Preparation method of monoclonal antibody of resisting treeshrew IgG

A monoclonal antibody, tree shrew technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of lack of anti-tree shrew IgG monoclonal antibodies and so on

Inactive Publication Date: 2010-01-27
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Also provided is the preparation method of the hybridoma cell line of the anti-tree shrew IgG monoclonal antibody, to solve the technical defect of the hybridoma cell line lacking the anti-tree shrew IgG monoclonal antibody in the prior art

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  • Preparation method of monoclonal antibody of resisting treeshrew IgG
  • Preparation method of monoclonal antibody of resisting treeshrew IgG
  • Preparation method of monoclonal antibody of resisting treeshrew IgG

Examples

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Embodiment 1

[0025] Isolation and purification of tree shrew IgG:

[0026] Isolation and purification of tree shrew IgG. Tree shrew serum was initially separated by octanoic acid-ammonium sulfate precipitation method, purified by Sephacry-S200 gel filtration chromatography and DEAE-Sephadex A-50 ion exchange chromatography, and then purified by SDS -PAGE electrophoresis to detect the separation and purification results.

[0027] (1) Preparation of tree shrew serum:

[0028] The blood was collected by neck dislocation, and the blood was placed at room temperature or 37°C for 1 hour, and then overnight in a refrigerator at 4°C. Collect the supernatant, centrifuge at 12000rpm for 10min. The obtained serum was immediately frozen and stored in a low-temperature refrigerator for future use.

[0029] (2) Isolation and purification of tree shrew IgG:

[0030] 1. Caprylic acid-ammonium sulfate precipitation method:

[0031] Take 1 part of serum and add 4 parts of 0.06mol / l pH 4.8 acetic acid b...

Embodiment 2

[0057] Establishment of anti-tree shrew IgG hybridoma cell line and preparation of its monoclonal antibody:

[0058] (1) Immunized animals:

[0059] BALB / C mice were immunized with isolated and purified tree shrew IgG four times. For the first immunization, the tree shrew IgG was mixed with an equal amount of Freund's complete adjuvant, vortexed and emulsified for half an hour, and the emulsified back and groin were subcutaneously injected for 6 ~8-week-old BALB / C mice were emulsified with tree shrew IgG and the same amount of incomplete Freund's adjuvant two weeks later for the second immunization in the same way, and two weeks after the second immunization, tree shrew IgG was used After the adjuvant was emulsified, it was injected intraperitoneally for the third immunization, and two weeks later for the fourth immunization (same as the third immunization). The injection volume for the first to fourth immunizations was 100 μg per mouse, and 0.3-0.4 ml was injected. Recall st...

Embodiment 3

[0066] Purification of monoclonal antibodies:

[0067] 1. Preliminary purification of antibodies by salting out method:

[0068] 1 part of ascitic fluid and the same volume of normal saline are fully mixed and placed on the ice-water mixture, then placed on a magnetic stirrer, and then 2 parts of 100% saturated ammonium sulfate are added dropwise, and the final concentration is 50% saturated. Ammonium sulfate was used for precipitation at 4°C overnight. High-speed centrifugation at 10000rpm, 4°C, 15min. Discard the supernatant and floating matter, and dissolve the precipitate in physiological saline twice the original volume of ascites. At the same time, add one volume of saturated ammonium sulfate, ie, the final concentration is 33%, and the operation steps are the same as 50% saturated ammonium sulfate for precipitation. After precipitation, overnight at 4°C, high-speed centrifugation at 10,000rpm, 4°C, 15min. Discard the supernatant and floating matter, and dissolve the ...

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Abstract

The invention relates to a preparation method of a monoclonal antibody of resisting treeshrew IgG, which specifically relates to the field of a monoclonal antibody and separation and purification of treeshrew IgG protein and belongs to the technical field of biology. The preparation method comprises the preparation steps: separating and purifying treeshrew IgG; immunizing a BALB / C rat; combining hybridoma; screening the hybridoma; cloning the hybridoma; preparing ascites of the monoclonal antibody; detecting the valence of the ascites of the monoclonal antibody; and preparing the monoclonal antibody.

Description

Technical field: [0001] The invention relates to a method for preparing an anti-tree shrew IgG monoclonal antibody, in particular to the field of monoclonal antibodies, and to the separation and purification of tree shrew IgG protein, belonging to the field of biotechnology. Background technique: [0002] The tree shrew (Tupaia belangeri, tree shrew) is a small mammal that looks like a squirrel. It is small in size, fast in growth and reproduction, easy to capture, domesticate and reproduce, and low in cost. Its metabolism and general anatomy are closer to humans than rodents. With the gradual reduction of non-human primate resources at home and abroad and the development trend of miniaturization of experimental animals, tree shrews, as a possible animal model for the study of human-related diseases, have received widespread attention and have been used in viruses, nerves, psychology, diabetes, Research on cerebral ischemia, blood vessels, tumors, parasites, etc. It has bee...

Claims

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Application Information

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IPC IPC(8): C12N15/06C12P21/08
Inventor 夏雪山温志刚冯悦祁立强刘丽魏大巧
Owner KUNMING UNIV OF SCI & TECH
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