CN114910641A - Detection kit for interleukin, detection method and application thereof - Google Patents
Detection kit for interleukin, detection method and application thereof Download PDFInfo
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Abstract
The invention provides a detection kit for interleukin, a detection method and application thereof, belonging to the technical field of biological detection. The kit comprises: a luminescent marker reagent which is an acridinium ester labeled interleukin antibody solution; the magnetic particle reagent is a streptavidin magnetic bead solution coated by a biotin-labeled interleukin antibody; the substrate solution is used for catalyzing the substrate to emit light. The invention provides a quantitative detection kit with wide detection range, high sensitivity and good precision and a detection method thereof by utilizing a magnetic particle chemiluminescence immunoassay method, is suitable for a full-automatic chemiluminescence apparatus, has simple operation, can simultaneously realize the detection of related interleukin contents in serum and plasma, can adapt to various clinical sample types, and has good practical application value.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a detection kit for interleukin, a detection method and application thereof.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Interleukins are a class of cytokines that are produced by and act on a wide variety of cells. At present, at least 38 interleukins are found, which are respectively named as IL-1-IL 38, have complex functions, form a network and are overlapped in a complex way; plays an important role in a series of processes such as maturation, activation, proliferation and immunoregulation of immune cells, and in addition, the immune cells also participate in various physiological and pathological reactions of organisms.
For example, the biological effects of IL-4 are characterized by pleiotropic, complex and double-effect properties, and the effects are different among different diseases. Research shows that the recombinant human IL-4 has multiple functions in the immune response of diseases such as mediated allergic diseases, autoimmune diseases, infectious diseases, tumors and the like, has treatment effect on the tumors, the autoimmune diseases, the infectious diseases and the like, and has a regulating effect on the immune response of vaccines by the IL-4. IL-5 can regulate the growth, activation and survival of eosinophils (leukocytes), and plays a crucial role in the migration of eosinophils from bone marrow to lung and other organs. IL-12 is a proinflammatory factor with multiple immunoregulation functions, and can promote the differentiation of Th0 cells to Th1 cells, induce the differentiated Th1 cells to generate a large amount of interleukins, form positive feedback regulation, enhance Th1 cells, inhibit the immune response of Th2 cells and secrete Th2 cytokines IL-4 and IL-5. IL-12p70 is the activation state of IL-12, and also has the effects of activating NK cells, promoting the differentiation of Th0 cells to Th1 cells, inhibiting the synthesis of IL-4 by Th2 cells, and selectively inhibiting the synthesis of IgE induced by IL-4, but can promote the synthesis of interleukin, TNF-alpha and IL-2, and has important effect in the medium secreted by Th 1. IL-15 is a pleiotropic cytokine with the function of activating T cells, B cells and NK cells and mediating the proliferation and survival of these cells. In addition, IL-15 is able to activate, maintain and expand CD8+ memory T cells, while not activating regulatory T lymphocytes (Tregs, with immunosuppressive functions). IL-17A is a proinflammatory cytokine secreted by a variety of cells. Not only can early diagnosis of inflammation be monitored, but also IL-17A plays a key role in a variety of tumors.
From the above, the expression level of interleukin is closely related to various diseases and body immunity. By detecting the expression level of the interleukin, certain related information can be utilized to obtain early diagnosis of certain diseases earlier. In the existing interleukin detection method, a commonly used ELISA kit is used for quantitative detection or RT-PCR is used for semi-quantitative detection. The ELISA kit is complex in operation, long in time, incapable of being automated and low in sensitivity. And RT-PCR is used to semi-quantify the interleukins in the sample. Because mRNA level is not in absolute linear relation, RT-PCR can not reflect the real level of interleukin of the reagent, and meanwhile RT-PCR is complex in operation, high in professional technical requirement, easy to be influenced by external conditions, expensive in price and not suitable for practical production.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a detection kit for interleukin, a detection method and application thereof. The invention provides a quantitative detection kit with wide detection range, high sensitivity and good precision and a detection method thereof by utilizing a magnetic particle chemiluminescence immunoassay method, is suitable for a full-automatic chemiluminescence apparatus, is simple to operate, can simultaneously realize the detection of related interleukin contents in serum and plasma, can adapt to various clinical sample types, and has good practical application value.
The invention provides a detection kit for interleukin, which comprises a luminescent marker reagent, a magnetic particle reagent, a calibrator, a quality control product and a substrate solution matched with the kit for use;
wherein the luminescent marker reagent comprises acridinium ester labeled interleukin antibody solution
The magnetic particle reagent comprises a biotin-labeled interleukin antibody coated streptavidin magnetic bead solution;
the calibrator and the quality control product comprise interleukin antigen;
the substrate solution is used for catalyzing the substrate to emit light.
The interleukins include, but are not limited to, IL-4, IL-5, IL-12p70, IL-15, and IL-17A.
In a second aspect of the present invention, there is provided a method for preparing the above-mentioned detection kit, the method at least comprising:
preparing a luminescent marker reagent;
preparing a magnetic particle reagent;
also comprises the preparation of a calibrator and a quality control product.
The third aspect of the invention provides the application of the kit in interleukin detection.
In a fourth aspect of the present invention, a method for detecting interleukin is provided, the method comprises detecting interleukin with the above kit.
The beneficial technical effects of one or more technical schemes are as follows:
1. the technical scheme combines the magnetic particles with the chemiluminescence technology, provides a quantitative detection method with wide detection range, high sensitivity and strong specificity, is suitable for a full-automatic chemiluminescence apparatus, is simple to operate, and meets the requirement of clinical large-scale detection.
2. According to the technical scheme, the analysis buffer solution components are added, so that different types of samples are treated, the detection of the interleukin content in serum and plasma can be realized at the same time, and the method can be suitable for various clinical sample types; and by optimizing and detecting the components of the analysis buffer solution, the sensitivity is improved, and meanwhile, the nonspecific binding is reduced.
3. The technical scheme adopts the high affinity and multistage amplification effect of biotin and streptavidin, and endows the analysis method with higher detection sensitivity, specificity and stability.
4. The technical scheme adopts a unique buffer system to improve the stability of the reagent and the repeatability of detection.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, the conventional detection for interleukin usually has the disadvantages of relatively complex whole detection process, long detection time, low sensitivity, poor precision and the like.
In view of this, the invention provides a detection kit for interleukin and a detection method thereof, which are carried out by adopting a double-antibody sandwich method, and the specific working principle is as follows: streptavidin magnetic beads of biotin-labeled interleukin antibodies are combined with interleukin in a calibrator, a quality control product or a sample to form an antigen-antibody complex, and then acridinium ester-labeled interleukin antibodies are added to form a sandwich complex of an antibody-antigen-antibody sandwich structure, and the complex is directly precipitated in an external magnetic field to separate the complex formed by immunoreaction from other unbound substances. Removing supernatant, cleaning precipitate complex, and adding chemiluminescent substrate solution A and B. The acridinium ester is catalytically cracked under alkaline conditions to form an unstable excited state intermediate, and when the excited state intermediate returns to a ground state, photons are emitted to form a luminescence reaction, namely, a luminometer is used for detecting the luminescence intensity of the reaction. The luminous intensity is in direct proportion to the content of the interleukin in the sample, and the concentration of the interleukin in the sample can be calculated by fitting a four-parameter Logistic equation.
In a typical embodiment of the present invention, there is provided a kit for detecting interleukin, the kit comprising: a luminescent marker reagent which is an acridinium ester labeled interleukin antibody solution;
the magnetic particle reagent is a streptavidin magnetic bead solution coated by biotin-labeled interleukin antibody;
the substrate solution is used for catalyzing the substrate to emit light. In one embodiment of the present invention, the substrate solution comprises a luminescent substrate A and a luminescent substrate B, wherein the luminescent substrate A may be H 2 O 2 The luminescent substrate B may be NaOH.
The interleukins include, but are not limited to, IL-4, IL-5, IL-12p70, IL-15, and IL-17A.
In another embodiment of the present invention, the antibody used above may be an interleukin monoclonal antibody; the antibody is two different interleukin monoclonal antibodies.
In another embodiment of the present invention, the kit further comprises a calibrator and a quality control, wherein the calibrator and the quality control comprise an interleukin antigen.
In another embodiment of the present invention, a method for preparing the above kit is provided, the method at least comprising:
preparing a luminescent marker reagent;
preparation of magnetic microparticle reagents.
Wherein the content of the first and second substances,
the preparation method of the luminescent marker reagent specifically comprises the following steps:
diluting an interleukin monoclonal labeled antibody by using a CB buffer solution, uniformly mixing a solution containing acridinium ester and the antibody according to the molar ratio of 10-40:1, and diluting by using a luminescent marker reagent buffer solution for later use after terminating the reaction;
the luminescent marker reagent buffer solution comprises the following specific components: adding 0.1-0.3M NaCl, 0.05-0.3% surfactant, 0.5-5% newborn calf serum, 0.5-5% trehalose and 0.1-2mM MgCl into 0.1-0.5M MOPS buffer system 2 0.5-1 ‰ antiseptic (such as NaN) 3 ) The pH value is 6.5-7.5.
The preparation method of the magnetic particle reagent comprises the following steps:
s1, coupling the interleukin monoclonal antibody and biotin to obtain an interleukin monoclonal antibody-biotin marker;
s2, coupling the interleukin monoclonal antibody-biotin label with streptavidin magnetic beads.
The specific preparation method of step S1 includes:
diluting the dialyzed interleukin monoclonal antibody to 1-5mg/mL by using a PBS buffer solution; dissolving biotin in DMSO to 2-20mg/mL, mixing the two solutions, reacting, and dialyzing.
Wherein, when mixing, the molar ratio of the interleukin monoclonal antibody to the biotin is 1: 10-40; mixing at room temperature, and reacting for 0.5-3 h.
The dialysis is carried out with 0.01-0.5M Tris-HCl (0.1-2% NaCl) buffer.
In the step S2, in the above step,
and (4) mixing the interleukin monoclonal antibody-biotin marker prepared in the step S1 and streptavidin magnetic beads according to the ratio of antibody: magnetic beads ═ (1-10 μ g): mixing 1mg of the reaction mixture according to a reaction ratio, reacting for 0.5-2h at room temperature, and finally diluting to a working concentration of 0.1-0.8mg/mL by using a magnetic particle buffer solution for later use;
wherein, the magnetic particle reagent buffer comprises the following components: 0.1-0.5M PBS buffer system, 0.1-0.3M NaCl, 0.01-0.3% Tween 20, 0.5-5% BSA, 0.5-5% trehalose, and 0.5-1 ‰ preservative (such as NaN 3 )，pH7.0-7.5。
The preparation method of the kit also comprises the preparation of a calibrator and a quality control material;
the preparation of the calibrator and the quality control material comprises the following steps: diluting interleukin antigen with calibrator buffer solution;
wherein the calibrator buffer specifically comprises: 0.05-0.1M PBS buffer system, adding 1% -5% bovine serum, 1-10% saccharide (such as trehalose), and 0.5-1 ‰ antiseptic (such as NaN) 3 )，pH 7.0-7.5。
In another embodiment of the present invention, the kit is used for interleukin detection.
The interleukins include, but are not limited to, IL-4, IL-5, IL-12p70, IL-15, and IL-17A.
In another embodiment of the present invention, a method for detecting interleukin is provided, which comprises detecting interleukin with the above kit.
Wherein the interleukin detection sample can be selected from blood, and further comprises serum and/or plasma.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. All components of the test kit of the present invention can be commercially obtained from biological or chemical reagents companies.
Examples
A kit for detecting the content of interleukin 4 comprises a luminous marker working solution (containing an acridinium ester labeled antibody solution with a certain concentration), a magnetic particle working solution (containing an antibody coated magnetic bead solution with a certain concentration), a calibrator (containing interleukin 4 with a series of concentrations and used for establishing a standard curve), a quality control product (containing interleukin 4 with a certain concentration) and a substrate solution (catalyzing substrate to emit light).
The method comprises the following steps: preparation of luminescent marker buffer:
adding 0.15M NaCl into 0.2M MOPS buffer system, adjusting pH to 6.5-7.5, adding 0.05% surfactant Tween-20, 1% newborn calf serum, 1% trehalose, and 0.1M MgCl 2 1% o preservative NaN 3 Fully and uniformly mixing until the mixture is completely dissolved, adding a proper amount of purified water for quantification, stirring and uniformly mixing for 0.5-2h, and verifying whether the pH value is between 7.0-7.5. Filtering with a filter at 2-8 deg.CAnd (5) storing for later use.
Compared with a common anti-reagent buffer system, the buffer solution is added with a protein protective agent and saccharides, so that the whole reaction system is more stable, and a detection result with good parallelism and accuracy can be obtained, thereby improving the sensitivity, precision and accuracy of the kit.
Step two: preparation of luminescent markers:
the interleukin-4 antibody was coupled to acridinium ester using the following procedure:
1. a certain amount of interleukin-4 antibody is weighed and diluted to 1mg/mL interleukin-4 antibody by using CB buffer solution (pH9.0-9.4). Acridinium ester was dissolved in DMSO beforehand to a solution of 5 mg/mL. Lysine was dissolved in water to 100 mg/mL.
2. And uniformly mixing the interleukin-4 antibody and the acridinium ester according to the molar ratio of 1:10, and uniformly mixing at room temperature for reaction for 1 h.
3. Lysine in an amount 10 times that of the antibody was added to the reaction mixture as a stop solution, and the mixture was mixed at room temperature and reacted for 15 min.
4. Finally, the reaction was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated three times.
5. Recovering dialyzed material into EP tube, and storing at 2-8 deg.C for use.
6. The acridinium ester-labeled antibody is diluted to 0.1. mu.g/mL by using a luminescent marker buffer solution to prepare a luminescent marker working solution for later use.
Step three, preparation of magnetic particle buffer solution
Adding 0.15M NaCl into 0.1M PBS buffer system, adjusting pH to 7.0-7.4, adding 0.1% Tween 20, 1% BSA, 3% trehalose, and 1 ‰ NaN as antiseptic 3 Fully and uniformly mixing until the mixture is completely dissolved, adding a proper amount of purified water for quantification, stirring and uniformly mixing for 0.5-2h, and verifying whether the pH value is between 7.0 and 7.4. Filtering with a filter, and storing at 2-8 deg.C.
Step four, biotin labeled antibody preparation
The coupling of interleukin 4 and biotin was carried out by the following steps:
1. the interleukin 4 monoclonal antibody is placed in a dialysis bag and placed in PBS buffer solution for dialysis, and the dialysate is replaced every 2h, and the operation is repeated for 3 times. The dialyzed IL-4 antibody was diluted to 1mg/mL with PBS.
2. Dissolving biotin with DMSO to 5mg/mL, mixing interleukin-4 antibody and biotin at a molar ratio of 1:30, and reacting at room temperature for 0.5-3 h.
3. Finally, the reaction was dialyzed against 0.05M Tris-HCl (0.15% NaCl) buffer using dialysis bags, and the dialysate was changed every two hours, 3 times.
4. Adding glycerol with the same volume to the solution obtained by dialysis, mixing uniformly, and storing at-20 ℃ for later use.
The coupling of biotin-labeled interleukin-4 and streptavidin magnetic beads is carried out by the following steps:
1. placing a certain amount of streptavidin magnetic beads on a magnetic frame for 1min, removing supernatant, adding PBS buffer solution, vortexing and mixing, placing on the magnetic frame for 1min, removing supernatant, repeating the operation for 3 times, and finally resuspending the magnetic beads with PBS.
2. Coupling 10 mu g of interleukin 4 antibody according to 1mg of magnetic beads, mixing the two, and reacting for 0.5h at room temperature.
3. Placing the reacted antibody coated magnetic beads on a magnetic frame for 1min, removing supernatant, adding PBS buffer, vortexing and mixing uniformly, placing on the magnetic frame for 1min, removing supernatant, repeating the operation for 3 times, finally resuspending the magnetic beads by using magnetic particle buffer, and keeping the magnetic beads at 4 ℃ for later use.
4. And diluting the coupled antibody-coated magnetic beads to 0.1mg/mL working solution for later use by using a magnetic particle buffer solution.
Step five: preparation of calibrator buffer
Adding 1% bovine serum albumin and 1% trehalose into 0.01M PBS buffer system, mixing for 1 hr, adjusting pH to 7.0-7.4, and adding 1 ‰ NaN as antiseptic 3 After mixing well for 1h, verifying pH to be 7.0-7.4, filtering with a filter, and storing at 2-8 deg.C.
Step six: preparation of calibrator
The calibrator was a solution of interleukin 4 purchased from an external source and diluted with calibrator buffer to concentrations of 0pg/mL, 2pg/mL, 5pg/mL, 20pg/mL, 100pg/mL, 500pg/mL and 2000pg/mL, respectively.
Step seven: luminescent substrate A is H 2 O 2 And the luminescent substrate B is NaOH.
Step eight: the use method of the kit for detecting the interleukin 4 content comprises the following steps:
the full-automatic magnetic particle chemiluminescence apparatus takes 50 mu L of antibody-coated magnetic bead working solution to an incubation cup, then 100 mu L of calibrator, quality control material or sample is added, incubation is carried out for 10min at 37 ℃, 100-fold-600 mu L of cleaning solution is used for cleaning for 3 times, then 50 mu L of acridine ester labeled antibody working solution is added, incubation is carried out for 10min at 37 ℃, after incubation, 100-fold-600 mu L of cleaning solution is used for cleaning for 3 times, finally, 100 mu L of each of chemiluminescence substrates A and B is added, the luminescence value is detected, and the concentration of interleukin 4 is calculated back according to the calibrator curve.
Step nine: the quality of the kit for detecting the interleukin 4 content prepared by the technology is detected.
(1.) precision: the interleukin 4 samples are respectively tested in the horizontal levels of 20pg/mL +/-10% and 1000pg/mL +/-10% by using three batches of kit in parallel for 10 times, and the test result is calculated, wherein the precision CV between batches is less than 10%.
Table 1: results of batch precision
As can be seen from the data in the table above, when three batches of kits are used for measuring a sample with the same precision, the precision among the batches is less than 10 percent, which shows that the kits in different batches have small difference, and the measurement result has repeatability
(2.) Linear Range: selecting an interleukin 4 sample with the concentration range of 2000 +/-200 pg/mL, diluting the sample into at least 7 diluted concentrations within the range of 2pg/mL to 2000pg/mL, detecting the sample on a full-automatic chemiluminescence instrument according to the specification requirement of the kit, repeatedly detecting the sample with each concentration for three times, calculating the average value, and calculating the linear correlation coefficient to be more than or equal to 0.99.
Table 2: linear range determination experimental results
From the linearity of the dose-response curve calculated in Table 2, the correlation coefficient is 0.9999 in the range of 2-2000 pg/mL. Therefore, the linear range of the interleukin 4 in the kit is specified to be 2-2000 pg/mL.
(3.) blank limit: and (3) simultaneously detecting 20 times of calibrator diluent or zero background value samples by using three batches of detection kits of different batches, calculating an average value (Mean) and a Standard Deviation (SD), calculating an RLU value by using a fitting equation Mean +2SD, and calculating a concentration value. Functional sensitivity: low concentration samples can be selected for multiple dilution to prepare a series of low concentration samples, and the batch-to-batch CV for each concentration sample is determined and calculated, with functional sensitivity being the analyte content of the sample having a CV closest to 20%.
As can be seen from the following Table 3, the blank limit of the kit is not more than 0.5pg/mL, and the concentration of interleukin 4 can be accurately detected to be not more than 2 pg/mL.
TABLE 3 sensitivity test results
A kit for detecting the content of interleukin-5 (IL-5) comprises a reagent A (containing streptavidin magnetic bead coated antibody solution with a certain concentration), a reagent B (containing acridinium ester labeled antibody solution with a certain concentration), a calibrator (containing IL-5 with a series of concentrations and used for establishing a standard curve), a quality control product (containing IL-5 with a certain concentration) and substrate liquid (catalyzing substrate luminescence).
The method comprises the following steps: preparation of a reagent buffer:
adding 0.15% NaCl into 0.05M MES buffer system, adjusting pH to 5.5-6.5, adding 0.1% surfactant Tween-20, 1% bovine serum albumin, 3% saccharide, and 1 ‰ preservative, mixing thoroughly until completely dissolving, adding appropriate amount of purified water for quantification, stirring and mixing for 0.5-2h, and verifying whether pH is 5.5-6.5. Filtering with a filter, and storing at 2-8 deg.C.
Compared with a common reagent buffer system, the buffer solution is added with a protein protective agent and saccharides, so that the whole reaction system is more stable, and a detection result with good parallelism and accuracy can be obtained, thereby improving the sensitivity, precision and accuracy of the kit.
Step two, preparation of reagent A
The magnetic particles and the coated antibody are coupled by the following steps:
1. the coated antibody was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated 3 times. The dialyzed coated antibody was diluted to 1mg/mL with PBS.
2. Dissolving biotin in DMSO to 5mg/mL, mixing IL-5 coated antibody and biotin at a molar ratio of 1:10, and reacting at room temperature for 0.5 h.
3. Finally, the reaction was dialyzed against 0.05M Tris-HCl (0.15% NaCl) buffer using dialysis bags, and the dialysate was changed every two hours, 3 times.
4. And uniformly mixing the solution obtained by dialysis and the magnetic particles according to the mass ratio of 1:5, and uniformly mixing at room temperature for reaction for 0.5 h.
5. And uniformly mixing the stock solution, diluting the stock solution to 0.2mg/mL by using a diluent which is prepared from 0.05M Tris buffer solution, 0.15% NaCl, 1% BSA and 0.1% Tween-20 and has the pH value of 8.0-8.5, preparing a reagent A, and storing at 2-8 ℃ for later use.
Step three: preparation of reagent B:
the labeled antibody is coupled with acridinium ester by the following steps:
1. 1mg of the labeled antibody was weighed and dissolved in CB buffer (pH9.0-10.0) to prepare a labeled antibody solution of 1 mg/mL. Acridinium ester was dissolved in DMSO beforehand to a solution of 5 mg/mL. Lysine was dissolved in water to 100 mg/mL.
2. And uniformly mixing the labeled antibody and the acridinium ester according to the molar ratio of 1:10, and uniformly mixing at room temperature for reaction for 0.5-4 h.
3. Lysine in an amount 10 times that of the antibody was added to the reaction mixture as a stop solution, and the mixture was mixed at room temperature and reacted for 15 min.
4. Finally, the reaction was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated three times.
5. Recovering dialyzed material into EP tube, and storing at 2-8 deg.C
6. The dialyzed solution was diluted to 0.25. mu.g/mL with a reagent buffer to prepare a reagent B for use.
Step four: preparation of calibrator buffer
Adding 0.15% NaCl and 1% trehalose into 0.05M Tris buffer system, mixing well for 1h, adjusting pH to 8.0-8.5, adding 1% bovine serum albumin and 1 ‰ NaN as antiseptic 3 After mixing for 1h, verifying pH to be 8.0-8.5, filtering with a filter, and storing at 2-8 deg.C.
Step five: preparation of calibrator
The calibrator was a solution of IL-5 purchased ex-situ, diluted with calibrator buffer, at concentrations of 0pg/mL,10pg/mL,50pg/mL,200pg/mL,1000pg/mL and 2000pg/mL, respectively.
Step six: luminescent substrate A is H 2 O 2 The luminescent substrate B is NaOH;
step seven: the use method of the kit for detecting the content of interleukin-5 (IL-5) comprises the following steps:
the full-automatic magnetic particle chemiluminescence apparatus takes 50 mu L of acridinium ester labeled antibody working solution to an incubation cup, then 50 mu L of magnetic particle coated antibody working solution is added, then 50 mu L of calibrator, quality control material or sample is added, incubation is carried out for 10min at 37 ℃, after incubation, 100-600 mu L of cleaning solution is used for cleaning for 3 times, finally 100 mu L of each of chemiluminescence substrates A and B is added, the luminescence value is detected, and the concentration of IL-5 is calculated according to the calibrator curve.
Step eight: quality detection of IL-5 content detection kit prepared by applying technology of the invention
(4.) precision: samples were taken at 20pg/mL + -10% and 1000pg/mL + -10% levels using three lots of kit IL-5, and assayed 10 times in parallel, and the assay results were calculated with a precision CV < 10% between lots.
TABLE 1 results of precision between lots
As can be seen from the data in the table, the precision of the same sample measured by the three batches of kits is less than 10% between batches, which shows that the difference between the kits of different batches is very small, and the measurement result has repeatability
(5.) Linear Range: selecting an IL-5 sample after separation with the concentration range of 2000 +/-200 pg/mL, diluting the sample into at least 7 dilution concentrations within the range of 0.5pg/mL to 1800pg/mL, detecting the sample on a full-automatic chemiluminescence instrument according to the specification requirement of the kit, repeatedly detecting the sample with each concentration for three times, calculating the average value, and calculating the linear correlation coefficient to be more than or equal to 0.99.
TABLE 2 Linear Range determination of Experimental results
From the linearity of the dose-response curve calculated in Table 2, the correlation coefficient is 0.9999 in the range of 0.5-1800 pg/mL. Therefore, the linear range of IL-5 in this kit is defined to be 0.5-1800 pg/mL.
(6.) blank limit: and (3) simultaneously detecting 20 times of calibrator diluent or zero background value samples by using three batches of detection kits of different batches, calculating an average value (Mean) and a Standard Deviation (SD), calculating an RLU value by using a fitting equation Mean +2SD, and calculating a concentration value. Functional sensitivity: low concentration samples can be selected for multiple dilution to prepare a series of low concentration samples, and the batch-to-batch CV of each concentration sample is measured and calculated, wherein the sample with the CV closest to 20% has the analyte content which is the functional sensitivity.
As can be seen from the following Table 3, the blank limit of the kit is not more than 0.05pg/mL, and the concentration of IL-5 can be accurately detected to be not more than 0.5 pg/mL.
TABLE 3 blank limit test results
A kit for detecting the content of interleukin-12 p70(IL-12p70) comprises a reagent A (containing streptavidin magnetic bead coated antibody solution with a certain concentration), a reagent B (containing acridinium ester labeled antibody solution with a certain concentration), a calibrator (containing IL-12p70 with a series of concentrations and used for establishing a standard curve), a quality control product (containing IL-12p70 with a certain concentration) and a substrate solution (catalyzing substrate luminescence).
The method comprises the following steps: preparation of a reagent buffer:
adding 0.15% NaCl into 0.05M MES buffer system, adjusting pH to 5.5-6.5, adding 0.1% surfactant Tween-20, 1% bovine serum albumin, 3% trehalose, and 1 ‰ preservative NaN 3 Fully and uniformly mixing until the mixture is completely dissolved, adding a proper amount of purified water for quantification, stirring and uniformly mixing for 0.5-2h, and verifying whether the pH value is between 5.5 and 6.5. Filtering with a filter, and storing at 2-8 deg.C.
Compared with a common reagent buffer system, the buffer solution is added with a protein protective agent and saccharides, so that the whole reaction system is more stable, and a detection result with good parallelism and accuracy can be obtained, thereby improving the sensitivity, precision and accuracy of the kit.
Step two, preparation of reagent A
The magnetic particles and the coated antibody are coupled by the following steps:
1. the coated antibody was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated 3 times. The dialyzed coated antibody was diluted to 1mg/mL with PBS.
2. Dissolving biotin in DMSO to 5mg/mL, mixing IL-12p70 coated antibody and biotin at a molar ratio of 1:10, and reacting at room temperature for 0.5 h.
3. Finally, the reaction was dialyzed against 0.05M Tris-HCl (0.15% NaCl) buffer using dialysis bags, and the dialysate was changed every two hours, 3 times.
4. And uniformly mixing the solution obtained by dialysis and the magnetic particles according to the mass ratio of 1:5, and uniformly mixing at room temperature for reaction for 0.5 h.
5. And uniformly mixing the stock solution, diluting the stock solution to 0.2mg/mL by using a diluent which is prepared from 0.05M Tris buffer solution, 0.15% NaCl, 1% BSA and 0.1% Tween-20 and has a pH value of 8.0-8.5, preparing a reagent A, and storing at 2-8 ℃ for later use.
Step three: preparation of reagent B:
the labeled antibody is coupled with acridinium ester by the following steps:
1. 1mg of the labeled antibody was weighed and dissolved in CB buffer (pH9.0-10.0) to prepare a labeled antibody solution of 1 mg/mL. Acridinium ester was dissolved in DMSO beforehand to a solution of 5 mg/mL. Lysine was dissolved in water to 100 mg/mL.
2. And uniformly mixing the labeled antibody and the acridinium ester according to the molar ratio of 1:10, and uniformly mixing at room temperature for reaction for 0.5-4 h.
3. Adding lysine as stop solution in an amount which is 10 times that of the antibody into the reactant, and uniformly mixing at room temperature for reaction for 15 min.
4. Finally, the reaction was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated three times.
5. Recovering dialyzed material into EP tube, and storing at 2-8 deg.C
6. The dialyzed solution was diluted to 0.1. mu.g/mL with a reagent buffer to prepare a reagent B for use.
Step four: preparation of calibrator buffer
Tris buffer at 0.05MAdding 0.15% NaCl and 1% trehalose, mixing for 1 hr, adjusting pH to 8.0-8.5, adding 1% bovine serum albumin and 1 ‰ NaN as antiseptic 3 After mixing for 1h, verifying pH to be 8.0-8.5, filtering with a filter, and storing at 2-8 deg.C.
Step five: preparation of calibrator
The calibrator was a commercially available solution of IL-12p70 antigen diluted in calibrator buffer at concentrations of IL-12p70 of 0pg/mL,10pg/mL,50pg/mL,200pg/mL,1000pg/mL and 2000pg/mL, respectively.
Step six: luminescent substrate A is H 2 O 2 The luminescent substrate B is NaOH;
step seven: the use method of the kit for detecting the content of interleukin 12p70(IL-12p70) comprises the following steps:
taking 50 mu L of acridinium ester labeled antibody working solution to an incubation cup by a full-automatic magnetic particle chemiluminescence apparatus, then adding 50 mu L of magnetic particle coated antibody working solution, then adding 50 mu L of calibrator, quality control material or sample, incubating for 10min at 37 ℃, cleaning for 3 times by using 100 mu L of cleaning solution after incubation, finally adding 100 mu L of each of chemiluminescence substrates A and B, detecting a luminescence value, and calculating the concentration of IL-12p70 according to a calibrator curve.
Step eight: quality detection of IL-12p70 content detection kit prepared by applying technology of the invention
(7.) precision: the results of 10 replicates using three batches of kit IL-12p70 at levels of 20pg/mL + -10% and 1000pg/mL + -10%, respectively, were calculated with an inter-batch precision CV of < 10%.
TABLE 1 results of precision between batches
| Precision degree | 3 batches (20pg/mL) | 3 batches of(1000pg/mL) |
| Mean value | 19.69 | 983.31 |
| SD | 0.87 | 22.36 |
| CV | 4.42％ | 2.27％ |
As can be seen from the data in the table, the precision of the same sample measured by the three batches of kits is less than 10% between batches, which shows that the difference between the kits of different batches is very small, and the measurement result has repeatability
(8.) Linear Range: selecting an isolated IL-12p70 sample with the concentration range of 2000 +/-200 pg/mL, diluting the sample into at least 7 diluted concentrations within the range of 0.5pg/mL to 1800pg/mL, detecting the sample on a full-automatic chemiluminescence instrument according to the specification requirement of the kit, repeatedly detecting the sample with each concentration for three times, calculating the average value, and calculating the linear correlation coefficient to be more than or equal to 0.99.
TABLE 2 Linear Range determination of Experimental results
From the linearity of the dose-response curve calculated in Table 2, the correlation coefficient is 0.9997 in the range of 0.5-1800 pg/mL. Therefore, the linear range of the IL-12p70 in the kit is defined as 0.5-1800 pg/mL.
(9.) blank limit: and (3) simultaneously detecting 20 times of calibrator diluent or zero background value samples by using three batches of detection kits of different batches, calculating an average value (Mean) and a Standard Deviation (SD), calculating an RLU value by using a fitting equation Mean +2SD, and calculating a concentration value. Functional sensitivity: low concentration samples can be selected for multiple dilution to prepare a series of low concentration samples, and the batch-to-batch CV for each concentration sample is determined and calculated, with functional sensitivity being the analyte content of the sample having a CV closest to 20%.
As can be seen from the following Table 3, the blank limit of the kit is not more than 0.05pg/mL, and the concentration of IL-12p70 which is not more than 0.5pg/mL can be accurately detected.
TABLE 3 sensitivity test results
A kit for detecting the content of IL-15 comprises a reagent A (containing acridinium ester labeled antibody solution with a certain concentration), a reagent B (containing biotin labeled IL-15 coated antibody solution with a certain concentration), a calibrator (containing IL-15 with a series of concentrations and used for establishing a standard curve), a quality control product (containing IL-15 with a certain concentration) and substrate liquid (catalyzing substrate luminescence).
The method comprises the following steps: preparing an antibody reagent buffer solution:
adding 0.9% NaCl, 0.1% triton, 1% protein protectant bovine serum albumin and 1% trehalose into a 0.01M PBS buffer system, fully mixing for 1h, adjusting the pH value to 7.0-7.4, fully mixing 1 ‰ preservative for 1h, verifying that the pH value is 7.0-7.4, filtering with a filter device, and storing at 2-8 ℃.
Compared with a common anti-reagent buffer system, the buffer solution is added with a protein protective agent and saccharides, so that the whole reaction system is more stable, and a detection result with good parallelism and accuracy can be obtained, thereby improving the sensitivity, precision and accuracy of the kit.
Step two: preparation of reagent A:
the labeled antibody is coupled with acridinium ester by the following steps:
1mg of the labeled antibody was weighed and dissolved in CB buffer (pH9.0-9.5) to prepare a labeled antibody solution of 1 mg/mL. Acridinium ester was dissolved in DMSO beforehand to a solution of 5 mg/mL. Lysine was dissolved in water to 100 mg/mL.
2. And uniformly mixing the labeled antibody and the acridinium ester according to the molar ratio of 1:10, and uniformly mixing at room temperature for reaction for 0.5-4 h.
3. Lysine in an amount 10 times that of the antibody was added to the reaction mixture as a stop solution, and the mixture was mixed at room temperature and reacted for 15 min.
4. Finally, the reaction was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated three times.
5. Recovering dialyzed material into EP tube, and storing at 2-8 deg.C
6. The dialyzed solution was diluted to 0.2. mu.g/mL with a reagent buffer to prepare a reagent B for use.
Step three, preparation of reagent B
Biotin was conjugated to the coating antibody using the following steps:
1. the coated antibody was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated 3 times. The dialyzed coated antibody was diluted to 1mg/mL with PBS.
2. Dissolving biotin in DMSO to 5mg/mL, mixing the coated antibody and biotin at a molar ratio of 1:10, and reacting at room temperature for 0.5 h.
3. Finally, the reaction was dialyzed in PBS buffer using dialysis bag, and the dialysate was changed every two hours for 3 times.
4. And (3) mixing the dialyzed biotin-IL-15 monoclonal coated antibody marker with streptavidin magnetic beads according to the antibody: magnetic beads ═ 8 μ g: mixing 1mg of reaction proportion, reacting for 30min at room temperature, washing for three times by using 0.01M PBS buffer solution, finally diluting the stock solution to 0.2mg/mL by using a diluent which is prepared from 0.05M Tris buffer solution, 0.15% NaCl, 1% BSA and 0.1% Tween-20 and has the pH value of 8.0-8.5, preparing a reagent B, and storing for later use at the temperature of 2-8 ℃.
Step four: preparation of calibrator buffer
Adding 0.9% NaCl and 3% trehalose into 0.05M Tris buffer system, mixing for 1 hr, adjusting pH to 8.0-8.5, and addingAdding 3% bovine serum albumin, 1 ‰ preservative NaN 3 After mixing for 1h, verifying pH to be 8.0-8.5, filtering with a filter, and storing at 2-8 deg.C.
Step five: preparation of calibrator
The calibrator was a commercially available IL-15 antigen, diluted in calibrator buffer, and had IL-15 solutions at concentrations of 0. mu.g/L, 10. mu.g/L, 50. mu.g/L, 200. mu.g/L, 1000. mu.g/L and 2000. mu.g/L, respectively.
Step six: luminescent substrate A is H 2 O 2 The luminescent substrate B is NaOH;
step seven: the use method of the kit for detecting the content of the IL-15 comprises the following steps:
taking 50 mu L of coating antibody, 50 mu L of calibrator, quality control material or sample and adding 50 mu L of labeled antibody by a full-automatic magnetic particle chemiluminescence apparatus, incubating for 10min at 37 ℃, adopting 100-600 mu L of cleaning solution to clean for 3 times, finally adding 100 mu L of each of chemiluminescence substrates A and B, detecting the luminescence value, and calculating the concentration of IL-15 according to the calibrator curve.
Step eight: quality detection of IL-15 content detection kit prepared by applying technology of the invention
(1) Precision: samples are respectively tested at 20ng/mL +/-10% and 1000ng/mL +/-10% levels by using three batches of kit IL-15 in parallel for 10 times, and the test result is calculated, wherein the CV between batches is less than 10%.
Table one: results of batch precision
| Precision degree | 3 batches of 20ng/mL | 3 batches 1000ng/mL |
| Mean value | 19.78 | 1013 |
| SD | 1.23 | 330.16 |
| CV | 6.22％ | 3.26％ |
From the data in the table, the density between the three batches of the kit for testing the same precision sample is less than 10%, which shows that the kit in different batches has small difference and the measurement result has repeatability.
(2) Linear range: selecting a separated sample with the concentration range of 2000 +/-200 ng/mL to be diluted into at least 7 diluted concentrations within the range of 0.5ng/mL to 1800ng/mL, detecting on a full-automatic chemiluminescence instrument according to the instruction requirements of the kit, repeatedly detecting the sample with each concentration for three times, calculating an average value, and calculating a linear correlation coefficient to be more than or equal to 0.99.
From the linearity of the dose-response curve calculated in Table 2, the correlation coefficient is 0.9994 in the range of 0.5-1800 ng/mL. The linear range of the kit is therefore specified to be 0.5-1800 ng/mL.
TABLE 2 Linear Range determination of Experimental results
(3) Blank limit: and (3) simultaneously detecting 20 times of calibrator diluted sample liquid or zero background value samples by using three batches of detection kits of different batches, calculating an average value (Mean) and a Standard Deviation (SD), calculating an RLU value by using a fitting equation Mean +2SD, and calculating a concentration value. Functional sensitivity: low concentration samples can be selected for multiple dilution to prepare a series of low concentration samples, and the batch-to-batch CV for each concentration sample is determined and calculated, with functional sensitivity being the analyte content of the sample having a CV closest to 20%.
As can be seen from the following table 3, the blank limit of the kit is not more than 0.05ng/mL, and the concentration of IL-15 can be accurately detected to be not more than 0.5 ng/mL.
TABLE 3 sensitivity test results
A kit for detecting the content of IL-17A comprises a reagent A (containing acridinium ester labeled antibody solution with a certain concentration), a reagent B (containing biotin labeled IL-17A coated antibody solution with a certain concentration), a calibrator (containing IL-17A with a series of concentrations and used for establishing a standard curve), a quality control product (containing IL-17A with a certain concentration) and substrate liquid (catalyzing substrate luminescence).
The method comprises the following steps: preparation of an anti-reagent buffer solution:
adding 1% bovine serum albumin and 3% trehalose into 0.01M PBS buffer system, mixing for 1 hr, adjusting pH to 7.0-7.4, and adding 1 ‰ NaN as antiseptic 3 After mixing well for 1h, verifying pH to be 7.0-7.4, filtering with a filter, and storing at 2-8 deg.C.
Compared with a common anti-reagent buffer system, the buffer solution is added with a protein protective agent and saccharides, so that the whole reaction system is more stable, and a detection result with good parallelism and accuracy can be obtained, thereby improving the sensitivity, precision and accuracy of the kit.
Step two: preparation of reagent A:
the labeled antibody is coupled with acridinium ester by the following steps:
1. 1mg of the labeled antibody was weighed and dissolved in CB buffer (pH9.0-9.5) to prepare a labeled antibody solution of 1 mg/mL. Acridinium ester was dissolved in DMSO beforehand to a solution of 5 mg/mL. Lysine was dissolved in water to 100 mg/mL.
2. And uniformly mixing the labeled antibody and the acridinium ester according to the molar ratio of 1:10, and uniformly mixing at room temperature for reaction for 0.5 h.
3. Lysine in an amount 10 times that of the antibody was added to the reaction mixture as a stop solution, and the mixture was mixed at room temperature and reacted for 15 min.
4. Finally, the reaction was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated three times.
5. Recovering dialyzed material into EP tube, and storing at 2-8 deg.C
6. The labeled antibody labeled with acridinium ester was diluted to 0.4. mu.g/mL with an anti-reagent buffer solution to prepare a reagent A for use.
Step three, preparation of reagent B
Biotin was conjugated to the coating antibody using the following steps:
1. the coated antibody was placed in a dialysis bag and dialyzed against PBS buffer, and the dialysate was changed every 2 hours, and this operation was repeated 3 times. The dialyzed coated antibody was diluted to 1mg/mL with PBS.
2. And dissolving biotin into DMSO at a concentration of 5mg/mL, mixing the coated antibody and biotin at a molar ratio of 1:10, and reacting at room temperature for 0.5 h.
3. Finally, the reaction was dialyzed against 0.05M Tris-HCl (1% NaCl) buffer using dialysis bags, and the dialysate was changed every two hours, 3 times.
4. And (3) mixing the dialyzed biotin-IL-17 monoclonal coated antibody marker with streptavidin magnetic beads according to the following antibody: magnetic beads 15 μ g: 1mg of the reaction mixture is mixed, the reaction mixture is reacted for 30min at room temperature, then the mixture is washed for three times by 0.01M PBS buffer solution, finally the stock solution is diluted to 0.2mg/mL by 0.01M PBS buffer solution, 0.9 percent NaCl, 1 percent BSA and 3 percent trehalose which are prepared into diluent with pH value of 7.2-7.6, and the reagent B is prepared and stored for standby at 2-8 ℃.
Step four: preparation of calibrator buffer
Adding 1% NaCl and 5% trehalose into 0.05M Tris buffer system, mixing for 1 hr, adjusting pH to 8.0-8.5, adding 1% bovine serum albumin and 1 ‰ NaN as antiseptic 3 After mixing for 1h, verifying pH to be 8.0-8.5, filtering with a filter, and storing at 2-8 deg.C.
Step five: preparation of calibrator
The calibrator was a commercially available IL-17A antigen, diluted in calibrator buffer, and had IL-17A concentrations of 0. mu.g/L, 10. mu.g/L, 50. mu.g/L, 200. mu.g/L, 1000. mu.g/L and 2000. mu.g/L, respectively.
Step six: luminescent substrate A is H 2 O 2 The luminescent substrate B is NaOH;
step seven: the use method of the kit for detecting the content of the IL-17A comprises the following steps:
50 mu L of coating antibody, 50 mu L of calibrator, quality control material or sample and 50 mu L of labeled antibody are taken by a full-automatic magnetic particle chemiluminescence apparatus, incubation is carried out for 10min at 37 ℃, 100-600 mu L of cleaning solution is adopted for cleaning for 3 times, and finally 100 mu L of each of chemiluminescence substrates A and B is added, the luminescence value is detected, and the concentration of IL-17A is calculated back according to the calibrator curve.
Step eight: quality detection of IL-17A content detection kit prepared by applying technology of the invention
(1) Precision: samples are respectively measured at 20ng/mL +/-10% and 1000ng/mL +/-10% levels by using three batches of kit IL-17A in parallel for 10 times, and the measurement result is calculated, wherein the precision CV among batches is less than 10%.
Table 1: results of batch precision
| Precision degree | 3 batches of 20ng/mL | 3 batches 1000ng/mL |
| Mean value | 19.89 | 1067 |
| SD | 1.13 | 315.16 |
| CV | 5.68％ | 2.95％ |
From the data in the table, the precision of the three batches of kits tested on the same precision sample is less than 10% between batches, which indicates that the difference between the kits of different batches is very small, and the measurement result has repeatability.
(2) Linear range: and (3) diluting the separated sample with the concentration range of 2000+ 10% to at least 7 diluted concentrations in the range of 0.5ng/mL to 1800ng/mL, detecting on a full-automatic chemiluminescence instrument according to the instruction requirements of the kit, repeatedly detecting the sample with each concentration for three times, calculating an average value, and calculating a linear correlation coefficient to be more than or equal to 0.99.
Table 2: linear range determination experimental results
From the linearity of the dose-response curve calculated in Table 2, the correlation coefficient is 0.9994 in the range of 0.5-1800 ng/mL. The linear range of the kit is therefore specified to be 0.5-1800 ng/mL.
(3) Blank limit: and (3) simultaneously detecting 20 times of calibrator diluted sample liquid or zero background value samples by using three batches of detection kits of different batches, calculating an average value (Mean) and a Standard Deviation (SD), calculating an RLU value by using a fitting equation Mean +2SD, and calculating a concentration value. Functional sensitivity: low concentration samples can be selected for multiple dilution to prepare a series of low concentration samples, and the batch-to-batch CV for each concentration sample is determined and calculated, with functional sensitivity being the analyte content of the sample having a CV closest to 20%.
As can be seen from the following table 3, the blank limit of the kit is not more than 0.05ng/mL, and the concentration of the IL-17A can be accurately detected to be not more than 0.5 ng/mL.
TABLE 3 sensitivity test results
Finally, it should be noted that, although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. An interleukin detection kit, characterized in that the kit comprises:
a luminescent marker reagent which is an acridinium ester labeled interleukin antibody solution;
the magnetic particle reagent is a streptavidin magnetic bead solution coated by biotin-labeled interleukin antibody;
the substrate solution is used for catalyzing the substrate to emit light;
the substrate solution comprises a luminescent substrate A and a luminescent substrate B, wherein the luminescent substrate A is H 2 O 2 The luminescent substrate B is NaOH;
the interleukins include IL-4, IL-5, IL-12p70, IL-15 and IL-17A.
2. The interleukin detection kit of claim 1, wherein the antibody is an interleukin monoclonal antibody.
3. The interleukin detection kit of claim 1, further comprising a calibrator and a quality control, the calibrator and quality control comprising an interleukin antigen.
4. A method for preparing a kit according to any one of claims 1 to 3, characterized in that it comprises at least:
preparing a luminescent marker reagent;
preparation of magnetic microparticle reagents.
5. The method according to claim 4,
the preparation method of the luminescent marker reagent comprises the following steps:
diluting an interleukin monoclonal labeled antibody by using a CB buffer solution, uniformly mixing a solution containing acridinium ester and the antibody according to the molar ratio of 10-40:1, and diluting by using a luminescent marker reagent buffer solution for later use after terminating the reaction;
the luminescent marker reagent buffer solution comprises the following specific components: adding 0.1-0.3M NaCl, 0.05-0.3% surfactant, 0.5-5% newborn calf serum, 0.5-5% trehalose and 0.1-2mM MgCl into 0.1-0.5M MOPS buffer system 2 0.5-1 ‰ preservative (including NaN3), and pH is 6.5-7.5.
6. The method of claim 4, wherein the magnetic microparticle reagent is prepared by a method comprising:
s1, coupling the interleukin monoclonal antibody and biotin to obtain an interleukin monoclonal antibody-biotin marker;
s2, coupling the interleukin monoclonal antibody-biotin label with streptavidin magnetic beads.
7. The method of claim 6, wherein the step S1 includes the following steps:
diluting the dialyzed interleukin monoclonal antibody to 1-5mg/mL by using a PBS buffer solution; dissolving biotin in DMSO to 2-20mg/mL, mixing the two solutions, reacting, and dialyzing to obtain the final product;
wherein, when mixing, the molar ratio of the interleukin monoclonal antibody to the biotin is 1: 10-40; uniformly mixing at room temperature and reacting for 0.5-3 h;
performing dialysis with 0.01-0.5M Tris-HCl (0.1-2% NaCl) buffer solution;
in the above-mentioned step S2, the step,
and (4) mixing the interleukin monoclonal antibody-biotin marker prepared in the step S1 and streptavidin magnetic beads according to the ratio of antibody: magnetic beads ═ (1-10 μ g): mixing 1mg of the reaction mixture according to a reaction ratio, reacting for 0.5-2h at room temperature, and finally diluting to a working concentration of 0.1-0.8mg/mL by using a magnetic particle buffer solution for later use;
wherein, the magnetic particle reagent buffer comprises the following components: 0.1-0.5M PBS buffer system, 0.1-0.3M NaCl, 0.01-0.3% Tween 20, 0.5-5% BSA, 0.5-5% trehalose, and 0.5-1 ‰ preservative (such as NaN 3 )，pH7.0-7.5。
8. The method of claim 5, wherein the method further comprises formulating a calibrator and a quality control;
the preparation of the calibrator and the quality control material comprises the following steps: diluting interleukin antigen with calibrator buffer solution;
wherein the calibrator buffer specifically comprises: 0.05-0.1M PBS buffer system, adding 1% -5% bovine serum, 1-10% saccharide (including trehalose), 0.5-1 ‰ antiseptic (including NaN) 3 )，pH 7.0-7.5。
9. Use of a kit according to any one of claims 1 to 3 for the detection of an interleukin;
the interleukins include IL-4, IL-5, IL-12p70, IL-15 and IL-17A.
10. A method for detecting interleukin, comprising detecting interleukin using the kit of any one of claims 1 to 3;
wherein the interleukin detection sample is selected from blood, and further comprises serum and/or plasma.
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