Polypeptide immunoassay kit and detection method thereof

A technology of immune detection and kit, applied in the biological field, can solve the problems of unfavorable promotion, high price, lack of candidate target detection methods, etc., and achieve the effect of low price, high specificity and high accuracy

Inactive Publication Date: 2011-05-18
BEIJING C & N INT SCI TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Serum peptidomics, which has emerged in recent years, has been successfully applied to the diagnosis of several cancers, such as breast cancer, ovarian cancer, prostate cancer, etc., mainly using surface-enhanced laser desorption ionization (SELDI) technology and immunomagnetic bead technology , but the system has the disadvantages of being expensive and unfavorable for popularization
However, there is no kit for the early diagnosis of liver cancer. The main reason is the lack of suitable candidate targets and suitable detection methods, which requires strong basic research support

Method used

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  • Polypeptide immunoassay kit and detection method thereof
  • Polypeptide immunoassay kit and detection method thereof
  • Polypeptide immunoassay kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 Preparation method of anti-synthetic peptide 5 antibody

[0054] 1) BALB / C mice were immunized with synthetic polypeptide 5 coupled to the carrier protein KLH as an immunogen; its full-length sequence is: Asn-Leu-Gly-His-Gly-His-Lys-His-Glu-Arg-Asp- Gln-Gly-His-Gly-His-Gln.

[0055] 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;

[0056] 3) Screening by ELISA method, cloning and purifying hybridoma cells positive for secreting antibodies by limiting dilution method;

[0057] 4) Select hybridoma cells targeting synthetic peptide 5, expand cell lines, prepare and purify monoclonal antibody ascites, detect monoclonal antibody titer, specificity, sensitivity, subtype identification, etc., and obtain anti-synthetic peptide 5 antibody.

Embodiment 2

[0058] The preparation method of embodiment 2 polypeptide immunoassay kit

[0059] Take 20 μl protein G-coated agarose particles (Protein G Agarose, Santa Cruz), put it in a 0.2mL Eppendorf tube, add 7.78 μg anti-synthetic peptide 5 antibody, rotate at 4°C (speed 5r / min), mix for 1 hour, After standing for 2 minutes, the supernatant was removed, and the obtained solid-phase carrier precipitate was washed 3 times with 100 μL of PBS buffer solution (0.01 mol / l, pH 7.4).

Embodiment 3

[0060] Example 3 The preparation method of the polypeptide immunoassay kit

[0061] Take 15 μl protein G-coated agarose particles (Protein G Agarose, Santa Cruz), put it in a 0.2mL Eppendorf tube, add 1.56 μg anti-synthetic peptide 5 antibody, rotate at 4°C (speed 5r / min), mix for 15 minutes, After standing for 1 minute, the supernatant was removed, and the obtained solid-phase carrier precipitate was washed 3 times with 100 μL of PBS buffer solution (0.01 mol / l, pH 7.4).

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Abstract

The invention relates to a polypeptide immunoassay kit, which comprises a protein G or protein A agarose serving as a solid phase carrier, a purified serum polypeptide antibody and PBS buffer solution. The invention also relates to a polypeptide immunomic spectrometry analysis method, which comprises the following steps: coupling a purified serum antibody with a proper solid carrier; allowing the coupled product to bond with a polypeptide marker antigen specifically, and separating the antigen from the carrier by using eluent; and finally, detecting polypeptide antigen in the eluent by high-pass matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Thus, the complete immunomic spectrometry analysis method is established.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a polypeptide immune 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 chance of radical surgery when th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/531G01N27/62C07K7/08C07K16/18
Inventor 魏开华原剑孙云波周晓明杨保安甄蓓张拓张馨月王东茂
Owner BEIJING C & N INT SCI TECH
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