the health management of patients.

[0080] The lowest detection limit of this kit can reach the fg / mL level, which means that it can detect extremely small amounts of interleukin 6 (IL-6). In the early stages of the disease, the concentration of IL-6 may be very low. High sensitivity can detect changes in its levels earlier, providing a key basis for early diagnosis of the disease. For example, in the early stages of infection or certain chronic diseases, traditional detection methods may not be able to detect small changes, but this kit can capture them keenly, so that timely intervention measures can be taken to increase the success rate of treatment and improve patient prognosis.

[0081] The detection range covers 0.1-2000pg / mL, which can meet the common IL-6 concentration range in clinical samples. Whether it is a mild inflammatory response with low IL-6 secretion or a severe condition with a large amount of IL-6 released, it can be accurately detected. This makes the kit suitable for different courses of various diseases and differences between different patients, providing strong support for comprehensive assessment of the patient's condition.

[0082] The double antibody sandwich fluorescent immunoassay is used to detect the specific epitope of IL-6 protein, reducing cross-reactions with other similar proteins. This means that the test results are more accurate and reliable, and will not be interfered by other cytokines or similar substances. It can provide clinicians with accurate diagnostic basis, avoid misdiagnosis and mistreatment, and ensure that patients receive targeted treatment plans.

[0083] By testing the company's reference products and samples of known concentration, the deviation between the measured value and the true value is within an acceptable range. Accurate test results help doctors make an objective assessment of the patient's condition and develop a reasonable treatment strategy. During the treatment process, accurate monitoring of changes in IL-6 levels can also adjust the treatment plan in a timely manner, improve treatment effects, and reduce unnecessary drug use and waste of medical resources.

[0084] The coefficient of variation (CV) of the same concentration of samples was less than 8% after repeated testing, indicating that the kit has good repeatability. This means that the results can be highly consistent when tested at different times, by different operators, and under different experimental conditions. Stable and reliable repeatability helps ensure the reliability and comparability of test results, providing a solid foundation for clinical diagnosis and research.

[0085] The test steps of the kit are relatively simple, the operation process is easy to master, and no complex instruments and equipment or professional technical training are required. This allows the kit to be widely used in medical institutions and laboratories at all levels, shortening the time for sample testing, improving testing efficiency, and providing a basis for clinical diagnosis and treatment in a timely manner, helping patients to receive accurate diagnosis and effective treatment as soon as possible.

[0086] It plays an important role in the diagnosis, treatment monitoring and prognosis evaluation of cardiovascular diseases, autoimmune diseases, infectious diseases and other diseases. It can assist doctors in judging the severity of the disease, the treatment effect and the patient's prognosis, and provide strong support for the formulation of personalized medical plans, thereby improving the treatment effect and quality of life of patients.

[0087] Through strict quality control and optimized production process, the stability and reliability of the test kit are guaranteed. At the same time, compared with other similar detection methods, under the premise of ensuring detection performance, it has certain cost advantages, reduces the cost of medical testing, reduces the economic burden of patients, and improves the utilization efficiency of medical resources.

[0088] The high sensitivity, wide detection range and good repeatability of this kit provide a powerful tool for medical research. Researchers can more accurately explore the role of IL-6 in the pathogenesis of diseases and gain a deeper understanding of its relationship with other cytokines and biomolecules. It will help to carry out large-scale clinical studies, reveal the distribution patterns and changing trends of IL-6 in different disease populations, provide data support for new therapeutic targets and drug development, and accelerate the innovation process of medical research.

[0089] This test kit is not only suitable for clinical laboratories in hospitals, but can also be used in primary medical institutions, mobile testing vehicles and other scenarios. Its portability and ease of operation make it possible to quickly test IL-6 in special situations such as emergency medical rescue and medical services in remote areas. It provides the possibility of accurate diagnosis of diseases in areas with relatively scarce medical resources, helps to achieve fairness and accessibility of medical services, and promotes the improvement of overall medical standards.

[0090] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A single molecule immunoassay kit for interleukin-6, characterized in that: Including IL-6 antibody-coated magnetic beads, biotin-labeled IL-6 antibody and SAPE-PE.

2. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: The magnetic beads are MS300 / Carboxyl magnetic beads with a particle size of 3um.

3. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: In the IL-6 antibody-coated magnetic beads, the mass ratio of antibody to magnetic beads was 1:50, and the concentration used was 40 μg / mL.

4. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: The biotin-labeled IL-6 antibody had a molar ratio of biotin to antibody of 20:1 and was used at a concentration of 1 μg / mL.

5. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: The preparation method of the SAPE-PE is: Step 1: Prepare BIO-PE by mixing biotin and PE in a molar ratio of 20:1; Step 2: Dilute SAPE and BIO-PE to 0.1 mg / mL, mix them in a volume ratio of 4:1, and react at 37°C for 1 h.

6. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: The reagent reaction steps are as follows: Step 1: Take a 96-well ELISA plate, add 50 μL IL-6 sample, 50 μL reagent 1 and 50 μL reagent 2 to each well, and incubate in a 37°C incubator for 30 min; Step 2: Magnetic aspiration, discard the supernatant, wash three times with 200 μL of PBST washing solution, and then add 50 μL of reagent 3; Step 3: Magnetic suction, discard the supernatant, wash three times with 200μL PBST cleaning solution, add 60μL PBS to each well to resuspend the magnetic beads, and load the sample on the single molecule detection liquid path for detection.

7. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: The minimum detection limit of the kit is at the fg / mL level.

8. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: The detection range of the kit is 0.1-2000pg / mL.

9. A single molecule immunoassay kit for interleukin-6 as claimed in claim 1, characterized in that: The precision of the kit is less than 8%.

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