CN110412274B - Application of C6ORF106 autoantibody detection reagent in preparation of lung cancer screening kit - Google Patents

Abstract

The invention relates to the field of in vitro diagnostic reagents, in particular to the use of a C6ORF106 autoantibody detection reagent in the preparation of a lung cancer screening kit. The present invention finds for the first time that the autoantibody level of C6ORF106 protein in the serum of lung cancer patients is significantly higher than that of healthy patients. In the present invention, the reagent for detecting the C6ORF106 protein autoantibody is used to prepare a lung cancer screening kit, which can realize the effective screening of lung cancer.

Description

The use of C6ORF106 autoantibody detection reagent in the preparation of lung cancer screening kit

technical field

The invention relates to the field of in vitro diagnostic reagents, in particular to the use of a C6ORF106 autoantibody detection reagent in the preparation of a lung cancer screening kit.

Background technique

Lung cancer is one of the most common malignant tumors in the world, and its morbidity and mortality are increasing year by year. Currently, the morbidity ranks first in the world, and it is a serious threat to human health and life.

Lung cancer is a kind of hidden disease, often showing clinical symptoms when the disease develops to the advanced stage. 70-80% of lung cancer patients are diagnosed with lung cancer symptoms in the middle or advanced stage, the cancer cells have spread, and missed the most advanced stage. The best time to cure, the five-year survival rate is low. For early-stage lung cancer patients, timely treatment can greatly improve the 5-year and above survival rate and quality of life of patients. Therefore, early diagnosis of lung cancer and effective screening are crucial.

Lung cancer screening refers to the routine physical examination of those without lung cancer-related symptoms to detect lung cancer before symptoms appear. If lung cancer molecular markers in plasma can be found, it is of great significance to prompt clinicians to take relevant treatment measures or decision-making for patients at an early stage.

Autoantibodies are antibodies produced by the body against its own organs, cells or cellular components. At present, autoantibodies of certain proteins have become markers of lung cancer, such as: p53, NY-ESO-1, CYFRA, etc. (Tang Z-M, Ling Z-G, WangC-M, Wu Y-B, Kong J-L (2017) Serum tumor- associated autoantibodies as diagnosticbiomarkers for lung cancer: Asystematic review and meta-analysis. PLoS ONE 12(7):e0182117).

C6ORF106 (Ensembl:ENSG00000196821), also known as ILRUN, is ubiquitously expressed in testis, adipose and other tissues. At present, there is no relevant report on the C6ORF106 protein autoantibody, nor is there any prior art related to lung cancer.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel autoantibody-based lung cancer marker, and the use of a detection reagent of the marker in the preparation of a lung cancer screening kit.

The technical scheme of the present invention includes:

Use of a reagent for detecting C6ORF106 protein autoantibody in the preparation of a lung cancer screening kit.

As mentioned above, the reagent for detecting C6ORF106 protein autoantibody is an enzyme-linked immunosorbent assay reagent or a linked immunoassay reagent.

As mentioned above, the reagent for detecting C6ORF106 protein autoantibody is a western blot reagent.

As mentioned above, the reagent for detecting C6ORF106 protein autoantibody is a reagent for a protein chip detection method.

As mentioned above, the reagent for detecting C6ORF106 protein autoantibody is a reagent for detecting C6ORF106 protein autoantibody in human serum.

A lung cancer screening kit, which includes reagents for detecting C6ORF106 protein autoantibodies.

As in the aforementioned kit, the reagent for detecting C6ORF106 protein autoantibody is an enzyme-linked immunosorbent assay reagent or an enzyme-linked immunosorbent assay reagent.

As in the aforementioned kit, the reagent for detecting C6ORF106 protein autoantibody is a western blot reagent.

As in the aforementioned kit, the reagent for detecting C6ORF106 protein autoantibody is a reagent for a protein chip detection method.

As in the aforementioned kit, the reagent for detecting C6ORF106 protein autoantibodies is a reagent for detecting C6ORF106 protein autoantibodies in human serum.

The invention provides a new lung cancer screening marker and a new lung cancer screening kit, which can realize the effective screening of lung cancer; and can use serum as a detection sample, and cause low harm to patients. The present invention has good application prospects.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Herein, "C6ORF106 autoantibody" refers to "C6ORF106 protein autoantibody".

Description of drawings

Figure 1: Comparison of C6ORF106 autoantibody levels in serum plasma of lung cancer patients (LC) and healthy controls (NC).

Figure 2: ROC analysis of lung cancer patients (LC) and healthy controls (NC).

Detailed ways

Example 1 The relationship between C6ORF106 autoantibody in plasma and lung cancer

1. Clinical data

30 lung cancer patients and 30 healthy controls were selected. The basic information is as follows:

Basic Information lung cancer patients healthy control number of people 30 30 age 58±10.5 42±8.9 male ratio 20 (66.7%) 13 (46.7%)

2. Detection principle

The C6ORF106 protein is immobilized on the HuProt ^TM human protein custom chip. After incubation with serum, C6ORF106 autoantibodies (mainly including IgG, IgM antibodies, and some other types of antibodies) in the serum will bind to it, and the unbound antibodies and other antibodies will be removed by washing. The protein was detected with anti-human IgM fluorescently labeled secondary antibody (cy5 labeled, showing red) and anti-human IgG fluorescent secondary antibody (cy3 labeled, showing green), and the signal was read by a fluorescence scanner. The intensity of the signal is related to the affinity of the antibody is positively correlated with quantity.

3. Methods

The reagents used in this section are as follows:

Specific steps are as follows:

1) Rewarming: Take the chip out of the -80°C refrigerator, place it in a 4°C refrigerator for half an hour, and then place it at room temperature for 15 minutes;

2) Sealing: After rewarming the chip, fix 14 blocks of fences. After fixing, add blocking solution to each block, place it on a side-swing shaker, and seal it at room temperature for 3 hours;

3) Serum sample incubation: After the blocking is completed, pour out the blocking solution, and then quickly add the pre-prepared serum incubation solution. Each chip can incubate 14 serum samples. The loading volume of each serum sample is 200 μL. Bed 20rpm, 4°C overnight incubation (serum samples were first placed in a 4°C chromatography cabinet to freeze and thaw, and the incubation solution was added to dilute at a ratio of 1:50 to obtain serum incubation solution);

4) Cleaning: Take out the chip and the chip fence together, aspirate the sample, and then quickly add an equal volume of PBST for several cycles to ensure that there is no cross-contamination between serum samples when the chip fence is removed. After removing the chip fence, place the chip in a chip cleaning box with cleaning solution, shake it horizontally, at room temperature 80 rpm, and clean it 3 times, 10 min each time;

5) Secondary antibody incubation: transfer the chip to an incubation box with 3 mL of secondary antibody incubation solution added, shake sideways at 40 rpm, protect from light, and stay at room temperature for 1 hr;

6) Cleaning: Take out the chip (be careful not to touch or scratch the top surface of the chip), place it in a chip cleaning box with cleaning solution, shake it horizontally, and clean it three times at 80 rpm at room temperature, 10 minutes each time. After completion, wash twice with ddH2O, 10min each time;

7) drying;

8) Scanning: Scan with Jingxin LuxScan 10K microarray chip scanner;

9) Data extraction: Open the corresponding GAL file (recording the location of the protein in the chip), align the chip image and each array of the GAL file as a whole, press the automatic alignment button, extract the data and save it.

4. Results

The average expression level of C6ORF106 autoantibody in the plasma of lung cancer patients was 91.3SNR (fluorescence signal relative quantitative ratio), and the average expression level of C6ORF106 autoantibody in the plasma of healthy controls was 52.8SNR. The lung cancer group was statistically significant compared with the healthy control group (p<0.05) (Figure 1). The ROC analysis results of the lung cancer group and healthy controls showed a specificity of 96.6% and a sensitivity of 27.6% (Fig. 2), indicating that the C6ORF106 autoantibody can specifically distinguish lung cancer from healthy controls.

From the above results, it can be seen that the levels of C6ORF106 autoantibodies in the serum of lung cancer patients and non-lung cancer patients are significantly different. The purpose of lung cancer screening can be achieved by detecting the level of C6ORF106 autoantibodies in serum.

Embodiment 2 The composition of the detection kit of the present invention and its use method

1. Composition of the kit

Test kit (14 people):

2. How to use the kit

Same as the third part of Example 1 - "Detection of C6ORF106 Autoantibody in Serum".

The kit of the present invention can detect the level of C6ORF106 autoantibody in serum, and can screen the risk of lung cancer in the population to be tested: if the level of C6ORF106 autoantibody is high (relative to healthy people), the risk of developing lung cancer is high. Low levels of C6ORF106 autoantibodies are associated with a low risk of lung cancer. It can be used for auxiliary diagnosis of clinical lung cancer, and provides effective basis for patients to take relevant treatment measures or decision-making, and has a good clinical application prospect.

Claims (4)

1. Use of a reagent for detecting C6ORF106 protein autoantibodies in the preparation of a lung cancer screening kit; the reagent for detecting C6ORF106 protein autoantibodies is a reagent for detecting C6ORF106 protein autoantibodies in human serum. 2 . The use according to claim 1 , wherein the reagent for detecting C6ORF106 protein autoantibodies is an enzyme-linked immunosorbent assay reagent. 3 . 3. purposes as claimed in claim 1 is characterized in that, the reagent that described detection C6ORF106 protein autoantibody is western blot reagent. 4. The use according to claim 1, wherein the reagent for detecting C6ORF106 protein autoantibody is a reagent for a protein chip detection method.

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