Anti-human IL-3 composition as well as application, detection kit and detection method thereof

Abstract

The invention provides an anti-human IL-3 composition as well as application, a detection kit and a detection method thereof, and relates to the technical field of monoclonal antibodies. The invention provides an anti-human IL-3 composition which comprises a first monoclonal antibody and a second monoclonal antibody, the first monoclonal antibody and the second monoclonal antibody are specifically combined with different epitopes of human IL-3. By adopting the kit containing the composition for resisting the human interleukin-3, the detection accuracy of the human interleukin-3 is improved, and the experimental operation is simplified.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to an anti-human IL-3 composition and application thereof, a detection kit and a detection method. Background Art

[0002] Interleukin-3 (IL-3), also known as multi-colony stimulating factor (multi-CSF), is an important member of the interleukin family. It is mainly produced by activated T cells, macrophages and bone marrow stromal stem cells. It plays an important role in the body's hematopoietic regulation and immune regulation by binding to its receptor IL-3R. IL-3 can not only induce the proliferation and maturation of multipotent hematopoietic progenitor cells, bone marrow cells, eosinophils, basophils, erythroid cells and megakaryocytes, but also activate the cytotoxic activity, bactericidal activity and tumoricidal activity of macrophages. In addition, IL-3 can also mobilize the response of peripheral blood stem cells and other cells to late activated cytokines such as G-CSF, thrombopoietin and erythropoietin.

[0003] As a very important hematopoietic stimulating factor, IL-3 shows good biological effects in stimulating hematopoiesis, anti-tumor, immune regulation, vaccines, gene therapy, etc., and shows good application prospects in clinical application. Therefore, the development of a highly sensitive IL-3 detection method is of great clinical significance.

[0004] Enzyme-linked immunosorbent assay (ELISA) is an immunoassay method that combines the high specificity of antigen-antibody reaction with the efficient catalytic action of enzymes. It is characterized by being easy to operate, rapid, effective, and highly specific. ELISA is widely used, such as in the clinical diagnosis and monitoring of diseases, the determination of harmful residues in food, and in combination with genetic engineering.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0006] In view of this, the present invention proposes an anti-human IL-3 composition and its application, detection kit and detection method. The composition provides a new product option for the detection of human interleukin-3 through a pair of monoclonal antibodies that can specifically bind to human IL-3.

[0007] The technical solution of the present invention is implemented as follows: the present invention provides an anti-human interleukin-3 composition, comprising a first monoclonal antibody and a second monoclonal antibody; the first monoclonal antibody and the second monoclonal antibody specifically bind to different antigenic epitopes of human interleukin-3;

[0008] The heavy chain complementary determining region of the first monoclonal antibody includes CDR1-H1, CDR1-H2 and CDR1-H3; the CDR1-H1 has an amino acid sequence with at least 90% homology to SEQ ID NO.1; the CDR1-H2 has an amino acid sequence with at least 90% homology to SEQ ID NO.2; the CDR1-H3 has an amino acid sequence with at least 90% homology to SEQ ID NO.3;

[0009] The light chain complementary determining region of the first monoclonal antibody includes CDR1-L1, CDR1-L2 and CDR1-L3; the CDR1-L1 has an amino acid sequence with at least 90% homology to SEQ ID NO.7; the CDR1-L2 is an amino acid sequence with at least 90% homology to SEQ ID NO.8; the CDR1-L3 is an amino acid sequence with at least 90% homology to SEQ ID NO.9;

[0010] The heavy chain complementary determining regions of the second monoclonal antibody include CDR2-H1, CDR2-H2 and CDR2-H3; the CDR2-H1 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.4; the CDR2-H2 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.5; the CDR2-H3 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.6;

[0011] The light chain complementary determining region of the second monoclonal antibody includes CDR2-L1, CDR2-L2 and CDR2-L3; the CDR2-L1 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.10; the CDR2-L2 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.11; and the CDR2-L3 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.12.

[0012] Based on the above technical solution, preferably, the V H The amino acid sequence of the first monoclonal antibody is as shown in SEQ ID NO.13; L The chain is shown in the amino acid sequence SEQ ID NO.15.

[0013] On the basis of the above technical solution, preferably, the V H The amino acid sequence of the second monoclonal antibody is as shown in SEQ ID NO.14; L The chain is shown in the amino acid sequence SEQ ID NO.16.

[0014] A summary of the sequence listing is shown in the following table:

[0015]

[0016] The present invention also provides the use of the anti-human interleukin-3 composition in preparing a product for detecting human interleukin-3.

[0017] The present invention also provides a human interleukin-3 detection kit, comprising the above-mentioned anti-human interleukin-3 composition;

[0018] Among them, the first monoclonal antibody is a capture antibody, and the second monoclonal antibody is a detection antibody.

[0019] On the basis of the above technical solution, preferably, the kit is an enzyme-linked immunosorbent assay kit.

[0020] The present invention also provides a method for detecting human interleukin-3, comprising:

[0021] Obtaining a gradient standard solution of a human interleukin-3 standard and a sample to be tested;

[0022] Adding the gradient standard solution and the sample to be tested to the ELISA plate coated with the first monoclonal antibody respectively;

[0023] Add enzyme-labeled second monoclonal antibody to each well of the ELISA plate, incubate at room temperature, wash the plate, add substrate for color development, and measure the OD value of each well;

[0024] According to the OD value of each well corresponding to the gradient standard solution, a standard curve expressing the relationship between the concentration and OD value of the gradient standard solution is obtained;

[0025] The concentration of interleukin-3 in the sample to be tested is obtained according to the standard curve and the OD value of the corresponding well of the sample to be tested.

[0026] On the basis of the above technical solution, preferably, the enzyme used for the enzyme-labeled second monoclonal antibody is horseradish peroxidase;

[0027] The substrate is 3,3',5,5'-tetramethylbenzidine.

[0028] The solution provided by the present invention has the following beneficial effects compared with the prior art:

[0029] 1. The present disclosure provides an optional antibody combination for the detection of IL-3 by providing a pair of specific monoclonal antibodies that can simultaneously bind to different antigenic epitopes on human interleukin-3, and the monoclonal antibodies have strong specificity and high binding affinity to IL-3.

[0030] 2. The IL-3 detection kit prepared by using the first monoclonal antibody as the capture antibody and the second monoclonal antibody as the detection antibody has high sensitivity and specificity for detecting IL-3 in the sample, and has small inter-plate variability, providing a better choice for clinical detection of IL-3.

[0031] 3. The ELISA detection method provided by the present disclosure adopts a double antibody sandwich method, using a first monoclonal antibody with strong specificity and a second monoclonal antibody, which improves the accuracy and precision of the detection method. At the same time, the sample and the detection antibody can be added to the detection plate at the same time, which reduces the detection time, simplifies the experimental operation steps, and can quickly provide reference test results for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0033] Figure 1 In one embodiment provided by the present invention, a schematic diagram of the experimental results of cross-reactions between the first monoclonal antibody (5G9) and the second monoclonal antibody (4C6) and various cytokines;

[0034] Figure 2 In one embodiment provided by the present invention, a standard curve diagram of IL-3 gradient standard solution and OD value. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present invention.If the specific conditions are not specified in the embodiment, they are carried out according to the conditions recommended by the normal conditions or the manufacturer.If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.The embodiments of the present disclosure are only exemplary and do not constitute any limitation to the scope of the present disclosure.It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure can be modified or replaced without departing from the spirit and scope of the present disclosure, but these modifications and replacements all fall within the protection scope of the present disclosure.

[0036] Example 1

[0037] (1) Animal immunization

[0038] Human IL-3 recombinant protein (purchased from Shanghai Jinan Biotechnology Co., Ltd., batch number: 0332540JE18) was dissolved in PBS buffer to obtain an IL-3 protein solution.

[0039] According to the volume ratio of 1:1, the prepared IL-3 protein solution and Freund's complete adjuvant (CFA) were mixed and fully emulsified to obtain the first antigen solution. Four female BALB / c mice were immunized with the first antigen solution, and 300uL of the prepared first antigen injection solution (containing 100ug antigen) was drawn and injected intraperitoneally.

[0040] After an interval of 14 days, the IL-3 protein solution was mixed with Freund's incomplete adjuvant (IFA) at a volume ratio of 1:1 to obtain a second antigen solution. The second antigen solution was intraperitoneally injected into the above 4 female BALB / c mice for the first booster immunization, 100ug antigen per mouse.

[0041] After an interval of 14 days after the first booster immunization, a second booster immunization was performed using a third antigen solution prepared with IL-3 protein solution without adjuvant, with 100 ug of antigen per animal.

[0042] After the second booster immunization, 7 days later, the third booster immunization was carried out by intraperitoneal injection using the third antigen solution prepared with IL-3 protein solution without adjuvant, 100 ug antigen per animal.

[0043] After the third booster immunization, the fourth booster immunization was carried out 7 days later using the third antigen solution prepared with IL-3 protein solution without adjuvant, with 100 ug antigen per animal through intraperitoneal injection.

[0044] (2) Hybridoma cell line screening

[0045] On the third day after the fourth booster immunization, spleens of four female BALB / c mice were collected, and cell fusion was performed according to conventional hybridoma fusion technology. The fused hybridoma cells were screened out using HAT selective medium, and the cell supernatant of the screened hybridoma cells was detected by indirect ELISA method, and the strongly positive cells were selected for cloning. Using the limiting dilution method, after 3 to 5 clonings, the strongly positive cell strains I, II, III, IV and V that stably secreted specific monoclonal antibodies were obtained.

[0046] Another BALB / c mouse was pretreated by intraperitoneal injection of 0.5 ml of liquid paraffin 10 days in advance. The suspension of the above 5 hybridoma cells was collected and intraperitoneally injected into the mouse. The ascites was collected 14 days later and purified by Protein-A Sepharose affinity chromatography system. The concentration of the purified protein was detected by Lowry method, and the results are shown in Table 1.

[0047] Table 1. Antibody protein concentration of each cell line

[0048] Cell lines Antibody Name Antibody protein concentration (mg / ml) Cell line Ⅰ First monoclonal antibody 3.62 Cell line II Secondary monoclonal antibody 5.36 Cell line III The third monoclonal antibody 2.48 Cell line IV The fourth monoclonal antibody 6.34 Cell line V The fifth monoclonal antibody 3.86

[0049] Example 2

[0050] The basic test procedure of double antibody sandwich ELISA method for IL-3 antigen was adopted, and screening was carried out through chessboard titration test.

[0051] The purified antibodies were taken to prepare the first monoclonal antibody solution, the second monoclonal antibody solution, the third monoclonal antibody solution, the fourth monoclonal antibody solution and the fifth monoclonal antibody solution. The purified antibodies were also taken to label HRP according to the sodium iodate method to prepare the first monoclonal enzyme-labeled antibody solution, the second monoclonal enzyme-labeled antibody solution, the third monoclonal enzyme-labeled antibody solution, the fourth monoclonal enzyme-labeled antibody solution and the fifth monoclonal enzyme-labeled antibody solution.

[0052] The first monoclonal antibody solution was prepared into solutions with concentrations of 1ug / mL, 2ug / mL, and 4ug / mL, respectively, and coated on the enzyme-labeled plate strips, and kept at 4°C overnight. After washing the plate, the plate was blocked with a blocking agent at 37°C for 1h to obtain the enzyme-labeled plate coated with the first monoclonal antibody. IL-3 purified antigen with a concentration of 1000pg / mL or 2000pg / mL was added to the enzyme-labeled plate coated with the first monoclonal antibody for reaction, and the second monoclonal enzyme-labeled antibody solution, the third monoclonal enzyme-labeled antibody solution, the fourth monoclonal enzyme-labeled antibody solution, or the fifth monoclonal enzyme-labeled antibody solution were added at the same time. The second monoclonal enzyme-labeled antibody solution, the third monoclonal enzyme-labeled antibody solution, the fourth monoclonal enzyme-labeled antibody solution, or the fifth monoclonal enzyme-labeled antibody solution all included three dilutions of 1:1000, 1:2000, and 1:4000. Incubate at room temperature for 1 h, wash the plate 5 times, develop color with TMB (3,3',5,5'-tetramethylbenzidine) substrate color solution for 15 min, add stop solution, and measure the OD value at 450 nm.

[0053] Similarly, the second monoclonal antibody is used as the coating antibody (capture antibody), and the enzyme-labeled antibody (detection antibody) combined with the second monoclonal antibody is the first monoclonal enzyme-labeled antibody solution, the third monoclonal enzyme-labeled antibody solution, the fourth monoclonal enzyme-labeled antibody solution, or the fifth monoclonal enzyme-labeled antibody solution.

[0054] The third monoclonal antibody is used as the coating antibody, and the enzyme-labeled antibody combined with the third monoclonal antibody is the first monoclonal enzyme-labeled antibody solution, the second monoclonal enzyme-labeled antibody solution, the fourth monoclonal enzyme-labeled antibody solution, or the fifth monoclonal enzyme-labeled antibody solution.

[0055] The fourth monoclonal antibody is used as the coating antibody, and the enzyme-labeled antibody of the fourth monoclonal antibody combination is the first monoclonal enzyme-labeled antibody solution, the second monoclonal enzyme-labeled antibody solution, the third monoclonal enzyme-labeled antibody solution, or the fifth monoclonal enzyme-labeled antibody solution.

[0056] The fifth monoclonal antibody is used as the coating antibody, and the enzyme-labeled antibody of the fifth monoclonal antibody combination is the first monoclonal enzyme-labeled antibody solution, the second monoclonal enzyme-labeled antibody solution, the third monoclonal enzyme-labeled antibody solution, or the fourth monoclonal enzyme-labeled antibody solution.

[0057] The measured OD values ​​are shown in Tables 2 and 3.

[0058] Table 2. Combinations of coated antibodies and enzyme-labeled antibodies

[0059]

[0060]

[0061] Table 3. Summary of measurement results for different combinations of coating concentration, antigen concentration, and enzyme-labeled antibody concentration

[0062]

[0063]

[0064] As shown in Table 2, the OD values ​​of combination ⑥ and combination ⑩ are relatively high. Preferably, the best combination of the coating antibody and the enzyme-labeled antibody is screened out as the coating antibody (capture antibody) is the first monoclonal antibody, and the enzyme-labeled antibody (detection antibody) is the second monoclonal antibody. The best combination of the coating antibody and the enzyme-labeled antibody is determined to be the coating antibody as the first monoclonal antibody and the enzyme-labeled antibody as the second monoclonal antibody; the concentration of the coating antibody is 2ug / mL, and the dilution of the enzyme-labeled antibody is 1:2000.

[0065] Example 3

[0066] The Sigma Mouse Monoclonal Antibody Isotyping Reagents antibody subclass kit was used for determination according to the method provided in the product manual. The results showed that the first monoclonal antibody and the second monoclonal antibody were both IgG1 class, and the light chain type was κ.

[0067] The detection of mouse monoclonal specificity against human interleukin-3 protein (IL-3) includes: using the first monoclonal antibody as the coating antibody and the second monoclonal antibody as the detection antibody, respectively detecting GM-CSF (Human), GM-CSF (Mouse), IL-1β (Human), IL-1β (Mouse), and mouse serum, all at a concentration of 5 ng / mL, wherein PBS buffer is used as a negative control, and 5 ng / mL human IL-3 recombinant protein is used as a control, and the above factors are prepared with standard diluent and cross-reactivity is determined, and no obvious cross-reaction or interference is observed. This shows that the antibody prepared by the present invention has good specificity. The results are as follows Figure 1 The standard diluent is a 0.01 mol / L PBS solution containing 1% BSA, pH 7.4.

[0068] See also Figure 1 GM-CSF (Human), GM-CSF (Mouse), IL-1β (Human), IL-1β (Mouse), and mouse serum were used as antigens, and the test results of the reaction with the first monoclonal antibody or the second monoclonal antibody were comparable to those of the PBS negative control, indicating that the first monoclonal antibody or the second monoclonal antibody had almost no specific binding with the antigen in the above experiment. The reaction results of the human IL-3 recombinant protein as an antigen with the first monoclonal antibody or the second monoclonal antibody showed that both the first monoclonal antibody and the second monoclonal antibody could specifically bind to IL-3, and the first monoclonal antibody and the second monoclonal antibody had high binding affinity to IL-3, providing feasibility for the application of the first monoclonal antibody or the second monoclonal antibody in the detection of IL-3.

[0069] Example 4

[0070] In this example, the coating antibody (capture antibody) is the first monoclonal antibody, the enzyme-labeled antibody (detection antibody) is the second monoclonal antibody, and a double antibody sandwich ELISA quantitative detection kit for IL-3 is established. The specific experimental steps include:

[0071] S1. Coat an ELISA plate with a first monoclonal antibody at a concentration of 2 ug / mL to obtain a coated ELISA plate.

[0072] S2. Use human IL-3 standard to prepare a gradient standard solution of human IL-3. The concentrations of the gradient standard solution are: 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL and 15.62 pg / mL. Use blank dilution (0 pg / mL) as a control.

[0073] S3. Add the gradient standard solution and the sample to be tested into the corresponding wells of the coated ELISA plate respectively.

[0074] S4. Add 1:1000 HPR enzyme-labeled secondary monoclonal antibody to each well of the ELISA plate, 50 μL / well, cover with sealing film, and incubate at room temperature for 1 hour.

[0075] S5. After the incubation is completed, discard the liquid in the wells, wash the plate five times with cleaning solution (0.01 mol / L PBST solution, pH 7.4), add 300 μL of sample each time, and pat dry the liquid in the wells on flat paper after washing.

[0076] S6. Add TMB colorimetric solution into the ELISA plate at 100uL / well, cover with sealing film, and incubate at room temperature in the dark for 15 minutes.

[0077] S7. After the incubation is completed, take out the ELISA plate, add 50uL of stop solution (2mol / L sulfuric acid solution) to each well, and immediately read the result at 450nm using an ELISA reader.

[0078] S8. Construct a standard curve based on the standard concentration of the gradient standard solution and the measured OD value: Use Origin software to fit and draw a four-parameter fitting curve graph of the standard curve, such as Figure 2 The standard curve equation is: y = (-0.042-4.007) / (1+(x / 382.65)^0.8876)+4.007, R 2 =0.99949.

[0079] The content of IL-3 in the unilateral sample was calculated according to the standard curve equation and the OD value of the sample to be tested.

[0080] Among them, the coating process in step S1 is: dilute the antibody 5G9 with pH9.5 0.05mol / L CBS solution to 2ug / mL, mix it with a vortexer, add it to a 96-well ELISA plate at 100μL / well, cover it with a sealing film, and incubate it in a 4°C refrigerator for 16-20h. After coating, use a blocking solution for blocking, specifically: add a blocking solution (0.01mol / L PBS+1%BSA+5% sucrose solution, pH7.4) to the plate well at 200ul / well, cover it with a cover film, and block it at 37°C for 1h. After the blocking is completed, discard the blocking solution, pat the ELISA plate dry, place it in a 37°C oven to dry for 0.5-2h, and then take it out for use.

[0081] The plate washing in step S5 is as follows: discard the liquid in the wells, wash the plate twice with a cleaning solution (0.01 mol / L PBST solution, wherein the Tween 20 content is 0.05%, pH 7.4), add 300 μL of sample each time, and after the plate washing is completed, pat the liquid in the wells dry on a flat paper.

[0082] Table 4. Summary of gradient standard solutions and corresponding OD values

[0083]

[0084]

[0085] Example 5

[0086] (1) This example adopts the ELISA detection method provided in Example 4. In step S3, the high value sample (523.96 pg / ml), the medium value sample (132.85 pg / ml) and the low value sample (15.23 pg / ml) are added to the corresponding wells of the same coated ELISA plate. 20 replicate test wells are set for each concentration. The results are shown in Table 5.

[0087] (2) This example adopts the ELISA detection method provided in Example 4. In step S3, the high value sample (523.96 pg / ml), the medium value sample (132.85 pg / ml) and the low value sample (15.23 pg / ml) are added to the corresponding wells of the coated ELISA plate. Twenty replicate test wells are set for each concentration, and the 20 wells corresponding to each concentration are set on three coated ELISA plates. The results are shown in Table 5.

[0088] Table 5. Intra-plate and inter-plate precision measurement results of ELISA plates

[0089]

[0090] As can be seen from Table 5, the intra-batch precision and inter-batch precision of the ELISA kit provided in the embodiments of the present disclosure are both less than 6%, indicating that the kit provided in the embodiments of the present disclosure, i.e., the measurement method, has good stability and high precision.

[0091] Example 6

[0092] The ELISA detection method provided in Example 4 was used to detect the recovery rate through spike recovery experiment and dilution recovery experiment.

[0093] The method of Example 4 is adopted, and the sample to be tested described in step S3 is: adding IL-3 of known concentration to serum, plasma, cell culture medium, and cell culture fluid to form serum samples, plasma samples, cell culture medium samples, and cell culture fluid samples. On the one hand, the actual results of ELISA detection of IL-3 using serum samples, plasma samples, cell culture medium samples, and cell culture fluid samples are calculated, and the results are shown in Table 6. On the other hand, serum samples, plasma samples, cell culture medium samples, and cell culture fluid samples are used for gradient dilution, dilution recovery experiments are performed, and the recovery rates are calculated. The results are shown in Table 7.

[0094] Table 6. Spike recovery test results

[0095]

[0096] Table 7. Recovery results of dilution recovery experiment

[0097]

[0098] Referring to Tables 6 and 7, the kit and the detection method provided by the embodiments of the present disclosure have a good recovery rate, indicating that the detection method provided by the embodiments of the present disclosure has high accuracy and strong specificity.

[0099] Example 7

[0100] The detection method provided in Example 4 was used, the sample diluent was used as the sample for detection, the measurement was repeated 20 times, the absorbance values ​​of the 20 measurement results were obtained, the sample concentration was calculated according to the standard curve, the average value and standard deviation were calculated, and the minimum detection limit of the kit was calculated by AV+2SD, and the results are shown in Table 8. Wherein, the sample diluent is a 0.01 mol / L PBS solution containing 1% BSA, pH 7.4.

[0101] Table 8. ELISA detection limit measurement results

[0102]

[0103]

[0104] According to the test results in Table 8, the detection sensitivity of human IL-3 is 3.8486 pg / ml, which is high in detection sensitivity and high in detection accuracy for detecting samples containing IL-3.

[0105] In summary, the present disclosure provides an anti-human interleukin-3 (IL-3) composition and a kit and a detection method comprising the composition, which is formed by coating a first monoclonal antibody of human IL-3 with an enzyme-labeled plate to form a solid phase carrier, binding to the IL-3 antigen in the biological sample to be tested, and then using a specific enzyme-labeled detection antibody (second monoclonal antibody) to form a solid phase antibody-antigen-enzyme-labeled antibody complex, and then adding a color-developing substrate solution for detection. The detection kit provided by the present disclosure adopts a one-step method, and the standard product / sample is added to the reaction together with the enzyme-labeled antibody during detection, and the reaction time is 1h. The entire experimental operation steps are few, the time is short, and the sensitivity is high, and the precision and accuracy are good.

[0106] 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 protection scope of the present invention.

Claims

1. A composition for resisting human interleukin-3, characterized in that: The method comprises a first monoclonal antibody and a second monoclonal antibody; the first monoclonal antibody and the second monoclonal antibody specifically bind to different antigenic epitopes of human interleukin-3; The heavy chain complementary determining region of the first monoclonal antibody includes CDR1-H1, CDR1-H2 and CDR1-H3; the CDR1-H1 has an amino acid sequence with at least 90% homology to SEQ ID NO.1; the CDR1-H2 has an amino acid sequence with at least 90% homology to SEQ ID NO.2; the CDR1-H3 has an amino acid sequence with at least 90% homology to SEQ ID NO.3; The light chain complementary determining region of the first monoclonal antibody includes CDR1-L1, CDR1-L2 and CDR1-L3; the CDR1-L1 has an amino acid sequence with at least 90% homology to SEQ ID NO.7; the CDR1-L2 is an amino acid sequence with at least 90% homology to SEQ ID NO.8; the CDR1-L3 is an amino acid sequence with at least 90% homology to SEQ ID NO.9; The heavy chain complementary determining regions of the second monoclonal antibody include CDR2-H1, CDR2-H2 and CDR2-H3; the CDR2-H1 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.4; the CDR2-H2 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.5; the CDR2-H3 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.6; The light chain complementary determining region of the second monoclonal antibody includes CDR2-L1, CDR2-L2 and CDR2-L3; the CDR2-L1 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.10; the CDR2-L2 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.11; and the CDR2-L3 has an amino acid sequence that is at least 90% homologous to SEQ ID NO.

12.

2. The anti-human interleukin-3 composition according to claim 1, characterized in that The V of the first monoclonal antibody H The amino acid sequence of the first monoclonal antibody is as shown in SEQ ID NO.13; L The chain is shown in the amino acid sequence SEQ ID NO.

15.

3. The anti-human interleukin-3 composition according to claim 1, characterized in that The V of the second monoclonal antibody H The amino acid sequence of the second monoclonal antibody is as shown in SEQ ID NO.14; L The chain is shown in the amino acid sequence SEQ ID NO.

16.

4. Use of the anti-human interleukin-3 composition according to any one of claims 1 to 3 in the preparation of a product for detecting human interleukin-3.

5. A human interleukin-3 detection kit, characterized in that: A composition comprising the anti-human interleukin-3 according to any one of claims 1 to 3; Among them, the first monoclonal antibody is a capture antibody, and the second monoclonal antibody is a detection antibody.

6. The detection kit for human interleukin-3 according to claim 5, characterized in that: The kit is an enzyme-linked immunosorbent assay kit.

7. A method for detecting human interleukin-3, characterized in that: include: Obtaining a gradient standard solution of a human interleukin-3 standard and a sample to be tested; Adding the gradient standard solution and the sample to be tested to the ELISA plate coated with the first monoclonal antibody respectively; Add enzyme-labeled second monoclonal antibody to each well of the ELISA plate, incubate at room temperature, wash the plate, add substrate for color development, and measure the OD value of each well; According to the OD value of each well corresponding to the gradient standard solution, a standard curve expressing the relationship between the concentration and OD value of the gradient standard solution is obtained; The concentration of interleukin-3 in the sample to be tested is obtained according to the standard curve and the OD value of the corresponding well of the sample to be tested.

8. The detection method according to claim 7, characterized in that: The enzyme used for the enzyme-labeled second monoclonal antibody is horseradish peroxidase; The substrate is 3,3',5,5'-tetramethylbenzidine.

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