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A sea anemone cytolysin gigantoxin-4 monoclonal antibody and its preparation and application

A monoclonal antibody and cytolysin technology, which is applied in the fields of antibodies, extracellular fluid diseases, biochemical equipment and methods, etc., and can solve the problems that there is no Gigantoxin-4 antibody for sea anemone cytolysin

Active Publication Date: 2019-02-01
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report about the anti-cytolysin Gigantoxin-4 in sea anemone

Method used

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  • A sea anemone cytolysin gigantoxin-4 monoclonal antibody and its preparation and application
  • A sea anemone cytolysin gigantoxin-4 monoclonal antibody and its preparation and application
  • A sea anemone cytolysin gigantoxin-4 monoclonal antibody and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1. The polypeptide sequence selection of anti-GT-4 monoclonal antibody and the preparation of polypeptide

[0030] Based on the analysis of the active site and transmembrane structure of GT-4, SEQ ID NO: 1 was selected as the epitope, and Abimate Biopharmaceutical (Shanghai) Co., Ltd. was entrusted to synthesize the polypeptide. The sequence is as follows:

[0031] Asp Val Ile Leu Pro Glu Phe Val Pro Asn Asn Lys (SEQ ID NO: 1)

[0032] Abimate Biomedical (Shanghai) Co., Ltd. was entrusted to synthesize the polypeptide, link the above polypeptide to the corresponding carrier, prepare and purify the antigen for subsequent immune response.

Embodiment 2

[0033] Example 2. Preparation and purification of anti-GT-4 monoclonal antibody

[0034] The polypeptide antigen purified in Example 1 was used for immunization, and 3 female BALB / c mice aged 8-12 weeks were immunized, and the mice were rapidly immunized. The initial immunization was performed subcutaneously under the skin around the shoulders and then double The legs were injected intramuscularly with approximately 1 / 8 of the immunogen per area and 1 / 2 of the remaining immunogen intraperitoneally. One week later, booster immunization with 50 μg, complete Freund's adjuvant, intraperitoneal injection. Two weeks later, booster immunization with 50 μg, incomplete Freund's adjuvant (IFA), intraperitoneal injection. For 3 weeks and later, 50 μg intraperitoneal injection was administered directly every week for booster immunization.

[0035] The spleen cells of the mouse with the best ELISA detection effect were fused with mouse myeloma cell SP2 / 0 cells (purchased from ATCC) at a ...

Embodiment 3

[0039] Example 3. Identification of Anti-GT-4 Monoclonal Antibodies

[0040] 1. Potency identification

[0041]Polypeptide antigen (4μg / ml) was coated on the ELISA plate, and the purified anti-GT-4 monoclonal antibody was carried out at 1:6.25K, 1:12.5K, 1:25K, 1:50K, 1:100K, 1:200K Dilute and add to the wells of the microtiter plate, select HIS antibody (HIS antibody 1mg / ml1:8K diluted with milk) as a positive control for system operation, and 5% milk as a negative control. After the reaction, goat anti-mouse IgG-HRP was added to develop the color. Positive judgment standard: the ratio (P / N) of the OD450 of the test well of the sample to the OD450 of the negative control well is ≥ 2.1, and the result shows that the antibody titer is 200K (Table 1).

[0042] Table 1 Antibody titer identification results

[0043] 6.25K

12.5K

25K

50K

100K

200K

Negative

Positive

potency

3.205

2.711

2.364

1.475

0.954

0.607 ...

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Abstract

The invention relates to the technical field of medical biological engineering, and provides a sea anemone cytolysin Gigantoxin-4 monoclonal antibody, a polypeptide used for preparing the monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody, and applications of the monoclonal antibody in detecting Gigantoxin-4 and neutralizing Gigantoxin-4 hemolysis.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to a sea anemone cytolysin Gigantoxin-4 monoclonal antibody, a polypeptide for preparing the monoclonal antibody, a hybridoma cell line secreting the monoclonal antibody, and the monoclonal antibody Use of antibodies for detection of Gigantoxin-4 and neutralization of Gigantoxin-4 hemolysis. Background technique [0002] my country has a vast sea area, rich marine biological resources, and frequent offshore operations. Workers are often injured by poisonous marine organisms, and poisonous sea anemone stings are a common marine organism injury. [0003] The tentacles and body of sea anemones contain a large number of nematocysts, each of which has a special sac-shaped organelle called nematocyst, which stores venom, and the nematocyst is coiled in the nematocyst. The tip is a thorn, and when mechanically or chemically stimulated, it releases a barbed wire to pierce t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K16/18C12N5/20G01N33/68G01N33/577A61K39/395A61P7/04
Inventor 胡波王梁华焦炳华郑欣高顺祥孙铭娟刘德婧
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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