CN112433051B - Application of platelet activating factor acetylhydrolase detection reagent in preparation of lung cancer screening kit - Google Patents
Application of platelet activating factor acetylhydrolase detection reagent in preparation of lung cancer screening kit Download PDFInfo
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Abstract
The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of a platelet activating factor acetylhydrolase (PLA2G7) detection reagent in preparation of a lung cancer screening kit. The invention discovers for the first time that the level of platelet activating factor acetylhydrolase (PLA2G7) in plasma exosomes of lung cancer patients is obviously higher than that of benign disease patients and healthy people in the lung. According to the invention, the reagent for detecting the platelet activating factor acetylhydrolase (PLA2G7) is used for preparing the lung cancer screening kit, so that the effective screening of the lung cancer can be realized.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of a platelet activating factor acetylhydrolase (PLA2G7) detection reagent in preparation of a lung cancer screening kit.
Background
Lung cancer is one of the most common malignant tumors in the world, the morbidity and mortality of the lung cancer are on the rising trend year by year, the morbidity is at the top of the world at present, and the health and the life of human beings are seriously threatened.
The lung cancer is a disease good in occult, clinical symptoms are often shown only when the disease develops to the advanced stage, 70-80% of lung cancer patients are already at the middle and advanced stages when the lung cancer symptoms are diagnosed, cancer cells are diffused, the best curing time is missed, and the five-year survival rate is low. For early-stage lung cancer patients, the survival rate and the survival quality of the patients can be greatly improved by 5 years and more through timely treatment. Early diagnosis and effective screening of lung cancer is therefore of great importance.
The screening of the lung cancer refers to that people without lung cancer related symptoms are subjected to routine physical examination, and the lung cancer is found in time before the symptoms appear. If the lung cancer molecular marker in the plasma can be found, the molecular marker has important significance for prompting a clinician to take relevant treatment measures or decisions for a patient at an early stage.
Platelet activating factor (PLA) is an endogenous phospholipid mediator with a wide range of biological activities, and has both physiological and pathological roles. PLA is synthesized and released by eosinophils, basophils, platelets, endothelial cells, leukocytes, lung, liver, kidney and other cells and organs in the body when stimulated by specific antigens. Platelet activating factor (PLAF) acts by binding to PLA receptors on the target cell membrane. Can cause platelet aggregation, neutrophil aggregation and release; produce a large amount of inflammatory mediators such as active oxygen, leukotriene and the like. Diseases associated with excess production of PLA include asthma, septic shock, and the like.
Platelet-activating factor acetylhydrolase (PLA2G7) (Unit number: Q13093) is called Platelet-activating factor acetylhydrolase, is a main degrading enzyme of Platelet-activating factor, and plays an important role in regulating the metabolism of Platelet-activating factor.
No existing technology related to lung cancer by platelet activating factor acetylhydrolase (PLA2G7) is available at present.
Disclosure of Invention
The invention aims to provide a novel autoantibody lung cancer marker and application of a detection reagent of the marker in preparation of a lung cancer screening kit.
The technical scheme of the invention comprises the following steps:
the application of a reagent for detecting platelet activating factor acetylhydrolase (PLA2G7) in preparing a lung cancer screening kit.
As the application, the reagent for detecting the platelet activating factor acetylhydrolase (PLA2G7) is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.
As for the application, the reagent for detecting the platelet activating factor acetylhydrolase (PLA2G7) is a western blot reagent.
As mentioned above, the reagent for detecting platelet activating factor acetylhydrolase (PLA2G7) is a reagent for a protein chip detection method.
As the aforementioned use, the reagent for detecting platelet-activating factor acetylhydrolase (PLA2G7) is a reagent for detecting platelet-activating factor acetylhydrolase (PLA2G7) in human plasma exosomes.
A lung cancer screening kit, which comprises a reagent for detecting platelet activating factor acetylhydrolase (PLA2G 7).
As the kit, the reagent for detecting the PAF acetylhydrolase (PLA2G7) is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay reagent.
As the kit, the reagent for detecting the platelet activating factor acetylhydrolase (PLA2G7) is a western blot reagent.
As the kit, the reagent for detecting the platelet activating factor acetylhydrolase (PLA2G7) is a reagent for a protein chip detection method.
As with the aforementioned kit, the reagent for detecting platelet-activating factor acetylhydrolase (PLA2G7) is a reagent for detecting platelet-activating factor acetylhydrolase (PLA2G7) in human plasma exosomes.
The key point of the invention is that the content of the platelet activating factor acetylhydrolase (PLA2G7) in the human plasma exosome is determined to be obviously related to the risk of suffering from the lung cancer, so the risk of suffering from the lung cancer can be judged by detecting the content of the platelet activating factor acetylhydrolase (PLA2G7) in the human plasma exosome, as for a means for specifically detecting the platelet activating factor acetylhydrolase (PLA2G7) in the human plasma exosome, various means disclosed in the prior art can be adopted, and the embodiment of the invention specifically adopts an enzyme-linked immunoassay method (protein chip) for detection, but is not limited to the means, and any method capable of detecting the content of the platelet activating factor acetylhydrolase (PLA2G7) can be used for screening the lung cancer.
The invention provides a new lung cancer screening marker and a new lung cancer screening kit, which can realize effective screening of lung cancer; and the plasma exosome can be used as a detection sample, so that the harm to a patient is low. The invention has good application prospect.
It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.
The above-mentioned aspects of the present invention will be further described in detail with reference to the following embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
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FIG. 1: lung cancer patients (LC), benign lung Disease (DC), and healthy control plasma exosomes were compared for their levels of platelet-activating factor acetylhydrolase (PLA2G 7).
FIG. 2: ROC analysis of lung cancer patients (LC) and benign lung Disease (DC).
FIG. 3: ROC analysis of Lung cancer patients (LC) and healthy controls (NC)
Detailed Description
EXAMPLE 1 relationship between platelet activating factor acetylhydrolase (PLA2G7) and Lung cancer in plasma
First, clinical data
40 lung cancer patients, 40 lung benign diseases (non-malignant tumors such as tuberculosis and hamartoma) and 40 healthy controls are selected, and the basic information is as follows:
| basic information | Patients with lung cancer | Benign lung disease | Healthy controls |
| Number of |
40 | 40 | 40 |
| Age (age) | 58.8±11.5 | 54.1±12.9 | 50.1±14.3 |
| Proportion of male | 33(82.5％) | 16(40％) | 17(44.7％) |
Second, detection principle
HuProtTMThe human protein custom chip is fixed with a protein antibody (ASLAFLQK amino acid sequence of specific recognition PLA2G7) of platelet activating factor acetylhydrolase (PLA2G7), after the human protein custom chip is incubated with plasma exosome protein, the platelet activating factor acetylhydrolase (PLA2G7) in serum is combined, the non-combined antibody and other proteins are washed and removed, then the fluorescence-labeled platelet activating factor acetylhydrolase (PLA2G7) antibody is combined with the platelet activating factor acetylhydrolase (PLA2G7) for detection, and a signal is read by a fluorescence scannerThe strength of the signal is positively correlated with the affinity and the quantity of the antibody.
Third, method
1. The preparation method of the plasma exosome protein comprises the following steps:
the plasma sample was removed from-80 ℃ and centrifuged at 12000g at 4 ℃ for 15 minutes, and the supernatant was transferred to a new centrifuge tube, filtered through a 0.22 μ M microfiltration membrane, and then separated using a commercially available exosome purification kit according to the instructions to obtain exosomes. Adding Urea with final concentration of 8M and a protease inhibitor for ultrasonic cleavage to obtain exosome protein, and utilizing a BCA kit to measure the protein concentration.
2. Detection of platelet activating factor acetylhydrolase (PLA2G7) in plasma exosome proteins
The reagents used in this section were as follows:
1) rewarming: taking out the chip from a refrigerator at-80 deg.C, putting in a refrigerator at 4 deg.C for rewarming for half an hour, and then putting in room temperature for rewarming for 15 min;
2) and (3) sealing: fixing 14 blocks in the rewarming chip, adding sealing liquid into each block after fixing, placing on a side swing bed, and sealing at room temperature for 3 hr;
3) incubation of plasma exosome protein samples: after sealing is finished, pouring the sealing liquid, then quickly adding a prepared plasma exosome protein incubation liquid, wherein each chip can incubate 14 plasma exosome protein samples, the sample loading volume of each plasma exosome protein sample is 200 mu L, and the shaking table is laterally swung at 20rpm and incubated overnight at 4 ℃ (the plasma exosome protein samples are frozen and thawed in a chromatography cabinet at 4 ℃, and the incubation liquid is added for dilution in a ratio of 1: 50 to obtain the plasma exosome protein incubation liquid);
4) cleaning: the chip and the chip fence are taken out together, the sample is sucked, then the PBST with the same volume is added rapidly, and the cycle is repeated for a plurality of times, so that no cross contamination exists among the serum samples when the chip fence is detached. After the chip fence is removed, the chip is placed in a chip cleaning box with cleaning solution, and is cleaned for 3 times (10 min each time) by a horizontal shaking table at room temperature of 80 rpm;
5) and (3) secondary antibody incubation: transferring the chip into an incubation box added with 3mL of secondary antibody incubation liquid, laterally swinging a shaker at 40rpm, keeping out of the sun, and keeping at room temperature for 1 hr;
6) cleaning: the chip was removed (note that the upper surface of the chip was not touched or scratched), and placed in a chip washing cassette containing a washing solution, and washed 3 times 10min each time, on a horizontal shaker at room temperature and 80 rpm. After completion, washing with ddH2O for 2 times, each for 10 min;
7) drying;
8) scanning: scanning by using a crystal core LuxScan 10K microarray chip scanner;
9) data extraction: opening the corresponding GAL file (recording the position of protein in the chip), aligning the chip image and each array of the GAL file integrally, pressing an automatic alignment button, extracting data and storing.
Fourthly, the result
The mean expression level of the platelet activating factor acetylhydrolase (PLA2G7) in the plasma of the lung cancer patients was 1.35 (protein relative quantitative ratio), the mean expression level of the platelet activating factor acetylhydrolase (PLA2G7) in the plasma exosome protein of the benign lung disease was 0.89, and the mean expression level of the platelet activating factor acetylhydrolase (PLA2G7) in the exosome protein of the healthy control was 0.72. The lung cancer group was statistically significant compared to both the benign lung disease group (p <0.01) and healthy controls (p <0.001) (fig. 1). The specificity of ROC analysis of the lung cancer group and benign diseases is 97.5%, and the sensitivity is 15.0% (figure 2); the lung cancer group and the healthy control have the ROC analysis result that the specificity is 97.5 percent and the sensitivity is 17.5 percent (figure 3); it shows that the platelet activating factor acetylhydrolase (PLA2G7) can specifically distinguish lung cancer from benign lung diseases.
From the above results, it is known that the difference in the level of platelet-activating factor acetylhydrolase (PLA2G7) in plasma exosome proteins of lung cancer patients and non-lung cancer patients is significant, and the purpose of lung cancer screening can be achieved by detecting the level of platelet-activating factor acetylhydrolase (PLA2G7) in plasma exosome proteins.
EXAMPLE 2 composition of the detection kit of the invention and method of use thereof
Kit composition
Detection kit (14 persons):
the reagent for preparing the plasma exosome protein needs to be self-prepared, and can be a commercial plasma exosome protein extraction kit.
Second, kit using method
The same as example 1, third part- "detection of platelet activating factor acetylhydrolase (PLA2G7) in plasma exosome protein".
The kit can screen the risk of lung cancer of a human to be detected by detecting the level of platelet activating factor acetylhydrolase (PLA2G7) in serum: the risk of lung cancer is high if the level of platelet activating factor acetylhydrolase (PLA2G7) is high (relative to benign patients and healthy people in the lung), and low if the level of platelet activating factor acetylhydrolase (PLA2G7) is low. Can be used for the auxiliary diagnosis of clinical lung cancer, provides effective basis for patients to take relevant treatment measures or decisions, and has good clinical application prospect.
SEQUENCE LISTING
<110> Sichuan university Hospital in western China
Application of <120> platelet activating factor acetylhydrolase detection reagent in preparation of lung cancer screening kit
<130> GYKH1094-2020P0111922CC20JS035
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Artificial Synthesis
<400> 1
Ala Ser Leu Ala Phe Leu Gln Lys
1 5
Claims (1)
1. The application of the reagent for detecting the platelet activating factor acetylhydrolase in the preparation of the lung cancer screening kit is characterized in that: the reagent for detecting the platelet-activating factor acetylhydrolase is a reagent for detecting the platelet-activating factor acetylhydrolase in human plasma exosomes;
the reagent for detecting the platelet activating factor acetylhydrolase is a reagent for a protein chip detection method, and the reagent contains an antibody specifically recognizing the ASLAFLQK amino acid sequence of the platelet activating factor acetylhydrolase.
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