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A preparation method of esophageal cancer immunomass spectrometry detection kit

A detection kit and mass spectrometry detection technology, which is applied in the field of biotechnology and mass spectrometry detection, can solve problems such as not being suitable for population censuses, and achieve the effects of strong binding specificity, simple operation, and low cost

Active Publication Date: 2014-10-15
BEIJING C & N INT SCI TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The premise of using tissue markers to diagnose esophageal cancer is to obtain tumor tissue or cells, which can only be achieved through invasive or even cumbersome operations, and is not suitable for population census

Method used

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  • A preparation method of esophageal cancer immunomass spectrometry detection kit
  • A preparation method of esophageal cancer immunomass spectrometry detection kit
  • A preparation method of esophageal cancer immunomass spectrometry detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 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 an immunogen; wherein, the full-length sequence of polypeptide 5 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 when it reached above 1:1000, splenocytes from BALB / C mice were fused with SP2 / 0 mouse myeloma cells under the action of PEG.

[0039] 3) Screen by ELISA method, clone and purify hybridoma cells positive for secreting antibodies by limited dilution method.

[0040] 4) Screen out 10 hybridoma cells against synthetic peptide 5, one of which is highly sensitive (1ng / well), expand the cell line, prepare and purify monoclonal antibody ascites, and test the titer of monoclonal antibody (1:200000 ), titer (0.0005μg / ml) and subtype identification (IgG2b), etc., to obtain specific monoclonal antibo...

Embodiment 2

[0041] Example 2 Esophageal cancer immune mass spectrometry detection method

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

[0043] 1) Take 25 μL 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 (speed 5r / min), mix for 1 hour, and let stand for 3 minutes , remove the supernatant, and wash Protein G Agarose 3 times with 100 μL PBS buffer (0.01mol / l, pH7.4);

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

[0045] 3) Leave it for 3 minutes, remove the supernatant, and wash Protein G Agarose 3 times with 200 μL PBS; transfer the suspension to another clean Eppendorf tu...

Embodiment 3

[0050] Example 3 Esophageal cancer immune mass spectrometry detection method

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

[0052] 1) Take 15 μL 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 (speed 5r / min), mix for 15 minutes, and place for 2 minutes. Remove the supernatant and wash Protein A Agarose 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.), 50 μL of PBS, mix with the Protein A Agarose washed in step 1), and mix at 4°C for 8 hours;

[0054] 3) Leave it for 2 minutes, remove the supernatant, and wash Protein A Agarose 3 times with 200 μL PBS; transfer the suspension to another clean Eppendorf tube for the last wash;

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Abstract

The invention provides an immuno-mass spectrometric detection kit for an esophagus cancer. The detection kit contains a monoclonal antibody secreted by a hybridoma cell strain of which the preservation number is CGMCC No. 5269, and a solid-phase carrier; and the monoclonal antibody is fixed on the solid-phase carrier. The monoclonal antibody related to the detection kit has strong binding specificity aiming at peptide marker antigens. Detection with high flux, high sensitivity and high accuracy of the esophagus cancer can be achieved by the combination of immunization and a mass-spectrometric technique. In addition, the immuno-mass spectrometric detection kit belongs to the technology of diagnosis of molecular level, and is simpler in operation and lower in cost compared with tumor iconography.

Description

technical field [0001] The invention relates to biotechnology and mass spectrometry detection, in particular to an immune mass spectrometry detection kit for esophageal cancer. Background technique [0002] Esophageal cancer is a common malignant tumor of the digestive tract. According to statistics, in 2008, there were 482,300 new cases of esophageal cancer worldwide, and 406,800 deaths. Esophageal cancer is divided into esophageal cancer and esophageal adenocarcinoma. In my country, esophageal cancer is the main type of esophageal cancer, especially in the northern region. In recent years, although some progress has been made in the treatment of esophageal cancer, the mortality rate of esophageal cancer is still high. The main reason is that the early symptoms are concealed and non-specific. Most of the patients with esophageal cancer discovered and treated clinically are in the middle or late stage. The prognosis of early esophageal cancer is very different from that in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/543
Inventor 魏开华肖汉族孙云波傅海媛侯利平黄亚娟宋纯艳杨保安甄蓓徐淑芳张拓王东茂周晓明原剑
Owner BEIJING C & N INT SCI TECH
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