Kit of high-effectively and quantitatively detecting Golgi apparatus 73

A technology for quantitative detection and Golgi apparatus, which is applied in the biological field, can solve the problems of high cost, and achieve the effects of low detection range, reduced false negatives, and strong specificity

Active Publication Date: 2016-11-23
RAYBIOTECH INC GUANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PET-CT examination is expensive, and invasi

Method used

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  • Kit of high-effectively and quantitatively detecting Golgi apparatus 73
  • Kit of high-effectively and quantitatively detecting Golgi apparatus 73
  • Kit of high-effectively and quantitatively detecting Golgi apparatus 73

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Secreted expression of GP73 gene

[0028] The GP73 gene sequence was inserted into the vector and transformed into Escherichia coli after successful sequencing. The expression product was verified by SDS-PADG to obtain a single band.

[0029] The sequence of GP73 antigen protein is consistent with GenBank accession number AAF44663.1.

Embodiment 2

[0030] Example 2: Preparation, purification and identification of anti-GP73 polyclonal antibody

[0031]The GP73 protein expressed by Escherichia coli described in Example 1 was used to immunize SPF-grade New Zealand rabbits (Guangdong Provincial Experimental Animal Center) for three times with an interval of half a month between each time. Three days after the last booster immunization, blood was collected from the heart. After the serum was precipitated, the immunoglobulin IgG was initially purified from the rabbit serum by ammonium sulfate precipitation, and then the anti-GP73 polyclonal antibody was further purified by ProteinG / A affinity column, SDS-PAGE and Western Blotting to identify purified antibodies.

Embodiment 3

[0032] Example 3: Preparation, purification and identification of anti-GP73 monoclonal antibody

[0033] SPF grade Balb / c mice (Guangdong Provincial Experimental Animal Center) were immunized with different KLH-coupled GP73 antigen polypeptides, and immunized three times with an interval of half a month between each time. Three days after the last booster immunization, the spleen cells were collected and fused with myeloma cells to obtain a hybridoma cell line, and the antibody secreted by the hybridoma cells was further purified with a ProteinG / A affinity column.

[0034] According to the GP73 protein sequence, the following three polypeptides were designed for experiments:

[0035] SEQ ID NO:1 CKEQCEERIEEVTKKGNEAV

[0036] SEQ ID NO:2 CDGQEEEQEAAGEGRNQQ

[0037] SEQ ID NO:3 CNMDENEAESETDKQAAL

[0038] Three hybridoma cell lines were obtained by using the above-mentioned 3-segment polypeptides. Next, the monoclonal antibodies secreted by these hybridoma cell lines were use...

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Abstract

The invention belongs to the technical field of bio-detection and particularly discloses a kit of high-effectively and quantitatively detecting Golgi apparatus 73 (GP73). In the invention, for the first time, an anti-GP73 polyclonal antibody and an anti-GP73 monoclonal antibody are mixed to obtain a combined capture antibody. The technology, instead of a conventional method in which only is one of the anti-GP73 polyclonal antibody or the anti-GP73 monoclonal antibody employed as a capture antibody, can significantly improve specificity and sensitivity of immune-detection and achieve a lower detection limit since the two antibodies hereinabove are mixed. The kits in the invention have strong specificity and sensitivity, lower detection limit and good linearity conditions.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for detecting GP73 and a kit thereof, in particular to an ELISA kit for quantitatively detecting GP73. Background technique [0002] Liver cancer is one of the most common clinical malignant tumors. The global incidence rate is increasing year by year, exceeding 626,000 / year, ranking fifth among malignant tumors; the death rate is close to 600,000 / year, ranking third among tumor-related deaths. At present, the number of cases in my country accounts for about 55% of the world; it ranks second only to lung cancer in tumor-related deaths. Therefore, liver cancer seriously threatens the health and life of our people. The liver is the largest substantial organ in the human body and undertakes various important metabolic functions of the human body. Therefore, once a malignant tumor occurs in the liver, it will lead to serious consequences that do not reach life. And becau...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 黄若磐张玉明胡洋罗树红
Owner RAYBIOTECH INC GUANGZHOU
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