Kit for detecting multiple cytokines in tumor immunity as well as preparation method and application of kit

Abstract

The invention belongs to the technical field of biological detection, and provides a multiple cell factor detection kit in tumor immunity and a preparation method and application thereof, and the detection kit comprises a capture antibody, a detection antibody, a mixed protein standard substance, a diluent and a washing buffer solution. The capture antibody is prepared by coupling a biotinylated antibody with a fluorescent microsphere coated with biotin to obtain a capture antibody coupled with a fluorescent encoding microsphere, and the detection antibody is prepared by coupling derivatized phycoerythrin PE-SMCC with a specific thioether bond of a sulfhydrylated antibody to obtain a fluorescent labeled antibody; the mixed protein standard substance adopts a freeze-drying protection system containing BSA, trehalose and the like. The single hole of the kit can synchronously detect various cell factors, the sample dosage is only 25 microliters, the kit is suitable for immune monitoring of iron overload and other chronic inflammation models, compared with a traditional ELISA method, the efficiency is improved by 10 times, the cost is reduced, and the kit has the remarkable advantages of being high in throughput, wide in linear range and high in stability.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a multiplex cytokine detection kit for tumor immunology, its preparation method, and its application. Background Technology

[0002] Tumors evolve gradually from normal cells. From a histopathological perspective, tumor tissue, like normal tissue, is composed of a large number of cells. Based on the degree of invasive growth, tumors can be divided into benign and malignant tumors; what is commonly referred to as "cancer" falls under the category of malignant tumors. Malignant tumors are characterized by their unlimited spread, infiltration, and metastasis to surrounding tissues, which can be figuratively understood as a state of "uncontrolled growth" and "immortality." Between normal tissue and highly malignant tumor tissue, there exist a series of intermediate transitional states with varying morphologies, reflecting that tumorigenesis is a multi-stage and highly complex evolutionary process. During this process, tumors exhibit dynamic changes at both the phenotypic and genetic levels. From a genetic origin perspective, tumors can be divided into two types: monoclonal and polyclonal. Monoclonal tumors originate from a single normal cell undergoing malignant transformation, subsequently proliferating to form an entire tumor population, with all cells originating from the same ancestor. Polyclonal tumors, on the other hand, involve multiple different cells undergoing malignant transformation simultaneously or sequentially, forming multiple cell subpopulations with significantly different genetic characteristics, which together constitute the tumor tissue. The tumor microenvironment (TME) is the internal environment in which tumor cells are produced and live. It is a local environment composed of tumor cells, various stromal cells, cytokines, chemokines, etc., which provide nutrition and support for the tumor. Essentially, it is the tumor's neighborhood.

[0003] Therefore, many basic scientific research topics involve the occurrence, development and treatment of tumors. The assessment of the tumor immune microenvironment is extremely important in various studies, which requires the detection of a variety of protein factors related to tumor immunity.

[0004] Currently, Western blotting (WB) and ELISA are commonly used to quantify multiple proteins. However, WB has low sensitivity, cannot identify conformational protein structures, and requires large sample sizes. ELISA, on the other hand, can only quantify one target per product, resulting in low efficiency and the need for large sample sizes. In current scientific research, which often requires multiple pathways for project implementation, obtaining samples is extremely difficult and precious, directly impacting the success or failure of the research project.

[0005] CN119470918A proposes a liquid-based chip technology-based inflammatory factor detection kit. This kit uses fluorescein to fluorescently encode microspheres and attaches capture antibodies to them via a chemical reaction. The fluorescent microspheres capture the target, and the detection antibody displays the signal, thus detecting inflammatory factors. This overcomes the drawbacks of previous methods, such as requiring large sample volumes, long testing times, and cumbersome procedures for soluble inflammatory factor detection, greatly facilitating researchers in hospitals and industry. However, this kit can only detect up to ten inflammatory factors, and products for detecting multiple protein factors related to tumor immunology research have been lacking in the market. Summary of the Invention

[0006] To address the aforementioned issues, this invention utilizes XMPlex multifactor detection based on encoded microspheres and antigen-antibody technology. Capture antibodies targeting different analytes are covalently coupled to specific encoded microspheres via a chemical reaction. Each encoded microsphere coupled with a specific capture antibody corresponds to one analyte. The microspheres are sequentially passed through red and green lasers by a flowing sheath fluid. The red laser is used to determine the fluorescent coding of the microspheres, thus distinguishing the magnetic beads and identifying the analyte. The green laser is used to measure the fluorescence intensity of the reporter molecule on the microsphere; this signal is proportional to the bound analyte. This achieves the goal of highly sensitive, rapid, accurate, and simultaneous quantification of multiple proteins.

[0007] Furthermore, a multi-factor kit for macrophage polarization research has been developed. This kit meets researchers' needs for timely assessment of macrophage status and simultaneous quantification of multiple macrophage-related protein factors, requiring only 25 µL of sample, significantly improving research throughput and efficiency. Simultaneously, the products utilize a dual monoclonal antibody design, ensuring specificity and accuracy in target identification, and overcoming the limitations of low sensitivity, inability to identify conformational protein structures, and large sample volume requirements of Western blotting and ELISA.

[0008] XMPlex multifactor detection utilizes encoded microspheres and antigen-antibody technology. Capture antibodies targeting different analytes are covalently coupled to specific encoded microspheres via a chemical reaction. Each encoded microsphere with a specific capture antibody corresponds to one analyte. The microspheres are sequentially passed through red and green lasers by a flowing sheath fluid. The red laser determines the fluorescence encoding of the microspheres, thus distinguishing them and identifying the analyte. The green laser measures the fluorescence intensity of the reporter molecule on the microsphere; this signal is proportional to the bound analyte. A single experiment can simultaneously quantify the levels of IL-12 p70, IL-6, IL-5, IL-2, IL-4, IL-17 / IL-17A, IL-22, CXCL13 / BCA-1, IL-1 beta / IL-1F2, IL-10, IL-1alpha / IL-1F1, CCL2 / MCP-1, CCL3 / MIP-1 alpha, CCL5 / RANTES, IL-12P40, CCL4 / MIP-1beta, IFN-gamma, IFN-beta, G-CSF, CCL11 / Eotaxin, VCAM-1 / CD106, GM-CSF, CXCL1 / GROalpha, EGF, TNF-alpha, and Acrp30 in a sample.

[0009] To achieve the above objectives, this invention proposes a method for preparing a multiplex cytokine detection kit for tumor immunology, comprising the following preparation steps: (1) Preparation of microsphere-coupled capture antibodies: n capture antibodies are biotinylated to obtain n biotinylated antibodies; fluorescently encoded microspheres with streptavidin covalently coupled to the surface are incubated with the n biotinylated antibodies respectively, so that each capture antibody is coupled one-to-one with microspheres with different internal fluorescent codes to form a mixture of n microsphere-coupled capture antibodies, and each capture antibody specifically recognizes one cytokine; (2) Preparation of detection antibodies: Thiolizing n detection antibodies to obtain thiolated antibodies, and then incubating the derivatized reporter fluorescent protein with the thiolated antibodies in the dark and then blocking them to form n fluorescently labeled detection antibody solutions; (3) Preparation of mixed protein standards: Mix n kinds of cytokines with lyophilization protection solution to obtain lyophilized powder containing n kinds of cytokines; (4) Kit assembly: Assemble the microsphere-conjugated capture antibody, detection antibody, mixed protein standard, standard diluent, sample diluent and washing buffer into a complete kit; The fluorescently encoded microspheres are combinations of fluorescently encoded microspheres with different energy levels formed by mixing two fluorescent dyes at different concentration ratios.

[0010] The n=26, and the 26 capture antibodies and detection antibodies specifically recognize the following cytokines: IL-12p70, IL-6, IL-5, IL-2, IL-4, IL-17 / IL-17A, IL-22, CXCL13 / BCA-1, IL-1 beta / IL-1F2, IL-10, IL-1 alpha / IL-1F1, CCL2 / MCP-1, CCL3 / MIP-1 alpha, CCL5 / RANTES, IL-12P40, CCL4 / MIP-1 beta, IFN-gamma, IFN-beta, G-CSF, CCL11 / Eotaxin, VCAM-1 / CD106, GM-CSF, CXCL1 / GRO alpha, EGF, TNF-alpha, and Acrp30.

[0011] The specific operation of biotin labeling in step (1) is as follows: the capture antibody and biotin-X succinimide ester are reacted at a molar ratio of 85:1 at pH 8.0-8.5 and room temperature for 2 hours or overnight at 4°C. The reaction product is purified by ultrafiltration, and the ultrafiltration washing is performed 3-4 times.

[0012] First, the sample and the microsphere suspension containing the capture antibody are mixed, and the analytes in the sample bind to the specific capture antibody on the microsphere. Then, the detection antibody is added, and the detection antibody specifically binds to the reaction product from the first step, ultimately forming a "capture antibody-antigen-fluorescein antibody" complex on the surface of the microsphere. When using flow cytometry to detect these microsphere complexes, the specific analyte types can be identified and their corresponding concentrations analyzed, enabling the quantitative detection of IL-12 p70, IL-6, IL-5, IL-2, IL-4, IL-17 / IL-17A, IL-22, CXCL13 / BCA-1, IL-1 beta / IL-1F2, IL-10, IL-1alpha / IL-1F1, CCL2 / MCP-1, CCL3 / MIP-1 alpha, CCL5 / RANTES, IL-12P40, CCL4 / MIP-1beta, IFN-gamma, IFN-beta, G-CSF, CCL11 / Eotaxin, VCAM-1 / CD106, GM-CSF, CXCL1 / GROalpha, EGF, TNF-alpha, and Acrp30 in the sample.

[0013] Before the antibody is coupled to the microspheres in step (1), the total concentration of the n-type capture microsphere mixture needs to be adjusted to 1,000,000 particles / µL; after the coupling reaction, it is blocked at 37°C for 30 minutes with TBST buffer containing 1% BSA.

[0014] The specific operation of the thiolization treatment in step (2) is as follows: the detection antibody and 2-iminothiacyclopentane hydrochloride are reacted at a mass ratio of 35:9 at pH 8.0-8.5, 37°C, and 1200 rpm for 40 minutes. The reaction product is purified by a 10 kDa ultrafiltration tube and washed 3-4 times.

[0015] In step (2), the fluorescent protein reported is phycoerythrin. The phycoerythrin derivatization process involves reacting phycoerythrin with SMCC at a molar ratio of 1:75-1:150 at room temperature for 60 minutes, and then purifying the PE-SMCC derivative using a 10 kDa ultrafiltration tube.

[0016] In step (2), the molar ratio of PE-SMCC derivative to thiolized antibody is 1.2-1.5:1, and the F / P ratio of the conjugated product after NEM blocking is 1.0-2.0.

[0017] The freeze-drying protectant in step (3) contains 1% w / v BSA, 5-10% w / v trehalose and 0.1% v / v ProClin300; the freeze-drying process is as follows: first, pre-freeze at -20°C for ≥12 hours, then pre-freeze at -80°C for ≥30 minutes, and finally freeze-dry under vacuum for ≥20 hours.

[0018] The detection sensitivity of the kit is <10 pg / mL, and the linear range is 10-700000 pg / mL.

[0019] Furthermore, this invention proposes a non-diagnostic application of the multiplex cytokine detection kit prepared according to the above method in an iron overload-induced chronic inflammation model, comprising the following steps: (1) The sample to be tested is co-incubated with the microsphere-conjugated capture antibody mixture so that the cytokines in the sample are captured by the corresponding encoded microspheres; (2) After washing, a detection antibody labeled with a fluorescent reporter protein is added to form a complex of microspheres-capture antibody-cytokine-detection antibody; (3) Detection by flow cytometry, wherein the flow cytometry instrument needs to collect at least 3000 microspheres / factors, identify the type of microspheres by internally encoded fluorescence, and quantify the concentration of cytokines by PE fluorescence intensity.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The microsphere surface achieves efficient and targeted coupling with the captured antibody, improving antibody activity and retention rate. (1) The chemical binding of the captured antibody is more stable. Antibody fixation is achieved through the biotin-streptavidin system (Biotin-AB and streptavidin-coated microspheres), which has extremely strong binding affinity with an affinity constant Ka=10.15 At mol / L, this concentration is tens of thousands of times stronger than the antigen-antibody binding force, equivalent to the strength of a covalent bond. After three washes with TBST, the binding rate is >95%. The binding reaction is rapid, requiring low concentrations of reaction reagents, and can be used at high concentrations, reducing experimental costs.

[0021] Conventional carboxyglobulin-conjugated antibodies form amide bonds through EDC / NHS, which are easily affected by pH fluctuations in the buffer solution and by external conditions such as buffer solution, temperature, and mechanical vibration. The labeled antibody is prone to detachment, with a washing detachment rate of 20-30%. In contrast, the binding method used in this invention is more efficient and stable.

[0022] (2) The binding efficiency of the captured antibody is higher.

[0023] Biotinylated antibodies react with SA microspheres at a ratio of 10-25 μg / million microspheres, reaching saturation binding within 1 hour. Each streptavidin molecule simultaneously binds 4 biotinylated antibodies, forming a three-dimensional network structure. The reaction concentration of biotinylated antibodies can be diluted to 0.1 μg / mL while maintaining high binding efficiency, whereas carboxyl sphere coupling requires an antibody concentration >1 mg / mL to approach saturation. Therefore, the antibody reagent consumption of this invention is reduced by 10 times.

[0024] 2. High sensitivity, stemming from multi-stage signal amplification and high-density capture.

[0025] Biotinylated antibodies form a "microsphere-SA-(Biotin-Ab)4" complex via SA bridging. In this method, there are 4 antibodies / SA, while existing carboxylated sphere-coupled antibodies have 1 antibody / carboxyl site. Each SA microsphere's avidin molecule can simultaneously bind 4 biotin molecules, forming a multivalent complex. Furthermore, after each capture antibody binds to the sample antigen, it then binds to the PE-labeled detection antibody (F / P=2). A single antigen molecule ultimately generates 8 PE molecule signals, thus achieving multi-stage signal amplification through two-stage amplification, significantly improving detection sensitivity and making it suitable for the detection of trace substances.

[0026] 4. The reaction process is faster and the operation is simpler.

[0027] After mixing SA microspheres with Biotin-AB, the coupling is completed in just 1 hour, without the need for 3-4 additional steps such as EDC activation, washing, and quenching (which takes 4-6 hours). Moreover, detection antibodies can be prepared simultaneously. When finally used, a "microsphere-capture antibody-antigen-PE detection antibody" complex is formed. All reagents can be added once and incubated for 1.5 hours before being directly used on the instrument.

[0028] The carboxylated sphere method requires stepwise incubation to capture antibodies, washing, adding SA-PE, and washing again, with a total time exceeding 4 hours. In contrast, the method of this invention has a simpler testing procedure, allowing for faster completion of experiments and data acquisition.

[0029] 5. Lower cost.

[0030] The microsphere conjugation of this invention uses SA microspheres to specifically conjugate with biotin-labeled antibodies. Each avidin molecule can simultaneously bind four biotin molecules, forming a multivalent complex. Compared with conventional carboxylated microsphere conjugated antibodies, the amount of labeled antibody used can be greatly reduced, significantly lowering costs.

[0031] 6. Multiplex detection capability is significantly superior to single-factor ELISA. It can simultaneously detect 26 cytokines in a single well, requiring only 25 μL of sample, saving 90% of sample and 80% of operation time compared to traditional ELISA. In an iron overload model, it successfully detected factors such as IL-6 and CCL5 within a wide dynamic range from 10,000 to 700,000 pg / mL, with a linear correlation coefficient R0. 2 >0.99, improving detection efficiency by more than 10 times.

[0032] Therefore, the kit prepared by this method shows significant and synergistic benefits in terms of antibody conjugation efficiency, detection sensitivity, standard stability, ease of operation, and cost-effectiveness. It provides a complete, reliable, and industrializable technical solution for multiplex cytokine detection, and has clear innovativeness and market application value. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the detection process for the standard protein in Example 2; Figure 2 Set / calibrator concentration values ​​for the standard curves of the 26 factors in Example 2; Figure 3 The standard curve MFI values / calibrator test MFI values ​​for the 26 factors in Example 2; Figure 4 This is a standard curve fitting graph for the 26 factors in Example 2; Figure 5 This is a scatter plot showing the test locations of the 26 factors in Example 2; Figure 6 The results are from blood tests conducted in week 3 of the mouse chronic iron overload model in Example 3. Detailed Implementation

[0034] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0035] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] Example 1 This embodiment provides a method for preparing capture antibodies and detection antibodies in immunoassay. The specific preparation process is as follows: I. Preparation of microsphere-conjugated capture antibodies.

[0037] 1. Antibody labeling Biotin (1) Antibody pretreatment. Take 50 micrograms of antibody (1 mg / mL, 50 uL) and use sodium bicarbonate buffer to prepare the antibody to 1 mg / mL.

[0038] (2) Biotin pretreatment and labeling. Take 2 mg / mL of Biotin-X SE (Biotin-X succinimide ester) and store it in an ultra-low temperature freezer. Take out one tube of Biotin-X SE (Biotin-X succinimide ester) solution, thaw it, and vortex it to mix. Take out 2 μL and add it to the antibody solution in step (1). Discard the remaining Biotin-X SE. React at room temperature for 2 h or at 4°C overnight.

[0039] (3) Biotin-AB ultrafiltration. The overnight labeled antibody from step (2) was transferred to a 0.5 mL ultrafiltration tube, and 300 μL of TBST was added to the reaction tube to wash the reaction tube and the solution was transferred to the ultrafiltration tube. The tube was centrifuged at 12,500 rpm, and finally the biotinylated antibody solution in the ultrafiltration tube was transferred to a 1.5 mL centrifuge tube.

[0040] (4) Biotin-AB storage. Take out 100uL of biotinylated antibody Biotin-AB from step (3), add TBST (containing 1% BSA) to 100uL to obtain 0.25 mg / mL of biotinylated antibody, and store it at 4 degrees for later use.

[0041] 2. Microsphere coupling with Biotin-AB (1) Take SA microspheres. Vortex the fluorescently coded SA microsphere suspension and transfer 500,000 magnetic fluorescently coded microspheres (10,000 microspheres / µL, 50µL) into a centrifuge tube using a pipette. The SA microspheres must be thoroughly vortexed before taking them; each vortexing should last 1.5 minutes. The fluorescently coded microspheres used in this step are Thermofish.

[0042] (2) Add 0.25 μg of biotinylated antibody Biotin-AB (0.25 mg / mL, 1 µL) to the tube containing the prepared SA microspheres, and shake at 37°C and 1200 rpm for 1 h. A microsphere SA-Biotin-AB complex is formed, and the four biotin binding sites of SA bind to the four Biotin-AB molecules.

[0043] (3) Add 1 μL of Biotin-X SE and incubate for 1 minute, then immediately perform magnetic separation for 1.5 minutes. Discard the supernatant and wash 5 times with TBST. While the microspheres are magnetically attracted, the supernatant is also attracted. Be careful not to touch the microspheres to avoid loss. The purpose of this step is to seal the microspheres.

[0044] (4) Finally, disperse the microspheres in 50 µL TBST (containing 1% BSA and 0.01% MB-1) and store at 4°C. Note that the coupled microspheres can only be stored at 4°C. During the above preparation process, the antibody retention rate is above 85%.

[0045] II. Preparation of fluorescein-conjugated antibodies in the detection system.

[0046] 1. PE-SMCC Derivatization Modification Succinimide-4-(n-methylmaleimide)cyclohexane-1-carbonate (SMCC) was dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 10 mg / ml stock solution. Phycoerythrin (PE) was then reacted with SMCC, sealed in aluminum foil, and rotated at room temperature for 60 minutes. This reaction allowed the amino groups on the phycoerythrin molecules to react with succinamide to generate derivatized phycoerythrin PE-SMCC, which was used for subsequent coupling with thiolized molecules. The specific steps are as follows: (1) Preparation of PE solution: Weigh 1.0 mg of PE powder, add 500 μL of PBS buffer, gently blow until completely dissolved, with a final concentration of 2 mg / mL, and then add 40 μL of 1M NaHCO3 solution dropwise to mix thoroughly.

[0047] (2) Prepare SMCC solution by weighing 10 mg of SMCC-NHS and dissolving it in 1 mL of anhydrous dimethyl sulfoxide (DMSO) and vortexing it thoroughly (10 mg / mL, prepare and use immediately). (3) Take 7.3 μL of the SMCC solution obtained in step (2) and add it to the centrifuge tube in step (1) under vortex. Shake at 37 degrees and 1200 rpm for 1 h.

[0048] (4) Transfer the reaction solution from step (3) to a 10KD ultrafiltration tube (Millipore 0.5 mL), centrifuge at 12500 rpm for 7 minutes and discard the filtrate in the bottom tube. Wash repeatedly with TBST 10 times. The specific washing method is as follows: use a pipette to add 0.35 mL of TBST suspended in the ultrafiltration tube (note that the pipette should not touch the ultrafiltration tube), centrifuge at 12500 rpm for 7 minutes and discard the filtrate in the bottom tube to remove free SMCC.

[0049] (5) Transfer the purified PE-SMCC concentrate in the ultrafiltration tube to a new 1.5 mL centrifuge tube and add TBST to 200 uL. The concentration of PE-SMCC is 2 mg / mL.

[0050] 2. Antibody thiolation This step involves introducing free thiol groups (-SH) onto the surface of the antibody molecule using 2-iminothiocyclopentane hydrochloride (2-IT), enabling site-specific coupling with maleimide-modified PE (PE-SMCC). The specific steps are as follows: Preparation of antibody solution: (1) Take 50 μg of antibody lyophilized powder and add it directly to 50 μL of PBS. Let it stand at 4°C for 5 minutes to dissolve it, and the final concentration is 1 mg / mL.

[0051] pH adjustment of the buffer solution: (2) Add 15 μL of 0.1 M NaHCO3 solution; (3) Add 5 μL of 0.1 M EDTA solution. At this point, the final volume of the antibody mixture is 70 μL, and the antibody concentration is approximately 0.71 mg / mL (50 μg / 70 μL).

[0052] Thiolization reaction: (4) Prepare a fresh 2-IT solution by weighing 6 mg of 2-iminothiacyclopentane hydrochloride and dissolving it in 1 mL of PBS (prepare immediately before use). (5) Take 4 μL of fresh 2-IT solution and add it dropwise to the antibody mixture obtained in step (3) above. Shake at 37 degrees and 1200 rpm for 40 min.

[0053] Purification and desalting: (6) Transfer the reaction solution from step (5) above to a 10KD ultrafiltration tube (Millipore 0.5 mL), centrifuge at 12500 rpm and 4 degrees Celsius for 5 minutes, and discard the filtrate in the bottom tube. Then wash repeatedly with TBST 10 times. The specific washing method is as follows: use a pipette to add 0.35 mL of TBST containing 10 mM EDTA into the ultrafiltration tube (note that the pipette should not touch the ultrafiltration tube), centrifuge at 12500 rpm for 5 minutes, and discard the solution in the bottom tube.

[0054] Product recycling: (7) After washing, invert the ultrafiltration tube into a new collection tube and measure the antibody concentration. The target value for controlling the antibody concentration in this step is between 0.5-1 mg / mL. If the concentration is too high, dilute it to the target concentration with washing buffer; if the concentration is too low, continue to concentrate it with the ultrafiltration tube. Store the final product at 4°C protected from light (best results are achieved within 24 hours).

[0055] 3. Antibody-conjugated PE-SMCC This step involves coupling a thiolated antibody with maleimide-activated PE (PE-SMCC) via a specific thioether bond to prepare a fluorescently labeled antibody for detection in flow cytometry and other methods. The specific steps are as follows: (1) Coupling reaction: Take 50 μg of the purified thiolized antibody (confirm its concentration is between 0.5-1 mg / mL) and transfer it to a 1.5 mL centrifuge tube (if the concentration is 0.8 mg / mL, take 62.5 μL). Add 50 μL of PE-SMCC (2 mg / mL, containing 100 μg of PE) under vortex conditions. React at 37 degrees Celsius and 1200 rpm for 0.5 h. Then transfer to room temperature (RT, about 25°CCT) and let it stand overnight (12-16 hours). Control the reaction time to avoid PE degradation due to excessive time.

[0056] (2) Thiol group blocking: After the above reaction is completed, add 1 μL NEM (20 mg / mL in DMSO) directly into the reaction tube and shake at 1200 rpm for 1 h at 37 degrees Celsius to block the unreacted thiol groups.

[0057] After the reaction, the solution was purified to remove uncoupled PE-SMCC. The reaction solution was transferred to a 50 kDa ultrafiltration tube and centrifuged at 12500 rpm at 4°C for 5 minutes. The filtrate was discarded. The solution was washed 4 times, with 0.4 mL of blocking / preservation buffer added each time, and centrifuged for 5 minutes. The solution was then recovered by inverting the tube and centrifuging at 1000 rpm for 2 minutes to collect the concentrate (approximately 50 μL).

[0058] (3) Product volume adjustment and storage: Finally, add 100uL TBST (containing 1% BSA and 0.1% proclin 300) to adjust the volume to 0.4 mg / mL (based on antibody).

[0059] In the above preparation process, microsphere-conjugated antibodies that specifically bind to the test sample (antigen) and fluorescein-conjugated detection antibodies that can generate detection signals and bind to antibodies in the capture system are obtained. These two products can be used to analyze the composition and corresponding concentration of the test sample.

[0060] Example 2 This embodiment provides a method for preparing mixed protein standards for immunoassay, as detailed below.

[0061] 1. Based on the detection requirements of the 26 proteins identified in Table 1 below, prepare the relevant raw material proteins and formulate the corresponding concentrations.

[0062] Table 1 2. Production Calculation: Case 1: Preparing standard products from stock solutions.

[0063] We have a 1.0 mg / mL protein stock solution and need to produce 100 lyophilized standard vials. Each standard vial contains 20 ng of protein and has a volume of 100 μL.

[0064] The calculation process for repackaging is as follows: (1) Total amount calculation: 20ng / vial × 100 vials = 2000 ng = 2μg, thus obtaining the total protein requirement.

[0065] (2) Take the volume of the mother liquor, 2μg÷(1μg / μL) = 2μL, that is, take a small volume from the concentrated mother liquor.

[0066] (3) Final volume preparation: 100μL / tube × 100 tubes = 10mL, which is the total amount of liquid dispensed into each lyophilized tube.

[0067] (4) Calculation of dilution solution: 10 mL - 0.002 mL = 9.998 mL. Add protective agents such as BSA and trehalose to the obtained diluted standard lyophilized solution.

[0068] The physical significance of the above preparation process is that 2 μL of high-concentration mother liquor is diluted to a 10 mL system and then dispensed into 100 lyophilized portions.

[0069] Case 2: Determine the output based on existing raw materials.

[0070] With 60 ng of protein raw material, a working solution is prepared by adding 200 uL of PBS. Based on a protein content of 1000 pg / vial and a standard volume of 100 μL / vial, 60 standard samples can be prepared.

[0071] The calculation process for repackaging is as follows: (1) Prepare the concentration: 60 ng ÷ 200 μL = 0.3 ng / μL, which is the working solution concentration.

[0072] (2) Number of refillable vials: 60 ng ÷ 1 ng / vial = 60 vials, 1000 pg / vial = 1 ng / vial.

[0073] (3) The required volume is 100μL / vial × 60 vials = 6 mL, which means the final volume is the volume of the lyophilized liquid containing the preservative.

[0074] The physical significance of the above preparation process is that the existing 60 ng protein raw material can be used to produce a maximum of 60 vials, with each vial of freeze-dried standard having a protein concentration of 1 ng / 100 μL / vial.

[0075] 3. Standard product preparation and packaging ① Prepare dispensing tools: sterilization pipette tip, vacuum lyophilization tube, 1.5 mL EP tube or 2 mL EP tube, 45 mL EP tube, standard lyophilized solution, pipette, test tube holder.

[0076] ② Locate the standard protein to be dispensed in the raw material inventory, calculate the volume of protein to be taken out and the volume to be prepared according to the production plan, transfer the corresponding volume into a 45 mL EP tube with a pipette, and mix it repeatedly by blowing and swirling with a pipette.

[0077] ③ After mixing thoroughly, the standard protein solution is dispensed into 100 μL vials. If high-concentration standards are available, attention should be paid to the appropriate concentration and dilution should be performed in stages.

[0078] After completing the aliquoting of the standard protein, place the aliquoted protein into a lyophilization box and affix a label to the outside of the lyophilization box. The label should include the following information: code, name, aliquoting date, quantity, etc.

[0079] ④ Freeze-drying of standard products: Place the packaged standard products in a -20℃ freezer overnight, and the next morning place them in an -80℃ ultra-low temperature freezer for pre-freezing for more than 30 minutes. Then, take them out and put them into a vacuum freeze dryer to start freeze-drying. The vacuum time is ≥20 hours. Do not cover the product during the entire process.

[0080] In this embodiment, the production of the mixed protein is an independent quantitative benchmark preparation step, which, together with the microsphere-conjugated antibody and the fluorescein-conjugated antibody, constitutes a complete detection system. Its core value lies in converting the fluorescence signal into concentration units (pg / mL or ng / mL), an essential step in upgrading the kit from qualitative to quantitative analysis. Case 1 represents the conventional production model, while Case 2 represents the inventory assessment model; the two models complement each other logically.

[0081] Using the raw material proteins listed in Table 1 and the method described in Example 1, capture and detection systems for 26 corresponding proteins were obtained. Simultaneously, calibrators for the 26 proteins were prepared according to the above method. Figure 2 The standard curve shown contains 26 proteins. Following... Figure 1 The procedure shown is used to perform the detection process for standard proteins and obtain... Figures 3 to 5 The detection results indicate that 26 factors were detected simultaneously with a linear range covering 10. 6 The results demonstrate that the microspheres are well-differentiated and free from cross-interference; they remain linear at high concentrations without signal quenching; low-expression factors (such as IL-1β, M. CCL2, and IL-12 p70) are detectable, and the sensitivity meets the standard; the data volume is reasonable (pg / mL) and can be used for subsequent standard curve conversion, indicating quantitative accuracy.

[0082] Example 3 The capture system and detection system obtained in Example 1, and the mixed protein standard obtained in Example 2 were assembled into an immunoassay kit. A mouse immunoassay model was established to evaluate the specificity and sensitivity of the kit constructed by this method.

[0083] I. Construction of a mouse model of chronic iron overload: 1. Laboratory animals and grouping Animals: Eighteen C57BL / 6 mice aged 6-8 weeks were selected. They were randomly divided into two groups: a model group (n=9 mice) which received intraperitoneal injection of iron dextran, and a control group (n=9 mice) which received intraperitoneal injection of the same volume of physiological saline.

[0084] Dynamic observation points: Three mice from each group were sacrificed at weeks 3, 5, and 7.

[0085] The number of repetitions of the above-mentioned in vitro cell experiments can be routinely adjusted based on the stability of the detection method and the expected effect.

[0086] In the control group, three biological replicates (control 1, control 2, and control 3) were set up to meet the statistical significance requirement (p<0.05); three technical replicates were set up in each control group to improve the baseline accuracy.

[0087] In the experimental groups, three biological replicates (experimental group 1, experimental group 2 and experimental group 3) were set up to meet the statistical significance requirement (p<0.05), and two technical replicates were set up in each experimental group to meet the statistical significance requirement (p<0.05). This design falls within the scope of routine experimental optimization in this field.

[0088] 2. Modeling scheme Iron dosage: Iron dextran was dissolved in physiological saline and the concentration was adjusted to 200 mg / mL. Each mouse was injected with 50 mg / kg of iron dextran according to its body weight.

[0089] Injection frequency and cycle: Injections were administered weekly for 3 weeks to establish a chronic iron overload model. This modeling method established a chronic inflammatory and fibrotic environment in the mouse liver.

[0090] 3. Model Indicator Validation After modeling, each group was analyzed at weeks 3, 5, and 7. Under this environment, macrophages in the liver are activated and polarized.

[0091] II. Sample Collection and Processing (1) Serum sample Collection: Blood was collected from mice via tail vein puncture. After proper disinfection, the vein was punctured, and the blood was allowed to flow naturally into the collection tube (avoiding forceful squeezing to prevent hemolysis). Blood volume: 0.5-1 mL.

[0092] Procedure: Place the blood collection tube upright at room temperature (20-25℃) for 30-60 minutes to allow the blood to coagulate naturally.

[0093] Storage: After aliquoting, quickly place the serum in a -20°C freezer to avoid repeated freeze-thaw cycles (repeated freeze-thaw cycles can lead to protein denaturation and loss of activity).

[0094] III. Reagent Kit Evaluation 1. Test kits and instruments The composition of the reagent kit is shown in Table 2.

[0095] Table 2 Instruments and equipment: constant temperature oscillator, vacuum pump, flow cytometer.

[0096] 2. Testing Procedure (96-well plate) Step 1: Add sample (25 μL of standard / sample).

[0097] (1) Gradient preparation of standard proteins Take the highest concentration standard of 10000 pg / mL and serially dilute it 2-fold in a 1.5 mL EP tube: Tube 1: 10000 pg / mL (stock solution); Tube 2: 5000 pg / mL (take 100 μL stock solution + 100 μL diluent); Tube 3: 2500 pg / mL (take 100 μL of tube 2 + 100 μL of diluent); Tube 4: 1250 pg / mL (take 100 μL of tube 3 + 100 μL of diluent); Tube 5: 625 pg / mL (take 100 μL of tube 4 + 100 μL of diluent); Tube 6: 312.5 pg / mL (take 100 μL of tube 5 + 100 μL of diluent); Tube 7: 156.25 pg / mL (take 100 μL of tube 6 + 100 μL of diluent); Tube 8: 78.125 pg / mL (take 100 μL of tube 7 + 100 μL of diluent).

[0098] (2) Sampling operation Using a multi-channel pipette (8 channels) or a single-channel pipette, slowly add 25 μL of standard / sample per well along the wall.

[0099] Avoid air bubbles: If air bubbles form, puncture them with a clean needle. Layout record: Mark the sample positions on the 96-well plate cap, take a photo for archiving (25 μL of standard / sample).

[0100] Step 2: Add the mixture of capture antibody-conjugated microspheres.

[0101] Add 10 μL of the mixture of capture antibody-conjugated microspheres (5000 particles of each factor) quickly to each well of a 96-well plate. Mix immediately after addition and premix by shaking at 800 rpm for 30 seconds.

[0102] First incubation: Seal the 96-well plate with a sealing film, transfer it to a constant temperature shaker, and incubate at 37°C, 1200 rpm, in the dark for 1 hour.

[0103] Step 3, First Wash. Place the 96-well plate in a plate centrifuge and centrifuge at 1500 rpm and 437°C for 5 minutes. The microspheres will precipitate at the bottom of the V-shaped wells, appearing as small pink clumps. Discard the supernatant. Using a multichannel pipette, quickly add 200 μL / well of pre-cooled TBST, adding it along the wall to avoid directly impacting the microsphere precipitate. After adding the liquid, shake at 800 rpm for 30 seconds to disperse the microspheres. Repeat the washing process three times, for a total of 3 cycles (centrifugation supernatant, discarding the liquid, adding the liquid).

[0104] Step 4: Add fluorescein-conjugated detection antibody.

[0105] Quickly add 10 μL / well using a multi-channel pipette until it reaches the middle of the well wall. Avoid air bubbles; if bubbles do form, do not pop them with the pipette tip (this will remove the antibody); allow them to stand for 1 minute to break naturally. Mix immediately and shake at 800 rpm for 30 seconds.

[0106] Second incubation: Replace with a new sealing film, transfer to a constant temperature shaker, and incubate at 37°C, 1200 rpm, in the dark for 30 hours.

[0107] Step 5, final wash. The procedure is the same as in step 3.

[0108] Step 6: Flow cytometry analysis. The MFI value of each microsphere subpopulation is obtained using flow cytometry, and then the concentration of the corresponding protein factor is calculated by substituting it into the standard curve equation.

[0109] 2. Evaluation Indicators like Figure 6 As shown, after sampling at week 3, 11 mouse cytokines were analyzed. M.CCL5 (chemokine) had the highest concentration, reaching 600,000 pg / mL, indicating macrophage and T cell recruitment and iron overload-induced activation of the inflammatory network. M.IL-6 (a pro-inflammatory core factor) had the second highest concentration, indicating an acute phase response, driving liver fibrosis, and interacting with hepcidin. M.IL-1α (alarm hormone) and MG-CSF (granulocyte colony-stimulating factor) also increased accordingly, reaching 200,000-300,000 pg / mL, indicating that hepatocellular injury releases M.IL-1α, activating Kupffer cells.

[0110] The test results showed that iron overload induces a tumor immune environment at various stages, which is consistent with the characteristics of chronic inflammation progressing to fibrosis.

[0111] 3. Reagent kit performance The above test results demonstrate that: simultaneous detection of 11 factors with a linear range covering 10... 5 The results demonstrate that the microspheres are well-differentiated with no cross-interference; they remain linear at high concentrations without signal quenching; low expression factors (such as IL-12p70) can be detected with adequate sensitivity; and the data volume (pg / mL) is reasonable and can be used for subsequent standard curve conversion, indicating quantitative accuracy.

[0112] The above embodiments should be understood as being used only to illustrate the present invention more clearly, and not to limit the scope of the present invention. After reading the present invention, any modifications of the present embodiments by those skilled in the art will fall within the scope defined by the appended claims.

Claims

1. A method for preparing a multiplex cytokine detection kit for tumor immunotherapy, characterized in that, The preparation steps include the following: (1) Preparation of microsphere-coupled capture antibodies: n capture antibodies are biotinylated to obtain n biotinylated antibodies; fluorescently encoded microspheres with streptavidin covalently coupled to the surface are incubated with the n biotinylated antibodies respectively, so that each capture antibody is coupled one-to-one with microspheres with different internal fluorescent codes to form a mixture of n microsphere-coupled capture antibodies, and each capture antibody specifically recognizes one cytokine; (2) Preparation of detection antibodies: Thiolizing n detection antibodies to obtain thiolated antibodies, and then incubating the derivatized reporter fluorescent protein with the thiolated antibodies in the dark and then blocking them to form n fluorescently labeled detection antibody solutions; (3) Preparation of mixed protein standards: Mix n kinds of cytokines with lyophilization protection solution to obtain lyophilized powder containing n kinds of cytokines; (4) Kit assembly: Assemble the microsphere-conjugated capture antibody, detection antibody, mixed protein standard, standard diluent, sample diluent and washing buffer into a complete kit; The fluorescently encoded microspheres are a combination of fluorescently encoded microspheres with different energy levels formed by mixing two fluorescent dyes in different concentration ratios. The n=26, and the 26 capture antibodies and detection antibodies specifically recognize the following cytokines: IL-12 p70, IL-6, IL-5, IL-2, IL-4, IL-17 / IL-17A, IL-22, CXCL13 / BCA-1, IL-1 beta / IL-1F2, IL-10, IL-1 alpha / IL-1F1, CCL2 / MCP-1, CCL3 / MIP-1 alpha, CCL5 / RANTES, IL-12P40, CCL4 / MIP-1beta, IFN-gamma, IFN-beta, G-CSF, CCL11 / Eotaxin, VCAM-1 / CD106, GM-CSF, CXCL1 / GROalpha, EGF, TNF-alpha, and Acrp30.

2. The preparation method according to claim 1, characterized in that, The specific operation of biotin labeling in step (1) is as follows: the capture antibody and biotin-X succinimide ester are reacted at a molar ratio of 85:1 at pH 8.0-8.5 and room temperature for 2 hours or overnight at 4°C. The reaction product is purified by ultrafiltration, and the ultrafiltration washing is performed 3-4 times.

3. The preparation method according to claim 1, characterized in that, Before the antibody is coupled to the microspheres in step (1), the total concentration of the n-type capture microsphere mixture needs to be adjusted to 1,000,000 particles / µL; after the coupling reaction, it is blocked at 37°C for 30 minutes with TBST buffer containing 1% BSA.

4. The preparation method according to claim 1, characterized in that, The specific operation of the thiolization treatment in step (2) is as follows: the detection antibody and 2-iminothiacyclopentane hydrochloride are reacted at a mass ratio of 35:9 at pH 8.0-8.5, 37°C, and 1200 rpm for 40 minutes. The reaction product is purified by a 10 kDa ultrafiltration tube and washed 3-4 times.

5. The preparation method according to claim 1, characterized in that, In step (2), the fluorescent protein reported is phycoerythrin. The phycoerythrin derivatization process involves reacting phycoerythrin with SMCC at a molar ratio of 1:75-1:150 at room temperature for 60 minutes, and then purifying the PE-SMCC derivative using a 10 kDa ultrafiltration tube.

6. The preparation method according to claim 5, characterized in that, In step (2), the molar ratio of PE-SMCC derivative to thiolized antibody is 1.2-1.5:1, and the F / P ratio of the conjugated product after NEM blocking is 1.0-2.

0.

7. The preparation method according to claim 1, characterized in that, The freeze-drying protectant in step (3) contains 1% w / v BSA, 5-10% w / v trehalose and 0.1% v / v ProClin 300; the freeze-drying process is as follows: first, pre-freeze at -20°C for ≥12 hours, then pre-freeze at -80°C for ≥30 minutes, and finally freeze-dry under vacuum for ≥20 hours.

8. The detection kit obtained by the preparation method according to any one of claims 1-7, characterized in that, The detection sensitivity of the kit is <10 pg / mL, and the linear range is 10-700000 pg / mL.

9. The non-diagnostic application of a multiplex cytokine detection kit prepared by the method according to any one of claims 1-7 in an iron overload-induced chronic inflammation model, characterized in that, include: (1) The sample to be tested is co-incubated with the microsphere-conjugated capture antibody mixture so that the cytokines in the sample are captured by the corresponding encoded microspheres; (2) After washing, a detection antibody labeled with a fluorescent reporter protein is added to form a complex of microspheres-capture antibody-cytokine-detection antibody; (3) Detection by flow cytometry, wherein the flow cytometry instrument needs to collect at least 3000 microspheres / factors, identify the type of microspheres by internally encoded fluorescence, and quantify the concentration of cytokines by PE fluorescence intensity.

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