Gastric cancer early screening ELISA kit and early screening detection method thereof

Abstract

The invention discloses a stomach cancer early screening ELISA kit and an early screening detection method thereof, and relates to the technical field of in vitro diagnosis. The kit comprises the following components: a pre-coated 96-well plate which is fixedly provided with methylated DNA fragments of an anti-5-methylcytosine monoclonal antibody (with the clone number of 33D3), cg07160992, cg13516541, cg02956093 and cg21496913; the detection assembly comprises a biotinylation detection probe and a streptavidin-HRP (horse radish peroxidase) conjugate; the pretreatment assembly comprises a hydrosulfite conversion reagent and MBD2 protein modified magnetic beads. According to the kit, through a double-index combined interpretation model, the sensitivity to early gastric cancer reaches 86%, the blank of non-invasive detection of stage I gastric cancer is filled, the total sensitivity is 90.5%, the international top-level cancer early screening product standard is met, operation is standardized, special instruments are not needed, and the kit has high clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnosis, in particular to a gastric cancer early screening ELISA kit and an early screening detection method thereof. BACKGROUND

[0002] Cancer refers to all malignant tumors. According to the statistics of the World Health Organization, there are more than 14 million new cancer patients worldwide every year. In China, cancer has ranked the second in various causes of death, and has ranked the first in cities. Among them, the mortality rate of lung cancer is rising the fastest, and has ranked the first in the mortality rate of malignant tumors. The second is liver cancer, gastric cancer and colorectal cancer. Therefore, early diagnosis of cancer is of great significance to prolong the survival time of patients and reduce the mortality rate of patients.

[0003] At present, the diagnosis of cancer mainly relies on laboratory examination, imaging examination and pathological examination. However, imaging examination and pathological examination cannot screen out early cancer patients on a large scale. Laboratory examination needs to use multiple tumor immune markers for detection to guide clinical diagnosis. At present, there are many tumor markers used in clinical practice, such as AFP, which is the most diagnostic index for liver cancer, serum carcinoembryonic antigen (CEA) for colon cancer, gastric juice sulfoglycoprotein (FSA), gastric cancer associated antigen (GCAA) for gastric cancer, and prostate specific antigen (PSA) for prostate cancer. However, according to recent research reports, various tumor markers have the problems of missed diagnosis and misdiagnosis. At present, there is no tumor marker with good specificity and high sensitivity that can be used for large-scale screening of various cancer patients. Therefore, highly sensitive specific markers for malignant tumors are still needed for the preliminary screening of large population of cancer patients, so as to improve the early diagnosis rate of malignant tumors.

[0004] The research on serum markers of malignant tumors has always been the focus of scientists, but most of the research results cannot meet the needs of practical application. It is particularly important to explore serum markers with high sensitivity and specificity. Therefore, we propose a gastric cancer early screening ELISA kit and an early screening detection method thereof to solve the problems mentioned in the background.

[0005] The above information disclosed in the background section is only intended to increase the understanding of the background of the present application, and therefore, it can include prior art known to those of ordinary skill in the art. SUMMARY

[0006] The purpose of the present application is to provide a gastric cancer early screening ELISA kit and an early screening detection method thereof to solve the problems in the background.

[0007] To achieve the above purpose, the present application provides the following technical solution: a gastric cancer early screening ELISA kit, comprising:

[0008] Pre-coat 96-well plates with anti-5-methylcytosine monoclonal antibody (clone number 33D3), methylated DNA fragments of cg07160992, cg13516541, cg02956093 and cg21496913 are fixed;

[0009] Detection components include biotinylated detection probes and streptavidin-HRP conjugates;

[0010] Pre-treatment components include bisulfite conversion reagents and MBD2 protein-modified magnetic beads.

[0011] Preferably, the capture antibody of the kit is anti-5-methylcytosine monoclonal antibody (clone number: 33D3), and the detection antibody is streptavidin-HRP conjugate.

[0012] Preferably, the bisulfite conversion reagent contains DNA denaturation buffer, 99% pure sodium bisulfite, and a DNA purification column, and the surface of the methylated DNA enrichment magnetic beads is modified with MBD2 protein.

[0013] A gastric cancer early screening ELISA kit early screening detection method, comprising the following steps:

[0014] a) Take 1 mL of serum sample to be tested, and treat it with bisulfite conversion;

[0015] b) Use enrichment magnetic beads to extract target methylated DNA;

[0016] c) Add the extracted DNA to the pre-coated 96-well plate and incubate at 37°C for 60 min;

[0017] d) Add detection antibody-enzyme conjugate, and measure the 450 nm absorbance after TMB color development;

[0018] e) When the signal value of cg07160992+cg13516541 is greater than or equal to 2.5 and the ratio of cg02956093 / cg21496913 is greater than or equal to 3, it is determined that the risk of gastric cancer is high.

[0019] Compared with the prior art, the beneficial effects of the present application are:

[0020] (1) The kit of the present application improves the early gastric cancer detection rate compared with the prior art under the premise of maintaining high specificity, and is suitable for conventional laboratory environment, and the operation complexity is relatively low compared with the traditional methylation PCR which requires special equipment and non-standard operation of polypeptide anti-chip. Only 4 hours of ELISA standardized process is needed.

[0021] (2) The kit of the present application fills the blank of non-invasive detection of I stage gastric cancer by a double-index combined interpretation model, with a sensitivity of 86% for early gastric cancer, a total sensitivity of 90.5%, reaching the international top standard of early cancer screening products, standardized operation, no special instrument is needed, and has high clinical application value.

[0022] The above summary is intended to illustrate only and is not intended to limit the application in any way. Further aspects, embodiments and features of the application will be apparent from the detailed description, taken in conjunction with the accompanying drawings and from the DETAILED DESCRIPTION which follows, and it is intended that such aspects, embodiments and features be included within the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A schematic diagram of the detection process of the present application is shown in the figure. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described in detail below with reference to the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0025] An early screening ELISA kit for gastric cancer comprises:

[0026] A 96-well plate is pre-coated and fixed with anti-5-methylcytosine monoclonal antibody (clone number 33D3), methylated DNA fragments of cg07160992, cg13516541, cg02956093 and cg21496913;

[0027] A detection assembly comprises biotinylated detection probes and streptavidin-HRP conjugates.

[0028] A pretreatment assembly comprises a bisulfite conversion reagent and MBD2 protein modified magnetic beads.

[0029] The capture antibody of the kit is anti-5-methylcytosine monoclonal antibody (clone number: 33D3), and the detection antibody is streptavidin-HRP conjugate.

[0030] The bisulfite conversion reagent comprises a DNA denaturation buffer, 99% pure sodium bisulfite, a DNA purification column, and the surface of the methylated DNA enrichment magnetic beads is modified with MBD2 protein.

[0031] An early screening detection method for an early screening ELISA kit for gastric cancer comprises the following steps:

[0032] a) Take 1 mL of serum sample to be tested, and treat it with bisulfite conversion; b) Add 1 mL of detection antibody to the serum sample, and incubate the mixture at 37°C for 1 hour;

[0033] b) using enrichment magnetic beads to extract target methylation DNA;

[0034] c) adding the extracted DNA into a pre-coated 96-well plate, and incubating at 37℃ for 60 min;

[0035] d) adding a detection antibody-enzyme conjugate, and determining the absorbance at 450 nm after TMB color development;

[0036] e) determining high risk of gastric cancer when the signal values of cg07160992 and cg13516541 are greater than or equal to 2.5, and the ratio of cg02956093 / cg21496913 is greater than or equal to 3.

[0037] I. Specificity and sensitivity evaluation of the gastric cancer early screening ELISA kit of the present application

[0038] Target detection: methylation levels of cg07160992, cg13516541, cg02956093 and cg21496913;

[0039] Determination criteria: cg07160992+cg13516541 is greater than or equal to 2.5, and the ratio of cg02956093 / cg21496913 is greater than or equal to 3;

[0040] Gold standard control: pathological results of gastroscopic biopsy.

[0041] 1. Specificity test

[0042] Group Sample size Sample type Kit detection result Specificity Healthy control group 100 cases Serum of healthy people 4 positive cases 96％ Other cancer group 100 cases Serum of colon cancer patients 5 positive cases 95％ Benign gastric disease group 100 cases Serum of gastric ulcer patients 6 positive cases 94％ Total 300 cases - - 95％

[0043] As can be seen from the above, the specificity of the kit of the present application to non-gastric cancer population reaches 95%, and has high specificity.

[0044] 2. Sensitivity test

[0045] Gastric cancer staging Sample size Number of positive cases diagnosed by pathology Number of positive cases detected by the kit Sensitivity Early stage (stage I-II) 100 cases Serum of healthy people 4 positive cases 86％ Late stage (stage III-IV) 100 cases Serum of colon cancer patients 5 positive cases 95％ Total 200 cases Serum of gastric ulcer patients 6 positive cases 90.5％

[0046] As can be seen from the above, the sensitivity of the kit of the present application to early gastric cancer reaches 86%, and the methylation level is low in the early stage of canceration, but the combination of cg07160992 / cg13516541 is sensitive to early epigenetic changes, and is significantly higher than the sensitivity of traditional serum markers.

[0047] II. Blood sample test cases

[0048] Case 1: low-risk sample

[0049]

[0050] Case 2: high-risk sample

[0051]

[0052] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0053] The embodiments of the present application are given for example and description, although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and modifications to the above embodiments within the scope of the present application.

Claims

1. A gastric cancer early screening ELISA kit, characterized by, The kit comprises: a pre-coated 96-well plate, which is fixed with anti-5-methylcytosine monoclonal antibody (clone number 33D3), methylated DNA fragments of cg07160992, cg13516541, cg02956093 and cg21496913; a detection component, which comprises biotinylated detection probes and streptavidin-HRP conjugate; a pretreatment component, which comprises a bisulfite conversion reagent and MBD2 protein modified magnetic beads.

2. The early screening ELISA kit for gastric cancer according to claim 1, characterized by: The capture antibody of the kit is anti-5-methylcytosine monoclonal antibody (clone number: 33D3), and the detection antibody is streptavidin-HRP conjugate.

3. The early screening ELISA kit for gastric cancer according to claim 1, characterized in that: The bisulfite conversion reagent comprises a DNA denaturation buffer, 99% pure sodium bisulfite and a DNA purification column, and the surface of the methylated DNA enrichment magnetic beads is modified with MBD2 protein.

4. The early screening detection method of the gastric cancer early screening ELISA kit as described in any one of claims 1-3, characterized in that, The kit comprises the following steps: a) 1 mL of the serum sample to be tested is subjected to bisulfite conversion treatment; b) the target methylated DNA is extracted using enrichment magnetic beads; c) the extracted DNA is added to the pre-coated 96-well plate, and incubated at 37°C for 60 min; d) the detection antibody-enzyme conjugate is added, and the absorbance at 450 nm is measured after TMB color development; e) when the signal values of cg07160992 and cg13516541 are greater than or equal to 2.5 and the ratio of cg02956093 / cg21496913 is greater than or equal to 3, it is determined that the subject has a high risk of gastric cancer.

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