A monoclonal antibody specifically binding to human GM-CSF and its detection kit and application

Abstract

The present invention provides a monoclonal antibody that specifically binds to human GM-CSF. The monoclonal antibody comprises a first antibody, 5G6, and a second antibody, 7F8. The heavy and light chain complementary determining region sequences of the 5G6 antibody are shown in SEQ ID NOs. 1 to 5, and the heavy and light chain complementary determining region sequences of the 7F8 antibody are shown in SEQ ID NOs. 6 to 10. Furthermore, a double-antibody sandwich ELISA quantitative detection kit for GM-CSF was developed using 5G6 as the coating antibody and 7F8 as the enzyme-labeled antibody. The developed kit utilizes a one-step method, in which the standard sample / test substance is added to the reaction along with the enzyme-labeled antibody. The reaction time is 1 hour, resulting in a short, minimal experimental procedure and high sensitivity, precision, and accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a monoclonal antibody specifically binding to human GM-CSF, a detection kit and an application thereof. Background Art

[0002] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an active immune effector with a wide range of immune activities in the body. GM-CSF is a proinflammatory cytokine that rapidly increases during infection or inflammation. At sites of inflammation, GM-CSF exerts proinflammatory effects by recruiting myeloid cells and enhancing their survival and activation. GM-CSF plays a key role in the pathogenesis of rheumatoid arthritis by influencing the activation, differentiation, and survival of macrophages, dendritic cells, and neutrophils. Studies have shown that a monoclonal antibody targeting GM-CSF is effective and well-tolerated in the treatment of rheumatoid arthritis (RA). Because GM-CSF is widely involved in the pathogenesis of allergic and inflammatory diseases, understanding the functions and mechanisms of these cytokines and their receptors through cytokine detection and other methods can help assess immune function and is crucial for disease diagnosis, disease progression, efficacy assessment, and treatment monitoring.

[0003] Currently, the most reliable quantitative method for detecting GM-CSF antibodies is the enzyme-linked immunosorbent assay (ELISA). ELISA uses an antigen or antibody coupled to an enzyme as a probe, and then uses the enzyme's activity to detect the presence of the antibody or antigen. However, the traditional ELISA method has limited sensitivity, a long reaction time, and is complex to perform.

[0004] In view of this, the GM-CSF detection method needs to be further improved, and there is an urgent need to provide a detection method with short reaction time, convenient operation and high sensitivity. Summary of the Invention

[0005] In view of this, the present invention provides a monoclonal antibody that specifically binds to human GM-CSF, and further provides a GM-CSF magnetic bead-enhanced direct ELISA detection kit. The kit comprises the above-mentioned monoclonal antibody and magnetic beads, and has the characteristics of high sensitivity, good specificity, short reaction time, and simple operation.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] One of the purposes of the present invention is to provide a monoclonal antibody that specifically binds to human GM-CSF, the monoclonal antibody comprising a first antibody 5G6 and a second antibody 7F8;

[0008] Among them, the complementary determining regions of the heavy chain and light chain of the first antibody 5G6 are as follows:

[0009] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO. 1: GFRSSFNY;

[0010] CDR-H2 comprises the amino acid sequence shown in SEQ ID NO. 2: INTDSGGT;

[0011] CDR-H3 comprises the amino acid sequence shown in SEQ ID NO. 3: ARNRRDYFSFDY;

[0012] CDR-L1 comprises the amino acid sequence shown in SEQ ID NO. 4: QNSVSA;

[0013] CDR-L2 comprises the amino acid sequence: SAS;

[0014] CDR-L3 comprises the amino acid sequence shown in SEQ ID NO. 5: QQYRDYPT;

[0015] The details of the heavy and light chain complementary determining regions of the second antibody 7F8 are as follows:

[0016] CDR-H1 comprises the amino acid sequence shown in SEQ ID NO. 6: GFRTSRSW;

[0017] CDR-H2 comprises the amino acid sequence shown in SEQ ID NO. 7: INDARISI;

[0018] CDR-H3 comprises the amino acid sequence shown in SEQ ID NO. 8: ARAGDVWFTFLFSY;

[0019] CDR-L1 comprises the amino acid sequence shown in SEQ ID NO. 9: QSVNDN;

[0020] CDR-L2 comprises the amino acid sequence: GAS;

[0021] CDR-L3 comprises the amino acid sequence shown in SEQ ID NO. 10: QQYSKGRDT.

[0022] Furthermore, the VH original sequence of the first antibody 5G6 is as shown in SEQ ID NO.11, or a sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO.11 and has equivalent function;

[0023] SEQ ID NO.11: DVKLVESGAALVKLGGSLKLSCAASGFRSSFNYMSWVRQTPEKRLELVAAINTDSGGTYYLDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALFYCARNRRDYFSFDYWGQGTSVTVSS;

[0024] The VL original sequence of the first antibody 5G6 is as shown in SEQ ID NO. 12, or a sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO. 12 and has equivalent function;

[0025] SEQ ID NO.12: DIVMTQSQRFMSTTVGDRVSITCKASQNSVSAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADFFCQQYRDYPTTFGGGTKLEIK;

[0026] The VH original sequence of the second antibody 7F8 is shown in SEQ ID NO. 13, or a sequence that has at least 90% identity to the amino acid sequence shown in SEQ ID NO. 13 and has equivalent function;

[0027] SEQ ID NO.13: EVQLVESGGGLVQPGGSLRLSCAASGFRTSRSWMHWLRQVPGKGPVWVSRINDARISITYADSVRGRFTISRDNANNTLFLQMNSLRADDTALYFCARAGDVWFTFLFSYWGQGTPVTVSS;

[0028] The VL original sequence of the second antibody 7F8 is as shown in SEQ ID NO. 14, or a sequence that has at least 90% identity to the amino acid sequence of SEQ ID NO. 14 and has equivalent function;

[0029] SEQ ID NO. 14: EIVLTQSPVTLSVSPGERVTLSCRASQSVNDNLAWYQQKLGQGPRLLIYGASTRATDIPARFSGSGSETEFTLTISSLQSEDFAVYYCQQYSKGRDTFGQGTKLEIK.

[0030] A second object of the present invention is to provide the use of the above monoclonal antibody in the detection of GM-CSF antibodies.

[0031] A third object of the present invention is to provide a GM-CSF antibody detection kit, which comprises the above-mentioned monoclonal antibody.

[0032] Furthermore, the first antibody 5G6 in the kit is labeled with Tosyl magnetic beads, and the second antibody is labeled with HRP enzyme.

[0033] In some specific embodiments, preferably, the preparation method of the first antibody 5G6-labeled Tosyl magnetic beads is as follows:

[0034] First, prepare a Magnosohere MS30 / Tosyl microsphere suspension; then add boric acid buffer and vortex; then add the antibody and vortex again to react; then add the blocking agent and continue the reaction; after the reaction is complete, remove the supernatant, wash with wash buffer, and then dry the washing solution; finally, suspend with blocking agent to prepare a magnetic bead coating solution.

[0035] Furthermore, the kit also includes an ELISA plate treated with a blocking agent, a freeze-dried standard, a standard, a sample diluent, a cleaning solution, a substrate display solution, and a reaction termination solution.

[0036] In some specific embodiments, preferably, the blocking agent is a buffer solution containing a blocking agent and a stabilizer;

[0037] The lyophilized standard is GM-CSF antigen;

[0038] The standard and sample diluents were buffered with 1% BSA;

[0039] The cleaning solution is 0.01 M PBS containing 0.05% Tween 20, pH 7.4;

[0040] The substrate display solution is TMB color developing solution;

[0041] The stop solution is a 2 mol / L sulfuric acid solution.

[0042] In some specific embodiments, preferably, the blocking agent is one of BSA or sodium caseinate, the stabilizer in the blocking agent is one or more of sucrose, trehalose, and PVP-10, and the buffer in the blocking agent is one of 0.01M PBS buffer or 0.05MTris buffer, with a pH of 6.0-8.0;

[0043] The buffer in the standard is 0.01 M PBS buffer or 0.05 M Tris buffer, with a pH of 7.4.

[0044] A fourth object of the present invention is to provide a detection method for the above-mentioned kit, comprising the following steps:

[0045] First, add the sample or standard to the sealed ELISA plate, then add Tosyl magnetic beads labeled with the primary antibody and the secondary antibody labeled with HRP enzyme, and incubate at room temperature; then place the ELISA plate in the magnetic plate, pour out the liquid in the ELISA plate, wash it with cleaning solution, and pat the plate dry; then remove the ELISA plate from the magnetic plate, add the substrate color development solution, and incubate in the dark; finally, add the stop solution and measure the OD value at 450nm to complete the detection.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The present invention provides monoclonal antibodies 5G6 and 7F8 that specifically bind to human GM-CSF. Furthermore, a double-antibody sandwich ELISA quantitative detection kit for GM-CSF was developed using 5G6 as the coating antibody and 7F8 as the enzyme-labeled antibody. The developed kit utilizes a one-step method, in which the standard sample / test substance and the enzyme-labeled antibody are added together for the reaction. The reaction time is one hour, resulting in a minimal number of steps and a short experimental time. It also exhibits high sensitivity (sensitivity of 1.1803 pg / mL), precision (within-assay CV% less than 10% and between-assay CV% less than 5%), and good accuracy (average recovery between 89% and 101%). BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a graph showing the antibody specificity test results in Example 2 of the present invention.

[0049] Figure 2 This is the winning curve in Example 3 of the present invention. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0051] Example 1

[0052] This example provides the preparation of GM-CSF-specific monoclonal antibodies, as follows:

[0053] 1.1 Animal immunization and hybridoma cell line screening

[0054] GM-CSF (purchased from R&D215-GM) was dissolved in PBS buffer and mixed with complete Freund's adjuvant (CFA) at a 1:1 volume ratio to emulsify the prepared antigen solution. Four female BALB / c mice were immunized with 300 µL of the prepared antigen injection solution (containing 100 µg of antigen) via intraperitoneal injection. A booster immunization was performed 14 days later with a 1:1 mixture of antigen and incomplete Freund's adjuvant (IFA) at a dose of 100 µg per mouse. A second booster immunization was performed 14 days later. A third booster immunization was performed 7 days after the second booster immunization, with a dose of 100 µg per mouse via intraperitoneal injection. A fourth booster immunization was performed 7 days after the third booster immunization, with a dose of 100 µg per mouse via intraperitoneal injection. Spleens were harvested three days after the fourth booster immunization and cell fusion was performed using conventional hybridoma fusion techniques. Hybridomas were selected using HAT selective medium. The supernatant of the selected hybridoma cells was tested by indirect ELISA, and the cells with strong positive results were selected for cloning. After 3-5 rounds of cloning using the limiting dilution method, strong positive cell lines that stably secreted specific mAbs were obtained and named 5G6, 7F8, and 4H9.

[0055] The three cell lines were further expanded: BALB / c mice were pretreated with an intraperitoneal injection of 0.5 mL of liquid paraffin 10 days in advance. The hybridoma cell suspensions were collected and injected intraperitoneally into the mice. Fourteen days later, ascites fluid was collected and purified using a Protein-A Sepharose affinity chromatography system. The purified protein concentration was determined using the Lowry method, as shown in Table 1.

[0056] Table 1 Antibody concentration test results

[0057]

[0058] 1.2 Screening of paired antibodies

[0059] The three purified antibodies were labeled with HRP using the sodium periodate method. The GM-CSF antigen was then detected using the basic ELISA double-antibody sandwich assay procedure, employing a checkerboard titration assay: enzyme-labeled plates were coated with each antibody at concentrations of 1 µg / mL, 2 µg / mL, and 4 µg / mL, incubated overnight at 4°C. After washing, the plates were blocked with blocking reagent for 1 hour at 37°C. The plates were then reacted with purified GM-CSF antigen at concentrations of 500 pg / mL and 1000 pg / mL. Two additional enzyme-labeled antibodies, different from the coated antibodies, were also added at dilutions of 1:1000, 1:2000, and 1:4000, respectively. The plates were incubated at room temperature for 1 hour. The plates were washed five times, developed with TMB substrate for 15 minutes, and then the stop solution was added. The OD value at 450 nm was measured.

[0060] Among them, the sodium periodate method for labeling HRP includes:

[0061] (1) Weigh 2 mg of HRP and dissolve it in 0.5 mL of distilled water.

[0062] (2) Add 0.2 mL of freshly prepared 0.1 M NaIO4 solution to the above solution, mix well, and stir in the dark at 4°C for 60 minutes.

[0063] (3) Add 0.2 mL of 0.16 M ethylene glycol aqueous solution, mix well, and stir at 4°C in the dark for 30 minutes.

[0064] (4) Add 2 mg of antibody (the antibody has been dialyzed overnight with pH = 9.5 carbonate buffer in advance), mix well and put into dialysis bag, dialyze against pH = 9.5 carbonate buffer at 4°C overnight.

[0065] (5) Add 0.2 mL of freshly prepared 4 mg / mL NaBH4 solution, mix well, and stir at 4°C in the dark for 30 minutes.

[0066] (6) Add saturated ammonium sulfate in an equal volume to the above liquid dropwise while stirring, mix well, and place in a 4°C refrigerator for 1 hour.

[0067] (7) Centrifuge at 3000 rpm for 30 min and discard the supernatant. Wash the precipitate twice with 50% saturated ammonium sulfate, 300 µL each time, and finally dissolve the precipitate in 200 µL of 0.01 mol / L PBS solution (pH = 7.4).

[0068] (8) The above solution was placed in a dialysis bag and dialyzed against 0.01 mol / L PBS buffer (pH = 7.4) at 4°C overnight. After removing the ammonium ions, the solution was centrifuged at 3000 rpm for 30 minutes to remove the precipitate. The supernatant was the enzyme conjugate. The obtained enzyme-labeled antibody was added to an equal volume of enzyme protectant and stored at 2-8°C.

[0069] Finally, based on the saturation concentrations of the coating antibody and enzyme-labeled antibody corresponding to the maximum OD value, the coating antibody concentration was determined to be 2 µg / mL, the enzyme-labeled antibody dilution was 1:1000, and the antigen concentration was 500 pg / mL. Preferably, the optimal combination of coating antibody and enzyme-labeled antibody was 5G6 for the coating antibody and 7F8 for the labeling antibody. Antibody combinations are shown in Table 2 and screening results in Table 3.

[0070] Table 2 Antibody combinations

[0071]

[0072] Table 3 Screening results of each antibody combination

[0073]

[0074] Example 2

[0075] This example provides the subtype and specificity identification of antibodies 5G6 and 7F8 screened in Example 1, as follows:

[0076] The antibody subclass was determined using the Sigma Mouse Monoclonal Antibody Isotyping Reagents kit according to the method provided in the product manual. The results showed that both antibody 5G6 and antibody 7F8 were IgG1 class, and the light chain type was κ.

[0077] GM-CSF (Human), G-CSF (Human), GM-CSF (Mouse), M-CSF (Human), and mouse serum were used as antigens to coat ELISA plates and reacted with antibodies 5G6 and 7F8. The OD values ​​of mAbs reacting with each antigen were determined by ELISA. 450 Among them, PBS buffer was used as negative control, and the results were as follows Figure 1 shown.

[0078] Depend on Figure 1 The results of the reaction between the first or second monoclonal antibody and G-CSF (Human), GM-CSF (Mouse), M-CSF (Human), and mouse serum as antigens were comparable to those of the PBS negative control, indicating that the first or second monoclonal antibody had little specific binding to the antigens in these experiments and no significant cross-reaction or interference with the human GM-CSF antigen. Furthermore, the results of the reaction between the first or second monoclonal antibody and the human GM-CSF recombinant protein as an antigen showed that both the first and second monoclonal antibodies specifically bound to GM-CSF, and that the first and second monoclonal antibodies had high binding affinities for GM-CSF, supporting the feasibility of using the first or second monoclonal antibody in the detection of GM-CSF.

[0079] Example 3

[0080] This embodiment provides a GM-CSF antibody detection kit and a detection method thereof, which are specifically as follows:

[0081] 3.1 The coated antibody is 5G6, and the enzyme-labeled antibody is 7F8. The double-antibody sandwich ELISA quantitative detection kit for GM-CSF includes:

[0082] (1) Take out the ELISA plate, reagents, etc. in the kit and rewarm them.

[0083] (2) Sample addition: Standard curve: GM-CSF antigen (purchased from RD) was serially diluted with diluent. The gradient concentrations after dilution were 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.62 pg / mL, and 7.81 pg / mL, respectively. Blank dilution was used as a control. Standards or samples were added to the ELISA plate at 50 μL / well. Then, 1:1000 diluted enzyme-labeled antibody 7F8 was added at 50 μL / well. The plate was covered with a sealing film and incubated at room temperature for 1 hour.

[0084] (3) Washing: After incubation, discard the liquid in the wells and wash the plate five times with cleaning solution (0.01M PBST solution, pH 7.4), adding 300 μL of sample each time. After washing, pat the liquid in the wells dry on flat paper.

[0085] (4) Color development: Add TMB color development solution into the ELISA plate at 100 μL / well, cover with sealing film, and incubate at room temperature in the dark for 15 minutes.

[0086] (5) Reading: After incubation, remove the ELISA plate, add 50uL of stop solution (2mol / L sulfuric acid solution) to each well, and immediately read the plate at 450nm using a ELISA reader.

[0087] The preparation method of the ELISA plate provided in the kit includes:

[0088] Coating: Dilute antibody 5G6 to 2 μg / mL with 0.05 M CBS solution at pH 9.5. Vortex mix and add 100 μL / well to a 96-well ELISA plate. Cover with sealing film and incubate at 4°C for 16-20 hours.

[0089] Wash the plate: discard the liquid in the wells and wash the plate twice with cleaning solution (0.01M PBST solution, pH 7.4), adding 300μL of sample each time. After washing, pat the liquid in the wells dry on flat paper.

[0090] Blocking: Add 200 μL / well of blocking solution (0.05 M Tris + 0.2% sodium caseinate + 30% sucrose solution, pH 8.0) to the plate, cover with cover film, and block at 37°C for 1 hour. After blocking, discard the blocking solution, pat the ELISA plate dry, and place it in a 37°C oven to dry for 4 hours before removing it for use.

[0091] 3.2 Bidding

[0092] Draw a standard curve based on the OD value and concentration of the standard sample: Use Origin software to fit and draw a four-parameter fitting curve of the standard curve (see Figure 2 ), the results are shown in Table 4, and the GM-CSF content in the samples was calculated based on the standard curve.

[0093] Curve equation: Fit: y = (-0.0393 - 5.4361) / (1 + (x / 532.4440) ^ 0.8463)+5.4361, R 2 =0.99992.

[0094] Table 4 OD measured under gradient concentrations 450 value

[0095]

[0096] 3.3 Kit performance test

[0097] 3.3.1 Precision test

[0098] Using the above experimental method, samples with high, medium, and low concentrations were selected and tested 20 times on one ELISA plate respectively; and samples with high, medium, and low concentrations were selected and tested 20 times on three ELISA plates respectively. The intra-plate precision and inter-plate precision were tested. The results are shown in Table 5.

[0099] Table 5 Intra-plate precision and inter-plate precision test results

[0100]

[0101] As can be seen from Table 5, the intra-batch precision CV% of the kit of the present invention is less than 10%, and the inter-batch accuracy CV% is less than 5%, reflecting that the measurement system of the kit has good stability.

[0102] 3.3.2 Recovery rate test

[0103] Using the above experimental method, GM-CSF of known concentrations was added to different samples, and spike recovery and dilution recovery experiments were performed. The spike recovery results are shown in Table 6, and the dilution recovery results are shown in Table 7.

[0104] Table 6 Spike recovery results

[0105]

[0106] Table 7 Dilution recovery results

[0107]

[0108] It can be seen from Tables 6 and 7 that the kit of the present invention has a good recovery rate, reflecting that the accuracy of the kit is high.

[0109] 3.3.3 Sensitivity test

[0110] Using the above experimental method, the sample diluent was used as the sample for detection, and the test was repeated 20 times to obtain the absorbance values ​​of the 20 measurement results. The sample concentration was calculated according to the standard curve, and the average value and standard deviation were calculated. The minimum detection limit of the kit was calculated by AV+2SD. The results are shown in Table 8.

[0111] Table 8 Average and standard deviation results of 20 repeated tests

[0112]

[0113] It can be seen from Table 8 that the detection sensitivity of the kit of the present invention for detecting human GM-CSF is 1.1803 pg / ml, which has high sensitivity.

[0114] In summary, the present invention provides an anti-human granulocyte-macrophage colony-stimulating factor (GM-CSF) monoclonal antibody, a kit containing the same, and a detection method thereof. The method involves coating a human GM-CSF antibody (5G6) on an ELISA plate to form a solid-phase carrier, which then binds to the GM-CSF antigen in the biological sample to be tested. A specific enzyme-labeled detection antibody (7F8) is then used to form a solid-phase antibody-antigen-enzyme-labeled antibody complex, which is then detected by intervening with a chromogenic substrate solution. The detection kit provided by the present invention utilizes a one-step method, in which the standard sample / test specimen is added to the reaction along with the enzyme-labeled antibody. The reaction time is one hour, resulting in a minimal number of steps and a short reaction time. The assay also offers high sensitivity, precision, and accuracy.

[0115] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0116] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, 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 monoclonal antibody that specifically binds to human GM-CSF, characterized in that: The monoclonal antibodies include a first antibody 5G6 and a second antibody 7F8; Among them, the complementary determining regions of the heavy chain and light chain of the first antibody 5G6 are as follows: The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1; The amino acid sequence of CDR-H2 is shown in SEQ ID NO. 2; The amino acid sequence of CDR-H3 is shown in SEQ ID NO. 3; The amino acid sequence of CDR-L1 is shown in SEQ ID NO.4; CDR-L2 amino acid sequence: SAS; The amino acid sequence of CDR-L3 is shown in SEQ ID NO.5; The details of the heavy and light chain complementary determining regions of the second antibody 7F8 are as follows: The amino acid sequence of CDR-H1 is shown in SEQ ID NO.6; The amino acid sequence of CDR-H2 is shown in SEQ ID NO.7; The amino acid sequence of CDR-H3 is shown in SEQ ID NO.8; The amino acid sequence of CDR-L1 is shown in SEQ ID NO.9; CDR-L2 amino acid sequence: GAS; The amino acid sequence of CDR-L3 is shown in SEQ ID NO.

10.

2. The monoclonal antibody according to claim 1, characterized in that The VH original sequence of the first antibody 5G6 is shown in SEQ ID NO.11; The VL original sequence of the first antibody 5G6 is shown in SEQ ID NO. 12; The VH original sequence of the second antibody 7F8 is shown in SEQ ID NO.13; The VL original sequence of the second antibody 7F8 is shown in SEQ ID NO.

14.

3. A human GM-CSF antigen detection kit, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2.

4. The kit according to claim 3, wherein In the kit, the first antibody 5G6 is labeled with Tosyl magnetic beads, and the second antibody is labeled with HRP enzyme.

5. The kit according to claim 4, characterized in that The preparation method of the primary antibody 5G6-labeled Tosyl magnetic beads is as follows: First, prepare a Magnosohere MS30 / Tosyl microsphere suspension; then add boric acid buffer and vortex; then add the antibody and vortex again to react; then add the blocking agent and continue the reaction; after the reaction is complete, remove the supernatant, wash with wash buffer, and then dry the washing solution; finally, suspend with blocking agent to prepare a magnetic bead coating solution.

6. The kit according to claim 3, characterized in that The kit also includes an ELISA plate treated with a blocking agent, a freeze-dried standard, a standard, a sample diluent, a cleaning solution, a substrate display solution, and a reaction stop solution; The lyophilized standard is GM-CSF antigen; The standard and sample diluents were buffered with 1% BSA.

7. The kit according to claim 6, characterized in that The blocking agent is a buffer solution containing a blocking agent and a stabilizer; The cleaning solution is 0.01 M PBS containing 0.05% Tween 20, pH 7.4; The substrate display solution is TMB color developing solution; The stop solution is a 2 mol / L sulfuric acid solution.

8. The kit according to claim 7, characterized in that The blocking agent is one of BSA or sodium caseinate, The stabilizer in the blocking agent is one or more of sucrose, trehalose, and PVP-10, and the buffer in the blocking agent is One of 0.01 M PBS buffer or 0.05 M Tris buffer, pH 6.0-8.0; The buffer in the standard is 0.01M PBS buffer or 0.05M Tris buffer, with a pH of 7.4.

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