Protein chip, protein chip diagnostic kit, preparation method and using method

A protein chip, diagnostic kit technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the lack of sensitivity and specificity markers or marker combination spectrum, not yet seen, low-dose spiral CT false positive rate Advanced problems, to achieve the effect of efficient early diagnosis or census screening

Active Publication Date: 2016-04-27
GUANGZHOU BIO BLUE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

therefore, The sensitivity needs to be further improved
Another product that uses autoantibodies for early detection of lung cancer is Paula’stest from GENESYSBIOLABSHAWAII. It is obviously different. By detecting the content of 3 kinds of tumor antigen proteins and 1 kind of lung cancer autoantibody to diagnose lung cancer, the preliminary report has a sensitivity of 74% and a specificity of 80%. The specificity of this kit needs to be further improved
[0018] To sum up, among the above-mentioned numerous diagnostic techniques for lung cancer, the currently commonly used diagnostic techniques for lung cancer screening in foreign countries are sputum cytology examination, X-ray film and other techniques, and the detection rate is still not high; while the false positive rate of low-dose spiral CT Too high, need other combined diagnostic methods or means
The technical method of detecting various molecular markers from blood is simple, economical, fast, non-invasive, painless and easy to accept, but there is still a lack of markers or marker combination profiles with high sensitivity and specificity
Changes in various genes and nucleic acid molecules involved in lung cancer are difficult to detect in serum
At present, there have been some reports on the use of multiple protein antigens to detect the corresponding antibody content in serum, and to judge whether the subject has lung cancer, but there is no protein chip for detecting the corresponding antibody in human serum. Most importantly, it is also necessary to further optimize the autoantibody marker spectrum to improve the sensitivity or specificity of detection and diagnosis of early lung cancer, so that it can be practically used for early clinical diagnosis of lung cancer or screening of lung cancer in high-risk groups of lung cancer

Method used

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  • Protein chip, protein chip diagnostic kit, preparation method and using method
  • Protein chip, protein chip diagnostic kit, preparation method and using method
  • Protein chip, protein chip diagnostic kit, preparation method and using method

Examples

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Embodiment 1

[0074] Prepare a new protein chip for early diagnosis of human lung cancer and detect the tested serum samples.

[0075] The new protein chip for diagnosing human lung cancer involved in the present invention can be implemented through a variety of specific technologies, and will be described in more detail below:

[0076] The six antigens involved in the method are adsorbed or covalently cross-linked on a suitable solid support. The six antigens can be purified natural proteins, or recombinantly expressed proteins, or recombinantly expressed recombinant fusion proteins containing the characteristic amino acid sequences of each antigen, or directly synthesized polypeptides with the characteristic amino acid sequences of each antigen. The solid phase support referred to can be glass slides, immunoblotting membranes, various microwell plates used in immunoadsorption, and the like. The antigen can be directly adsorbed on the solid support by electrostatic interaction, or cross-l...

Embodiment 2

[0082] Preparation of 6 corresponding antigens.

[0083]The DNA fragment of (1) P53 antigen fragment, (2) SOX2 antigen fragment, (3) COPB1 antigen fragment, (4) EFHD2 antigen fragment, (5) EIF4G3 antigen fragment, (6) PCNA antigen fragment described in the present invention has been Pre-cloned in pBluescriptSK(+ / -) prokaryotic cloning vector using EcoRI and XhoI enzymes. The recombinant expression vector pET30b(+) was double-digested with EcoRI and XhoI, and a double-digested fragment of about 5.4kb was recovered for future use. EcoRI and XhoI double-digested the pBluescriptSK(+ / -) vectors containing the genes of each antigen fragment, recovered the double-digested fragments corresponding to each antigen, and cloned them into the recovered pET30b(+) expression vectors respectively. Recombinant clones were screened by EcoRI and XhoI double enzyme digestion respectively. A large number of plasmids were extracted by alkaline lysis method, and 0.1 μg of DNA was used to transform...

Embodiment 3

[0085] Preparation of Derivative Kit of Protein Chip for Early Diagnosis of Lung Cancer (Protein Chip Diagnostic Kit)

[0086] The composition of the kit:

[0087] Protein chips with pre-spotted antigens;

[0088] sample diluent;

[0089] washing liquid concentrate;

[0090] Fluorescently labeled secondary antibody concentrate;

[0091] Preparation of each component of the kit:

[0092] 1. Preparation of protein chips with pre-spotted antigens: buy commercial BSA-coated and aldehyde-treated carrier slides, such as CSS-100 slides from CELAssociates, USA. Adopt the purified recombinant protein of 6 kinds of antigens prepared in Example 2, and human IgG (0.2mg / ml), human IgM (0.2mg / ml) standards as samples, add an appropriate amount of biotinylated BSA and tween-20, The above 14 kinds of samples were spotted on the carrier glass slides to form a microarray by using a biochip spotting machine, and 10 parallel microarrays were spotted on each slide, and each microarray was sep...

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Abstract

The invention relates to a protein chip, a protein chip diagnostic kit, a preparation method and a using method. The protein chip contains the following protein markers of autologous antigen protein: a P53 antigen fragment, an SOX2 antigen fragment, a COPB1 antigen fragment, an EFHD2 antigen fragment, an EIF4G3 antigen fragment and a PCNA antigen fragment, wherein the characteristic amino acid sequence of the P53 antigen fragment is as shown in ID.1sequence; the characteristic amino acid sequence of the SOX2 antigen fragment is as shown in ID.2sequence; the characteristic amino acid sequence of the COPB1 antigen fragment is as shown in ID.3sequence; the characteristic amino acid sequence of the EFHD2 fragment is as shown in picture 4seeqeunce; the characteristic amino acid sequence of the EIF4G3 antigen fragment is as shown in ID.5seqeunce; the characteristic amino acid sequence of the PCNA antigen fragment is as shown in ID.6sequence. Early diagnosis or general investigation screening for suspected patients of lung cancer or high-risk population of lung cancer can be carried out extremely conveniently, quickly, noninvasively and efficiently.

Description

technical field [0001] The invention relates to a protein chip for early diagnosis of lung cancer based on autoantibody detection of six lung cancer-related autoantigens, and a diagnostic kit derived from the protein chip. The present invention further provides the application of the protein chip and the diagnostic kit derived from the protein chip in early diagnosis of lung cancer, early lung cancer screening of healthy people or high-risk groups of lung cancer. [0002] Specifically, the present invention relates to a protein chip that can be used for serological detection and early diagnosis of human lung cancer. The protein chip can detect the respective IgG and IgM types of six lung cancer-related autoantigens in the blood samples of the subjects. The relative content of autoantibodies and the results of the corresponding antibody detection of these 6 autoantigens are jointly analyzed to determine whether the subject has lung cancer. Furthermore, the present invention al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/552
CPCG01N33/552G01N33/57423G01N33/57473G01N33/57484
Inventor 胡海
Owner GUANGZHOU BIO BLUE TECH CO LTD
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