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Neutralized monoclone antibody 4C13 of antiricin, its production and use

A monoclonal antibody, ricin technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of simultaneous recognition of ricin monoclonal antibodies, etc., and achieve broad application Foreground effect

Active Publication Date: 2007-06-20
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports of neutralizing monoclonal antibodies that simultaneously recognize the linear epitopes of ricin A chain and B chain

Method used

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  • Neutralized monoclone antibody 4C13 of antiricin, its production and use
  • Neutralized monoclone antibody 4C13 of antiricin, its production and use
  • Neutralized monoclone antibody 4C13 of antiricin, its production and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The construction of embodiment one anti-ricin monoclonal antibody hybridoma cell line

[0035] 1. Materials:

[0036] Freund's complete adjuvant and incomplete adjuvant, TMB is a product of Sigma, 20% fetal bovine serum is a product of Beijing Yuanheng Shengma Biotechnology Research Institute, serum-free RPMI 1640 is a product of Gibco, and SP2 / 0 cells are imported from ATCC , preserved in our laboratory, and Balb / c mice and Kunming mice were purchased from the Experimental Animal Center of the Academy of Military Medical Sciences. The rest of the reagents were purchased from the market.

[0037] 2. Method results:

[0038] 1. The attenuation treatment of ricinus in the purified ricin (Nicolson, G.L., Blaustein, J., 1972.The interaction of Ricinus com-munis agglutinin with normal and tumor cell surfaces.Biochim.Biophys.Acta 266,543 Formaldehyde with a final concentration of 1% was added to -547.) 2mg / ml, treated at 37°C for 72 hours, dialyzed in PH7.20.01M PBS for 72...

Embodiment 2

[0046] Example 2 Screening and identification of anti-ricin neutralizing monoclonal antibody hybridoma cell line

[0047] 1. Materials:

[0048] Ditto.

[0049] 2. Method results:

[0050] 1. Determination of the median lethal dose (IC50) of ricin on mouse myeloma cell SP2 / 0 Dilute ricin with RPMI-1640 so that the concentrations are 0ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL mL, 1.25ng / mL, 0.625ng / mL, 0.3125ng / mL, 0.156ng / mL, 0.078ng / mL were added to 96-well cell culture plate respectively, each concentration was replicated in 3 wells, 100μl / well, logarithmic adjustment The concentration of SP2 / 0 cells in the growth phase was adjusted to 5 × 10 5 / ml, 100 μl / well. Cultivate in a 5% CO2 incubator at 37°C for 24 hours, add 10 μl MTT to each well, incubate in a 5% CO2 incubator at 37°C for 6 hours, centrifuge at 2000 r / min for 10 minutes, discard the supernatant, add 100 μl DMSO to dissolve the crystals, and measure the A value at a wavelength of 570 nm . According to the fo...

Embodiment 3

[0056] The Protein A purification of embodiment 3 monoclonal antibody 4C13 and

[0057] Protective Experiments on BALB / C Mice

[0058] 1. Materials:

[0059] Protein A Sepharose CL 4B column protein column: product of Beijing Benyuan Zhengyang Biotechnology Co., Ltd.; same as above.

[0060] 2. Method results:

[0061] 1. Add 1 ml of pH8.0, 0.1moL / L phosphate buffer to 2 ml of mouse ascites and adjust the pH to 9 with pH9.0, 1moL / L TRIS-HCL. Add mouse ascites to the Protein A Sepharose CL 4B column protein column that has been equilibrated with 0.1moL / L phosphate buffer solution pH 8.0, and wash the column with the above buffer solution until no foreign protein is detected in the effluent. Elute with pH 3.0 citrate buffer, collect the effluent, and immediately neutralize with 1moL / L TRIS-HCL pH 8.5 buffer, and dialyze with pH 7.2, 0.01M PBS for 72h. Samples were taken to measure OD260 and OD280 on a UV spectrophotometer, the protein content was ...

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Abstract

An anti-recin neutralizing monoclonal antibody 4C13, its production and use are disclosed. The process is carried out by cloning gene of antibody light and heavy chain variable region from 4C13 hybrid tumor cell, encoding mouse antibody variable region, constructing and expressing multiple small-molecular gene engineering antibody based on light and heavy chain variable region gene and cross-linking multiple biological active molecules based on encoded polypeptide or protein. It can be used to inspect recin, diagnose recin and immunotoxin poisoning and prepare related medicines.

Description

technical field [0001] The present invention relates to an anti-ricin neutralizing monoclonal antibody 4C13, and also relates to the preparation method of the monoclonal antibody and the application in preparing ricin vaccine and ricin detection, diagnosis and treatment drugs. Background technique [0002] Ricin is a ribosome-inactivating protein that can bind to almost all eukaryotic cells and exhibit cytotoxicity. One molecule of ricin into the cell is enough to stop the protein synthesis of the whole cell and cause death. Its toxicity is 6000 times that of cyanide. When ricin aerosol enters the body, it can cause severe alveolar inflammation and pulmonary edema, and eventually cause death due to pulmonary obstruction and hypoxia (1. GriffithsGD, Lindsay CD, Allenby AC, et al. Protection against inhalation toxicity of ricinand abrin by immunization . Hum Exp Toxicol 1995; 14: 155-64.2. Brown RFR, White DE. Ultrastructure of rat lung following inhalation of ricin aerosol....

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N5/18A61K39/395G01N33/53
Inventor 郭建巍沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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