CN110412280B

CN110412280B - Application of SLC25A25 autoantibody detection reagent in preparation of lung cancer screening kit

Info

Publication number: CN110412280B
Application number: CN201910572197.9A
Authority: CN
Inventors: 郭婷婷; 张立
Original assignee: West China Precision Medicine Industrial Technology Institute; West China Hospital of Sichuan University
Current assignee: West China Precision Medicine Industrial Technology Institute; West China Hospital of Sichuan University
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2022-09-09
Anticipated expiration: 2039-06-26
Also published as: CN110412280A

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of an SLC25A25 autoantibody detection reagent in preparing a lung cancer screening kit. The invention discovers for the first time that the autoantibody level of SLC25A25 protein in serum of a lung cancer patient is obviously higher than that of a healthy patient. According to the invention, the reagent for detecting the SLC25A25 protein autoantibody is used for preparing the lung cancer screening kit, so that effective screening of lung cancer can be realized.

Description

Application of SLC25A25 autoantibody detection reagent in preparation of lung cancer screening kit

Technical Field

The invention relates to the field of in-vitro diagnostic reagents, in particular to an application of an SLC25A25 autoantibody detection reagent in preparing 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 the conventional physical examination is carried out on people without lung cancer related symptoms, and the lung cancer is found in time before the symptoms appear. If the lung cancer molecular marker in the blood plasma can be found, it is of great significance to prompt 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 tune-associated autoimmune polymers for lung cancer: A systematic review and meta-analysis. PLoS ONE 12(7): e 0182117).

The SLC25A25 gene (Ensembl: ENSG00000148339) is No. 25 member of solute transporter family 25 (solute carrier family 25), and the SLC25A25 protein encoded by the gene belongs to the calcium-binding mitochondrial vector family and has a characteristic mitochondrial vector domain at the C-terminus. The protein is present in the inner membrane of mitochondria and functions as a transporter. SLC25A25 protein has been reported to be associated with colorectal cancer; however, no reports related to the SLC25A25 protein autoantibody are found at present, and no prior art related to the SLC25A25 protein autoantibody and lung cancer is found.

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 SLC25A25 protein autoantibody in preparing a lung cancer screening kit.

As the application, the reagent for detecting the SLC25A25 protein autoantibody is a reagent for enzyme-linked immunosorbent assay.

As for the application, the reagent for detecting the SLC25A25 protein autoantibody is a western blot reagent.

As mentioned above, the reagent for detecting SLC25A25 protein autoantibody is a reagent for protein chip detection method.

As mentioned above, the reagent for detecting SLC25A25 protein autoantibody is a reagent for detecting SLC25A25 protein autoantibody in human serum.

A lung cancer screening kit comprises a reagent for detecting SLC25A25 protein autoantibody.

As the kit, the reagent for detecting the SLC25A25 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.

The kit is characterized in that the reagent for detecting the SLC25A25 protein autoantibody is a western blot reagent.

As the kit, the reagent for detecting the SLC25A25 protein autoantibody is a reagent for a protein chip detection method.

As the kit, the reagent for detecting the SLC25A25 protein autoantibody is a reagent for detecting the SLC25A25 protein autoantibody in human serum.

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 foregoing aspects of the present invention are explained in further detail below with reference to specific 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, "SLC 25a25 autoantibody" refers to "SLC 25a25 protein autoantibody".

Drawings

FIG. 1: lung cancer patients (LC), benign lung Disease (DC), healthy control (NC) plasma SLC25a25 autoantibody levels were compared.

FIG. 2 is a schematic diagram: lung cancer patients (LC) were analyzed by ROC with healthy controls (NC).

Detailed Description

Example 1 relationship of SLC25A25 autoantibodies to Lung cancer in plasma

First, clinical data

30 lung cancer patients and 30 healthy controls are selected, and basic information is as follows:

second, detection principle

HuProt ^TM SLC25A25 protein is fixed on a human protein customization chip, after serum is added for incubation, SLC25A25 autoantibodies (mainly including IgG and IgM antibodies and other types of antibodies) in the serum can be combined, the unbound antibodies and other proteins are removed by cleaning, anti-human IgM fluorescent labeled secondary antibody (cy5 labeled and red) and anti-human IgG fluorescent secondary antibody (cy3 labeled and green) are used for detection, signals are read by a fluorescence scanner, and the strength of the signals is positively correlated with the affinity and the quantity of the antibodies.

Third, method

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 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 serum samples: after sealing is finished, pouring the sealing liquid completely, then quickly adding the prepared serum incubation liquid, wherein each chip can incubate 14 serum samples, the sample loading volume of each serum sample is 200 mu L, shaking the table at 20rpm laterally, and incubating overnight at 4 ℃ (the serum samples are frozen and thawed in a chromatography cabinet at 4 ℃, and the incubation liquid is added to dilute in a ratio of 1: 50 to obtain the serum 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 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 3 times, each time for 10 min;

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 the completion, the mixture is washed for 2 times for 10min by ddH 2O;

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 SLC25a25 autoantibody in the plasma of lung cancer patients was 10.3SNR (fluorescence signal versus quantitative ratio), and the mean expression level of SLC25a25 autoantibody in healthy control plasma was 7.0 SNR. The lung cancer group was statistically significant compared to healthy controls (p <0.05) (fig. 1). The ROC analysis results for lung cancer group and healthy control showed 96.6% specificity and 24.1% sensitivity (fig. 2), indicating that SLC25a25 autoantibody can specifically distinguish lung cancer from healthy control.

The results show that the level difference of the SLC25A25 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 SLC25A25 autoantibody in the serum.

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

Kit composition

Detection kit (14 persons):

second, kit using method

Same as example 1, third part- "detection of SLC25A25 autoantibodies in serum".

The kit can screen the risk of the patient to be detected for the lung cancer by detecting the level of SLC25A25 autoantibody in serum: if the level of SLC25a25 autoantibodies is high (relative to healthy humans) then the risk for lung cancer is high, and if the level of SLC25a25 autoantibodies is low then the risk for 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. The application of a reagent for detecting the SLC25A25 protein autoantibody in preparing a lung cancer screening kit is characterized in that the reagent for detecting the SLC25A25 protein autoantibody is a reagent for detecting the SLC25A25 protein autoantibody in human serum.

2. The use of claim 1, wherein said reagent for detecting SLC25A25 protein autoantibody is an enzyme-linked immunosorbent assay reagent.

3. The use of claim 1, wherein the reagent for detecting SLC25A25 protein autoantibodies is a western blot reagent.

4. The use of claim 1, wherein said reagent for detecting SLC25A25 protein autoantibody is a reagent for a protein chip detection method.