An ELISA kit for combined detection of autoantibodies for early-stage cardia adenocarcinoma screening

A technology of combined detection of autoantibodies, applied in the fields of molecular biology and oncology, to achieve strong balance, reduce misdiagnosis rate, and improve specificity

Active Publication Date: 2020-10-09
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of six TAAs, P53, CDH1, NRG1, MDM2, RHOA and IVL, to detect the expression of corresponding autoantibodies in the serum of patients with cardiac adenocarcinoma has not been reported.

Method used

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  • An ELISA kit for combined detection of autoantibodies for early-stage cardia adenocarcinoma screening
  • An ELISA kit for combined detection of autoantibodies for early-stage cardia adenocarcinoma screening
  • An ELISA kit for combined detection of autoantibodies for early-stage cardia adenocarcinoma screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of tumor-associated antigens

[0030] By using a prokaryotic expression system, six tumor-associated antigens (P53, CDH1, NRG1, MDM2, RHOA, and IVL) were prokaryotically expressed and purified in order to prepare for the next experiment. The specific antigen preparation process is as follows:

[0031] (1) Using gene cloning technology to construct recombinant prokaryotic expression plasmids of six tumor-associated antigen proteins (P53, CDH1, NRG1, MDM2, RHOA and IVL);

[0032] (2) Expressing the target protein: Transform the constructed recombinant prokaryotic expression plasmids into Escherichia coli BL21(DE3) and use IPTG (isopropylthiogalactopyranoside) to induce the expression of the target protein;

[0033] (3) Purify the target protein: according to the tag carried by the target protein, use the traditional corresponding purification scheme to purify the target protein;

[0034] (4) The protein concentration was measured by Bradford metho...

Embodiment 2

[0036] Embodiment 2: the preparation of kit

[0037] According to the principle of indirect ELISA, the invention prepares an autoantibody combined detection ELISA kit for screening and diagnosing early cardia adenocarcinoma. The principle of indirect enzyme-linked immunoassay is to connect the antigen to a solid-phase carrier, and the antibody to be tested in the sample combines with it to form a solid-phase antigen-test antibody complex, and then use the enzyme-labeled secondary antibody and the solid-phase antigen-test antibody to The antibody in the complex is combined to form a solid-phase antigen-test antibody-enzyme-labeled secondary antibody complex, and then measure the degree of color development after adding the substrate to determine the content of the antibody to be tested (see figure 2 ).

[0038] 1. Experimental materials and reagents:

[0039] (1) 6 tumor-associated antigen proteins (P53, CDH1, NRG1, MDM2, RHOA and IVL);

[0040] (2) 48-well ELISA plate: pur...

Embodiment 3

[0073] Embodiment 3: the usage method of kit

[0074] 1. Serum sample incubation:

[0075] Dilute the serum sample to be tested with the sample diluent at a ratio of 1:100, and then add the diluted serum sample into the reaction wells of the 48-well microplate plate coated with antigen, with a sample volume of 100 μl / well, Place in a constant temperature incubator at 37°C and incubate for 1 h, then discard the liquid in the reaction well, and wash with washing solution 5 times, each time for 3 min.

[0076] 2. Enzyme-labeled secondary antibody incubation:

[0077] Dilute the horseradish peroxidase-labeled RecA protein with the secondary antibody diluent at a ratio of 1:80000, and then add the diluted horseradish peroxidase-labeled RecA protein to the reaction wells of the 48-well microtiter plate In this method, the sample volume was 100 μl / well, and incubated in a constant temperature incubator at 37°C for 1 hour, then the liquid in the sample well was discarded, and washed...

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Abstract

The invention belongs to the technical field of medical biology, and specifically discloses an autoantibody joint detection ELISA kit for early cardia adenocarcinoma screening. The kit comprises a solid phase carrier and a tumor-associated antigen coated on the solid phase carrier, and the tumor-associated antigen is composed of P53, CDH1, NRG1, MDM2, RHOA and IVL. Furthermore, the kit also comprises a sample diluent, a second antibody, a second antibody diluent, positive control serum, negative control serum, a developing solution, a stop solution and a washing solution. The ELISA kit provided by the invention can effectively detect cardia adenocarcinoma, especially early cardia adenocarcinoma, has detection sensitivity as high as 86% and specificity as high as 88%, can be used for large-scale screening of asymptomatic people in cardia adenocarcinoma high-incidence areas, and is beneficial to screening and early discovery of asymptomatic high-risk people.

Description

technical field [0001] The invention belongs to the fields of molecular biology and oncology, and in particular relates to an ELISA kit for combined detection of autoantibodies for early cardia adenocarcinoma screening. Background technique [0002] The cardia is located below the esophagogastric junction and most adjacent to the stomach, extending approximately 2 cm downward from the squamous-columnar junction. Stein et al. divided adenocarcinoma of the esophagogastric junction (AEG) into three types. Type I AEG is distal esophageal adenocarcinoma originating from special intestinal metaplasia; type II is true cardiac adenocarcinoma originating from Short segments of gastric cardia epithelium or intestinal metaplasia at the esophagogastric junction; type III is subcardia adenocarcinoma involving the esophagogastric junction and distal esophagus. Type II is the main type in my country, and the cardiac adenocarcinomas mentioned in this patent are all type II cardiac adenocar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/574G01N33/543
CPCG01N33/543G01N33/57446
Inventor 王立东陈瑶宋昕赵学科李贝韩雪娜范宗民王苒杨苗苗王盼盼
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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