CN113092780A - Marker for identifying neocoronary pneumonia and application thereof - Google Patents
Marker for identifying neocoronary pneumonia and application thereof Download PDFInfo
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- CN113092780A CN113092780A CN202110345853.9A CN202110345853A CN113092780A CN 113092780 A CN113092780 A CN 113092780A CN 202110345853 A CN202110345853 A CN 202110345853A CN 113092780 A CN113092780 A CN 113092780A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/55—IL-2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The invention discloses a marker for identifying neocoronary pneumonia and application thereof, belonging to the field of medical biology. The invention discovers that the IL-2 level of blood of a new coronary rehabilitation patient is obviously improved relative to a healthy contrast after the blood of the new coronary rehabilitation patient is stimulated by SARS-CoV-2 antigen peptide, and the IL-2 detection index is suitable for clinical rapid detection, can be used as a new index for detecting whether SARS-CoV-2 is infected, can be used for identifying asymptomatic infectors, and is also beneficial to clinical epidemiological investigation. The invention uses the expression level of IL-2 as an index for detecting whether SARS-CoV-2 is infected, and has the advantages of simple detection method, high detection speed, strong sensitivity (86.4%) and high specificity (96.7%).
Description
Technical Field
The invention belongs to the field of medical biology, and relates to a marker for identifying new coronary pneumonia and application thereof, in particular to a method for identifying existing SARS-CoV-2 infection by detecting IL-2 level.
Background
Cytokines are a generic term for polypeptide cell-regulating substances, including interleukins, interferons, growth factors, cell stimulating factors, tumor necrosis factors, and the like. Cytokines are mainly synthesized by peripheral immune cells (e.g., macrophages, lymphocytes, fibroblasts). Factors such as IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, INF-gamma, TNF-alpha, IFN-alpha and the like are common cell inflammation factors in human immunity and play an important role in human immune regulation. For example, IL-1 beta, IL-6 and TNF-alpha play a role in osteoarthritis, coronary atherosclerosis syndrome and cerebral infarction, IL-2, IL-4, IL-5, IL-6, IL-10, INF-gamma and the like are secreted by helper T cells Th1 and Th2 cells, participate in regulating the dynamic balance of the functions of Th1 and Th2 cells, and maintain the normal cellular immunity and humoral immunity functions of the organism, when the organism is attacked by alloantigen to cause the change of the balance of the immune functions of the organism, one subgroup of Th1 and Th2 cells is increased in function, the other subgroup is reduced in function, and therefore the concentration of each cytokine secreted by Th1 and Th2 is correspondingly changed. IL-17 is mainly involved in the generation of innate immunity and certain inflammations, and IL-17 has the unique characteristic of powerfully recruiting neutrophils and the function of promoting the release of various cytokines and is involved in the generation of various inflammatory diseases of the body. It has strong inflammatory effect, is a fine-tuning factor of inflammatory reaction, and can promote the local production of chemotactic factors such as IL-8, monocyte chemotactic protein (MCP-1) and growth regulatory factor-a, so that the rapid increase of monocytes and neutrophils can stimulate the production of IL-6 and prostaglandin-2, and enhance local inflammation. The expression levels of IL-8, IL-12p70, TNF-alpha and the like are obviously increased in the serum of patients with virus row infection and condyloma acuminatum.
The global spread of new coronary pneumonia seriously threatens human health, and the research and development of convenient, quick and effective biomarkers are vital to predict new coronary patients in time.
Disclosure of Invention
The present invention aims at finding the marker of SARS-CoV-2 infection for identifying the existing SARS-CoV-2 infection. The invention discovers that the cell factor IL-2 can be used as an index for whether SARS-CoV-2 is infected by research.
According to the invention, the IL-2 level of blood of a new coronary rehabilitation patient is obviously increased relative to a healthy control after the blood is stimulated by SARS-CoV-2 antigen peptide, so that the IL-2 detection index is suitable for clinical rapid detection, can be used as a new index for detecting whether SARS-CoV-2 is infected, can be used for identifying asymptomatic infectors, and is also beneficial to clinical epidemiological investigation.
The invention provides the following technical scheme:
the reagent for detecting the expression level of IL-2 has the application of preparing a kit for detecting whether SARS-CoV-2 is infected or not.
The SARS-CoV-2 antigen peptide and the reagent for detecting IL-2 expression level have the application of preparing the reagent kit for predicting whether SARS-CoV-2 is infected or not.
A kit for detecting whether SARS-CoV-2 is infected or not, comprising reagents for detecting the expression level of IL-2; further, it also contains SARS-CoV-2 antigen peptide.
Further, the reagent for detecting the expression level of IL-2 includes reagents used in ELISA, chemiluminescence, and the like.
Further, the detection sample of the kit is a blood sample taken from a subject to be detected.
The invention has the following advantages and beneficial effects: the invention uses the expression level of IL-2 as an index for detecting whether SARS-CoV-2 is infected, and has the advantages of simple detection method, high detection speed, strong sensitivity (86.4%) and high specificity (96.7%).
Drawings
FIG. 1 is a graph showing the expression of IL-2 in convalescent patients and healthy controls after stimulation with negative control culture medium (N), positive control culture medium (P), test culture medium (T), and SARS-CoV-2 antigen peptide.
FIG. 2 is a ROC graph.
Detailed Description
The following examples are intended to further illustrate the present invention and should not be construed as limiting the present invention, and any other changes, modifications, substitutions, combinations and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and shall be included within the scope of the present invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
1. Collection of specimens
1.1 origin of specimen
The study examined blood from 124 COVID-19 convalescent patients (62 males, 62 females) who recovered after SARS-CoV-2 infection in admission to south Hospital, Wuhan university, median age 52 years (23-83 years), and an additional 30 healthy donors enrolled. These sample assays were performed on a flow cytometer (BD, FACSCanto II, usa).
1.2 specimen requirements
1.2.1 specimen: whole blood
1.2.2 Collection:
whole blood: venous blood samples were collected using EDTA anticoagulation tubes.
1.2.3 preservation and transportation: and (3) processing and detecting the sample to be detected within 4 hours after blood drawing, if the sample cannot be detected within 4 hours, standing for no more than 24 hours at 4 ℃ after the processing is finished. The whole collected blood is required to be stored at-20 ℃ for more than 24 hours for not more than one year. Repeated freeze-thawing (no more than three times) is not suitable for sample preservation. Transporting at 2-8 deg.C.
2. Inspection method
2.1 preparation before experiment
3-well cell culture plates;
negative control culture (N): RPMI-1640 medium;
positive control culture (P): contains phytohemagglutinin (PHA, phytohemagglutinin) 1.6 mg/mL;
test medium (T): contains SARS-CoV-2 antigen peptide mixed peptide, SARS-CoV nucleoprotein (N) protein, 0.015 mu g/mL, receptor-binding domain (RBD) of the Spike (S) protein, 0.03 mu g/mL, S1(N-terminal immunogenic binding the RBD), 0.015 mu g/mL, and N-S1, 0.03 mu g/mL.
Twelve cytokine detection kits (flow-type fluorescence method) (purchased from Jiangxi Saiki Biotechnology Co., Ltd.) mainly comprise capture microsphere mixed liquor, quantitative standard substance, fluorescence detection reagent, sample diluent and the like, and are shown in Table 1 below.
TABLE 1
The capture microsphere mixed solution in the kit comprises 12 capture microspheres with different fluorescence intensities, the surfaces of the capture microspheres are respectively coated with IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, INF-y, TNF-alpha and IFN-alpha specific antibodies, the capture microspheres are respectively combined with IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, INF-gamma, TNF-alpha and IFN-alpha in a sample to be detected in a specific way and then combined with a fluorescence detection reagent marked by PE to capture the microspheres, the sample to be detected and detect the antibodies to form a double-antibody sandwich complex (capture microspheres + sample to be detected + antibody to be detected marked by PE), and analyzing the fluorescence intensity of the double-antibody sandwich compound to obtain the contents of IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, INF-gamma, TNF-alpha and IFN-alpha of the sample to be detected.
2.2 in vitro Release of cytokines
1) Collecting: the whole blood collection amount is not less than 4 mL;
2) subpackaging culture solution: adding a negative control culture solution (N), a positive control culture solution (P) and a test culture solution (T) into corresponding holes of a cell culture plate from left to right in sequence in a biological safety cabinet according to the direction of a handle of the culture plate, wherein each hole is 100 mu L;
3) subpackaging whole blood: 1mL of the uniformly mixed whole blood is respectively subpackaged into corresponding holes of the cell culture plate within eight hours, wherein the whole blood is 1 mL/hole.
4) Culturing: covering the upper cover of the culture plate tightly, quickly putting the culture plate into an electric heating constant temperature incubator at 37 ℃ for culturing for 16-24 hours, and keeping the cell culture plate horizontally placed in the culture process, wherein the cell culture plate cannot be turned upside down.
5) Collecting: after the culture is finished, placing the cell culture plate on a flat desktop, standing for 1min, carefully opening the cover, and sucking the supernatant from each cell culture plate hole for detection; note that: can not absorb cell layer to prevent hemolysis.
2.3 Standard tube preparation
1) Opening the quantitative standard (B), transferring the standard to a centrifuge tube, and labeling the tube with the highest concentration;
2)2mL of sample diluent (D) is used for resuspending the standard substance, and the sample is placed for 15min at room temperature;
3) gently mixing the standard substance by using a suction head to avoid violent shaking; taking 9 experimental sample tubes which are respectively marked as 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 and 1:512, and adding 300 mu L of sample diluent (D) into each tube;
4) sucking 300 mu L of liquid from the highest concentration standard sample tube into a 1:2 tube, blowing and stirring uniformly, sucking 300 mu L of liquid from the 1:2 tube into a 1:4 tube, blowing and stirring uniformly, and repeating the steps until the volume reaches 1:512 tube; as shown in table 2 below:
TABLE 2
| Pipe number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Concentration (pg/mL) | 10 | 20 | 40 | 80 | 156 | 312 | 625 | 1250 | 2500 | 5000 |
| Dilution factor of standard | 1:512 | 1:256 | 1:128 | 1:64 | 1:32 | 1:16 | 1:8 | 1:4 | 1:2 | Maximum concentration |
5) Adding 25 mu L of the standard substance which is diluted in a gradient manner into the standard substance tube;
2.4 cytokine expression test:
1) adding 25 mu L of sample to be detected into each sample tube, and adding 25 mu L of physiological saline into the negative control tube;
2) vortex and mix the capture microsphere mixture (A), add 25 μ L in each experimental tube;
3) all tubes were dosed with 25. mu.L of fluorescence detection reagent (C);
4) after the experiment tube is fully and uniformly mixed by vortex, incubating for 2.5 hours at room temperature in a dark place;
5) adding 1mL of PBS solution into each experimental tube, centrifuging 200g for 5min, and carefully sucking off the supernatant;
6) add 100. mu.L PBS solution to each tube and stand for detection.
7) Fluorescence detection: and (3) carrying out fluorescence detection on the experimental tube on the flow cytometer in a calibrated state in sequence according to the sequence of the standard tube, the negative control tube and the sample tube. Vortex each tube and mix for 3-5 seconds immediately before testing.
3. Analysis of results
The results show that the new crown rehabilitation and the healthy control are stimulated by a negative control culture solution (N), a positive control culture solution (P) and a test culture solution (T) respectively, the IL-2 of the two groups is increased after the P (containing phytohemagglutinin) stimulation, but the IL-2 of the two groups is obviously different after the T (containing SARS-CoV-2 antigen peptide) stimulation, and the IL-2 level of the new crown rehabilitation patient is obviously increased (figure 1); using SPSS software, ROC curves were generated using healthy control lg (T/N) and neocoronal convalescence lg (T/N), with an area under the curve (AUC) of 0.935, a sensitivity of 86.4%, a specificity of 96.7%, and a cut-off value of 2.99(T/N >2.99) (FIG. 2, Table 3).
TABLE 3
| Area under curve | 95% confidence interval | P value | Joden index | Sensitivity (%) | Degree of specificity (%) |
| 0.94 | 0.89-0.99 | <0.0001 | 0.476 | 86.4 | 96.7 |
Claims (6)
1. The application of the reagent for detecting the expression level of IL-2 in preparing a kit for detecting whether SARS-CoV-2 is infected or not.
Use of SARS-CoV-2 antigen peptide and a reagent for detecting IL-2 expression level in the preparation of a kit for detecting whether or not SARS-CoV-2 is infected.
3. A kit for detecting whether SARS-CoV-2 is infected, characterized in that: comprising reagents for detecting the level of IL-2 expression.
4. The kit of claim 3, wherein: also included are SARS-CoV-2 antigen peptides.
5. The use according to claim 1 or 2 or the kit according to claim 3 or 4, characterized in that: the reagent for detecting the IL-2 expression level comprises a reagent used in an ELISA method and a chemiluminescence method.
6. The use according to claim 1 or 2 or the kit according to claim 3 or 4, characterized in that: the detection sample of the kit is a blood sample taken from a subject to be detected.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113820494A (en) * | 2021-07-19 | 2021-12-21 | 广州医科大学附属市八医院 | Blood polypeptide group new coronary biomarker |
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| WO2020234833A1 (en) * | 2019-05-22 | 2020-11-26 | Geistlich Pharma Ag | Method for treating, preventing, inhibiting or reducing cytokine release |
| CN111999508A (en) * | 2020-05-15 | 2020-11-27 | 上海交通大学 | Diagnostic marker and application thereof in COVID-19 diagnosis and coronavirus past infection detection |
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Cited By (1)
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| CN113820494A (en) * | 2021-07-19 | 2021-12-21 | 广州医科大学附属市八医院 | Blood polypeptide group new coronary biomarker |
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