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Hepatocirrhosis immuno-mass spectrometric detection reagent kit

A detection kit and mass spectrometry detection technology, which is applied in the field of liver cirrhosis immune mass spectrometry detection kits, to achieve the effect of strong early warning ability, low cost and strong specificity

Active Publication Date: 2012-06-20
BEIJING C & N INT SCI TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no suitable detection kit for liver cirrhosis polypeptide markers that can accurately, specifically and easily detect liver cirrhosis, so as to better serve as an early warning and early prevention of liver cancer

Method used

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Examples

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Embodiment 1

[0036]The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Example 1 Preparation method of polypeptide marker antigen monoclonal antibody

[0037] 1) BALB / C mice were immunized with synthetic polypeptide 5 coupled to carrier protein KLH as immunogen; its full-length sequence was: Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp -Gln-Gly-His-Gly-His-Gln.

[0038] 2) After 2 weeks, the tail blood titer was detected, and after reaching more than 1:1000, BALB / C mouse spleen cells were fused with SP2 / 0 mouse myeloma cells under the action of PEG;

[0039] 3) Screen by ELISA method, clone and purify the hybridoma cells that secrete antibody positive by limiting dilution method;

[0040] 4) 10 hybridomas against synthetic peptide 5 were screened out, and unexpectedly one of them was found to be more sensitive (1ng / well), the cell line was expanded, the monoclonal antibody ascites was prepared and purified, and the ...

Embodiment 2

[0041] Example 2 Immuno-mass spectrometry detection method for liver cirrhosis

[0042] The process of the polypeptide immunoassay mass spectrometry detection method of the present invention is as follows: figure 1 shown. Specifically:

[0043] 1) Take 25 μL of protein G-coated agarose particles (Protein G Agarose, Santa Cruz), put it in a 0.6 mL Eppendorf tube, add 7.78 μg AP0105, rotate at 4°C (rotation speed 5 r / min), mix for 1 hour, and leave for 3 min , remove the supernatant, wash Protein G Agarose 3 times with 100 μL PBS buffer (0.01mol / l, pH7.4);

[0044] 2) Take 24 μg of synthetic peptide 5 polypeptide standard (solid-phase chemical synthesis, Beijing Zhongke Yaguang Biotechnology Co., Ltd.) and 50 μL of PBS, mix with the washed Protein G Agarose in step 1), and rotate and mix at 4°C for 8 hours;

[0045] 3) Place for 3 min, remove the supernatant, wash Protein G Agarose 3 times with 200 μL PBS; move the suspension to another clean Eppendorf tube during the last wa...

Embodiment 3

[0050] Example 3 Immuno-mass spectrometry detection method for liver cirrhosis

[0051] The process of the polypeptide immunoassay mass spectrometry detection method of the present invention is as follows: figure 1 shown. Specifically:

[0052] 1) Take 15 μL of protein A-coated agarose particles (Protein A Agarose, Santa Cruz), put it in a 0.2 mL Eppendorf tube, add 1.56 μg AP0105, rotate at 4°C (rotation speed 5 r / min), mix for 15 minutes, and leave for 2 minutes , remove the supernatant, wash Protein AAgarose 3 times with 100 μL PBS buffer (0.01mol / l, pH7.4);

[0053] 2) Take 24 μg of synthetic peptide 5 polypeptide standard (solid-phase chemical synthesis, Beijing Zhongke Yaguang Biotechnology Co., Ltd.) and 50 μL of PBS, mix with the washed Protein A Agarose in step 1), and rotate and mix at 4°C for 8 hours;

[0054] 3) Place for 2 min, remove the supernatant, wash Protein A Agarose 3 times with 200 μL PBS; move the suspension to another clean Eppendorf tube during the ...

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Abstract

The invention relates to a hepatocirrhosis immuno-mass spectrometric detection reagent kit, which comprises a monoclonal antibody and a solid carrier, which are secreted by Hybridoma cell strain with a preserving number of CGMCC No.5269. The monoclonal antibody has strong specificity for polypeptide marker antigens; and by adopting the immunity technology to be combined with the mass spectrum technology, high flux, high sensitivity and high precision on detecting the hepatocirrhosis can be realized, so an early warning effect on the liver cancer can be better played. In addition, the reagent kit belongs to the diagnosis in molecular level, so the early warning capacity is stronger compared with the tumor imaging, and the cost is low.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a liver cirrhosis immune mass spectrometry detection kit and a detection method thereof. Background technique [0002] With the maturity and development of liver cancer surgery, the treatment methods and means of liver cancer are becoming more and more abundant, and are no longer limited to a single surgical treatment. Liver resection and liver transplantation are still the main treatment methods for patients with liver cancer, but minimally invasive treatment, percutaneous hepatic arterial chemoembolization and other technical methods, as an important supplement to surgical treatment, have also been widely used. Surgical treatment opportunities, reducing or reducing tumor recurrence and metastasis have all played a certain role, prolonging the survival time of patients. Even so, HCC patients have insidious onset and no obvious symptoms, and most patients have lost the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N27/64
Inventor 魏开华孙云波原剑周晓明付海媛侯利平黄亚娟杨保安郑俊杰甄蓓张拓王东茂
Owner BEIJING C & N INT SCI TECH
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