CN118130788A - Thrombomodulin immunochromatography test strip, kit and application thereof in sepsis - Google Patents
Thrombomodulin immunochromatography test strip, kit and application thereof in sepsis Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- 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
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Abstract
The invention provides a thrombomodulin immunochromatography test strip, a kit and application thereof in sepsis, and relates to the technical field of biology. The thrombus regulation protein immunochromatography test strip comprises a sample pad, a marking pad, a detection pad and an absorption pad which are sequentially arranged on a fixed bottom plate along the immunochromatography direction, wherein a double-antibody sandwich principle is adopted, and a monoclonal antibody capable of combining thrombus regulation protein is respectively coated on the detection line and the marking pad. The rapid sepsis screening and monitoring can be realized by applying the kit to the detection of thrombomodulin.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a thrombomodulin immunochromatography test strip, a kit and application thereof in sepsis.
Background
Sepsis (sepsis) is a life-threatening organ dysfunction caused by deregulation of the body's response to infection, and is associated with manifestations of inflammatory factor storms, organ damage, etc. Sepsis patients often show signs of excessive inflammation and immune dysfunction. Early in sepsis, the pathological process is associated with a strong activation of the innate immune system, and a storm of cytokines caused by the release of a large number of pro-inflammatory factors will lead to activation of a large number of immune cells and activation of the complement system, the coagulation system and vascular endothelium, leading to extensive microthrombosis and impairment of organ function.
Vascular endothelial injury is one of the main mechanisms of coagulation disorders in sepsis. Thrombomodulin (thrombomodulin, TM) is an endothelial cell membrane glycoprotein that plays an important role in protecting endothelial cells, maintaining vascular homeostasis, etc., which are the primary target of attack by pathogens and toxins when sepsis occurs. Thrombomodulin becomes soluble thrombomodulin (soluble thrombomodulin, sTM) after entering blood circulation from damaged endothelial cells when sepsis occurs, which is a currently recognized biomarker of vascular endothelial injury, and can be significantly elevated in sepsis. At present, the blood plasma thrombomodulin level is mainly monitored clinically, and repeated blood drawing is needed for dynamic monitoring, so that the development of a product for noninvasively monitoring the blood plasma thrombomodulin level of a sepsis patient is very important for dynamic evaluation of the illness state and reduction of iatrogenic blood loss.
In view of this, the present invention has been made.
Disclosure of Invention
The object of the present invention is to provide the use of a substance or device for detecting thrombomodulin in urine for the manufacture of a product for detecting sepsis, in order to reduce the problem of repeated blood draws required for dynamic monitoring of sepsis. The invention also aims at providing a thrombomodulin immunochromatographic test strip.
In order to solve the technical problems, the invention adopts the following technical scheme:
The invention provides a thrombomodulin immunochromatography test strip, which comprises a sample pad, a marking pad, a detection pad and an absorption pad which are sequentially arranged on a bottom plate along the immunochromatography direction;
the detection pad is provided with a quality control line and a detection line;
The detection line is coated with a first thrombomodulin monoclonal antibody, and the marking pad is coated with a second thrombomodulin monoclonal antibody; the first thrombomodulin monoclonal antibody and the second thrombomodulin monoclonal antibody bind different epitopes of thrombomodulin;
The second thrombomodulin monoclonal antibody is labeled with a tracer marker.
In an alternative embodiment, the quality control line is coated with an antibody that specifically binds to the second thrombomodulin monoclonal antibody.
In alternative embodiments, the marking pad comprises a glass cellulose film, a polyester fiber film, a nonwoven fabric or a nitrocellulose film, preferably an untreated glass cellulose film.
In an alternative embodiment, the detection pad comprises a nitrocellulose membrane.
In alternative embodiments, the tracer labels include one or more of latex particles, fluorescent molecular markers, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal selenium, colloidal silver, and colloidal carbon.
In an alternative embodiment, the thrombomodulin immunochromatographic test strip further comprises a housing.
The invention also provides a thrombomodulin detection kit, which comprises the thrombomodulin immunochromatographic test strip.
In an alternative embodiment, the kit further comprises a urine collection device.
The invention also provides application of a substance or device for detecting thrombomodulin in preparing a product for detecting sepsis, wherein the thrombomodulin comprises thrombomodulin in urine.
In an alternative embodiment, the thrombomodulin further comprises thrombomodulin in the blood.
In alternative embodiments, the substance or device for detecting thrombomodulin comprises a substance or device for nucleic acid amplification reaction, ELISA detection method, immunoblotting method, immunomagnetic bead detection, chemiluminescent method or immunochromatographic test strip detection.
In an alternative embodiment, the device for detecting thrombomodulin comprises a thrombomodulin immunochromatographic test strip as described above, or a kit as described above.
Compared with the prior art, the invention has the following beneficial effects:
The invention discovers that the level of the thrombomodulin of the patient with sepsis is obviously reduced compared with the healthy person or the patient with non-sepsis, and when the boundary value of the thrombomodulin is 15.45TU/ml, the sensitivity is 57 percent and the specificity is 80 percent. So that the detection of thrombomodulin in urine can be used for sensitively, simply and noninvasively screening sepsis. The invention also provides a thrombomodulin immunochromatography test strip, when in use, urine is sampled once, and the urine is proved to be negative after the reaction, so that the content of thrombomodulin in urine is obviously reduced, the possibility of sepsis is prompted, and the defect that repeated blood drawing is needed for dynamically monitoring the plasma thrombomodulin level is overcome.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the following description will briefly explain the drawings needed in the embodiments or the prior art description, and it is obvious that the drawings in the following description are some embodiments of the invention and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a thrombomodulin immunochromatographic test strip provided in example 1;
FIG. 2 is a schematic diagram of a shell of a thrombomodulin immunochromatographic test strip provided in example 1;
FIG. 3 shows the results of measurement of thrombomodulin content in blood of sepsis group and healthy control group;
FIG. 4 shows the results of thrombomodulin content measurements in urine from sepsis group and healthy control group;
FIG. 5 is a ROC curve of thrombomodulin in blood and thrombomodulin in urine as diagnostic markers of sepsis.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The inventor detects the level of the thrombomodulin in the patient with sepsis, and discovers that the level of the thrombomodulin in the patient with sepsis is obviously reduced compared with the healthy patient or the patient with non-sepsis, and when the boundary value of the thrombomodulin is 15.45TU/ml, the sensitivity is 57 percent, and the specificity is 80 percent. Based on this finding, the present invention provides the use of a substance or device for detecting thrombomodulin, including thrombomodulin in urine, in the preparation of a product for detecting sepsis.
In alternative embodiments, the detection comprises one or more of sepsis screening, sepsis diagnosis, sepsis recurrence diagnosis, and sepsis prognosis.
In an alternative embodiment, the thrombomodulin further comprises thrombomodulin in blood, and experiments show that the area under ROC curve (AUC) of the thrombomodulin in blood and the thrombomodulin in urine in combination detection is 0.83 (95% CI 0.772-0.897, P < 0.001), and when the thrombomodulin in blood is not less than 9.05TU/ml and the thrombomodulin in urine is not more than 15.45TU/ml, the sensitivity is 77.5%, and the specificity is 85.5%.
In alternative embodiments, the substance or device for detecting thrombomodulin includes, but is not limited to, reagents and/or apparatus for detecting thrombomodulin, and/or reagents and/or apparatus for detecting nucleic acid encoding thrombomodulin. Including but not limited to substances or devices for nucleic acid amplification reactions, such as but not limited to substances or devices for reverse transcription, PCR amplification reactions, qPCR amplification reactions, or RT-PCR amplification reactions; or substances or devices for ELISA detection methods, immunoblotting methods, immunomagnetic bead detection, chemiluminescent methods or immunochromatographic test paper detection. Depending on the specific detection means, the person skilled in the art may select a substance for detecting thrombomodulin according to the methods described in general and more specific textbooks, references, process manuals, commodity descriptions, standard documents, etc., which the present invention is not limited to.
In an alternative embodiment, the device for detecting thrombomodulin in urine comprises immunochromatographic test paper, and when the device is used, a urine sample is taken once, and the urine sample is negative in response, namely, the content of thrombomodulin in urine is obviously reduced, so that the possibility of sepsis is prompted.
The invention also provides a thrombomodulin immunochromatography test strip, which is convenient for detecting thrombomodulin in urine. The test strip comprises a sample pad, a marking pad, a detection pad and an absorption pad which are sequentially arranged on a bottom plate along the immunochromatography direction, wherein a quality control line and a detection line are arranged on the detection pad.
The detection line of the thrombomodulin immunochromatographic test strip is coated with a first thrombomodulin monoclonal antibody, the label pad is coated with a second thrombomodulin monoclonal antibody, and the second thrombomodulin monoclonal antibody is labeled with a tracer marker; the first thrombomodulin monoclonal antibody and the second thrombomodulin monoclonal antibody bind different epitopes of thrombomodulin to enable simultaneous binding to thrombomodulin.
The quality control line can be provided with components to be coated according to the general principle of immunochromatography detection, and can be selected by a person skilled in the art according to a well-known and conventional manner, and the invention is not limited thereto.
In an alternative embodiment, the composition of the coating of the quality control line comprises an antibody that is capable of specifically binding to said second thrombomodulin monoclonal antibody. For example, when the second thrombomodulin monoclonal antibody is a murine antibody, the quality control line is coated with an antibody that binds to the murine antibody, exemplary antibodies include, but are not limited to, goat anti-murine antibodies.
The thrombomodulin immunochromatography test strip adopts a double-antibody sandwich principle to detect thrombomodulin in a sample to be detected, when the thrombomodulin exists in the sample to be detected, a second thrombomodulin monoclonal antibody in a labeling pad captures the thrombomodulin to form a tracer marker-second thrombomodulin monoclonal antibody-thrombomodulin immune complex, and when the immune complex flows through a detection line of the detection pad, the first thrombomodulin monoclonal antibody on the detection pad captures the detection line to form a tracer marker-second thrombomodulin monoclonal antibody-thrombomodulin-first thrombomodulin monoclonal antibody immune complex, and a detectable signal is formed on the detection line.
The tracer labels are used to provide an observable detection signal that can be visually observed (e.g., a color change) or detected by an instrument (e.g., fluoresced), and the labels can be selected from labels conventional in the art, to which the present invention is not limited, exemplary labels include, but are not limited to, one or more of latex particles, fluorescent molecular markers, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal selenium, colloidal silver, and colloidal carbon.
The materials constituting the various parts of the immunochromatographic test strip for detecting thrombomodulin may be selected from materials acceptable in the art and known in the art. Sample pad materials illustratively include, but are not limited to, glass cellulose films, polyester fiber films, or nonwoven fabrics, preferably including glass cellulose films. Exemplary materials for the marking pad include, but are not limited to, a glass cellulose film, a polyester fiber film, or a nonwoven fabric, preferably an untreated glass cellulose film, which refers to a glass cellulose film that has not been labeled with an antibody or washed. Absorbent pad materials illustratively include, but are not limited to, absorbent paper. Exemplary detection pad materials include, but are not limited to, nitrocellulose membranes.
In an alternative embodiment, the thrombomodulin immunochromatographic test strip further comprises a housing. The housing may be selected from those conventional in the art, and the present invention is not limited thereto.
In an optional embodiment, a window for observing the detection line and the quality control line is arranged on the shell; the housing is further provided with a through hole for applying a sample to the sample pad.
The invention also provides a kit for detecting thrombomodulin, which comprises the thrombomodulin immunochromatographic test strip. The test kit also optionally includes conventional reagents and consumables for testing, including, but not limited to, one or more of sample lysates, dilutions, color cards, samplers, and the like.
In an alternative embodiment, the thrombomodulin detection kit further comprises a urine collection device.
It will be appreciated that the thrombomodulin immunochromatographic test strip or the kit comprising the same provided by the present invention can detect any conventional sample in the art, besides urine, can also detect lysate including, but not limited to, blood, plasma, serum, body fluid, cells or tissues, etc., and the use of the thrombomodulin immunochromatographic test strip or the kit comprising the same is not limited.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1
The embodiment provides a thrombomodulin immunochromatographic test strip, as shown in fig. 1 and 2. In fig. 1 and 2, 100 is a sample pad, 200 is a label pad, 300 is a detection pad, 310 is a detection line, 320 is a quality control line, 400 is an absorption pad, 500 is a housing, 510 is a sample application hole, and 520 is a window.
The immunochromatographic test strip comprises a sample pad 100, a label pad 200, a detection pad 300 and an absorption pad 400 which are fixed on a bottom plate along the chromatographic direction; the detection line 310 and the quality control line 320 are arranged on the detection pad 300 along the chromatographic direction. The sample pad is made of untreated glass cellulose membrane; the marking pad is made of untreated glass cellulose film; the detection pad is made of Nitrocellulose (NC) membrane; the absorbent pad is made of cellulose filter paper.
The marking pad 200 is coated with a mouse anti-thrombomodulin monoclonal antibody, and a second thrombomodulin monoclonal antibody is marked by colloidal gold; the concentration of the colloidal gold-labeled second thrombomodulin monoclonal antibody was 20. Mu.g/ml and the spray amount was 1. Mu.L/cm.
The detection line 310 is coated with a mouse anti-thrombomodulin monoclonal antibody, the concentration of the first thrombomodulin monoclonal antibody is 100. Mu.g/ml, and the spraying amount is 1. Mu.L/cm.
The quality control line 320 is coated with goat anti-mouse antibody, the concentration of the goat anti-mouse antibody is 100 mug/ml, and the spraying amount is 1 mug/cm.
The sample pad 100, the marking pad 200, the detection pad 300, and the absorption pad 400 are sequentially adhered to the base plate, and then assembled with the housing 500, and the housing 500 is provided with a sample loading hole 510 corresponding to the sample pad 100 and a window 520 corresponding to the detection line 310 and the quality control line 320.
And (3) result judgment: adding 0.1-0.2 ml urine into the sample adding hole, and taking two red lines on the cellulose membrane of the test strip as positive thrombomodulin; only one red line (near the end of the bibulous filter paper, control line) appears as negative. Reagent failure is considered if no red line appears.
The result determination time: the strong positive specimen can be aggregated by red colloidal gold particles in a detection line within 2 minutes; weak positive specimens (1-2 ng/ml) require about 10 minutes to determine the results. The sensitivity of detecting thrombomodulin level by colloidal gold immunochromatography can reach 1ng/ml.
Clinical significance: for healthy people, the level of the thrombomodulin is more than 1ng/ml, and the detection is negative; for sepsis patients, urinary thrombus regulating protein levels were routinely <1ng/ml, detected as positive. The reagent can also be used for detecting the level of the thrombomodulin in blood plasma. For healthy people, the plasma thrombomodulin level is <1ng/ml, and the detection is negative; for sepsis patients, plasma thrombomodulin levels were routinely >1ng/ml, detected as positive.
Example 2
Materials and methods:
102 sepsis patients and 55 healthy physical examination patients with synchronous physical examination, which are collected and treated by the department of severe medicine 2022, 12 months and 2023, 7 months of the 908 th hospital of the joint care army, are selected. The sepsis patients were taken from the patients in 2h of the department and the healthy physical examination patients were taken from 2ml of peripheral venous blood and 2ml of urine, and the thrombomodulin level was detected by a chemiluminescent immunoassay.
(II) experimental results:
(1) Comparison of baseline data for sepsis and healthy controls:
The age and sex differences of the two groups of patients were statistically significant (P > 0.05). Compared with healthy controls, the sepsis group has an extended Prothrombin Time (PT), international Normalized Ratio (INR), activated Partial Thromboplastin Time (APTT), elevated fibrinogen, fibrin Degradation Products (FDP), D-dimer, white blood cell count, neutrophil count, glutamate-pyruvate transaminase, glutamate-oxaloacetate and creatinine, shortened Thrombin Time (TT), reduced antithrombin, lymphocyte count, platelet count, total protein and albumin, and statistically significant differences (P < 0.05).
Table 1 baseline data for healthy control and sepsis groups
(2) Comparing the sepsis group with the healthy control group plasma thrombomodulin and urinary thrombomodulin, the results are shown in fig. 3 and 4, and it can be seen that the sepsis group plasma thrombomodulin level and the increase, the urinary thrombomodulin level decrease, and the differences are all statistically significant (P < 0.001).
(3) ROC curve analysis plasma thrombomodulin, urinary thrombomodulin and plasma thrombomodulin in combination with urinary thrombomodulin diagnostic efficacy against sepsis the ROC curve is shown in fig. 5. The area under ROC curve (AUC) for plasma thrombomodulin to diagnose sepsis is 0.82 (95% ci 0.759-0.887, p < 0.001); when the limit value of the thrombomodulin in the blood plasma is 9.05TU/ml, the sensitivity is 73% and the specificity is 89%. The area under ROC curve (AUC) of the urothrombomodulin for diagnosing sepsis is 0.67 (95% CI 0.590-0.758, P < 0.001); when the boundary value of the thrombomodulin was 15.45TU/ml, the sensitivity was 57%, and the specificity was 80%. The area under ROC curve (AUC) of plasma thrombomodulin combined with urinary thrombomodulin for diagnosing sepsis is 0.83 (95% CI 0.772-0.897, P < 0.001), and when the plasma thrombomodulin is not less than 9.05TU/ml and the urinary thrombomodulin is not more than 15.45TU/ml, the sensitivity is 77.5%, and the specificity is 85.5%.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. The thrombus regulating protein immunochromatography test strip is characterized by comprising a sample pad, a marking pad, a detection pad and an absorption pad which are sequentially arranged on a bottom plate along the immunochromatography direction;
the detection pad is provided with a quality control line and a detection line;
The detection line is coated with a first thrombomodulin monoclonal antibody, and the marking pad is coated with a second thrombomodulin monoclonal antibody; the first thrombomodulin monoclonal antibody and the second thrombomodulin monoclonal antibody bind different epitopes of thrombomodulin;
The second thrombomodulin monoclonal antibody is labeled with a tracer marker.
2. The thrombomodulin immunochromatographic test strip according to claim 1, wherein said quality control line is coated with an antibody which specifically binds to said second thrombomodulin monoclonal antibody.
3. The thrombomodulin immunochromatographic test strip according to claim 1, characterized in that said marking pad comprises a glass cellulose membrane, a polyester fiber membrane, a nonwoven fabric or a nitrocellulose membrane, preferably an untreated glass cellulose membrane;
preferably, the detection pad comprises a nitrocellulose membrane.
4. The thrombomodulin immunochromatographic test strip according to claim 1, wherein said tracer marker comprises one or more of latex particles, fluorescent molecular markers, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal selenium, colloidal silver and colloidal carbon;
Preferably, the tracer label is colloidal gold;
Preferably, the thrombomodulin immunochromatographic test strip further comprises a housing.
5. The thrombomodulin detection kit, characterized by comprising the thrombomodulin immunochromatographic test strip according to any one of claims 1 to 4.
6. The thrombomodulin assay kit of claim 5, further comprising a urine collection device.
7. Use of a substance or device for detecting thrombomodulin, including thrombomodulin in urine, for the preparation of a product for detecting sepsis.
8. The use of claim 7, wherein the thrombomodulin further comprises thrombomodulin in the blood.
9. The use according to claim 7 or 8, wherein the substance or device for detecting thrombomodulin comprises a substance or device for nucleic acid amplification reaction, ELISA detection method, immunoblotting method, immunomagnetic bead detection, chemiluminescent method or immunochromatographic test strip detection.
10. The use according to any one of claims 7 to 9, wherein the means for detecting thrombomodulin comprises a thrombomodulin immunochromatographic test strip according to any one of claims 1 to 4, or a kit according to claim 5 or 6.
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