Method for preparing bacillus coli 0157:H7 antigen monoclonal antibody by subtractive immunization

A monoclonal antibody, Escherichia coli technology, applied in the direction of antibacterial immunoglobulin, etc., can solve the problems of low expression yield, high technical difficulty, limitation of natural protein structure and function detection of pathogenic microorganisms, etc., to reduce workload and improve immunity. effect of effect

Inactive Publication Date: 2008-12-17
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this type of antibody can be used in Western Blot experiments, it is very limited to study the structure and function of natural proteins and the detection of pathogenic microorganisms
However, using eukaryotic expression systems such as yeast, insect, and mammalian cells to express proteins has problems such as longer experimental periods, lower expression yields, and greater technical difficulty.

Method used

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  • Method for preparing bacillus coli 0157:H7 antigen monoclonal antibody by subtractive immunization
  • Method for preparing bacillus coli 0157:H7 antigen monoclonal antibody by subtractive immunization
  • Method for preparing bacillus coli 0157:H7 antigen monoclonal antibody by subtractive immunization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Prepare antigen

[0025] Dissolve freeze-dried strains E. coli O157:H7 and E. coli 44752 in normal saline, inoculate them in nutrient broth medium and incubate them at 37°C for 18 hours, then inoculate them in selective medium (Endo medium) and incubate them at 37°C for 18 hours. The typical colonies were inoculated on nutrient agar medium, cultured at 37°C for 18 hours, washed with PBS, centrifuged at 4000r / min for 20min, washed twice with PBS, fixed with 0.4% formaldehyde, and prepared with an antigen concentration of 10 8 cfu / mL bacterial suspension, stored at 4°C.

[0026] Subtractive immunization to immunize mice

[0027] Steps for subtractive immunization to immunize BALB / c mice: Subtractive immunization: use Escherichia coli 44752 as the tolerogen and Escherichia coli O157:H7 as the immunogen, and use 5-6 week-old female BALB / c mice. First, inject 0.5mL antigen intraperitoneally with the tolerogen to immunize the mice, and then inject cyclophosphamide (Cy) (100...

Embodiment 2

[0057] Subtractive immunization method prepares the method for Escherichia coli O157: H7 antigen monoclonal antibody, comprises the steps:

[0058] (1) Select 4-week-old female BALB / c mice;

[0059] (2) Escherichia coli 44186 was used as the tolerance source to immunize the BALB / c mice by intraperitoneal injection, and the BALB / c mice were injected intraperitoneally at 6 minutes, 18 hours and 40 hours after the injection of the tolerance source, according to the body weight of the BALB / c mice per kilogram Cyclophosphamide 50mg, use indirect ELISA to detect the tolerance of mice;

[0060] (3) Repeat step (2) every two weeks until the BALB / c mice are tolerant to the tolerogen;

[0061] (4) 7d, 14d, and 28d after the last immunization that made the BALB / c mice tolerant, the BALB / c tolerant to the tolerogen was immunized by intraperitoneal injection with Escherichia coli O157:H7 as the immunogen For the mice, after the last immunization with the Escherichia coli O157:H7, the mou...

Embodiment 3

[0063] Subtractive immunization method prepares the method for Escherichia coli O157: H7 antigen monoclonal antibody, comprises the steps:

[0064] (1) Select 8-week-old female BALB / c mice;

[0065] (2) Escherichia coli 44824 was used as the tolerance source to immunize the BALB / c mice by intraperitoneal injection, and the BALB / c mice were injected intraperitoneally at 16 minutes, 30 hours and 60 hours after the injection of the tolerance source, according to the body weight of the BALB / c mice per kilogram Cyclophosphamide 80mg, use indirect ELISA to detect the tolerance of mice;

[0066] (3) Repeat step (2) every two weeks until the BALB / c mice are tolerant to the tolerogen;

[0067] (4) 14dd, 21d, and 35d after the last immunization that made the BALB / c mice tolerant, the BALB / c tolerant to the tolerogen was immunized by intraperitoneal injection with Escherichia coli O157:H7 as the immunogen For the mice, after the last immunization with the Escherichia coli O157:H7, the ...

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Abstract

The invention discloses a method for preparing Escherichia coli O157:H7 antigen monoclonal antibody by using a subtractive immunization method. The method includes the steps that: (1) a BALB / c mouse is selected; (2) Escherichia coli, except Escherichia coli O157:H7, is used as toleragen for immunizing the BALB / c mouse and after the mouse is injected with the toleragen and then the abdominal cavity of the mouse is injected with cyclophosphamide to test the tolerance of the mouse; (3) the step (2) is repeated two weeks till the BALB / c mouse is tolerant toward the toleragen; (4) after the tolerance occurs in the BALB / c mouse, the Escherichia coli O157:H7 is used for immunizing the BALB / c mouse. The mouse which has low reaction titer with the toleragen while high reaction titer with the Escherichia coli O157:H7 is taken out and then the monoclonal antibody of the Escherichia coli O157:H7 is prepared according to the conventional hybridoma technique. Based on that antigen is not sublimated, the method of the invention is easier to obtain the monoclonal antibody of bacterial antigen so as to improve the immune effects and reduce the filtration workload of the monoclonal antibody.

Description

technical field [0001] The invention relates to an immunization method for preparing monoclonal antibodies, in particular to a method for preparing Escherichia coli O157:H7 antigen monoclonal antibodies by subtractive immunization. Background technique [0002] In 1975, Kohler and Milstein published the paper "Continuous Culture of Fusion Cells Secreting Predetermined Specific Antibodies", and monoclonal antibody technology came out. Monoclonal antibody technology has become an important tool in modern life science research. Conventional hybridoma technology usually cannot obtain monoclonal antibodies to specific antigens of interest. To prepare monoclonal antibodies against bacterial antigens by conventional techniques, biochemical methods are usually used to separate and purify natural proteins as immunogens, and then the spleen cells of immunized mice are fused with myeloma cells, and the cell lines secreting antibodies against the target protein are screened out with im...

Claims

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Application Information

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IPC IPC(8): C07K16/12
Inventor 李君文郎婧金敏谌志强王新为陈照立邱志刚王景峰
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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