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High-specificity and high-sensitivity monoclonal antibody for detecting human alpha-fetoprotein as well as reagent kit and application

A technology of monoclonal antibody and alpha-fetoprotein, applied in the direction of immunoglobulin, anti-animal/human immunoglobulin, specific peptide, etc., can solve the problem that the linear range and sensitivity need to be further improved, has no clinical application value, and cannot be used Issues such as kit development and production to achieve good detection results

Active Publication Date: 2017-08-08
广州普锐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although anti-hAFP monoclonal antibodies have been successfully prepared at home and abroad, most of them do not have clinical application value and cannot be used for kit development and production
Among the hAFP detection ELISA kit products sold at home and abroad, the linear detection range is within 20-400ng / ml, and the overall quality of imported kits is obviously better than that of domestic products, but its linear range and sensitivity still need to be further improved

Method used

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  • High-specificity and high-sensitivity monoclonal antibody for detecting human alpha-fetoprotein as well as reagent kit and application
  • High-specificity and high-sensitivity monoclonal antibody for detecting human alpha-fetoprotein as well as reagent kit and application
  • High-specificity and high-sensitivity monoclonal antibody for detecting human alpha-fetoprotein as well as reagent kit and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation of embodiment 1 monoclonal antibody

[0050] Antigen human alpha-fetoprotein (Human Myoglobin) was purchased from Guilin Inmate Biotechnology Co., Ltd., isolated and purified from human umbilical cord blood, and its concentration was 7.6mg / ml. composed of amino acid residues. hAFP is a multifunctional transporter that is highly expressed in the fetus and silenced after birth. Serum hAFP concentration is a direct or indirect evaluation index of many diseases, especially common in prenatal diagnosis and liver cancer diagnosis and condition monitoring.

[0051] The preparation method of anti-human alpha-fetoprotein mouse monoclonal antibody is as follows:

[0052] (1) Animal immunity

[0053] 1) Initial immunization: 30 μg of antigen plus an equal volume of complete Freund's adjuvant is fully emulsified and injected subcutaneously into mice at multiple points;

[0054] 2) Secondary immunization: 2 weeks later, the same amount of antigen as the first imm...

Embodiment 2

[0095] The specificity analysis of embodiment 2 monoclonal antibody

[0096] The four important monoclonal antibodies Ab 1-A b 4 screened above all have very high affinity to the immunogen hAFP. In order to further identify their specificity and verify whether they can specifically bind to naturally occurring hAFP, they were tested in cells The following two experiments were designed at the level and organizational level.

[0097] 1) Immunofluorescence staining of HepG2 human liver cancer cells

[0098] It is known that human liver cancer cells HepG2 can synthesize a large amount of hAFP in the cells, and the four monoclonal antibodies Ab1-Ab 4 were used for cell fluorescence staining experiments.

[0099] Experimental method: ① One day before the experiment, take HepG2 cells in good condition to make a cell suspension, and count the cells at a cell concentration of 5×10 5 Place the amount of cells / well into the wells of a six-well plate with coverslips placed; ②Cultivate in t...

Embodiment 3

[0110] Example 3 Sequence and structure analysis of monoclonal antibodies Ab1-Ab3

[0111] After further sequence and structural analysis of the obtained monoclonal antibodies Ab1-Ab3, it was found that the variable regions of Ab1-Ab3 all include a heavy chain variable region and a light chain variable region.

[0112] (1) From the perspective of gene sequence:

[0113] The gene sequence of the heavy chain variable region of the monoclonal antibody Ab1 is shown in SEQ ID NO.1, and the gene sequence of the light chain variable region is shown in SEQ ID NO.2;

[0114] The gene sequence of the heavy chain variable region of the monoclonal antibody Ab2 is shown in SEQ ID NO.3, and the gene sequence of the light chain variable region is shown in SEQ ID NO.4;

[0115] The gene sequence of the heavy chain variable region of the monoclonal antibody Ab3 is shown in SEQ ID NO.5, and the gene sequence of the light chain variable region is shown in SEQ ID NO.6.

[0116] (2) From the per...

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Abstract

The invention discloses a high-specificity and high-sensitivity monoclonal antibody for detecting human alpha-fetoprotein as well as a reagent kit and application. Three monoclonal antibodies containing monoclonal antibodies Ab1 to Ab3 can be used for specifically identifying human-derived natural hAFP (human Alpha-fetoprotein) in human hepatocellular carcinoma cells and hepatocellular carcinoma tissues and have very good specificity. When double-antibody sandwich ELISA (Enzyme-linked Immunosorbent Assay) detection is carried out on a sample by utilizing an antibody matched combination Ab1+Ab3 / HRP-Ab2, a linear detection range is 5ng / ml to 250ng / ml, the lowest detection limit is 2ng / ml and the upper detection limit is 400ng / ml; the linear detection range and the lowest detection limit are better than a linear range of 5ng / ml to 200ng / ml and the upper detection limit of 5ng / ml of an imported kit; a clinical sample detection result shows that a detection method has the positive blood serum accuracy of 100 percent and the negative blood serum accuracy of 100 percent. Therefore, an established double-antibody sandwich ELISA method has a larger linear range and better popularization and application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a group of monoclonal antibodies with high affinity and high specificity binding to different epitopes of human alpha-fetoprotein and applications thereof. Background technique [0002] Human alpha fetoprotein (hAFP) is an embryonic glycoprotein synthesized by fetal liver cells and yolk sac, its function is similar to that of human serum albumin (HSA), and it can be regarded as fetal HSA replacement during developmental period. Under normal circumstances, hAFP only exists in fetal serum, and the concentration of hAFP can drop back to the normal level (<5ng / ml) after one year of fetal birth, and the concentration in normal human serum is generally not higher than 20ng / ml. When primary liver cancer occurs in the body, the liver cancer cells can secrete a large amount of hAFP in the peripheral blood, causing an abnormal increase in blood hAFP concentration. [0003] hAFP...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/577G01N33/574
CPCC07K16/18C07K2317/33C07K2317/565C07K2317/92G01N33/57438G01N33/57484G01N33/577G01N2333/471
Inventor 向军俭黄建芳孙一帆
Owner 广州普锐生物科技有限公司
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