CN110596391B

CN110596391B - Application of FCRL2 autoantibody detection reagent in preparation of lung cancer screening kit

Info

Publication number: CN110596391B
Application number: CN201910897115.8A
Authority: CN
Inventors: 何彦琪; 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: 2022-11-11
Anticipated expiration: 2039-09-20
Also published as: CN110596391A

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of an FCRL2 autoantibody detection reagent in preparation of a lung cancer screening kit. The invention discovers for the first time that the autoantibody level of the FCRL2 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 FCRL2 protein autoantibody is used for preparing the lung cancer screening kit, so that effective screening of lung cancer can be realized.

Description

Application of FCRL2 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 an FCRL2 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 advanced stage, 70-80% of lung cancer patients are in the middle and advanced 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 biomakers for lung cancer: A systematic review and meta-analysis. PLoS ONE 12 (7): e 0182117).

The FCRL2 protein encoded by the FCRL2 gene (Ensembl: ENSG 00000132704) is a member of the immunoglobulin receptor superfamily, and is one of several Fc receptor-like glycoproteins gathered on the long arm of chromosome 1. At present, the FCRL2 protein is reported to be used as a diagnostic marker of chronic lymphocytic leukemia; however, no report related to the FCRL2 protein autoantibody exists at present, and no prior art related to the lung cancer by the FCRL2 protein autoantibody 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 FCRL2 protein autoantibody in preparing a lung cancer screening kit.

As the application, the reagent for detecting the FCRL2 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.

As for the application, the reagent for detecting the FCRL2 protein autoantibody is a western blot reagent.

As mentioned above, the reagent for detecting the FCRL2 protein autoantibody is a reagent for a protein chip detection method.

As the application, the reagent for detecting the FCRL2 protein autoantibody is a reagent for detecting the FCRL2 protein autoantibody in human serum.

A lung cancer screening kit, which comprises a reagent for detecting an FCRL2 protein autoantibody.

As the kit, the reagent for detecting the FCRL2 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunosorbent assay reagent.

As the kit, the reagent for detecting the FCRL2 protein autoantibody is a western blot reagent.

As the kit, the reagent for detecting the FCRL2 protein autoantibody is a reagent for a protein chip detection method.

As the kit, the reagent for detecting the FCRL2 protein autoantibody is a reagent for detecting the FCRL2 protein autoantibody in human serum.

The key point of the invention is that the content of the FCRL2 autoantibody in the human blood is determined to be significantly related to the risk of suffering from lung cancer, so the risk of suffering from lung cancer can be judged by detecting the content of the FCRL2 autoantibody in the human blood, and as for a means for specifically detecting the FCRL2 autoantibody in the 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.

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 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.

Hereinafter, "FCRL2 autoantibody" refers to "FCRL2 protein autoantibody".

Drawings

FIG. 1: lung cancer patients (LC), healthy control (NC) serum levels were compared for FCRL2 autoantibodies.

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

Detailed Description

Example 1 correlation of FCRL2 autoantibodies with lung cancer in plasma

1. Clinical data

59 lung cancer patients and 30 healthy controls were selected, and the basic information was as follows:

basic information	Patients with lung cancer	Healthy controls
			Number of people	59	30
Age (age)	54.5±7.6	42±8.9
			Proportion of male	37(62.7％)	13(46.7％)

2. Principle of detection

HuProt ^TM FCRL2 protein (full-length protein, with the Uniprot number: Q96LA 5-2) is fixed on a human protein customization chip, after serum is added for incubation, FCRL2 autoantibodies (mainly including IgG and IgM antibodies and also some 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 labeled and red) and an anti-human IgG fluorescent secondary antibody (cy 3 labeled and green) 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 for producing a composite material

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, completely pouring the sealing liquid, then quickly adding a prepared serum incubation liquid, wherein each chip can incubate 14 serum samples, the sample loading volume of each serum sample is 200 mu L, and the side shaking table is used for incubation at 20rpm and 4 ℃ overnight (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, 10min 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 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 the completion, the mixture is washed by ddH20 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 corresponding GAL file (recording the position of protein in the chip), aligning the chip image and GAL file array, pressing the automatic alignment button, extracting data and storing. 4. Results

The mean expression level of FCRL2 autoantibodies in the plasma of lung cancer patients was 44.7SNR (fluorescence signal to quantitative ratio), and the mean expression level of FCRL2 autoantibodies in the plasma of healthy controls was 61.2SNR. The lung cancer group was statistically significant compared to healthy controls (p < 0.01) (FIG. 1). The ROC analysis results of the lung cancer group and the healthy control showed 96.6% specificity and 23.7% sensitivity (fig. 2), indicating that the FCRL2 autoantibody can specifically distinguish lung cancer from the healthy control.

From the results, the differences of the FCRL2 autoantibodies in the serum of the lung cancer patients and the serum of the non-lung cancer patients are obvious, and the purpose of screening the lung cancer can be achieved by detecting the FCRL2 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. method for using kit

Same as example 1 third part- "detection of FCRL2 autoantibodies in serum".

The kit can screen the risk of the to-be-detected people suffering from lung cancer by detecting the level of the FCRL2 autoantibody in serum: if the FCRL2 autoantibody level is low (relative to healthy humans), the risk of lung cancer is high, and if the FCRL2 autoantibody level is high, 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 the reagent for detecting the FCRL2 protein autoantibody in preparing the kit for screening the risk of the lung cancer is characterized in that the reagent for detecting the FCRL2 protein autoantibody is the reagent for detecting the FCRL2 protein autoantibody in human serum.

2. The use according to claim 1, wherein the reagent for detecting the FCRL2 protein autoantibody is a reagent for an enzyme-linked immunosorbent assay.

3. The use according to claim 1, wherein the reagent for detecting an autoantibody to the FCRL2 protein is a western blot reagent.

4. The use according to claim 1, wherein the reagent for detecting the FCRL2 protein autoantibody is a reagent for a protein chip detection method.