A method for improving the sensitivity of human IL-1β target detection and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to a method for improving the sensitivity of Human IL-1β target detection and its application. The present invention provides an ELISA double antibody sandwich method for improving the sensitivity of Human IL-1β target detection. Compared with the traditional one-step ELISA sandwich method in which the capture antibody is bound to an ELISA plate, the method provided by the present invention incubates the antigen with the double antibody in the entire liquid phase, which is beneficial to the full exposure of the antibody binding site and the increase in the collision probability between the antigen and the antibody. Compared with the case of directly fixing the antibody on the ELISA plate, Cy3 is used as a capture label to improve the target antibody binding efficiency and the detection sensitivity of the target. In addition, the present invention carries out a freeze-drying process on the reagent for detecting Human IL-1β. By optimizing the freeze-dried liquid, the stability of the reagent is increased, so that the reagent can be transported at room temperature, saving transportation costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for improving the sensitivity of Human IL-1β target detection and an application thereof. Background Art

[0002] Interleukin-1, also known as lymphocyte-activating factor, is composed of two forms of polypeptide cytokines, IL-1α and IL-1β. It is associated with many cell activities, including proliferation, differentiation, and apoptosis. It is mainly produced by monocytes and macrophages in the blood. Various epithelial cells, endothelial cells, and interstitial cells can also produce IL-1β, but the IL-1β in the blood is mainly produced by monocytes and macrophages.

[0003] IL-1β, produced by activated macrophages, is enzymatically activated by proteinase-1. The mature IL-1 molecule can induce the release of IL-2, promote B cell maturation and proliferation, and induce fibroblast growth factor activity. These effects can stimulate thymocyte proliferation, thereby influencing the body's immune response. Research has shown that IL-1 protein is associated with the initial inflammatory response, acting as a regulatory factor and generally considered an endogenous pyrogen. Bacterial endotoxins and some non-bacterial inflammatory factors can induce IL-1 production, which is then released into local tissues. Elevated IL-1β levels indicate tissue damage or infection, such as sepsis. Research on IL-1β has primarily focused on various physiological and pathological immune and inflammatory processes. Currently, conventional ELISA kits for detecting IL-1β suffer from insufficient sensitivity.

[0004] Therefore, a method to improve the sensitivity of Human IL-1β target detection is urgently needed. Summary of the Invention

[0005] In view of this, the present invention proposes a method for detecting IL-1β, comprising:

[0006] A. Coat the anti-Cy3 antibody onto an ELISA plate to obtain a Cy3 antibody-coated ELISA plate;

[0007] B. Label IL-1β antibody with Cy3 to obtain antibody 1;

[0008] C. Labeling IL-1β antibody with biological enzyme to obtain antibody 2;

[0009] D. mixing antibody 1 and antibody 2 to obtain mixed antibody 3;

[0010] E. co-incubating the sample to be tested with the mixed antibody 3 for the first time to obtain an immune complex 1;

[0011] F. adding the immune complex to the ELISA plate coated with Cy3 antibody in step A for a second incubation to obtain immune complex 2;

[0012] G. Adding luminescent substrate solution to the immune complex 2 and detecting the luminescent intensity to obtain the IL-1β content in the sample to be tested.

[0013] Conventional IL-1β detection kits coat the capture antibody on the solid phase of the ELISA plate. However, when the capture antibody is directly coated on the ELISA plate, there is site blocking for binding to the antigen, and the probability of antigen-antibody collision is low, and it cannot fully bind. Therefore, the antibody's ability to capture the antigen is weak and the sensitivity is low. The present application increases the probability of collision binding and improves the target antibody binding efficiency by allowing the antigen and antibody to react in a pure liquid phase environment, thereby improving the sensitivity of detecting Human IL-1β and increasing the sample detection rate.

[0014] According to a specific embodiment of the present invention, the method further comprises:

[0015] A standard curve of IL-1β concentration-luminescence value is drawn using a standard substance, wherein the standard substance contains IL-1β with different concentration gradients.

[0016] According to a specific embodiment of the present invention, the detection method further comprises:

[0017] (1) using a standard as a test sample, performing steps (A) to (G) to measure the luminescence intensity corresponding to different concentrations, wherein the standard comprises IL-1β at different concentration gradients;

[0018] (2) drawing a standard curve of IL-1β concentration-luminescence value according to the luminescence intensity;

[0019] (3) Obtaining the IL-1β content in the unknown sample according to the IL-1β concentration-luminescence value standard curve.

[0020] According to a specific embodiment of the present invention, the IL-1β antibody is a mouse anti-human IL-1β antibody.

[0021] According to a specific embodiment of the present invention, Antibody 1 or Antibody 2 can be prepared fresh or freeze-dried for storage after preparation.

[0022] According to a specific embodiment of the present invention, in step C, the biological enzyme includes at least one of horseradish peroxidase and alkaline phosphatase.

[0023] According to a specific embodiment of the present invention, in step D, the antibody 1 and the antibody 2 are mixed in an equal mass ratio.

[0024] According to a specific embodiment of the present invention, in step E, the container during the first incubation does not contain Cy3 antibody, the temperature during the first incubation is 35-40° C., and the incubation time is 25-40 min.

[0025] According to a specific embodiment of the present invention, in step F, the temperature of the second incubation is 35-40° C., and the incubation time is 25-40 min.

[0026] According to a specific embodiment of the present invention, in step G, the luminescent substrate solution includes at least one of acridinium ester, terpyridine ruthenium, isoluminol and derivatives thereof.

[0027] The second aspect of the present invention provides a lyoprotectant, wherein the components of the lyoprotectant include at least one of amino acids, polymers, sugars, inorganic salt ions, surfactants, and buffers.

[0028] According to a specific embodiment of the present invention, the amino acids include at least one of BSA, hydrolyzed casein, and fish skin gelatin.

[0029] According to a specific embodiment of the present invention, the polymer includes at least one of sodium hydroxymethyl cellulose, sodium hydroxymethyl starch, and PVP.

[0030] According to a specific embodiment of the present invention, the carbohydrate includes at least one of rhamnose, chitosan oligosaccharide, β-cyclodextrin, dextran, and trehalose.

[0031] According to a specific embodiment of the present invention, the inorganic salt ions include at least one of NaCl, KCl, CaCl2, MgCl2, and ZnCl2.

[0032] According to a specific embodiment of the present invention, the buffer comprises at least one of PBS, Tris, Mes, and Heps.

[0033] According to a specific embodiment of the present invention, the surfactant includes tween 20 and / or Surfactant 10G.

[0034] According to a specific embodiment of the present invention, the lyoprotectant further comprises a preservative.

[0035] According to a specific embodiment of the present invention, the preservative comprises ProClin TM 300.

[0036] The present invention uses freeze-drying reagent technology to increase reagent stability and achieve room temperature transportation and storage.

[0037] The inventors further optimized the freeze-dried protective agent and screened out the protective solution with the best protective effect. The component formulation is as follows:

[0038] PBS 10MM, BSA 0.1%-5%, trehalose 1%-5%, chitosan oligosaccharide 0.2%-5%, sodium hydroxymethylcellulose 1%-5%, PC300 0.05%.

[0039] According to a specific embodiment of the present invention, the mass percentage of the BSA, hydrolyzed casein, chitosan oligosaccharide, rhamnose, β-cyclodextrin or fish skin gelatin in the lyoprotectant is 0.1%-5%;

[0040] The mass percentage of sodium hydroxymethyl cellulose, sodium hydroxymethyl starch, trehalose or glucose in the freeze-drying protective agent is 1%-5%;

[0041] The mass percentage of the inorganic salt ions in the freeze-drying protective agent is 0.9%-1%;

[0042] The mass percentage of the surfactant in the freeze-drying protective agent is 0.1%-0.5%.

[0043] The mass percentage of the preservative in the freeze-drying protective agent is 0.01-0.1%.

[0044] The third aspect of the present invention provides use of the lyoprotectant described in the second aspect in preparing a lyophilized powder, wherein the lyophilized powder is an IL-1β antibody or a compound having 80% homology with an IL-1β antibody, such as a labeled or modified IL-1β antibody.

[0045] The fourth aspect of the present invention provides an IL-1β detection kit, characterized in that it includes an anti-Cy3 antibody, a reagent A, a reagent B, and a luminescent substrate solution.

[0046] in,

[0047] The reagent A is a Cy3-labeled IL-1β antibody.

[0048] Reagent B is an enzyme-labeled IL-1β antibody.

[0049] The luminescent substrate solution is a substrate buffer solution that can catalyze the luminescence of reagent B.

[0050] According to a specific embodiment of the present invention, the dosage form of reagent A and / or reagent B is lyophilized powder.

[0051] According to a specific embodiment of the present invention, the components of the lyophilization protectant of the lyophilized powder include at least one of amino acids, polymers, sugars, inorganic salt ions, surfactants, and buffers.

[0052] According to a specific embodiment of the present invention, the amino acids include at least one of BSA, hydrolyzed casein, and fish skin gelatin.

[0053] According to a specific embodiment of the present invention, the polymer includes at least one of sodium hydroxymethyl cellulose, sodium hydroxymethyl starch, and PVP.

[0054] According to a specific embodiment of the present invention, the carbohydrate includes at least one of rhamnose, chitosan oligosaccharide, β-cyclodextrin, dextran, and trehalose.

[0055] According to a specific embodiment of the present invention, the inorganic salt ions include at least one of NaCl, KCl, CaCl2, MgCl2, and ZnCl2.

[0056] According to a specific embodiment of the present invention, the buffer comprises at least one of PBS, Tris, Mes, and Heps.

[0057] According to a specific embodiment of the present invention, the surfactant includes tween 20 and / or Surfactant 10G.

[0058] The present invention uses freeze-drying reagent technology to increase reagent stability and achieve room temperature transportation and storage.

[0059] According to a specific embodiment of the present invention, the mass percentage of the BSA, hydrolyzed casein, chitosan oligosaccharide, rhamnose, β-cyclodextrin or fish skin gelatin in the lyoprotectant is 0.1%-5%;

[0060] The mass percentage of sodium hydroxymethyl cellulose, sodium hydroxymethyl starch, trehalose or glucose in the freeze-drying protective agent is 1%-5%;

[0061] The mass percentage of the inorganic salt ions in the freeze-drying protective agent is 0.9%-1%;

[0062] The mass percentage of the surfactant in the freeze-drying protective agent is 0.1%-0.5%.

[0063] The inventors further optimized the freeze-dried protective agent and screened out the protective solution with the best protective effect. The component formulation is as follows:

[0064] PBS 10MM, BSA 0.1%-5%, trehalose 1%-5%, chitosan oligosaccharide 0.2%-5%, sodium hydroxymethylcellulose 1%-5%, PC300 0.05%.

[0065] The fifth aspect of the present invention provides the lyoprotectant described in the third aspect, and the fourth aspect provides the use of the aforementioned kit in detecting IL-1β.

[0066] The method for detecting IL-1β provided by the present invention, and the kit designed for the detection method of the present invention, have the following beneficial effects compared with the prior art:

[0067] 1. In the present invention, the antigen and antibody react in a pure liquid phase environment, which increases the probability of collision binding and improves the sensitivity of IL-1β detection;

[0068] 2. The most suitable freeze-dried protective agent has been screened out. The use of freeze-dried reagents can increase the stability of the reagents and achieve transportation and storage at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0070] Figure 1 is the chemical formula of Cy3 after NHS ester activation in the embodiment of the present invention;

[0071] Figure 2 This is the reaction process of Cy3-labeled IL-1β antibody activated by NHS ester in the examples of the present invention. DETAILED DESCRIPTION

[0072] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0073] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0074] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0075] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0076] In this article, the term "N,N-dimethylformamide" refers to an organic compound with the chemical formula C3H7NO, a colorless, transparent liquid. It is both a widely used chemical raw material and an excellent solvent. It is miscible with water and most organic solvents and has excellent solubility for a wide range of organic and inorganic compounds.

[0077] In this article, the term "labeled antibody" refers to an antibody that has been labeled with an enzyme, ferritin, or colloidal gold. It is a method for preparing samples for immunoelectron microscopy and is used to observe antigen-antibody immune complexes. Immunolabeling technology involves labeling specific antigens or antibody molecules with substances that are both easy to measure and highly sensitive. The enhanced amplification effect of these markers is used to display the properties and content of the antigen or antibody in the reaction system. Antibody labeling is mainly used for the localization analysis of antigens. In some cases, it can also be used to quantitatively detect antigens in samples mixed with a large number of other molecules. Because antibodies have a high affinity for their corresponding antigens, antibodies with easily recognizable markers can locate and analyze antigens and are an ideal, rapid, and inexpensive quantitative measurement method.

[0078] According to a specific embodiment of the present invention, the present invention provides an ELISA kit for improving the sensitivity of human IL-1β target detection and a preparation method thereof. The reagents of the kit sequentially include Cy3 antibody coating of the ELISA plate, ELISA plate blocking, HRP-labeled antibody, and Cy3 antibody and HRP antibody are packaged and freeze-dried in EP tubes using intermediate concentrations.

[0079] The present invention provides a method for pre-mixing and lyophilizing a capture antibody and a detection antibody. In some embodiments, the capture antibody and the detection antibody are pre-mixed and not lyophilized before reacting with a standard / sample.

[0080] In the present invention, all samples used are human serum samples.

[0081] In the present invention, PC300 is the same as ProClin TM 300, is one of the common preservatives.

[0082] In the present invention, the purchase sources and product numbers of the test freeze-dried protective solution original reagents are shown in Table 1.

[0083] Table 1

[0084] Reagents Purchase Source Item No. PBS Phosphate Buffered Saline Tablets (pH 7.4) Wokai 72013560 BSA sigma V900933 D(+)-Trehalose, dihydrate Shanghai Test 63011536 Chitosan Shanghai Test 69047438 Sodium carboxymethyl cellulose Shanghai Test 30036328 PC300 sigma 48914-U Fish skin gelatin sigma G7041 β-cyclodextrin Shanghai Test 69009360 Rhamnose Wokai XW01615535706 tween20 Shanghai Test 30189328 hydrolyzed casein sigma C7290-250G TrisBase sigma T6687-100G NaCl Shanghai Test 10019308 Glucan Shanghai Test 68006736

[0085] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention provides drawings, which are part of the disclosure of the present invention. They are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. By referring to these contents, ordinary technicians in this field should be able to understand other possible implementation methods and the advantages of the present invention.

[0086] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0087] Example 1

[0088] This embodiment provides a method for detecting Human IL-1β with improved sensitivity. The experimental steps of the method are as follows.

[0089] Step 1: Coat the ELISA plate with Anti-Cy3 antibody (abcam ab52060) at a concentration of 1 μg / mL in 0.05 mol / L carbonate buffer. Block the plate with blocking buffer for 1 hour, shake, and dry.

[0090] Step 2: Use Cy3 NHS ester (aladdin C651286) to label mouse anti-human IL-1beta Antibody (R&D MAB201). Dissolve 1 mg of Cy3 NHS ester in 1 ml of DMF (N,N-dimethylformamide) (The chemical formula after NHS ester activation of Cy3 is shown in Figure 1 , NHS ester labeled antibody reaction process see Figure 2 ), add 10 μl to 50 μg of mouse anti-human IL-1 beta antibody, and rehydrate with PBS to a concentration of 1.5 mg / ml. Incubate at 4°C in the dark overnight. The next day, dialyze four times with TBS and rehydrate with TBS to 1 mg / ml. Add glycerol in a 1:1 ratio and store at -20°C.

[0091] Step 3: Use HRP labeling (HRP conjugation kit -Lightning- (ab102890) Mouse anti-human IL-1 beta Antibody (R&D MAB601); adjust the antibody concentration to 0.5 mg / ml according to the manufacturer's instructions.

[0092] Step 4: Mix the labeled 0.5 mg / ml Cy3-IL-1beta Antibody and HRP-IL-1beta Antibody at a volume ratio of 1:1 and dilute 3000-fold in diluent (20 mm PBST, 0.5% BSA, 0.1% PC300). Dispense 50 μl into EP tubes.

[0093] Step 5: Prepare serial dilutions (20 mm PBST, 0.5% BSA, 0.1% PC300) of IL-1β standard (R&D, Cat. No. 201-LB-025 / CF). Add 50 μl of the standard / sample to the EP tube containing the antibody in Step 4. Vortex and centrifuge, then incubate in a 37°C incubator for 30 min.

[0094] Step 6: Take out the liquid in the EP tube, add it to the ELISA plate prepared in step 1, and incubate it in a 37°C incubator for 30 minutes;

[0095] Step 7: Remove the incubated ELISA plate and wash it five times with 300 μl of TBS washing solution (Elabscience, Cat. No. E-IR-R116, diluted to 1×), each time for 1 min, and pat dry.

[0096] Step 8: Add substrate solution (Elabscience, Cat. No. E-IR-R201) to the plate and incubate at 37°C for 15 min. Remove the plate and add 2M color stop solution (Elabscience, Cat. No. E-ELIR-006). Measure the light at 450 nm.

[0097] Example 2

[0098] This example provides a method for detecting Human IL-1β with improved sensitivity. The experimental steps of the method are as follows:

[0099] Steps 1 to 3 are the same as those in Example 1;

[0100] Step 4: Serially dilute the standard sample and add 50 μl of the standard sample / sample to the ELISA plate prepared in step 1.

[0101] Step 5: Dilute the labeled Cy3-IL-1betaAntibody and HRP-IL-1betaAntibody 1500 times with diluent, take 25ul of each and add them to the ELISA plate in step 4;

[0102] Step 6: Place in a 37°C incubator and incubate for 60 minutes.

[0103] Step 7: Take out the incubated ELISA plate, wash it 5 times with TBS solution, and pat dry;

[0104] Step 8: Add substrate solution to the plate, incubate at 37°C for 15 minutes, remove from the plate, add stop solution, and measure light.

[0105] Comparative Example 1

[0106] This experiment provides a commonly used Human IL-1β ELISA sandwich detection method

[0107] Step 1: Coat the ELISA plate with mouse anti-human IL-1beta Antibody (R&DMAB201) at a concentration of 1 μg / ml, then block the plate with blocking solution for 1 hour, shake the plate, and dry it for later use;

[0108] Step 2: Use HRP labeling (HRP conjugation kit -Lightning- (ab102890)) Mouse anti-human IL-1betaAntibody (R&D MAB601);

[0109] Step 3: Serially dilute the standard sample and add 50 μl of the standard sample / sample to the ELISA plate prepared in step 1.

[0110] Step 4: Dilute the labeled HRP-IL-1betaAntibody 3000 times with diluent, take 50ul and add it to the ELISA plate in step 3, and incubate it in a 37℃ incubator for 60min;

[0111] Step 5: Take out the incubated ELISA plate, wash it 5 times with TBS solution, and pat dry;

[0112] Step 6: Add substrate solution to the plate and incubate at 37°C for 15 minutes. Remove the plate, add stop solution, and measure the light.

[0113] The dilution gradients of the standards, the number of samples measured, and the OD values ​​of the standards and samples measured in Examples 1-3 are shown in Table 2.

[0114] Table 2

[0115]

[0116]

[0117] As can be seen from Table 2, using the method of Example 1 of the present application, at the same OD value, the concentration of the standard that can be distinguished is lower, and the detection is more sensitive. According to the sensitivity, the order from high to low is: Example 1, Example 2, and Example 3. The reason why the result of Example 1 is better than that of Example 2 is that after the sample and two antibodies are added to the ELISA plate in Example 2, the Cy3-IL-1beta Antibody antibody will bind to the anti-Cy3 antibody coated on the bottom of the plate and be fixed in the bottom area. Compared with Example 1, the probability of collision with the antigen and the other pair of antibodies is reduced, and there is a large steric effect, so the ability to capture the antigen is weak and the sensitivity is low. This shows that the method provided in Example 1 of the present application can improve the sensitivity of Human IL-1β and improve the sample detection rate.

[0118] Example 4

[0119] This example provides a method for detecting Human IL-1β with improved sensitivity, and the reagent is freeze-dried. The experimental steps of the method are as follows:

[0120] Step 1: Coat the ELISA plate with Anti-Cy3 antibody (abcam ab52060) at a concentration of 1 μg / mL in 0.05 mol / L carbonate buffer. Block the plate with blocking buffer for 1 hour, shake, and dry.

[0121] Step 2: Mouse anti-human IL-1beta Antibody (R&D MAB201) was labeled with Cy3 NHS ester (aladdin C651286);

[0122] Step 3: Use HRP to label mouse anti-human IL-1betaAntibody (R&D MAB601);

[0123] Step 4: Dilute the labeled Cy3-IL-1betaAntibody and HRP-IL-1betaAntibody 5000 times in lyophilization protection solution. Dispense 100ul each into EP tubes;

[0124] Step 5: Freeze-dry the EP tube containing the mixed antibody (Xinzhi freeze dryer temperature: -45°C, time: 18 hours, pressure <15Pa);

[0125] Step 6: Serially dilute the standard. Take 100ul of the standard / sample and add it to the EP tube containing the lyophilized antibody in step 5. Vortex and centrifuge, then incubate in a 37℃ incubator for 30min.

[0126] Step 7: Take out the liquid in the EP tube, add it to the ELISA plate prepared in step 1, and incubate it in a 37°C incubator for 30 minutes;

[0127] Step 8: Take out the incubated ELISA plate, wash it 5 times with TBS solution, and pat dry;

[0128] Step 9: Add substrate solution to the plate and incubate at 37°C for 15 minutes. Remove the plate, add stop solution, and measure the light.

[0129] The freeze-dried protective solution used in Example 4 contains the following components: PBS 10MM, BSA 0.1%-5%, trehalose 1%-5%, chitosan oligosaccharide 0.2%-5%, sodium hydroxymethylcellulose 1%-5%, fish skin gelatin 0.2%-1%, and PC300 0.05%.

[0130] The inventors compared the differences between the embodiment 4 (lyophilized reagent) and the reagent in Example 1 (non-lyophilized reagent) in terms of precision, dilution recovery rate, and thermal stability.

[0131] The experimental results are as follows:

[0132] The precision test results are shown in Table 3, which shows that the values ​​of the reagent tested each time are not much different and the reagent is relatively stable:

[0133] Table 3

[0134]

[0135] The dilution recovery test results are shown in Table 4, indicating that the reagent has high accuracy:

[0136] Table 4

[0137]

[0138] The results of the thermal stability test are shown in Table 5, indicating that the reagents can be transported at room temperature:

[0139] Table 5

[0140]

[0141] The results showed that there was little difference between freeze-dried reagents and non-freeze-dried reagents, and the use of freeze-dried reagents could increase the stability of the reagents.

[0142] Example 5

[0143] Lyophilization protection solution 1: PBS 10MM, BSA 0.1%-5%, trehalose 1%-5%, chitosan oligosaccharide 0.2%-5%, sodium hydroxymethylcellulose 1%-5%, PC3000.05%;

[0144] Lyophilization protection solution 2: PBS 10MM, fish skin gelatin 0.1%-5%, β-cyclodextrin 0.1%-0.5%, rhamnose 0.5%-5%, tween 20 0.1%-0.5%, PC300 0.05%;

[0145] Freeze-dried protective solution 3: PBS 10MM, fish skin gelatin 0.1%-5%, hydrolyzed casein 0.1%-5%, chitosan oligosaccharide 0.1%-5%, PC3000.05%;

[0146] Lyophilization protection solution 4: Tris-HCl 50mM, NaCl 0.9%, BSA 0.1%-5%, trehalose 1%-5%, chitosan oligosaccharide 0.2%-5%, β-cyclodextrin 0.1%-1%, PC3000.05%;

[0147] Lyophilization protection solution 5: Tris-HCl 50mM, NaCl 0.9%, fish skin gelatin 0.1%-5%, dextran 1%-5%, sodium hydroxymethylcellulose 1%-5%, PC3000.05%;

[0148] Freeze-dried protective solution 6: PBS 10MM, BSA 0.1%-5%, trehalose 1%-5%, chitosan oligosaccharide 0.2%-5%, sodium hydroxymethylcellulose 1%-5%, fish skin gelatin 0.2%-1%, PC300 0.05%.

[0149] The freeze-dried protective solutions 1-6 were placed at 37°C for 30 days, 60 days and at -20°C to compare the heat retention rate data. The results are shown in Table 6:

[0150] Table 6

[0151]

[0152]

[0153] The freeze-dried protective solutions 1-6 were placed at room temperature for 90 days, 180 days and at -20°C for comparison. The heat retention rate data are shown in Table 7:

[0154] Table 7

[0155]

[0156] The data showed that after being placed at room temperature for 180 days, the heat retention rate was above 85%. The freeze-dried protective solutions 1-6 provided by the present invention can all be transported and stored at room temperature, and the freeze-dried protective solution 1 is the most stable.

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting IL-1β for non-disease diagnosis purposes, characterized in that: include: A. Coat the anti-Cy3 antibody onto an ELISA plate to obtain a Cy3 antibody-coated ELISA plate; B. Label IL-1β antibody with Cy3 to obtain antibody 1; C. Labeling IL-1β antibody with biological enzyme to obtain antibody 2; D. mixing antibody 1 and antibody 2 in an equal mass ratio to obtain mixed antibody 3; E. co-incubating the sample to be tested with the mixed antibody 3 for the first time to obtain an immune complex 1, wherein the container during the first incubation does not contain Cy3 antibody; F. adding the immune complex 1 to the ELISA plate coated with Cy3 antibody in step A for a second incubation to obtain immune complex 2; G. Adding luminescent substrate solution to the immune complex 2 and detecting the luminescent intensity to obtain the IL-1β content in the sample to be tested.

2. The method according to claim 1, characterized in that The method for detecting IL-1β further comprises: (1) Using a standard as a test sample, perform steps (A) to (G) to measure the luminescence intensity corresponding to different concentrations, wherein the standard comprises IL-1β at different concentration gradients; (2) drawing a standard curve of IL-1β concentration-luminescence value according to the luminescence intensity; (3) Obtaining the IL-1β content in the unknown sample according to the IL-1β concentration-luminescence value standard curve.

3. The method according to claim 1, characterized in that The IL-1β antibody is a mouse anti-human IL-1β antibody; In step C, the biological enzyme is horseradish peroxidase or alkaline phosphatase; In step E, the temperature of the first incubation is 35-40° C., and the incubation time is 25-40 minutes; In step F, the temperature of the second incubation is 35-40° C., and the incubation time is 25-40 minutes; In step G, the luminescent substrate liquid is selected from one of acridinium ester, terpyridine ruthenium, isoluminol and their derivatives.

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