CN110456065B

CN110456065B - Application of CCDC130 autoantibody detection reagent in preparation of lung cancer screening kit

Info

Publication number: CN110456065B
Application number: CN201910702833.5A
Authority: CN
Inventors: 李蕊岑; 唐怀蓉; 黄燕; 李文钰
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: 2019-07-31
Filing date: 2019-07-31
Publication date: 2022-09-09
Anticipated expiration: 2039-07-31
Also published as: CN110456065A

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a CCDC130 autoantibody detection reagent in preparing a lung cancer screening kit. The invention discovers for the first time that the autoantibody level of the CCDC130 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 CCDC130 protein autoantibody is used for preparing the lung cancer screening kit, so that effective screening of lung cancer can be realized.

Description

Application of CCDC130 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 a CCDC130 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 late stage, 70-80% of lung cancer patients are in the middle and late 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 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 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 tune-associated autoimmune polymers for lung cancer: A systematic review and meta-analysis. PLoS ONE 12(7): e 0182117).

CCDC130(Ensembl: ENSG00000104957) was ubiquitously expressed in spleen (RPKM 12.4), endometrium (RPKM 9.8) and 25 other tissues. At present, no report related to the CCDC130 protein autoantibody exists, and no prior art related to lung cancer exists.

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 CCDC130 protein autoantibody in preparing a lung cancer screening kit.

As the application, the reagent for detecting the CCDC130 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.

As for the application, the reagent for detecting the CCDC130 protein autoantibody is a western blot reagent.

As mentioned above, the reagent for detecting the CCDC130 protein autoantibody is a reagent for a protein chip detection method.

As for the aforementioned use, the reagent for detecting the CCDC130 protein autoantibody is a reagent for detecting the CCDC130 protein autoantibody in human serum.

A lung cancer screening kit, which comprises a reagent for detecting a CCDC130 protein autoantibody.

As the kit, the reagent for detecting the CCDC130 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.

As the kit, the reagent for detecting the CCDC130 protein autoantibody is a western blot reagent.

As the kit, the reagent for detecting the CCDC130 protein autoantibody is a reagent for a protein chip detection method.

As the kit, the reagent for detecting the CCDC130 protein autoantibody is a reagent for detecting the CCDC130 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.

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.

Hereinafter, "CCDC 130 autoantibody" refers to "CCDC 130 protein autoantibody".

Drawings

FIG. 1: lung cancer patients (LC), benign lung Disease (DC), healthy control (NC) plasma levels of CCDC130 autoantibodies were compared.

FIG. 2 is a schematic diagram: ROC analysis of lung cancer patients (LC) and benign lung Disease (DC).

FIG. 3: lung cancer patients (LC) were analyzed by ROC with healthy controls (NC).

Detailed Description

Example 1 correlation of CCDC130 autoantibodies with Lung cancer in plasma

First, clinical data

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

basic information	Lung cancer patients	Benign lung disease	Healthy controls
				Number of people	30	29	30
Age (age)	58±10.5	46.5±10.0	42±8.9
				Proportion of male	20(66.7)	12(41.4％)	13(46.7％)

Second, detection principle

HuProt ^TM The human protein customizing chip is fixed with CCDC130 protein, after being incubated by adding serum, CCDC130 autoantibodies (mainly including IgG and IgM antibodies and also some other types of antibodies) in the serum can be combined, the unbound antibodies and other proteins are removed by cleaning, and then an anti-human IgM fluorescent labeled secondary antibody (cy5 labeled and showing red color) and an anti-human IgG fluorescent labeled secondary antibody (cy5 labeled and showing red color) are usedAnd detecting a secondary antibody (cy3 labeled and showing green color), reading a signal by a fluorescence scanner, wherein the strength of the signal is in positive correlation with the affinity and the quantity of the antibody.

Third, method

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 serum samples: after sealing is finished, pouring the sealing liquid completely, then quickly adding a serum incubation liquid prepared in advance, wherein each chip can incubate 14 serum samples, the sample loading volume of each serum sample is 200 mu L, and the shaking table is laterally swung at 20rpm and incubated 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: taking out the chip and the chip fence together, sucking out the sample, then quickly adding the PBST with the same volume, and repeating the steps for a plurality of times to ensure that no cross contamination exists among the serum samples when the chip fence is dismantled. 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 second antibody incubation liquid, laterally swinging a shaker at 40rpm, keeping out of the light, 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 CCDC130 autoantibodies in the plasma of lung cancer patients was 21.3SNR (fluorescence signal relative quantitative ratio), the mean expression level of CCDC130 autoantibodies in the plasma of benign lung disease was 12.9SNR, and the mean expression level of CCDC130 autoantibodies in the plasma of healthy control was 14.5 SNR. The lung cancer group was statistically significant compared to both the benign lung disease group (p <0.05) and the healthy control group (p <0.001) (fig. 1). The ROC analysis result of the lung cancer group and benign diseases shows that the specificity is 93.1%, and the sensitivity is 24.1% (figure 2); the results of ROC analysis of lung cancer groups versus healthy controls showed specificity of 96.4% and sensitivity of 27.6% (FIG. 3), indicating that CCDC130 autoantibodies can specifically distinguish lung cancer from benign lung disease and healthy controls.

The results show that the level difference of the CCDC130 autoantibody in the serum of the lung cancer patient and the non-lung cancer patient is obvious, and the aim of screening the lung cancer can be achieved by detecting the level of the CCDC130 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

The same as example 1, third part- "detection of CCDC130 autoantibodies in serum".

The kit can screen the risk of lung cancer of the people to be detected by detecting the level of the CCDC130 autoantibody in serum: if the level of CCDC130 autoantibodies is high (relative to healthy humans), the risk of lung cancer is high, and if the level of CCDC130 autoantibodies is low, 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. The application of a reagent for detecting the CCDC130 protein autoantibody in preparing a lung cancer screening kit; the reagent for detecting the CCDC130 protein autoantibody is a reagent for detecting the CCDC130 protein autoantibody in human serum.

2. The use according to claim 1, wherein the reagent for detecting the autoantibody to the CCDC130 protein is an enzyme-linked immunosorbent assay reagent.

3. The use according to claim 1, wherein the reagent for detecting autoantibodies to the CCDC130 protein is a western blot reagent.

4. The use according to claim 1, wherein the reagent for detecting the autoantibody to the CCDC130 protein is a reagent for a protein chip detection method.