A kit for detecting cytokines in a blood sample

Abstract

The present application belongs to the technical field of biological detection, and provides a kit for detecting cytokines in blood samples. The kit comprises fluorescent coded polystyrene magnetic microspheres coupled with capture antibodies, biotin-labeled paired detection antibodies, streptavidin-labeled phycoerythrin with a final concentration of 0.5-2 ug / mL, a washing buffer containing 0.05%-0.1% Tween-20 or triton-100, an experimental buffer containing 0.5%-2% bovine serum albumin, and a sample diluent. The sample diluent comprises the experimental buffer and treated human serum. The treated human serum is obtained by first heat inactivation and carbon adsorption treatment of human serum, and then centrifugal separation using an ultrafiltration centrifugal tube. By using the kit provided by the present application, quantitative results of 7 cytokines can be obtained in a single detection of a single sample, and the detection range can reach 0.977-128000 pg / mL.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a kit for detecting cytokines in a blood sample. BACKGROUND

[0002] Cytokines are a general term for polypeptide cell regulatory substances, including interleukins, interferons, growth factors, cell stimulating factors, tumor necrosis factors, etc., which are mainly synthesized by peripheral immune cells (such as macrophages, lymphocytes, and fibroblasts). Factors such as IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and IFN-γ are common cytokines in human immunity, which are secreted by helper T cells Th1 and Th2 cells, participate in regulating the dynamic balance of Th1 and Th2 cell functions, and maintain the normal cellular and humoral immune functions of the body. The concentration level of cytokines in human samples is closely related to inflammation, viral infection, autoimmune diseases, etc., and is currently an early sensitive "warning" marker and even a quantitative marker for various diseases.

[0003] A single cytokine can be used as a diagnostic basis for diseases such as inflammation, but its sensitivity and specificity cannot meet the clinical needs and has great limitations. The combined detection of multiple cytokines can more clearly show the immune function and state of the human body, better assist in the diagnosis of various diseases, and also play a monitoring role in the treatment process to guide clinical medication and solve the pain point of difficult-to-obtain clinical samples, which has important clinical significance. However, due to the great difference in the content of each factor in different individuals in different states, the detection range is required to be wide. For example, the above-mentioned cytokines have very low content in healthy individuals, about 1-10 pg / mL; in individuals with cytokine storm, the content is very high, about 10000-100000 pg / mL, and the concentration difference between the two can reach 5-6 orders of magnitude. However, there is no reported detection method and kit for multiple cytokines on the market, especially for IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and IFN-γ.

[0004] The commonly used cytokine detection methods at present include enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), Western blot, flow cytometry fluorescence detection method (CBA), etc. The detection range of the above detection methods is generally between 5 pg / mL and 5000 pg / mL. Although the detection range can be widened by diluting the sample by 5-10 times, it still cannot completely meet the detection needs of the above-mentioned cytokines. Moreover, further increasing the dilution multiple of the sample will exceed the acceptance standard of the recovery rate, and cannot guarantee the accuracy of the results.

[0005] Therefore, it is urgent to develop a detection method and kit for detecting multiple cytokines rapidly, with high specificity, high sensitivity and wide detection range, so as to overcome the problems in the prior art. SUMMARY

[0006] The present application aims to provide a kit for detecting cytokines in blood samples, which solves the technical problems of complicated operation, single detection index and insufficient detection range in the prior art, and realizes quantitative detection of multiple targets in a single sample.

[0007] To achieve the above-mentioned object, the technical scheme adopted by the present application is as follows: a kit for detecting cytokines in blood samples, comprising fluorescent coded polystyrene magnetic microspheres coupled with capture antibodies, biotin-labeled paired detection antibodies, streptavidin-labeled phycoerythrin (SA-PE) with a final concentration of 0.5-2 ug / mL, a washing buffer containing 0.05%-0.1% Tween-20 or triton-100, an experimental buffer containing 0.5%-2% bovine serum albumin (BSA), and a sample diluent, wherein the sample diluent comprises the experimental buffer and a treated serum, and the treated serum is obtained by centrifugal separation of the serum using an ultrafiltration centrifugal tube after heat inactivation and carbon adsorption treatment.

[0008] The detection principle of the application is that the capture antibody coupled on the fluorescence coded polystyrene magnetic microspheres and the biotin labeled pairing detection antibody are respectively combined with the target points of the target substances in the human samples such as plasma, serum and the like, to form a double antibody sandwich immune complex, the complex is combined with the streptavidin labeled phycoerythrin (PE) on the biotin on the pairing detection antibody to form a final immune detection complex (coupling capture antibody fluorescence coded polystyrene magnetic microspheres-target substance-biotin labeled pairing detection antibody-streptavidin labeled phycoerythrin). On the detection platform such as a multi-color flow cytometer or a liquid suspension chip detector, the detection instrument generates two beams of lasers with different wavelengths, one beam with a wavelength of 635 nm is called red laser, and one beam with a wavelength of 532 nm is called green laser. The red laser excites the microspheres in the final immune detection complex to produce two beams of excitation light with different fluorescence intensities, which are received by the classification laser detector (APC channel and APC-Cy7 channel) in the detection platform, so as to determine the type of the microspheres and then distinguish different detection items. The green laser excites the phycoerythrin (PE) in the final immune detection complex to produce a third excitation light, which is received by the reporting laser detector (PE channel) in the detection platform. The fluorescence intensity of the excitation light is positively related to the concentration of the target substance, and the concentration of the target substance in the sample can be obtained by establishing a fitting curve of the standard or calibration sample. Through the above-mentioned manner, the computer of the detection platform analyzes the three beams of excitation light which are mutually free of crosstalk and completely different, so as to complete the quantitative detection of the cytokines in the sample.

[0009] When the sample concentration exceeds the original linear range of the kit, the sample is diluted by a suitable multiple within the allowable multiple through the sample diluent, and the accurate concentration of the sample is obtained after the calculation.

[0010] However, the conventional kit on the market can dilute the sample by a certain multiple to expand the detection range, but the maximum dilution multiple of the sample is low. Too high dilution multiple will completely change the matrix environment of the target protein, which may affect the structure of the target protein, thereby causing serious deviation of the detection result, and even unable to detect. It still cannot completely meet the detection of cytokines with large content difference under different individuals and different states.

[0011] In view of this problem, the inventors have made improvement schemes from different directions, optimized the treatment of the sample, screened the antibody, optimized the microsphere coupling process, and optimized the buffer composition and addition concentration, but the detection range cannot be greatly expanded. When the sample diluent is replaced by serum, the detection range changes by an order of magnitude.

[0012] In the actual test process, the dilution range of the serum without human serum is 4-16 times, and the dilution range of the serum with human serum is 8-64 times, and the detection range is significantly expanded. However, the situation of different projects is not the same, and it cannot completely meet the requirements, and the stability of detection is poor (different samples, different batches of the same sample detection, the maximum dilution factor is unstable), and the inventors expect to further expand the detection range and improve the detection stability.

[0013] The inventors treat the serum by first heat inactivation and carbon adsorption treatment to remove hormones, growth factors, complement factors and other substances, and then centrifugal separation by using ultrafiltration centrifuge tubes with different pore sizes to remove endogenous cytokines. The mixed human serum under this treatment removes substances that interfere with detection, while partially preserving the matrix environment, avoiding changes in protein properties due to environmental changes during gradient dilution, thereby improving the accuracy of the detection results. The maximum dilution factor can reach 32-128 times.

[0014] As preferred, the heat inactivation condition is 55℃ to 56℃ water bath, 25min to 30min; the carbon adsorption operation is using 50 mesh to 100 mesh activated carbon to adsorb the serum at 2℃ to 8℃ overnight, followed by low speed centrifugal separation and suction of the supernatant.

[0015] As preferred, the ultrafiltration centrifuge tube is an ultrafiltration centrifuge tube with a pore size greater than 50K.

[0016] As preferred, the addition amount of the treated serum is 20% to 50% of the sample diluent.

[0017] As preferred, the ultrafiltration centrifuge tube is a 100K ultrafiltration centrifuge tube. The 100kD ultrafiltration retentate is mixed with the experimental buffer at different ratios to prepare a sample diluent, and after adjustment, the maximum dilution factor can reach 128 times, but in the actual experiment process, it is found that the sensitivity decreases to 3.908 pg / mL.

[0018] Therefore, as preferred, after obtaining the retentate, it is also subjected to dilution and renaturation treatment, and is used as a treated serum.

[0019] As preferred, the retentate is added with 0.01%~0.05% SDS and 0.5%~2% PEG-8000 during the dilution and renaturation process. In the experimental buffer, 100kD ultrafiltration retentate is added as a sample diluent, and based on this result, the inventors guess that it may be because the ultrafiltration tube concentrates the protein during centrifugal ultrafiltration, resulting in too high concentration of macromolecular proteins, changes in protein structure, and decreased solubility, which is not conducive to the reaction, resulting in a significant decrease in sensitivity.

[0020] Thus, the diluent is added to the retentate to reduce the protein concentration, and the diluent contains a certain amount of SDS and PEG, so that the protein is dissolved by denaturation and returns to the normal state.

[0021] The serum subjected to the above treatment is mixed with the experimental buffer to configure a sample diluent, and the maximum dilution ratio can reach 128-256 times, and the sensitivity can reach 0.977 pg / mL.

[0022] Preferably, the added amount of the treated serum is 20-50% of the sample diluent. Preferably, the kit further comprises a working solution containing 0.5-2% bovine serum albumin (BSA), 0.2-0.4 mg / mL mouse IgG, and 0.1-0.2 mg / mL mouse anti-human IgG.

[0023] Preferably, in the working solution, a plurality of surfactants are added, including any one or several of 0.02% Tween-20, 0.02% polysorbate, 0.01% SP-20, and 0.03% BASF 2520.

[0024] Preferably, the capture antibody is a mouse anti-human monoclonal antibody.

[0025] Preferably, the capture antibody is a mouse anti-human IL-2 monoclonal capture antibody, a mouse anti-human IL-4 monoclonal capture antibody, a mouse anti-human IL-6 monoclonal capture antibody, a mouse anti-human IL-8 monoclonal capture antibody, a mouse anti-human IL-10 monoclonal capture antibody, a mouse anti-human TNF-α monoclonal capture antibody, and a mouse anti-human IFN-γ monoclonal capture antibody; and the paired detection antibody is a biotin-labeled mouse anti-human IL-2 monoclonal paired detection antibody, a biotin-labeled mouse anti-human IL-4 monoclonal paired detection antibody, a biotin-labeled mouse anti-human IL-6 monoclonal paired detection antibody, a biotin-labeled mouse anti-human IL-8 monoclonal paired detection antibody, a biotin-labeled mouse anti-human IL-10 monoclonal paired detection antibody, a biotin-labeled mouse anti-human TNF-α monoclonal paired detection antibody, and a biotin-labeled mouse anti-human IFN-γ monoclonal paired detection antibody.

[0026] The above kit provided by the present application can reach a maximum dilution ratio of 256 times and a sensitivity of 0.977 pg / mL by mixing the serum diluted and stored after heat inactivation, carbon adsorption, and 100K ultrafiltration treatment with the experimental buffer as a sample diluent, and optimizing the added amount of the treated serum and the subsequent preparation steps of the treated serum.

[0027] In another aspect of the present application, a preparation method of the above kit is provided, which specifically comprises the following steps:

[0028] 1) The fluorescently encoded polystyrene magnetic microspheres are activated using EDC and NHS, and the activation pH is 5.0-6.0.

[0029] 2) The activated polystyrene magnetic microspheres are coupled with the mouse anti-human monoclonal capture antibody.

[0030] 3) The coupled polystyrene magnetic microspheres are blocked using a blocking solution containing bovine serum albumin (BSA) and glycine, washed with a washing buffer, and finally resuspended using a protein stabilizing solution (containing 2% BSA and 2% trehalose) and stored at 2-8°C.

[0031] 4) The paired detection antibody is labeled by adding biotin, and the molar ratio of the paired detection antibody to biotin is 1:20-1:50.

[0032] 5) The labeled antibody is purified and enriched by dialysis or centrifugal ultrafiltration to obtain the biotin-labeled paired detection antibody, which is stored at 2-8°C for short-term storage and at -20°C for long-term storage.

[0033] Another aspect of the present application also provides a method for using the kit, which specifically comprises the following steps:

[0034] 1) The fluorescently encoded polystyrene magnetic microspheres coupled with the capture antibody are diluted 30-50 times using the experimental buffer to obtain mixed capture microspheres.

[0035] 2) The biotin-labeled paired detection antibody is diluted 50-150 times using the experimental buffer to obtain mixed detection antibody.

[0036] 3) The mixed capture microspheres are added to the reaction well.

[0037] 4) The reaction well plate is placed on a magnetic separator, and the supernatant is shaken off.

[0038] 5) The working solution, standard or quality control or human sample, mixed detection antibody are sequentially added to the reaction well, and shaken and incubated.

[0039] 6) Step 4 is repeated, and the washing buffer is added to each well for repeated washing.

[0040] 7) Streptavidin-labeled phycoerythrin (SA-PE) is added to the reaction well, and shaken and incubated.

[0041] 8) Step 6 is repeated, and finally the washing buffer is added to each well.

[0042] 9) After shaking at room temperature, detection is performed on a multicolor flow cytometer or a liquid suspension chip detection platform.

[0043] If the test result exceeds the detection upper limit of the kit, the sample is diluted by a suitable dilution factor within 256 times using a sample diluent, and then the detection is performed according to steps 1-9, and after recalculation, the accurate concentration result of the sample is obtained.

[0044] Another aspect of the present application also provides the use of the above-mentioned kit for quantitative detection of cytokines in blood samples, and in particular for simultaneous quantitative detection of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha and IFN-gamma in human blood samples.

[0045] The detection instrument used in the kit of the present application includes but is not limited to a multicolor flow cytometer or a liquid suspension chip detection platform.

[0046] Compared with the prior art, the present application has the beneficial effects that the detection sensitivity is 0.977 pg / mL, and the sample dilution factor can be up to 256 times, so that the detection range can reach 0.977 pg / mL to 128000 pg / mL, basically meeting the current detection requirements. In particular, it can be used for simultaneous quantitative detection of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha and IFN-gamma, and has a wide dilution factor range and excellent specificity and reproducibility. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Scatter plot of polystyrene magnetic microspheres with fluorescence encoding corresponding to the seven cytokines of the present application.

[0048] Figure 2 Standard curve of IL-2 in the kit of one embodiment of the present application.

[0049] Figure 3 Standard curve of IL-4 in the kit of one embodiment of the present application.

[0050] Figure 4 Standard curve of IL-6 in the kit of one embodiment of the present application.

[0051] Figure 5 Standard curve of IL-8 in the kit of one embodiment of the present application.

[0052] Figure 6 Standard curve of IL-10 in the kit of one embodiment of the present application.

[0053] Figure 7 Standard curve of TNF-alpha in the kit of one embodiment of the present application. (Note: TNF-alpha and TNF-alpha have the same meaning)

[0054] Figure 8 Standard curve of IFN-γ in the kit of one embodiment of the present application. (Note: IFN-γ is equivalent to IFN-gamma) DETAILED DESCRIPTION

[0055] The technical solutions of the present application will be described clearly and completely in combination with the drawings and examples of the specification.

[0056] Brief description of the development process of the cytokine kit

[0057] 1. Research goal

[0058] The linear range of the current cytokine detection kit is generally 2.5-5000 pg / mL, the maximum allowed dilution multiple of the sample is 5-10 times, and thus the allowable detection range is 2.5-50000 pg / mL. The research goal of the present application is a linear range of 0.977-500 pg / mL, a maximum allowed dilution multiple of 256 times, and an allowable detection range of 0.977-128000 pg / mL.

[0059] 2. Development process

[0060] 2.1. Screening of antibody pairs

[0061] 2.1.1: Determination of antibody type

[0062] Since the polyclonal antibody can recognize multiple sites of the target protein, the detection sensitivity is high and the specificity is weak. The imported Siemens kit uses polyclonal antibodies, and on the chemiluminescence detection platform, the detection sensitivity can reach 1.5 pg / mL. However, since the present kit is a 7-item cytokine combined detection kit, and there is a certain similarity in sequence and structure between cytokines, the use of polyclonal antibodies will produce unknown reactions, resulting in false positives in the detection results. After comprehensive comparison, it is believed that non-specific results are unacceptable, so monoclonal antibodies are selected to reduce sensitivity and enhance detection specificity to avoid non-specific results.

[0063] 2.1.2: Antibody screening test-cross reaction

[0064] In the antibody pairing test, each item only reacts to the corresponding target protein and does not react with other cytokine proteins below 500000 pg / mL.

[0065] 2.1.3: Antibody screening test-sensitivity

[0066] In the development process, each project is screened with more than 40 antibody pairs to select the optimal antibody pair combination in a pure buffer system. The sensitivity of the test can reach 0.488 pg / mL.

[0067] 2.2, Buffer adjustment

[0068] 2.2.1: Serum test - false positive

[0069] In human serum, there may be human anti-mouse antibodies (HAMA), general anti-animal antibodies, heterophilic antibodies, and rheumatoid factors that interfere with detection, leading to false positive test results. Different individuals differ greatly, and in order to reduce the impact of this factor, a certain concentration of mouse IgG and mouse anti-human IgG is added to the experimental buffer of the kit.

[0070] Adding mouse IgG can bind to interfering substances such as HAMA, thereby reducing the probability of contact between HAMA and other interfering substances and the capture antibodies and detection antibodies in the kit, thereby reducing the impact and achieving passive protection.

[0071] Adding mouse anti-human IgG can actively bind to interfering substances such as HAMA, thereby blocking the interference ability of HAMA and other interfering substances, thereby eliminating the impact and achieving active protection.

[0072] After a series of concentration and formula adjustments, 200 cases of healthy human serum were tested, and there was no false positive reaction. If the above substances are not added, the false positive rate is 30%.

[0073] 2.2.2: Serum test - false negative

[0074] In human serum, there are a large number of proteins such as IgG, complement, and receptors that may block the binding sites of target proteins or the binding sites of capture antibodies or detection antibodies in the kit, leading to false negative test results. To eliminate this impact, first, in the microsphere coupling process, a variety of molecular weight proteins are added to protect the capture antibodies on the microsphere end after coupling; second, in the experimental buffer, a variety of surfactants are added and the concentration is adjusted to reduce non-specific adsorption; third, the amount of detection antibody is adjusted so that the serum cannot completely block it; finally, the reaction volume is adjusted to dilute the interfering substances to a certain extent, thereby reducing the interference.

[0075] After a series of adjustments, 10 cases of healthy human serum were mixed with standard samples to make their concentrations around 2-5 pg / mL, and the relative deviation was between ±20%.

[0076] 2.3, Maximum dilution factor of serum

[0077] The linear range of the detection method is generally 3-4 orders of magnitude, and in order to test the extremely high concentration of sample concentration, the sample is diluted in advance before detection by using diluent. The maximum dilution factor of general kit is between 5-10 times. Too high dilution factor will completely change the matrix environment of target protein, which may affect the structure of target protein, thus causing serious deviation of detection results. In theory, there is no problem of using human serum as diluent. However, the content of protein, lipid and hormone in different individuals is different and the difference is significant. The use of single or mixed human serum as diluent cannot guarantee its applicability to all samples. In actual test process, the maximum dilution factor without using human serum is 4-16 times, and the maximum dilution factor with using human serum is 8-64 times. The situation is different for different projects, which cannot fully meet the requirements.

[0078] The following optimization process is carried out according to the results:

[0079] 1. Since the diluent is mixed with human serum, there are at least two individual interferences in the detection process, one is the individual interference of the sample, and the other is the individual interference in the diluent. The individual interference in the diluent cannot be accepted and should be completely removed. Therefore, the serum treatment method commonly used in cell culture, carbon adsorption and heat inactivation, is adopted to remove complement, hormone and lipid, etc.

[0080] 2. The mixed human serum should cover as many individual matrix environments as possible to provide the corresponding matrix environment in the detection process and avoid the fundamental change of the matrix environment after high dilution. Therefore, mixed human serum is selected, and the mixed human serum should contain different age groups, different genders, and at least 20 individuals.

[0081] 3. Since the detection environment of the standard is pure Buffer, which is significantly different from the serum environment, serum is introduced into the Buffer.

[0082] After the above optimization, the maximum dilution factor is still 8-64 times, and the detection sensitivity is 0.977 pg / mL.

[0083] Since the above results do not meet the expectations, separation and purification experiments are carried out to find the influencing factors affecting the dilution factor.

[0084] The human serum is fractionated using ultrafiltration centrifuge tubes with 100K, 50K, 30K and 3K, so as to obtain solutions with different molecular weights, which are tested, and it is found that the ultrafiltration retentate of 100k and 50K affects the detection sensitivity, but after sample dilution using the liquid, the maximum dilution factor can be increased to 32-128 times. The 30K retentate also affects the detection sensitivity, but the maximum dilution factor of different projects is increased to different degrees. The 3K retentate significantly affects the detection sensitivity, and the maximum dilution factor is not increased.

[0085] According to the above results, it is considered that the macromolecular protein above 100kD may be the key to increase the maximum dilution factor, and the protein with a molecular weight between 3-50kD is one of the sources of interference.

[0086] According to this result, the 100kD ultrafiltration retentate is mixed into the experimental buffer and sample diluent according to different ratios, and after adjustment, the maximum dilution factor can reach 128 times, and the sensitivity is reduced to 3.908pg / mL.

[0087] According to this result, it is speculated that because the ultrafiltration tube concentrates the protein during centrifugal ultrafiltration, the concentration of macromolecular protein is too high, the protein structure is changed, the solubility is reduced, and the overall matrix environment is not conducive to the reaction, resulting in a significant decrease in sensitivity.

[0088] Therefore, the diluent is added to the retentate to reduce the protein concentration, and the diluent contains a certain amount of SDS and PEG to restore the normal state of the protein.

[0089] The serum subjected to the above treatment is mixed into the experimental buffer to configure a sample diluent, and preliminary test shows that the maximum dilution factor can reach 128-256 times, and the sensitivity is increased to 0.977pg / mL.

[0090] According to this result, the subsequent processing serum preparation steps are refined, such as adjusting and fixing the retentate ratio, increasing the ultrafiltration washing step to more completely remove the interfering substances, refining the dilution and denaturation step to obtain more stable treated serum, and optimizing the addition amount of the treated serum, so as to finally output a stable process, and the maximum dilution factor can reach 256 times, and the sensitivity is 0.977pg / mL.

[0091] The present application develops a kit and uses the kit to detect IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha and IFN-gamma 7 kinds of cytokines in human blood samples.

[0092] Example 1: Fluorescently encoded polystyrene magnetic microspheres coupled with capture antibodies

[0093] 1.1 Take 7 different fluorescently encoded carboxylated polystyrene magnetic microspheres 1 mL, add to centrifuge tube, stand on magnetic separator for 2 min, discard supernatant, and the specific reagent information is shown in Table 1.

[0094]

[0095] 1.2 Add 1 mL of activation buffer (50 mM MES, pH 6.0), vortex to mix, stand on magnetic separator for 2 min, discard supernatant, and repeat washing 3 times;

[0096] 1.3 Add 900 μL of activation buffer (50 mM MES, pH 6.0) to resuspend the microspheres, vortex to mix;

[0097] 1.4 Add 50 μL of EDC (40 mg / ml) and 50 μL of NHS (40 mg / ml) solution, vortex to mix, and incubate at 37°C for 30 min in the dark;

[0098] 1.5 After incubation, stand on magnetic separator for 2 min, discard supernatant;

[0099] 1.6 Add 1 mL of activation buffer (50 mM MES, pH 6.0), vortex to mix, stand on magnetic separator for 2 min, discard supernatant, and repeat washing 3 times;

[0100] 1.7 Add 500 μl of activation buffer (50 mM MES, pH 6.0), vortex to mix, and add 100 μg of cytokine capture antibody per mL of microspheres, finally add activation buffer to a total volume of 1 mL, and incubate at 37°C for 2 h in the dark;

[0101] 1.8 After incubation, stand on magnetic separator for 2 min, discard supernatant;

[0102] 1.9 Add 1 mL of blocking solution (containing 5% BSA and 4% glycine), vortex to mix, and incubate at 37°C for 1 h in the dark;

[0103] 1.10 After incubation, stand on magnetic separator for 2 min, discard supernatant;

[0104] 1.11 Add 1 mL of washing buffer (containing 0.1% Tween-20), vortex to mix, stand on magnetic separator for 2 min, discard supernatant, and repeat washing 2 times;

[0105] 1.12 Add 1 mL of protein stabilization solution (containing 2% BSA and 2% trehalose), vortex to mix, and store at 2-8°C in the dark, to obtain 7 kinds of fluorescently encoded polystyrene magnetic microspheres coated with different cytokine capture antibodies.

[0106] Example 2: Biotin labeling of detection antibodies

[0107] 2.1 Biotin was pre-dissolved in DMSO to a final concentration of 10 mg / ml;

[0108] 2.2 The cytokine detection antibody pairs were added to each desalting column respectively, centrifuged at 1000 g for 2 min, and the cytokine antibody in the centrifuge tube was collected;

[0109] 2.3 Biotin / DMSO solution was added according to the molar ratio of 1:20 of the cytokine antibody to biotin, and mixed with the antibody to be labeled;

[0110] 2.4 Incubate overnight at 4°C in the dark;

[0111] 2.5 After incubation, add the same volume of Tris-HCl, and incubate at room temperature for 1 h in the dark;

[0112] 2.6 After incubation, remove the unreacted biotin with a desalting column or ultrafiltration centrifuge tube;

[0113] 2.7 The concentration of the desalted antibody was determined by ultraviolet spectrophotometry, and seven cytokine biotin-labeled detection antibodies were obtained respectively, and stored at -20°C.

[0114] Example 3: Preparation of experimental buffer

[0115] To 1000 mL of pure water, add 3.392 ± 0.034 g of disodium hydrogen phosphate dodecahydrate, 0.083 ± 0.001 g of sodium dihydrogen phosphate dihydrate, 8.766 ± 0.088 g of sodium chloride, and 20.0000 ± 0.2 g of BSA, then add 0.3 mL of ProClin™ 300, and dissolve thoroughly. After sterilization filtration, store at room temperature.

[0116] Example 4: Preparation of sample diluent

[0117] To 500 mL of the experimental buffer prepared in Example 3, add 500 mL of treated serum.

[0118] Preparation of treated serum:

[0119] 4.1 Heat inactivation: heat the serum in a water bath at 56°C for 30 minutes.

[0120] 4.2 Activated carbon adsorption: use 50 mesh activated carbon to adsorb the heat-inactivated serum overnight at 4°C, then perform low-speed centrifugal separation, and aspirate the supernatant to remove the activated carbon.

[0121] 4.3 Ultrafiltration Retention: The above serum was centrifuged and filtered using a 100K ultrafiltration centrifuge tube, and the retentate was collected. The retention ratio was set to 1:3, i.e., the retentate:filtrate ratio was 1:3.

[0122] 4.4 Washing: The retentate was diluted and mixed with 3 times the volume of the retentate with 10 mM PBS (pH 7.2), and then centrifuged and filtered using a 100K ultrafiltration centrifuge tube. The retention ratio was still set to 1:3.

[0123] 4.5 Dilution and Storage: 3 times the volume of the retentate was added with a storage solution, which was a 10 mM PBS (pH 7.2) solution containing 0.02% SDS and 2% PEG-8000, to obtain treated serum, which was stored at -20°C.

[0124] Example 5: Preparation of a Washing Buffer

[0125] To 1000 mL of pure water, 33.92 ± 0.34 g of disodium hydrogen phosphate dodecahydrate, 0.827 ± 0.008 g of sodium dihydrogen phosphate dihydrate, 8.766 ± 0.088 g of sodium chloride, and 0.5 mL of triton-100 were sequentially added, and then thoroughly dissolved and mixed. After sterilization filtration, it was stored at room temperature.

[0126] Example 6: Preparation of a Working Solution

[0127] To 100 mL of the experimental buffer prepared in Example 3, 30 mg of mouse IgG and 20 mg of mouse anti-human IgG were added, and then 0.2 mL of Tween-20, 0.2 mL of polysorbate, 0.1 mL of SP-20, and 0.3 mL of BASF 2520 were added, and then thoroughly dissolved and mixed.

[0128] Example 7: Preparation of a Cytokine Composite Standard

[0129] 7.1 After the freeze-dried powders of the recombinant proteins of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and IFN-γ cytokines were reconstituted with pure water, they were thoroughly mixed.

[0130] 7.2 The mixed solution of the recombinant proteins of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and IFN-γ cytokines was calibrated by NIBSC standards using the kit of the present application;

[0131] 7.3 After calibration, the mixed solution of the recombinant proteins was diluted with a protein stabilizing solution (containing 2% BSA and 2% trehalose) to a final concentration of 10,000 pg / mL, and then stored at -70°C or freeze-dried using a freeze-dryer. The above is a cytokine composite standard.

[0132] Example 8: Establishment of a Kit Standard Curve

[0133] 8.1 Reconstitute or reconstitute the cytokine complex standard with pure water

[0134] 8.2 Use sample diluent to pre-dilute the cytokine complex standard to a concentration of 500 pg / mL for each cytokine, and then perform a 2-fold gradient dilution, so that the concentration range of the standard curve is 500.000 pg / mL, 250.000 pg / mL, 125.000 pg / mL, 62.500 pg / mL, 31.250 pg / mL, 15.625 pg / mL, 7.813 pg / mL, 3.906 pg / mL, 1.953 pg / mL, 0.977 pg / mL, and the sample diluent is used as a blank sample (Blank);

[0135] 8.3 Mix 7 kinds of fluorescently encoded polystyrene magnetic microspheres coupled with different capture antibodies, dilute with experimental buffer by 40 times to obtain mixed capture microspheres.

[0136] 8.4 After mixing 7 kinds of biotin-labeled paired detection antibodies, dilute with experimental buffer by 150 times to obtain mixed detection antibodies.

[0137] 8.5 Dilute the quality control and the sample to be tested by 2 times with sample diluent and reserve.

[0138] 8.6 Add 25 μL of mixed capture microspheres to the reaction well;

[0139] 8.7 Place the reaction well plate on the magnetic separator for 2 min, and discard the supernatant;

[0140] 8.8 Add 25 μL of mixed detection antibodies, 25 μL of standard or diluted quality control or diluted sample to be tested, and 25 μL of working solution to the reaction well in turn, and incubate at 37°C for 120 min with 1000 rpm shaking;

[0141] 8.9 Repeat step 4, add 150 μL of washing buffer to each well, and repeat the washing for a total of 3 times;

[0142] 8.10 Add 50 μL of streptavidin-labeled phycoerythrin (SA-PE) to the reaction well, and incubate at 37°C for 30 min with 1000 rpm shaking;

[0143] 8.11 Repeat step 6, and finally add 100 μL of washing buffer to each well;

[0144] 8.12 After shaking at room temperature for 30 s at 1000 rpm, detect on a multicolor flow cytometer or a liquid suspension chip detection platform, and the detection results are shown in Figure 1, showed that 7 cytokines could be distinguished obviously and did not interfere with each other

[0145] 8.13 Using SoftMax software, 5 parameters were used to fit the standard curve with weight factor of 1 / Y^2. The fitting curve was shown in Figure 2-1, and the data analysis results after fitting the standard curve of 7 cytokines were shown in Table 2-8. Figures 2-8

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[0153] 8.14 The relative deviation of back-calculation of each concentration point of the standard curve was within ±15%, and R 2 ≥0.990. The detection range of each cytokine in the kit for detecting 7 cytokines in human samples was 0.977-500.000 pg / mL.

[0154] Example 9: Precision and accuracy test

[0155] 9.1 The cytokine complex standard was diluted to the theoretical concentration of 160.000 pg / mL and 4.000 pg / mL of each cytokine using the verified healthy human serum, i.e. two concentration levels of quality control were obtained.

[0156] 9.2 The quality control at the above two concentration levels was tested for 5 days, one batch per day, and each level was tested for 3 times, which was completed by 2 experimenters.

[0157] 9.3 The test results showed that the detection precision and accuracy of each cytokine in the kit for detecting 7 cytokines in human samples were within ±10%, which indicated that the kit had excellent precision and accuracy. The test results were shown in Tables 9-15.

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[0165] Example 10: Confirmation of maximum dilution factor of samples

[0166] 10.1 Two different healthy human serum samples were mixed with cytokine complex standard samples respectively, so that the concentration of each cytokine was between 1200 pg / mL and 500 pg / mL, and the healthy human serum content in the mixed solution was more than 95%.

[0167] 10.2 Two kinds of diluents were used to gradient dilute the above samples respectively, group A diluent was sample diluent of example 3, group B diluent was experimental buffer containing 50% serum, and the serum was not treated, and the samples were detected and compared. The sample dilution control table is as follows Table 16.

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[0169] 10.3 The qualified standard is that the concentration of each sample after recovery is compared with the recovery concentration of sample 1, and the recovery rate is between 85% and 115%.

[0170] 10.4 The test results show that the sample diluent used in the kit for detecting 7 cytokines in human samples can dilute the maximum dilution factor of each cytokine to 256 times, and the sample buffer without treated serum cannot achieve this effect, which shows that the kit can expand the detection upper limit to 128000 pg / mL. The comparison of test results is shown in Tables 17-23 below, and the concentration unit is pg / mL.

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[0178] From the above comparative test results, it can be seen that using the sample diluent of group A: the sample diluent of example 3 of the present application, the test results of each sample are stable, and the recovery rate of the calculated concentration after recovery is between 85% and 115% of the calculated concentration of sample 1, which meets the requirements.

[0179] However, using group B: the experimental buffer containing 50% untreated serum to dilute the sample, after more than 64 times dilution, it cannot be guaranteed that the test results of the diluted sample meet the requirements, and the recovery rate is not between 85% and 115%.

[0180] Example 11 Comparison of maximum dilution multiples of different sample diluents

[0181] 11.1 Use one verified healthy human serum to dilute the cytokine complex standard to a theoretical concentration of 900.000 pg / mL for each cytokine, and the healthy human serum content is 95%.

[0182] 11.2 Use different sample diluents a-g to dilute the above samples by gradient, and then detect and compare. The sample dilution control table is shown in Table 24.

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[0184] Sample diluent a: sample diluent of example 3.

[0185] Sample diluent b: different from the sample diluent of example 3 in that during the treatment process, 100K ultrafiltration centrifuge tube is used for centrifugation, and the addition amount of the cut-off liquid is 10%.

[0186] Sample diluent c: different from the sample diluent of example 3 in that 100K ultrafiltration centrifuge tube is used for centrifugation, and the addition amount of the cut-off liquid is 60%.

[0187] Sample diluent d: different from the sample diluent of example 3 in that during the treatment process, 100K ultrafiltration centrifuge tube is used for centrifugation, and the addition amount of the cut-off liquid is 50%.

[0188] Sample diluent e: different from the sample diluent of example 3 in that during the treatment process, 50K ultrafiltration centrifuge tube is used for centrifugation, and the addition amount of the cut-off liquid is 50%.

[0189] Sample diluent f: different from the sample diluent of example 3 in that during the treatment process, 30K ultrafiltration centrifuge tube is used for centrifugation, and the addition amount of the cut-off liquid is 50%.

[0190] Sample diluent g: Different from the sample diluent of Example 3, during the processing, centrifugation was performed using a 3K ultrafiltration centrifuge tube, and the cut-off liquid was taken, with an addition amount of 50%.

[0191] 11.3 The qualified standard is that the concentration of each sample after the calculation is compared with the calculated concentration of sample 1, and the recovery rate is between 85% and 115%.

[0192] 11.4 The test results show that the kit for detecting 7 cytokines in human samples according to the application uses a sample diluent to dilute the sample, and the maximum dilution multiple of each cytokine can reach 256 times, which is the optimal choice. After the sample is diluted 256 times by other sample buffers, the recovery rate does not meet the requirements, the accuracy of the test results is low, and the expected effect cannot be achieved. The comparison of the test results is shown in Table 25 below:

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[0196] Example 12 Sensitivity comparison of different sample diluents

[0197] 12.1 According to the method of Example 7, standard curves are established using different sample diluents a-c.

[0198] Sample diluent a: The sample diluent of Example 3

[0199] Sample diluent b: Different from the sample diluent of Example 3, during the dilution and preservation of the cut-off liquid, SDS and PEG-8000 are not added.

[0200] Sample diluent c: Different from the sample diluent of Example 3, the serum is not treated, and the addition amount is 50%.

[0201] 12.2 The above experiment is repeated 3 times, and the relative deviation of the calculated concentration of each point of the standard curve is within ± 15%, R2≥0.990 is the standard, and the minimum standard curve concentration that meets the requirements is set as the sensitivity.

[0202] 12.3 A verified healthy human serum is used to dilute the cytokine composite standard to the preset sensitivity of each cytokine theoretical concentration, and the healthy human serum content is 95%.

[0203] 12.4 The above sample is detected, repeated 10 times, CV is within 10%, then the preset sensitivity is the sensitivity. If it does not meet the standard, the preset sensitivity is increased, the sample is reconfigured and detected until the requirements are met. To maintain unity, the worst sensitivity of the 7 projects is set as the final sensitivity of the 7 projects.

[0204] 12.5 The test results show that the sensitivity decreases after the ultrafiltration treatment of the serum, and the sensitivity decrease can be avoided by increasing SDS and PEG-8000 in the dilution and preservation step. The test results of each sample diluent are as follows in Table 26.

[0205]

[0206] The above describes the present application and its embodiments, which are not limited, and the drawings only show one of the embodiments of the present application, and the actual structure is not limited thereto. In general, if a person skilled in the art is inspired, without departing from the purpose of the present application, without creative design, similar structure and embodiments of the technical solution can be designed, which should belong to the protection scope of the present application.

Claims

1. A kit for simultaneously detecting IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and IFN-γ in blood samples, characterized in that: The apparatus comprises fluorescently encoded polystyrene magnetic microspheres conjugated with capture antibodies, biotin-labeled paired detection antibodies, streptavidin-labeled phycoerythrin at a final concentration of 0.5–2 μg / mL, a wash buffer containing 0.05%–0.1% Tween-20 or Triton-100, a test buffer containing 0.5%–2% bovine serum albumin, and a sample diluent. The sample diluent includes the test buffer and treated human serum, the amount of which is 20%–50% of the sample diluent volume. The treated human serum is obtained by first heat-inactivating and carbonizing human serum, then centrifuging it using a 100K ultrafiltration centrifuge tube to obtain a retentate, to which 0.01%–0.05% SDS and 0.5%–2% PEG-8000 are added. The test buffer is obtained by adding 3.392 ± 0.034 g of the solution to 1000 mL of pure water. Disodium hydrogen phosphate dodecahydrate, 0.083±0.001g sodium dihydrogen phosphate dihydrate, 8.766±0.088g sodium chloride and 20.0000±0.2g BSA were added to 0.3 mL ProClin™ 300, thoroughly dissolved and mixed, and then filtered through a sterile filter to obtain the final product.

2. The kit according to claim 1, characterized in that: The heat inactivation conditions are a water bath at 55°C to 56°C for 25 to 30 minutes; the carbon adsorption operation involves using 50-60 mesh activated carbon to adsorb serum overnight at 2°C to 8°C, followed by low-speed centrifugation and aspirating the supernatant.

3. The kit as described in claim 1, characterized in that: It also includes a working solution containing 0.5% to 2% bovine serum albumin, 0.2 to 0.4 mg / mL mouse IgG, and 0.1 to 0.2 mg / mL mouse anti-human IgG.

4. The kit as described in claim 3, characterized in that: The working fluid contains a variety of surfactants.

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