CN110412279B

CN110412279B - Application of KLC3 autoantibody detection reagent in preparation of lung cancer screening kit

Info

Publication number: CN110412279B
Application number: CN201910570875.8A
Authority: CN
Inventors: 刘志强; 张立
Original assignee: West China Precision Medicine Industrial Technology Institute; West China Hospital of Sichuan University
Current assignee: West China Precision Medicine Industrial Technology Institute; West China Hospital of Sichuan University
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2022-09-09
Anticipated expiration: 2039-06-26
Also published as: CN110412279A

Abstract

The invention relates to the field of in-vitro diagnostic reagents, in particular to application of a KLC3 autoantibody detection reagent in preparing a lung cancer screening kit. The invention discovers for the first time that the level of autoantibodies of KLC3 protein in serum of a lung cancer patient is obviously higher than that of a healthy patient. According to the invention, the reagent for detecting the KLC3 protein autoantibody is used for preparing the lung cancer screening kit, so that effective screening of lung cancer can be realized.

Description

Application of KLC3 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 a KLC3 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 people without lung cancer related symptoms are subjected to routine physical examination, 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 tune-associated autoimmune polymers for lung cancer: A systematic review and meta-analysis. PLoS ONE 12 (7): e 0182117).

The KLC3 gene (kinesin light chain 3, Ensembl: ENSG00000104892) encodes a member of the kinesin light chain gene family. Kinesins are molecular motors involved in the transport of cargo along microtubules and are composed of two Kinesin Heavy Chain (KHC) and two Kinesin Light Chain (KLC) molecules. No reports exist about KLC3 protein as a cancer marker; the reports of the KLC3 protein autoantibody related to lung cancer are not found.

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:

application of a reagent for detecting KLC3 protein autoantibody in preparing a lung cancer screening kit.

As the application, the reagent for detecting the KLC3 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or a combined immunoassay reagent.

As the application, the reagent for detecting the KLC3 protein autoantibody is a western blot reagent.

As mentioned above, the reagent for detecting the KLC3 protein autoantibody is a reagent for a protein chip detection method.

As the aforementioned application, the reagent for detecting the KLC3 protein autoantibody is a reagent for detecting the KLC3 protein autoantibody in human serum.

A lung cancer screening kit, which comprises a reagent for detecting KLC3 protein autoantibody.

As the kit, the reagent for detecting the KLC3 protein autoantibody is a reagent for enzyme-linked immunosorbent assay or an enzyme-linked immunoassay reagent.

The kit is characterized in that the reagent for detecting the KLC3 protein autoantibody is a western blot reagent.

As the kit, the reagent for detecting the KLC3 protein autoantibody is a reagent for a protein chip detection method.

As the kit is used, the reagent for detecting the KLC3 protein autoantibody is a reagent for detecting the KLC3 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 above-mentioned aspects of the present invention will be further described in detail with reference to the following 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, "KLC 3 autoantibody" refers to "KLC 3 protein autoantibody".

Drawings

FIG. 1: comparison of KLC3 autoantibody levels in serum of lung cancer patients (LC), healthy controls (NC).

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

Detailed Description

EXAMPLE 1 relationship of KLC3 autoantibodies to Lung cancer in plasma

First, clinical data

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

basic information	Patients with lung cancer	Healthy controls
			Number of people	30	30
Age (age)	58±10.5	42±8.9
			Proportion of males	20(66.7％)	13(46.7％)

Second, detection principle

HuProt ^TM KLC3 protein is fixed on a human protein customization chip, after the human protein customization chip is incubated by adding serum, KLC3 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 (cy5 labeled and red) and an anti-human IgG fluorescent secondary antibody (cy3 labeled and green) are used for detection, a signal is read by a fluorescence scanner, and the strength of the signal is in positive correlation with the affinity and the quantity of the antibodies.

Third, method

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 the completion, the mixture is washed for 2 times for 10min by ddH 2O;

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.

Fourthly, the result

The mean expression level of KLC3 autoantibodies in the plasma of lung cancer patients was 51.8SNR (fluorescence signal versus quantitative ratio), and the mean expression level of KLC3 autoantibodies in healthy control plasma was 33.5 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 group and healthy control showed specificity of 96.6% and sensitivity of 20.7% (FIG. 2), indicating that KLC3 autoantibody can specifically distinguish lung cancer from healthy control.

The results show that the level difference of the KLC3 autoantibody in the serum of the lung cancer patient and the serum of the non-lung cancer patient is obvious, and the aim of screening the lung cancer can be achieved by detecting the level of the KLC3 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 KLC3 autoantibodies in serum".

The kit can screen the risk of the lung cancer of the people to be detected by detecting the level of the KLC3 autoantibody in serum: if KLC3 autoantibody levels are high (relative to healthy humans), the risk of lung cancer is high, and if KLC3 autoantibody levels are low, the risk of lung cancer is low. 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 KLC3 protein autoantibody in preparing a lung cancer screening kit is disclosed, and the reagent for detecting the KLC3 protein autoantibody is a reagent for detecting the KLC3 protein autoantibody in human serum.

2. The use of claim 1, wherein the reagent for detecting the KLC3 protein autoantibody is a reagent for an enzyme-linked immunosorbent assay.

3. The use according to claim 1, characterized in that the reagent for detecting autoantibodies to KLC3 protein is a western blot reagent.

4. The use of claim 1, wherein the reagent for detecting the KLC3 protein autoantibody is a reagent for a protein chip detection method.