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Autoantibody joint detection ELISA kit for screening early esophageal cancer

A technology of combined detection of autoantibodies, applied in the fields of molecular biology and oncology, to achieve the effect of high cost performance, increase detection rate, and reduce mortality

Active Publication Date: 2019-08-30
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no esophageal cancer specific related antigen kit for clinical diagnosis

Method used

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  • Autoantibody joint detection ELISA kit for screening early esophageal cancer
  • Autoantibody joint detection ELISA kit for screening early esophageal cancer
  • Autoantibody joint detection ELISA kit for screening early esophageal cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation of 8 tumor-associated antigens

[0029] By using a prokaryotic expression system, 8 tumor-associated antigens (EYA4, ABL1, ATF2, CD38, NOTCH1, ZBTB20, BCL2L1, and P53) were prokaryotically expressed and purified to prepare for the next experiment. The specific antigen preparation process is as follows:

[0030] (1) Using gene cloning technology to construct recombinant prokaryotic expression plasmids of 8 tumor-associated antigen proteins (EYA4, ABL1, ATF2, CD38, NOTCH1, ZBTB20, BCL2L1 and P53);

[0031] (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;

[0032] (3) Purify the target protein: Purify the target protein according to the tag carried by the target protein using a traditional corresponding purification scheme;

[0033] (4) The protein concentration was me...

Embodiment 2

[0035] Embodiment 2: the preparation of kit

[0036] According to the principle of indirect ELISA, the invention prepares an autoantibody joint detection ELISA kit for early esophageal cancer screening. The principle of indirect enzyme-linked immunoassay is to link 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 antibodies in the complex combine 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.

[0037] 1. Reagents and materials:

[0038] (1) 8 tumor-associated antigen proteins (EYA4, ABL1, ATF2, CD38, NOTCH1, ZBTB20, BCL2L1 and P53);

[0039] (2) 96-well ELISA plate: purchased from Corning Company;

[0040] ...

Embodiment 3

[0071] Embodiment 3: the detection method of kit of the present invention

[0072] 1. Serum sample incubation:

[0073] Dilute the serum sample to be tested with the sample diluent at a ratio of 1:100, and then add the diluted serum sample to the spotting well of the antigen-coated 96-well microplate plate, and the sample volume is 100 μl / well , placed in a constant temperature incubator at 37°C for 1 h, then discarded the liquid in the spotting well, and washed 5 times with washing solution.

[0074] 2. Enzyme-labeled secondary antibody incubation:

[0075] Dilute the horseradish peroxidase-labeled RecA protein with the secondary antibody diluent at a ratio of 1:8000, and then add the diluted horseradish peroxidase-labeled RecA protein to the 96-well microtiter plate for spotting In the wells, the sample volume was 100 μl / well, placed in a 37°C constant temperature incubator and incubated for 1 hour, then the liquid in the sample wells was discarded, and washed 5 times with...

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Abstract

The invention belongs to the technical field of tumor medicine, and particularly discloses an autoantibody joint detection ELISA kit for screening early esophageal cancer. The kit comprises a solid-phase carrier and tumor-associated antigens coated on the solid-phase carrier, wherein the tumor-associated antigens are EYA4, ABL1, ATF2, CD38, NOTCH1, ZBTB20, BCL2L1 and P53. Furthermore, the kit alsocomprises a sample diluent, a second antibody, a second antibody diluent, positive control serum, negative control serum, a color developing solution, a stop solution and a washing solution. The ELISA kit provided by the invention can effectively detect esophageal cancer, especially early esophageal cancer, has the detection sensitivity of 94% and the specificity of 79%, can be used for large-scale screening of asymptomatic people in high-incidence areas of esophageal cancer, and is beneficial to screening and early discovery of asymptomatic high-risk population.

Description

technical field [0001] The invention relates to the fields of molecular biology and oncology, in particular to an autoantibody combined detection ELISA kit for screening early esophageal cancer. Background technique [0002] Esophageal cancer (the esophageal cancer shown in this patent is esophageal squamous cell carcinoma) is one of the six major malignant tumors in the world. There are about 500,000 new cases of esophageal cancer every year in the world, and more than half of them occur in China, which is 100% higher than that in Western countries. times. According to the latest national cancer report of the National Cancer Center in 2018, the number of new cases of esophageal cancer in my country in 2014 was 18.85 / 100,000, ranking sixth among malignant tumors, while the death rate of esophageal cancer was 14.11 / 100,000, ranking first among malignant tumors. fourth place. The Taihang Mountain area at the junction of Henan, Hebei and Shanxi provinces in my country is the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N33/57407
Inventor 王立东宋昕赵学科周福有高社干范宗民李学民岳文彬周英发王苒李贝韩雪娜冯常炜任景丽
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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