CN110596386A

CN110596386A - Application of EGFLAM autoantibody detection reagent in preparation of lung cancer screening kit

Info

Publication number: CN110596386A
Application number: CN201910894446.6A
Authority: CN
Inventors: 张立; 李为民
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2019-12-20
Anticipated expiration: 2039-09-20
Also published as: CN110596386B

Abstract

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

Description

Application of EGFLAM 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 EGFLAM 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.

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 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, WangC-M, Wu Y-B, Kong J-L (2017) Serum tune-associated autoimmune agents as diagnostic biologics for lung cancer: A systematic review and meta-analysis. PLoS ONE 12(7): e 0182117).

The EGFLAM gene (gene sequence number Ensembl: ENSG00000164318) is widely distributed in tissues such as placenta, fat and the like. At present, no report related to the EGFLAM 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 EGFLAM protein autoantibody in preparing a lung cancer screening kit.

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

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

As the application, the reagent for detecting the EGFLAM protein autoantibody is a reagent for a protein chip detection method.

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

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

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

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

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

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

The key point of the invention is that the content of the EGFL AM autoantibody in human blood is determined to be obviously related to the risk of suffering from lung cancer, so that the risk of suffering from lung cancer can be judged by detecting the content of the EGFL AM autoantibody in human blood, and as for a means for specifically detecting the EGFL autoantibody in human blood, various means disclosed in the prior art can be adopted.

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.

"EGFRAM autoantibody" herein refers to "EGFRAM protein autoantibody".

Drawings

FIG. 1: lung cancer patients (LC), healthy control (NC) serum levels of egfram autoantibodies were compared.

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

Detailed Description

Example 1 correlation of EGFLAM autoantibodies in plasma with Lung cancer

First, 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 (age)	49.5±5.7	42.0±8.9
			Proportion of male	20(66.7％)	13(44.8％)

Second, detection principle

HuProt^TMHuman protein custom on-chip immobilizationThe EGFL protein is determined (the adopted EGFL protein is full-length protein, the protein serial number is Ensembl: ENSP00000423228), serum is added for incubation, the EGFL autoantibodies (mainly including IgG and IgM antibodies and other antibodies) in the serum can be combined, the unbound antibodies and other proteins are cleaned and removed, an anti-human IgM fluorescent labeled secondary antibody (cy5 labeled and shown in red) and an anti-human IgG fluorescent secondary antibody (cy3 labeled and shown in green) are used for detection, signals are read through a fluorescence scanner, and the strength of the signals is positively correlated with the affinity and the quantity of the antibodies.

Third, method (detection of EGFLAM autoantibody 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-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: 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 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 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 completion with ddH₂O 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.

Fourthly, the result

The mean expression level of egfl autoantibodies in the plasma of lung cancer patients was 36.8SNR (fluorescence signal to quantitative ratio), and the mean expression level of egfl autoantibodies in the plasma of healthy controls was 19.8 SNR. The lung cancer group was statistically significant compared to healthy controls (p <0.05) (fig. 1). The results of ROC analysis of lung cancer groups and healthy controls showed specificity of 96.6% and sensitivity of 13.3% (FIG. 2), indicating that EGFLAM autoantibodies can specifically distinguish lung cancer from healthy controls.

The results show that the level difference of the EGFLAM 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 EGFLAM 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 EGFL AM autoantibodies in serum".

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

2. The use of claim 1, wherein the reagent for detecting the EGFLAM protein autoantibody is a reagent for an enzyme-linked immunosorbent assay or a combined immunoassay reagent.

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

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

5. The use of any one of claims 1 to 4, wherein the reagent for detecting the EGFLAM protein autoantibody is a reagent for detecting the EGFLAM protein autoantibody in human serum.

6. A lung cancer screening kit is characterized by comprising a reagent for detecting an EGFLAM protein autoantibody.

7. The kit of claim 6, wherein the reagent for detecting the EGFLAM protein autoantibody is an enzyme-linked immunosorbent assay reagent or an enzyme-linked immunoassay reagent.

8. The kit of claim 6, wherein the reagent for detecting the EGFRAM protein autoantibody is a western blot reagent.

9. The kit of claim 6, wherein the reagent for detecting the EGFLAM protein autoantibody is a reagent for a protein chip detection method.

10. The kit of any one of claims 6 to 9, wherein the reagent for detecting the EGFLAM protein autoantibody is a reagent for detecting the EGFLAM protein autoantibody in human serum.