CN110412299B

CN110412299B - Application of HECTD3 autoantibody detection reagent in preparation of lung cancer screening kit

Info

Publication number: CN110412299B
Application number: CN201910838256.2A
Authority: CN
Inventors: 王巍
Original assignee: West China Second University Hospital of Sichuan University
Current assignee: West China Second University Hospital of Sichuan University
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2022-11-04
Anticipated expiration: 2039-09-05
Also published as: CN110412299A

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of an HECTD3 autoantibody detection reagent in preparation of a lung cancer screening kit. The invention discovers for the first time that the autoantibody level of the HECTD3 protein in the serum of a lung cancer patient is obviously lower than that of a healthy patient. According to the invention, the reagent for detecting the HECTD3 protein autoantibody is used for preparing the lung cancer screening kit, so that the effective screening of the lung cancer can be realized.

Description

Application of HECTD3 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 application of an HECTD3 autoantibody detection reagent in preparation of 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.

Lung cancer is a disease good in hiding, and often shows clinical symptoms when the disease develops to the advanced stage, 70-80% of lung cancer patients are in the middle and advanced stages when being diagnosed with lung cancer symptoms, cancer cells are diffused, the best cure 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 tumor-associated autoimmune tumors for lung cancer: A systematic review and meta-analysis. PLoS ONE 12 (7): e 0182117).

HECTD3 (Ensembl: ENSG 00000126107) is an E3 ubiquitin ligase. At present, no report related to the HECTD3 protein autoantibody exists, and no prior art related to lung cancer exists.

Disclosure of Invention

The invention aims to provide a novel lung cancer marker of autoantibody and application of a detection reagent of the marker in preparing a lung cancer screening kit.

The technical scheme of the invention comprises the following steps:

application of a reagent for detecting the HECTD3 protein autoantibody in preparing a lung cancer screening kit.

As the application, the reagent for detecting the HECTD3 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.

As for the application, the reagent for detecting the HECTD3 protein autoantibody is a western blot reagent.

As mentioned above, the reagent for detecting the HECTD3 protein autoantibody is a reagent for a protein chip detection method.

As for the application, the reagent for detecting the HECTD3 protein autoantibody is a reagent for detecting the HECTD3 protein autoantibody in human serum.

A lung cancer screening kit comprises a reagent for detecting an autoantibody of HECTD3 protein.

As the kit, the reagent for detecting the HECTD3 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay reagent.

According to the kit, the reagent for detecting the HECTD3 protein autoantibody is a western blot reagent.

As the kit, the reagent for detecting the HECTD3 protein autoantibody is a reagent for a protein chip detection method.

As the kit, the reagent for detecting the HECTD3 protein autoantibody is a reagent for detecting the HECTD3 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.

The term "HECTD3 autoantibody" as used herein refers to a "HECTD3 protein autoantibody".

Drawings

FIG. 1: comparison of HECTD3 autoantibody levels in serum of lung cancer patients (LC), healthy controls (NC).

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

Detailed Description

Example 1 correlation of HECTD3 autoantibodies in plasma with Lung cancer

1. Clinical data

30 lung cancer patients and 29 healthy controls are selected, and the basic information is as follows:

basic information	Patients with lung cancer	Healthy controls
			Number of people	30	29
Age(s)	49.5±5.7	42.0±8.9
			Proportion of male	20(66.7％)	13(44.8％)

2. Principle of detection

HuProt ^TM The human protein customization chip is fixed with HECTD3 protein, after the human protein customization chip is incubated by adding serum, HECTD3 autoantibodies (mainly comprising IgG and IgM antibodies and other antibodies) in the serum can be combined, the unbound antibodies and other proteins are removed by cleaning, an anti-human IgM fluorescent labeled secondary antibody (cy 5 label, showing red color) and an anti-human IgG fluorescent secondary antibody (cy 3 label, showing green color) are used for detection, a signal is read by a fluorescence scanner, and the strength of the signal is positively correlated with the affinity and the quantity of the antibodies.

3. Method (detection of HECTD3 autoantibodies in serum)

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 the temperature of-80 ℃, putting the chip in the refrigerator at the temperature of 4 ℃ for rewarming for half an hour, and then putting the chip in the refrigerator at the room temperature for rewarming for 15min;

2) And (3) sealing: fixing 14 blocks in the re-warmed 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 loading volume of each serum sample is 200 mu L, and performing side-shaking at 20rpm at 4 ℃ for overnight incubation (the serum samples are frozen and thawed in a 4 ℃ chromatography cabinet, and the incubation solution is added to dilute in a ratio of 1;

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 secondary antibody incubation liquid, laterally swinging a shaker at 40rpm, keeping out of the sun, 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. After completion with ddH ₂ O cleaning for 2 times, 10min each time;

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 HECTD3 autoantibodies in the plasma of lung cancer patients was 7.1SNR (fluorescence signal to quantitative ratio), and the mean expression level of HECTD3 autoantibodies in healthy control plasma was 9.6SNR. Lung cancer groups were statistically significant compared to healthy controls (p < 0.05) (figure 1). The ROC analysis result of the lung cancer group and the healthy control shows that the specificity is 96.6% and the sensitivity is 16.7% (figure 2), which shows that the HECTD3 autoantibody can specifically distinguish the lung cancer from the healthy control.

The results show that the level difference of the HECTD3 autoantibodies in the serum of the lung cancer patients and the serum of the non-lung cancer patients is obvious, and the aim of screening the lung cancer can be fulfilled by detecting the level of the HECTD3 autoantibodies 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. kit using method

Same as example 1, third part- "detection of HECTD3 autoantibodies in serum".

The kit can screen the risk of lung cancer of the people to be detected by detecting the level of the HECTD3 autoantibody in serum: if the level of HECTD3 autoantibodies is low (relative to healthy people), the risk of developing lung cancer is high, and if the level of HECTD3 autoantibodies is high, the risk of developing 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 the reagent for detecting the HECTD3 protein autoantibody in preparing the kit for screening the risk of suffering from lung cancer is characterized in that the reagent for detecting the HECTD3 protein autoantibody is a reagent for detecting the HECTD3 protein autoantibody in human serum.

2. The use according to claim 1, wherein the reagent for detecting the HECTD3 protein autoantibody is a reagent for enzyme-linked immunosorbent assay.

3. The use according to claim 1, wherein the reagent for detecting an autoantibody to HECTD3 protein is a western blot reagent.

4. The use according to claim 1, wherein the reagent for detecting the HECTD3 protein autoantibody is a reagent for a protein chip detection method.