CN116027048A - Cytokine storm syndrome diagnosis kit and application thereof - Google Patents
Cytokine storm syndrome diagnosis kit and application thereof Download PDFInfo
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- CN116027048A CN116027048A CN202310067903.0A CN202310067903A CN116027048A CN 116027048 A CN116027048 A CN 116027048A CN 202310067903 A CN202310067903 A CN 202310067903A CN 116027048 A CN116027048 A CN 116027048A
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Abstract
The invention provides a cytokine storm syndrome diagnosis kit and application thereof, wherein the kit comprises 4 biotin-labeled detection antibodies of inflammatory factors TNF-alpha, IL-6, PGE2 and PAF. The kit can be used for diagnosing the cytokine storm syndrome through the combined quantitative detection of 4 inflammatory factors, has the advantages of comprehensive index coverage, high sensitivity, good specificity and the like, and can improve the accuracy of the cytokine storm syndrome diagnosis through the combined detection of 4 inflammatory factors, and the clinical use is more convenient and faster.
Description
Technical Field
The invention belongs to the technical field of biological diagnosis, and particularly relates to a cytokine storm syndrome diagnosis kit and application thereof.
Background
In many diseases, including influenza, sepsis or parts of the case of immunotherapy, a special class of clinical syndrome may occur, namely rapid high production of various cytokines in the body fluids with fever, hypotension, hypoxia, a series of clinical and experimental symptoms called cytokine release syndrome, also called cytokine storm (cytoki ne storm).
Cytokine storm-related cytokines are released by immune cells when activated, interacting with target cells. Related cytokines (interleukins, interferons, etc.) are mainly derived from activated immune cells and re-act on the activation of immune cells (including T-cells, monocytes, macrophages, etc.), triggering an acute inflammatory response to induce epithelial and tissue damage, leading to microvascular leakage, heart failure, and even death. The phenomenon of rapid and massive production of multiple cytokines is also a significant cause of acute respiratory distress syndrome and multiple organ failure. The detection of cytokine storm-related cytokines in patient serum samples before serious side effects occur has important guiding significance for clinical diagnosis and treatment.
According to the literature, increases in serum, such as partial cytokine levels, have a clear correlation with the occurrence of cytokine storms in patients. For example, chinese patented invention patent CN112034187B discloses a set of markers for predicting 2019 coronavirus cytokine release syndrome and thrombostorm, and uses thereof, kits and preparation methods of kits, the markers being VEGF D, tnfα, SCF, LI F, I L2, I L4, I L6, I L8, I L10, I L15, I L17A, I L18, I L1 β and ifnγ, respectively. The VEGF D, TNF alpha, SCF, LI F, I L2, I L4, I L6, I L8, I L10, I L15, I L17A, I L18, I L1 beta, I FN gamma or a kit prepared by the kit provided by the invention is used for predicting 2019 coronavirus (COVI D19) cytokine release syndrome and thrombotic storm, and has the advantages of rapid and accurate detection, low cost and the like, and has wide application prospect. However, the above patent relates to the detection of single relevant indexes, which can only reflect a certain aspect of the cytokine storm, lack of comprehensiveness, and can not accurately diagnose the cytokine storm, and the highest prediction accuracy is only 83.6%.
In view of the above problems, the chinese patent application CN107870233a discloses a detection of cytokine storm-related cytokines, which is a prognostic detection index combination for performing immunotherapy or other diseases producing cytokine storm. The cytokine combination covers 9 indexes (IFN-gamma, TNF-alpha, IL-2R, IL-6, IL-10, GM-CSF, CRP, MIP-1 alpha and MIP-1 beta) capable of detecting the cytokine storm, high-sensitivity detection of serum trace samples can be realized by a solid-phase chip/liquid-phase chip/ELISA kit method, and the sensitivity of the cytokine storm diagnosis can reach 90% and the specificity can reach 100% by the joint detection of the 9 indexes and drawing an ROC curve.
However, the above detection requires simultaneous detection of 9 indexes, and has the defects of high operation difficulty and high detection cost. Therefore, the invention can accurately diagnose the cytokine storm, reduce the diagnosis cost and improve the diagnosis efficiency by optimizing the index for detecting the cytokine storm.
Disclosure of Invention
The invention mainly provides a cytokine storm syndrome diagnosis kit and application thereof. In order to achieve the purpose of the invention, the following technical scheme is adopted:
in one aspect, the invention relates to a cytokine storm syndrome diagnostic kit comprising biotin-labeled detection antibodies for 4 inflammatory factors TNF-alpha, IL-6, PGE2 and PAF.
In a preferred embodiment of the invention, the kit is a solid phase chip, a liquid phase chip or an ELISA kit.
In a preferred embodiment of the invention, the kit can quantitatively detect the levels of the 4 inflammatory factors TNF- α, IL-6, PGE2 and PAF in serum.
In a preferred embodiment of the present invention, the kit does not include detection antibodies for other inflammatory factors than the 4 inflammatory factors described above.
Another aspect of the invention relates to a cytokine storm syndrome diagnostic marker comprising 4 inflammatory factors TNF- α, IL-6, PGE2 and PAF.
In a preferred embodiment of the invention, the diagnostic marker does not comprise other inflammatory factors.
The invention also relates to application of the kit or the marker in preparation of a cytokine storm syndrome diagnosis kit.
The invention also relates to application of the kit or the marker in preparation of a kit for diagnosing the predicted cytokine storm syndrome.
The beneficial effects of the invention are that
The kit can be used for diagnosing the cytokine storm syndrome through the combined quantitative detection of 4 inflammatory factors, has the advantages of comprehensive index coverage, high sensitivity, good specificity and the like, and can improve the accuracy of the cytokine storm syndrome diagnosis through the combined detection of 4 inflammatory factors, and the clinical use is more convenient and faster.
Drawings
FIG. 1 is a graph showing the results of cytokine storm-related factor indicators (based on 100% of the average value of the monitoring results of healthy volunteers as a calculation basis);
fig. 2 is a graph of analysis of the joint index ROC curve.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments in order to make the objects, technical solutions and advantages of the present invention more apparent.
Example 1
1. Solid phase chip for cytokine storm diagnosis
Chips comprising detection of TNF-alpha (tumor necrosis factor-alpha), IL-6 (interleukin-6), PGE2 (prostaglandin E2) and PAF (platelet activating factor) indicators, antibodies capturing the indicators being spotted on the chips;
4 biotin-labeled detection antibodies corresponding to TNF- α, IL-6, PGE2 and PAF;
streptavidin-fluorescence (SA-Cy 3);
mating reagents: standard mixture, sample diluent, sealing liquid, eluent I and eluent I I;
other equipment consumables: centrifuge tube, centrifuge at 4deg.C, normal temperature centrifuge, enzyme labeling instrument, vortex oscillator, shaker, chip small-sized centrifuge, chip scanner, software GenePix Pro 6.0.
2. The solid phase chip is used for detecting the 4 index combinations
Preparation before chip test
1. All reagents were removed from the-80 ℃ refrigerator in advance and allowed to equilibrate to room temperature.
2. Sample composition and preparation
Sample composition: serum was collected in standardized fashion in 35 healthy subjects, 70 subjects receiving immunotherapy.
Sample preparation: serum samples were centrifuged at 10,000rpm and 15 min at 4℃to obtain the supernatant. After centrifugation the samples were diluted with sample according to 1:1 dilution to a final volume of 100 uL.
3. Standard preparation
To the cytokine standard mixture was added 500 μl of sample dilution and gently mixed to dissolve the powder thoroughly. Tubes were labeled Std1. The 6 clean empty centrifuge tubes were labeled Std2-Std7. 200. Mu.L of sample diluent was added to each tube. 100. Mu.L of Std1 was pipetted into Std2 and gently mixed. Further 100 μl was aspirated from Std2 to Std3, and so on. Another centrifuge tube was added with 100. Mu.L of sample dilution, labeled CNTRL. No cytokine standard or sample was added to CNTRL as a negative control.
(II) chip detection operation
1. Sample incubation
The solid phase chip was taken out of the refrigerator at 4℃and dried for 2 hours at room temperature before use.
100. Mu.L of blocking solution was added to each well and the wells were blocked at room temperature for 30 min.
2. Sample hybridization and washing
Completely removing the blocking fluid in each well; 100 μl of diluted corresponding standard and sample was added to each well, followed by adhesive tape and placed on a horizontal shaker at 4deg.C overnight.
The sample in the well was removed completely, 150. Mu.L of eluent I was added, and washed 5 times with 2 min each at room temperature with gentle shaking.
The chip was placed in a washing box, and a sufficient amount of eluent I was added, followed by washing with shaking at room temperature for 2 times, 5 min each time.
The eluent I was removed completely, 150. Mu.L of eluent I I was added and washed 2 times with 5 min each at room temperature with gentle shaking.
Taking out the biotin-labeled detection antibody, adding 1400 mu L of sample diluent for dilution after short centrifugation, and mixing by light shaking. 80. Mu.L of the diluted biotin-labeled detection antibody was added to each well, and incubated at room temperature for 2hr.
The biotin-labeled detection antibody was removed completely from each well and washed in steps 2-4.
Taking out streptavidin-fluorescence, after short centrifugation, adding 1400 mu L of blocking solution, and mixing by light shaking.
Adding 80 μl of streptavidin-fluorescence into each well, attaching adhesive tape, and incubating at room temperature for 2hr in dark place with aluminum foil.
Streptavidin-fluorescence was removed completely from each well and washed as per step 2.
3. Detection of
The eluent I was completely removed and the chip was removed from the frame.
A sufficient amount of eluent I was added to the washing cassette and washed with gentle shaking at room temperature for 2 times, 10min each.
The eluent I was removed, a sufficient amount of eluent II was added to the washing cassette and washed with gentle shaking at room temperature for 10min.
Eluent II was removed, a sufficient amount of Milli-Q water was added to a 30mL centrifuge tube and washed.
Centrifugal at 1000rpm for 3min, spin-dry the chip.
(III) chip data acquisition
After the sample and the standard substance detected in the experiment are detected by a scanner, the fluorescence obtained by detection is automatically calculated and optimized by software GenePix Pro 6.0 to form an output file.
(IV) data analysis and result display
1. Standard curve
Fitting a logarithmic standard curve according to the signal values detected by the standard substance, wherein in the experiment, standard curves R of 4 indexes 2 > 0.95, indicating a good fit of the standard curve.
2. Cytokine storm related index data analysis for immunotherapy patients
Comparison and statistical analysis of cytokine storm related indicators of 70 immunotherapy patients and 35 healthy volunteers showed that 42 of 70 patients developed symptoms after 1 week, cytokine storm related cytokines were significantly different (P < 0.05) from healthy control group and other 28 patients not developed symptoms, and expression level was significantly increased, as shown in fig. 1.
3. Evaluation of cytokine storm-related factor diagnostic Effect
By the joint detection of 4 indexes, the sensitivity of the cytokine storm diagnosis can reach 92% and the specificity can reach 100% by drawing an ROC curve, as shown in figure 2.
The experimental results show that the risk of the occurrence of the cytokine storm can be judged by detecting the change of 4 indexes related to the cytokine storm in serum and carrying out joint analysis on the 4 indexes, thereby providing basis for targeted treatment. Can prevent further deterioration of cytokine storm, improve prognosis of patients, and has important significance for clinical application.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (8)
1. A kit for diagnosing a cytokine storm syndrome, said kit comprising biotin-labeled detection antibodies for 4 inflammatory factors TNF- α, IL-6, PGE2 and PAF.
2. The kit of claim 1, which is a solid phase chip, a liquid phase chip or an ELISA kit.
3. The kit according to claim 1, which can quantitatively detect the contents of 4 inflammatory factors TNF- α, IL-6, PGE2 and PAF in serum.
4. The kit according to claim 1, which does not comprise a detection antibody for inflammatory factors other than the above 4 inflammatory factors.
5. A cytokine storm syndrome diagnostic marker comprising 4 inflammatory factors TNF- α, IL-6, PGE2 and PAF.
6. The diagnostic marker of claim 5, which does not include other inflammatory factors.
7. Use of a kit according to any one of claims 1 to 4 or a diagnostic marker according to claim 5 or 6 for the preparation of a diagnostic kit for diagnosing cytokine storm syndrome.
8. Use of a kit according to any one of claims 1 to 4 or a diagnostic marker according to claim 5 or 6 for the preparation of a diagnostic kit for predicting cytokine storm syndrome.
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| CN107870233A (en) * | 2017-10-27 | 2018-04-03 | 上海华盈生物医药科技有限公司 | One kind can be used for the combination of cytokines and detection method of " cytokine storm " monitoring |
| US20190175649A1 (en) * | 2016-02-18 | 2019-06-13 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
| CN110167589A (en) * | 2016-12-14 | 2019-08-23 | 普罗根尼蒂公司 | Enterogastric diseases are treated using integrin inhibitor |
| US20200262908A1 (en) * | 2016-12-14 | 2020-08-20 | Progenity, Inc. | Treatment of a disease of the gastrointestinal tract with an il-12/il-23 inhibitor released using an ingestible device |
| CN112034187A (en) * | 2020-06-04 | 2020-12-04 | 北京臻知医学科技有限责任公司 | Marker for predicting 2019 coronavirus disease cytokines and thrombus storm, application and kit |
| US20210393741A1 (en) * | 2020-06-22 | 2021-12-23 | Imunami Laboratories Pte. Ltd. | Recombinant polypeptides and methods for use in the treatment of cancer |
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2023
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190175649A1 (en) * | 2016-02-18 | 2019-06-13 | Enlivex Therapeutics Ltd | Combination immune therapy and cytokine control therapy for cancer treatment |
| CN110167589A (en) * | 2016-12-14 | 2019-08-23 | 普罗根尼蒂公司 | Enterogastric diseases are treated using integrin inhibitor |
| US20200262908A1 (en) * | 2016-12-14 | 2020-08-20 | Progenity, Inc. | Treatment of a disease of the gastrointestinal tract with an il-12/il-23 inhibitor released using an ingestible device |
| CN107870233A (en) * | 2017-10-27 | 2018-04-03 | 上海华盈生物医药科技有限公司 | One kind can be used for the combination of cytokines and detection method of " cytokine storm " monitoring |
| CN112034187A (en) * | 2020-06-04 | 2020-12-04 | 北京臻知医学科技有限责任公司 | Marker for predicting 2019 coronavirus disease cytokines and thrombus storm, application and kit |
| US20210393741A1 (en) * | 2020-06-22 | 2021-12-23 | Imunami Laboratories Pte. Ltd. | Recombinant polypeptides and methods for use in the treatment of cancer |
Non-Patent Citations (5)
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| LI-MI HUANG 等: "Preconditioning rats with three lipid emulsions prior to acute lung injury affects cytokine production and cell apoptosis in the lung and liver", 《LIPIDS IN HEALTH AND DISEASE》, vol. 19 * |
| UNDURTI N DAS: "Bioactive Lipids as Mediators of the Beneficial Action(s) of Mesenchymal Stem Cells in COVID-19", 《AGING AND DISEASE》, vol. 11, no. 4 * |
| VALERIE A. FADOK 等: "Macrophages That Have Ingested Apoptotic Cells In Vitro Inhibit Proinflammatory Cytokine Production Through Autocrine/Paracrine Mechanisms Involving TGFb , PGE2, and PAF", 《THE JOURNAL OF CLINICAL INVESTIGATION》, vol. 101, no. 4 * |
| 朱淑敏 等: "新冠肺炎寒湿疫小鼠模型的建立及其评价", 《中国比较医学杂志》, vol. 32, no. 6 * |
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