CN111257572B - HRAS protein autoantibodies and their applications - Google Patents

Abstract

The invention discloses HRAS protein autoantibody and its application, including its application in the preparation of cancer detection products and in cancer detection. The invention can realize effective detection and auxiliary diagnosis of cancer, and can more accurately distinguish lung cancer patients from healthy people; and the invention can use serum as a detection sample, with simple operation, low cost, high accuracy and no trauma. The present invention has good application prospects.

Description

HRAS protein autoantibodies and their applications

technical field

The invention belongs to the field of biomedicine, and specifically relates to HRAS protein autoantibodies and their application in the preparation of cancer detection products and application in cancer detection.

Background technique

According to data from the World Health Organization, in 2018, the number of new cancer patients worldwide reached 18.1 million, 9.6 million people died of cancer, and 2.09 million new cases of lung cancer occurred globally, ranking first among all cancer types. According to the statistics of the National Cancer Center in 2017, the incidence and mortality of lung cancer ranked first among malignant tumors, with the incidence rate of 20.27% in men, and the incidence rate of 14.94% in women. breast cancer (17.07%); the mortality rate was the first in both males and females, 23.89% and 17.70%, respectively. It is predicted that between 2015 and 2030, lung cancer mortality in China may increase by about 40%.

Lung cancer can be histologically divided into two main categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 79% of lung cancers diagnosed and includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Currently, there have been tremendous advances in targeted therapy and immunotherapy for lung cancer, but surgical resection, adjuvant radiotherapy and/or chemotherapy remain the preferred methods for early treatment of NSCLC patients. Although survival rates for all cancers have improved in recent years, lung cancer survival rates remain low. In 2012-2015, the survival rate for lung cancer in men was 16.8%, which was 62.5% lower than that of thyroid cancer, which has the highest survival rate, and the survival rate for women was only 25.1%. The low survival rate of lung cancer is related to the limitations of diagnostic techniques. Most lung cancer patients are found at an advanced stage at the time of diagnosis, losing the best time for treatment. Currently, high-resolution (or low-dose) computed tomography (LDCT) of the chest is the only screening test that is effective in reducing early mortality from lung cancer. Programs for free screening of high-risk groups with LDCT have been established in many regions. A study in the United States has shown that LDCT screening has a good potential for early lung cancer screening, and may reduce the mortality rate of high-risk lung cancer groups by 20%. But clinical translation remains an issue. The problem with LDCT is that the high false-positive rate can lead to overdiagnosis to some extent. Repeated exposure to radiation may also lead to potential health hazards. Therefore, the discovery of non-invasive serum biomarkers for early lung cancer diagnosis will greatly benefit lung cancer intervention and prevention. Using biomarker-based initial screening followed by LDCT or combining biomarker testing with LDCT has become a better means of lung cancer diagnosis.

People have noticed that the body's immune system may have a preventive effect on tumors more than 100 years ago. The growth of tumors is accompanied by the production of some substances with antigenic properties. These substances are no longer treated as "self" by the body, but as antigens. The immune system produces antibodies to destroy these antigens. These antibodies are called autoantibodies. Since their discovery in the 1970s, hundreds of autoantibodies have been identified. Not all tumor-associated antigens elicit an immune response in all individuals, and not all immune responses are tumor-specific. Tumor-associated autoantibody testing offers an alternative approach to cancer detection. This approach is expected to be achieved by the detection of autoantibodies that are highly expressed (relative to healthy people and benign tumors) in the serum of cancer patients. Currently, tumor-associated antigens, such as carbohydrate antigen (CA) 125, CA19-9, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP), are currently used in clinical practice. The content of these markers in serum increases with the growth of tumor size, so the sensitivity and specificity of tumor-associated antigens in the early stage of cancer are very poor. Autoantibodies are produced early in tumorigenesis and can be detected five years before clinical symptoms appear. In the early stage of cancer, the detected signal of tumor-associated antigens is very weak, but since the concentration of autoantibodies is much higher than that of antigens, the detection of autoantibodies is equivalent to an amplified signal. The half-life of autoantibodies in the blood circulation reaches 30 days, and it is also more stable than other markers in vitro. Autoantibody detection has the advantages of economical cost-effectiveness and simple operation. Autoantibody detection can be implemented directly on existing clinical testing platforms using archived sera.

HRAS (Ensembl:ENSG00000174775MIM:190020) is encoded by the HRAS gene on chromosome 11 and is ubiquitously expressed in skin (RPKM 24.4), brain (RPKM 14.8) and 25 other tissues. The HRAS protein encoded by HRAS is GTPase, and HRas is a small G protein belonging to the small GTPase superfamily. When HRas binds to guanosine triphosphate, it will bind to Raf kinases such as c-Raf, and further activate MAPK/ERK path. Mutations in this gene are associated with a variety of cancers, including bladder cancer, follicular thyroid cancer, and oral squamous cell carcinoma. Patent application WO/2018/187228 discloses early diagnosis of lung cancer by serological markers. At present, although anti-HRAS autoantibodies have been studied, their sensitivity is low and their clinical application value is limited.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing cancer detection technology, especially lung cancer, and provide an autoantibody with high sensitivity and specificity in the preparation of cancer detection products and the application in cancer detection.

One aspect of the present invention provides the use of autoantibodies in the preparation of products for cancer detection, wherein the autoantibodies include HRAS protein autoantibodies.

Preferably, the HRAS protein autoantibodies include IgG and/or IgA autoantibodies.

Preferably, the cancer includes lung cancer, bladder cancer, follicular thyroid cancer, and oral squamous cell carcinoma. More preferably, the cancer is lung cancer.

Preferably, the detection comprises coating the antigen on a solid phase carrier.

In a specific embodiment of the present invention, the antigen is HRAS protein antigen.

Specifically, the HRAS protein antigens are IgG and IgA.

Preferably, the coating includes direct coating and indirect coating, wherein the direct coating is to coat the antigen directly on the solid phase carrier, and the indirect coating is to pass between biotin and streptavidin. The specific reaction coats the antigen indirectly on the solid support.

In a specific embodiment of the present invention, the coating is an indirect coating method.

Preferably, the solid phase carrier includes enzyme-labeled microplates, microparticles, microspheres, affinity membranes, liquid phase chips, glass slides, test strips and plastic spheres.

In a specific embodiment of the present invention, the solid phase carrier is an enzyme-labeled microplate.

Preferably, the detection includes visible light chromogenic method, chemiluminescence method, and fluorescence luminescence method.

In a specific embodiment of the present invention, the detection is a fluorescence luminescence method.

Specifically, the fluorescent luminescence method is an enzyme-linked immunosorbent assay.

Preferably, the product includes reagents used to prepare the product, such as labeled secondary antibody, dilution buffer, coating buffer, blocking buffer, washing buffer, chromogenic substrate, and stop solution.

In a specific embodiment of the present invention, the labeled secondary antibodies are anti-human IgA horseradish peroxidase-labeled secondary antibodies and anti-human IgG horseradish peroxidase-labeled secondary antibodies.

In a specific embodiment of the present invention, the dilution buffer is PBST containing BSA.

Specifically, the dilution buffer is 1×PBST containing 1% BSA.

In a specific embodiment of the present invention, the coating buffer is a carbonate buffer.

Specifically, the coating buffer is 0.05M carbonate buffer at pH 9.6.

In a specific embodiment of the present invention, the blocking buffer is PBST containing BSA.

Specifically, the blocking buffer is 1×PBST containing 3% BSA.

In a specific embodiment of the present invention, the washing buffer is PBST.

Specifically, the washing buffer is 1×PBST at pH 7.4.

In a specific embodiment of the present invention, the chromogenic substrate is tetramethylbenzidine.

In a specific embodiment of the present invention, the stop solution is H ₂ SO ₄ .

Specifically, the stop solution is 2M H ₂ SO _{4 .}

Preferably, the product is used for qualitative or quantitative detection of the HRAS protein autoantibody in the sample to be tested.

Preferably, the samples to be tested include blood, serum, plasma, milk, tears, saliva, sweat, gastrointestinal fluid, respiratory secretions, and urine. More preferably, the sample is blood. Particularly preferably, the sample is serum.

Another aspect of the present invention provides the use of autoantibodies in cancer detection, the autoantibodies include HRAS protein autoantibodies.

Preferably, the HRAS protein autoantibodies include IgG and/or IgA autoantibodies.

In a specific embodiment of the present invention, the HRAS protein autoantibodies are IgG and IgA.

In a specific embodiment of the present invention, the cancer detection is lung cancer detection.

Specifically, the lung cancer detection can accurately identify lung cancer patients and healthy people.

Specifically, the lung cancer patients and healthy people were subjected to ROC curve analysis.

Specifically, among the lung cancer patients and healthy people, the specificity and sensitivity of HRAS IgG autoantibody ROC analysis were 92.52% and 8.91%, and the specificity and sensitivity of HRAS IgA autoantibody ROC analysis were 91.59% and 20.30%.

The invention provides the application of HRAS protein autoantibody in the preparation of cancer detection products and cancer detection, which can realize effective detection and auxiliary diagnosis of cancer, and can more accurately distinguish lung cancer patients from healthy people; and can use serum as detection Sample, simple operation, low cost, high accuracy, non-invasive. The present invention has good application prospects.

Description of drawings

Figure 1: Comparison of HRAS IgG-type autoantibodies in serum of lung cancer patients and healthy controls.

Figure 2: Comparison of HRAS IgA autoantibody levels in serum of lung cancer patients and healthy controls.

Figure 3: Comparison of HRAS IgG autoantibody levels in serum of patients with stage I lung cancer and healthy controls.

Figure 4: Comparison of HRAS IgA autoantibody levels in serum of patients with stage I lung cancer and healthy controls.

Figure 5: Comparison of HRAS IgG autoantibody levels in serum of patients with stage II lung cancer and healthy controls.

Figure 6: Comparison of HRAS IgA autoantibody levels in serum of patients with stage II lung cancer and healthy controls.

Figure 7: Comparison of HRAS IgG autoantibody levels in serum of patients with stage III lung cancer and healthy controls.

Figure 8: Comparison of HRAS IgA autoantibody levels in serum of patients with stage III lung cancer and healthy controls.

Figure 9: Comparison of HRAS IgG autoantibody levels in serum of patients with stage IV lung cancer and healthy controls.

Figure 10: Comparison of HRAS IgA autoantibody levels in serum of patients with stage IV lung cancer and healthy controls.

Figure 11: ROC analysis of HRAS IgG-type autoantibodies in sera of lung cancer patients and healthy controls.

Figure 12: ROC analysis of HRAS IgA type autoantibodies in sera of lung cancer patients and healthy controls.

Detailed ways

Unless otherwise defined, professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. like:

The HRAS autoantibodies in the present invention refer to HRAS protein autoantibodies.

The ROC curve in the present invention refers to the receiver operating characteristic curve.

The TMB in the present invention refers to tetramethylbenzidine.

The BSA in the present invention refers to bovine serum albumin.

PSBT in the present invention refers to phosphate Tween buffer.

The present invention will be further described below with reference to the accompanying drawings and specific embodiments of the description. These examples are only intended to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in the following examples are usually in accordance with the conventional conditions in the art or in accordance with the conditions suggested by the manufacturer. Unless otherwise specified, all are conventional methods.

Example 1 Application of HRAS autoantibodies in serum in the detection of lung cancer

1. Clinical samples

202 lung cancer patients and 107 healthy controls were selected. The basic information is shown in Table 1:

Table 1: Basic Information Sheet for Lung Cancer Patients

2. Rationale

The HRAS protein is immobilized on the enzyme-labeled well plate. After incubation with serum, HRAS autoantibodies (mainly including IgG, IgA antibodies, and some other types of antibodies) in the serum will bind to it, and the unbound antibodies and other proteins will be removed by washing. Then add anti-human IgA horseradish peroxidase-labeled secondary antibody or anti-human IgG horseradish peroxidase-labeled secondary antibody for incubation, wash off the unbound secondary antibody, add horseradish peroxidase substrate, wait for the substrate After color development, detected by a microplate reader, the intensity of the signal is positively correlated with the number of HRAS autoantibodies.

3. Method

(1) Coating HRAS protein: Dilute the protein with 0.05M PH9.6 carbonate buffer, add 50ng/well of the diluted protein to the reaction well of the ELISA plate, overnight at 4°C;

(2) Rewarming: The ELISA plate in step (1) was taken out from the 4°C refrigerator, placed at room temperature for half an hour, and washed four times with 1×PBST of pH 7.4.

(3) Blocking: Add 1×PBST blocking solution containing 3% BSA to the enzyme label in step (2), incubate at 37°C for 1 hour, and wash twice;

(4) Add serum samples: Add serum samples diluted 1:200 to the ELISA plate in step (3), place them on a side-swing shaker at 12 rpm, incubate for 2.5 hours at room temperature, and wash four times (serum needs to be frozen at 4°C in advance Fusion, the diluted serum is first shaken and mixed before sampling);

(5) Add enzyme-labeled antibody: Add horseradish peroxidase-labeled anti-human IgG or anti-human IgA antibody (4 μg/ml) to each reaction well of the enzyme-labeled plate in step (4), and place it on a side rocker. Bed 12rpm, incubate for 1 hour at room temperature in the dark, wash twice;

(6) Add chromogenic substrate: add 50 μl of temporarily prepared TMB substrate solution to each reaction well of the ELISA plate in step (5);

(7) Termination: After the liquid in the well of the ELISA plate in step (6) turns blue, add 25 μl 2MH2SO4 to each reaction well;

(8) On-machine reading: place the microplate plate of step (7) in a TECAN F50 microplate reader to measure the reading of OD 450, and save the data.

4. Experimental results

The data measured by the microplate reader was analyzed by statistical software, and the ROC curve analysis of HRAS IgG and IgA autoantibodies in the lung cancer group and the healthy group was performed. The results are as follows:

It can be seen from Figure 1 that the expression level of HRAS IgG autoantibodies in the serum of lung cancer patients was not statistically different from that of healthy controls.

As can be seen from Figure 2, the expression level of HRAS IgA autoantibodies in the serum of lung cancer patients was significantly different from that of the healthy control group.

It can be seen from Figure 3 and Figure 4 that the levels of IgG-type autoantibodies and IgA-type autoantibodies in stage I lung cancer patients were not statistically different from those in healthy people.

It can be seen from Figure 5 that there is no statistical difference between the levels of IgG autoantibodies in stage II lung cancer patients and healthy controls; as can be seen from Figure 6, there is a statistically significant difference between IgA autoantibodies in stage II lung cancer patients and healthy controls.

It can be seen from Figures 7 and 8 that the levels of IgG-type autoantibodies and IgA-type autoantibodies in stage III lung cancer patients were not statistically different from those in healthy people.

It can be seen from Figure 9 that there is no statistical difference in the level of IgA type autoantibodies between patients with stage IV lung cancer and healthy controls. As can be seen from Figure 10, there is a significant difference in the level of IgG type autoantibodies between lung cancer patients and healthy controls.

As can be seen from FIG. 11 , the ROC analysis specificity of the HRAS IgG autoantibody in the lung cancer group and the healthy group was 92.52%, and the sensitivity was 8.91%.

12 , in the ROC analysis of the HRAS IgA type autoantibody in the lung cancer group and the healthy group, the specificity was 91.59% and the sensitivity was 20.30%.

Based on the above results, it can be seen that the levels of HRAS autoantibodies in the serum of lung cancer patients and healthy people are significantly different. By detecting the level of HRAS autoantibodies in serum, the purpose of lung cancer detection can be achieved.

The descriptions of the above-described embodiments are only for understanding the method of the present invention, however, the present invention is not limited to the specific details of the above-described embodiments. For those skilled in the art, without departing from the principles of the present invention, the present invention can also be improved and modified, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims (4)

1. the application of HRAS protein autoantibody as a marker in the preparation of a product for cancer detection, wherein the HRAS protein autoantibody is an IgA antibody, the cancer is lung cancer, and the detection comprises an antigen. Coated on a solid phase carrier, the antigen is HRAS protein antigen. 2. application according to claim 1 is characterized in that, described coating comprises direct coating and indirect coating, wherein, direct coating is to coat antigen directly on the solid phase carrier, and indirect coating is The antigen is indirectly coated on the solid support by the specific reaction between biotin and streptavidin. 3. The application according to claim 1, wherein the solid phase carrier comprises an enzyme-labeled microplate, a microsphere, an affinity membrane and a glass slide. 4 . The application according to claim 1 , wherein the detection comprises visible light colorimetry, chemiluminescence or fluorescence luminescence. 5 .

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