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Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer

A technology of autoantibody and combined detection, applied in the field of biomedicine, can solve the problems of high false positive detection of tumor marker protein chip detection system, inability to judge the specific type of tumor, and lack of liver cancer detection markers, so as to improve the efficiency of detection and diagnosis , Improve the efficiency of clinical testing and reduce the effect of false positive rate

Active Publication Date: 2016-10-26
厦门生迪生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the multi-tumor marker protein chip detection system (C12 chip) has high detection false positives, and there are no targeted liver cancer detection markers, and the detection efficiency is low
Tumor gene mutation detection reagents rely on complicated operations such as polymerase chain reaction (PCR), and are prone to false positives, which is an unreliable analytical method
Six tumor marker assay kits and human malignant tumor-specific growth factor (TSGF) enzyme-linked immunoassay (ELISA) kits can only detect tumors in patients, but cannot determine the specific type of tumors
The mechanism is not well understood, however, in recent years, the importance of 14-3-3zeta protein in cancer has become increasingly evident

Method used

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  • Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer
  • Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer
  • Multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation of eight tumor-associated antigens

[0028] By using a prokaryotic expression system, 8 tumor-associated antigens (CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta, and p62) were prokaryotically expressed and purified in order to prepare for the next experiment. The specific antigen preparation process is as follows:

[0029] 1) The recombinant prokaryotic expression plasmids of eight tumor-related proteins (CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62) were constructed using gene cloning technology.

[0030] 2) Expression of the target protein: the constructed recombinant prokaryotic expression plasmids were respectively transformed into Escherichia coli Bl21(DE3), and IPTG (isopropylthiogalactoside) was used to induce the expression of the target protein.

[0031] 3) Purification of the target protein: according to the tag carried by the target protein, the target protein is purified using a traditional corresponding purification ...

Embodiment 2

[0034] Embodiment 2: the preparation of kit

[0035] According to the principle of indirect ELISA, the invention prepares an ELISA kit for combined detection of various autoantibodies that can be used for early screening and diagnosis of liver cancer. 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 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.

[0036] 1. Reagents and materials

[0037] (1) Eight tumor-associated proteins (CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62 proteins);

[0038] (2) Anti...

Embodiment 3

[0058] Embodiment 3: the detection method of kit of the present invention

[0059] 1. Serum sample incubation:

[0060] Dilute the serum sample to be tested with 1% BSA solution at a ratio of 1:100, and then dilute the diluted serum sample according to figure 2 Add the layout shown in the sample wells of the 96-well ELISA plate that has been coated with the antigen. Wash with washing solution 5 times.

[0061] 2. Enzyme-labeled secondary antibody incubation:

[0062] Dilute the horseradish peroxidase-labeled RecA protein with 1% BSA solution at a ratio of 1:8000, and then add the diluted horseradish peroxidase-labeled RecA protein to the wells of the 96-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 5 times with washing solution.

[0063] 3. Color development and termination reaction

[0064]Mix the chromogenic soluti...

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Abstract

The invention discloses a multi-autoantibody joint detection ELISA kit for early screening and diagnosis on liver cancer. The kit contains a solid carrier and tumor-associated antigen, wherein the solid carrier is coated by the tumor-associated antigen; the tumor-associated antigen is CyclinB1, p90, c-myc, p53, NPM1, HCC1, 14-3-3zeta and p62. Furthermore, the kit further contains sample diluent, a second antibody, second antibody diluent, positive control serum, negative control serum, a developing liquid, a termination liquid and a washing liquid. The ELIS kit disclosed by the invention is applied to early screening on liver cancer and has the advantages of high sensitivity, good stability, simplicity and convenience in operation and the like. In addition, the ELISA kit disclosed by the invention is capable of detecting 11 blood samples simultaneously, so that the cost can be lowered, the efficiency can be improved, and patients suffering from liver cancer can be diagnosed and treated before occurrence of clinical symptoms.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an ELISA kit for combined detection of multiple autoantibodies used for early screening and diagnosis of liver cancer. Background technique [0002] Primary liver cancer is one of the most common malignant tumors in the world, and more than half of the new cases of liver cancer each year are in China, which seriously threatens people's lives and health. The onset of liver cancer is very insidious, and the prognosis is poor. Surgical resection and liver transplantation are considered to be the means of radical treatment for liver cancer, but most patients have already entered the advanced stage of liver cancer when they are first diagnosed, thus losing the opportunity for treatment. The main reason is that patients with early liver cancer usually have no obvious symptoms, or the size is small, and it is difficult to detect by imaging examinations. As the main serum marker, al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574
CPCG01N33/57438
Inventor 张建营叶华王晓
Owner 厦门生迪生物技术有限公司
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