CN110596390B

CN110596390B - Application of EEF2K autoantibody detection reagent in preparation of lung cancer detection kit

Info

Publication number: CN110596390B
Application number: CN201910896747.2A
Authority: CN
Inventors: 李蕊岑; 唐怀蓉; 王婷
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2023-01-06
Anticipated expiration: 2039-09-20
Also published as: CN110596390A

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of an EEF2K autoantibody detection reagent in preparing a lung cancer screening kit. The invention discovers for the first time that the level of the autoantibody of EEF2K protein in the serum of a lung cancer patient is obviously higher than that of a healthy patient. According to the invention, the reagent for detecting the EEF2K protein autoantibody is used for preparing the lung cancer screening kit, so that effective screening of lung cancer can be realized.

Description

Application of EEF2K autoantibody detection reagent in preparation of lung cancer detection kit

Technical Field

The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of an EEF2K autoantibody detection reagent in preparing a lung cancer screening kit.

Background

The lung cancer is one of the most common malignant tumors in the world, the morbidity and the mortality of the lung cancer are on the rising trend year by year, the morbidity is at the top in the world at present, and the health and the life of human beings are seriously threatened.

The lung cancer is a disease good in hiding, clinical symptoms are usually shown only when the disease is developed to the advanced stage, 70-80% of lung cancer patients are in the middle and advanced stages when being diagnosed with the lung cancer symptoms, 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 of lung cancer and effective screening are therefore of paramount 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.

Autoantibodies are antibodies produced by the body to self-organs, cells or cellular components. At present, autoantibodies to certain proteins have become markers for lung cancer, such as: p53, NY-ESO-1, CYFRA, etc. (Tang Z-M, link Z-G, wang C-M, wu Y-B, kong J-L (2017) Serum tumor-associated autoimmune tumors for lung cancer: A systematic review and meta-analysis. PLoS ONE 12 (7): e 0182117).

The EEF2K protein (Ensemble number of its gene: ENSG 00000103319) is a highly conserved protein kinase in calmodulin-mediated signaling pathways, associated with cell division. High EEF2K protein expression has now been found to be associated with a variety of cancers, including lung cancer (Eukaryotic activation factors 2 kinase inhibitors of the expression of proteins in cells transplantation and cancer cell metabolism. Int J cancer.2018May 1 (9): 1865-1877). However, reports related to EEF2K protein autoantibodies are not found at present, and EEF2K protein autoantibodies and lung cancer are not reported.

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 the EEF2K protein autoantibody in preparing a lung cancer screening kit.

As the application, the reagent for detecting the EEF2K protein autoantibody is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.

As the application, the reagent for detecting the EEF2K protein autoantibody is a western blot reagent.

As mentioned above, the reagent for detecting the EEF2K protein autoantibody is a reagent for a protein chip detection method.

As mentioned above, the reagent for detecting EEF2K protein autoantibodies is a reagent for detecting EEF2K protein autoantibodies in human serum.

A lung cancer screening kit comprises a reagent for detecting EEF2K protein autoantibody.

As the kit, the reagent for detecting the EEF2K protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay reagent.

As the kit, the reagent for detecting the EEF2K protein autoantibody is a western blot reagent.

As the kit, the reagent for detecting the EEF2K protein autoantibody is a reagent for a protein chip detection method.

As the kit, the reagent for detecting the EEF2K protein autoantibody is a reagent for detecting the EEF2K protein autoantibody in human serum.

The key point of the invention is that the content of the EEF2K autoantibody in the human blood is determined to be obviously related to the risk of lung cancer, so that the risk of lung cancer can be judged by detecting the content of the EEF2K autoantibody in the human blood, and as for a means for specifically detecting the EEF2K autoantibody in the human blood, various means disclosed in the prior art can be adopted.

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 serum can be used as a detection sample, so that the harm to a patient is low. The invention has good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

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.

Hereinafter, "EEF2K autoantibody" refers to "EEF2K protein autoantibody".

Drawings

FIG. 1: lung cancer patients (LC), benign lung Disease (DC), healthy control (NC) serum levels of EEF2K autoantibodies were compared.

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 correlation of EEF2K autoantibodies in plasma with Lung cancer

1. Clinical data

30 lung cancer patients, 29 lung benign diseases (non-malignant tumors such as tuberculosis and hamartoma) and 29 healthy controls are selected, and basic information is as follows:

2. principle of detection

HuProt ^TM EEF2K protein (full-length protein, ensemble number is ENSP 00000456243) is fixed on a human protein customization chip, after serum is added for incubation, EEF2K autoantibodies (mainly including IgG and IgM antibodies and also some other types of antibodies) in the serum are combined, the unbound antibodies and other proteins are cleaned and removed, an anti-human IgM fluorescent labeled secondary antibody (cy 5 label, showing red) and an anti-human IgG fluorescent secondary antibody (cy 3 label, showing green) are used for detection, signals are read through a fluorescence scanner, and the strength of the signals is positively correlated with the affinity and the quantity of the antibodies.

3. Method of producing a composite material

The reagents used in this section were as follows:

the method comprises the following specific steps:

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 15min;

2) And (3) sealing: fixing 14 blocks in the rewarming chip, adding sealing liquid into each block after fixing, placing on a side swing table, and sealing at room temperature for 3hr;

3) Incubation of serum samples: after sealing is finished, pouring the sealing solution completely, then quickly adding the prepared serum incubation solution, wherein each chip can incubate 14 serum samples, the sample loading volume of each serum sample is 200 mu L, laterally swinging a shaker at 20rpm, and incubating overnight at 4 ℃ (the serum samples are frozen and thawed in a chromatography cabinet at 4 ℃, and the incubation solution is added to dilute at a ratio of 1: 50 to obtain the serum incubation solution);

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 removing the chip fence, putting the chip into a chip cleaning box with cleaning solution, horizontally shaking at room temperature of 80rpm, and cleaning for 10min for 3 times;

5) And (3) secondary antibody incubation: transferring the chip into an incubation box added with 3mL of second antibody incubation liquid, laterally swinging a shaker at 40rpm, keeping out of the light, and keeping at room temperature for 1hr;

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 for 10min each time, on a horizontal shaker at room temperature and 80 rpm. Cleaning with ddH2O for 2 times, each time for 10min;

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.

4. Results

The mean expression level of EEF2K autoantibodies in the plasma of lung cancer patients was 203.9SNR (fluorescence signal to quantitative ratio), the mean expression level of EEF2K autoantibodies in the plasma of benign lung disease was 260.3SNR, and the mean expression level of EEF2K autoantibodies in the plasma of healthy control was 258.7 SNR. The lung cancer group was statistically significant compared to the benign lung disease group (p < 0.05) and the healthy control group (p < 0.05) (FIG. 1). The specificity of ROC analysis of the lung cancer group and benign diseases is 82.1%, and the sensitivity is 56.7% (FIG. 2); ROC analysis of the lung cancer group and healthy controls resulted in 89.7% specificity and 33.3% sensitivity (FIG. 3), indicating that EEF2K autoantibodies can specifically distinguish lung cancer from benign lung disease and healthy controls.

The results show that the level difference of the EEF2K autoantibody in the serum of the lung cancer patient and the non-lung cancer patient is obvious, and the purpose of screening the lung cancer can be achieved by detecting the level of the EEF2K autoantibody in the serum.

Example 2 composition of the detection kit of the invention and method of use thereof

1. Kit composition

Detection kit (14 persons):

2. method for using kit

Same as example 1, third part- "detection of EEF2K autoantibodies in serum".

The kit can screen the risk of lung cancer of the people to be detected by detecting the level of the EEF2K autoantibody in serum: if the level of EEF2K autoantibodies is low (relative to healthy humans), the risk of lung cancer is high, and if the level of EEF2K autoantibodies is high, the risk of lung cancer is low. The method 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.

Claims

1. Use of a reagent for detecting EEF2K protein autoantibodies in human serum in the preparation of a kit for screening lung cancer risk.

2. The use of claim 1, wherein the reagent for detecting EEF2K protein autoantibodies is a reagent for enzyme-linked immunosorbent assay.

3. The use according to claim 1, wherein the reagent for detecting the autoantibodies to the EEF2K protein is a western blot reagent.

4. The use of claim 1, wherein the reagent for detecting the EEF2K protein autoantibody is a reagent for a protein chip detection method.