Method for preparing ZEN (Zearalenone) monoclonal antibody by utilizing rabbit fetal fibroblast

A technology based on fibroblasts and zearalenone, which is applied in the field of preparing zearalenone monoclonal antibodies, can solve the problems of unfavorable animal protection and waste of resources, and achieve the effect of simple method, low cost and huge market value

Inactive Publication Date: 2018-11-13
WUXI ZODOLABS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The basis of immunological methods lies in the acquisition of antibodies. At present, the preparation technology of such antibodies in China is still the traditional hybridoma cell technology. The feeder layer cells are mainly various cells derived from mice (such as peritoneal macrophages, mouse adult cells, etc.). Fibroblasts, etc.), each use needs to raise and kill a large number of mice, which wastes resources and is not conducive to animal protection. Therefore, providing a new feeder layer cell of mouse hybridoma cells has broad application prospects and a huge market value

Method used

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  • Method for preparing ZEN (Zearalenone) monoclonal antibody by utilizing rabbit fetal fibroblast
  • Method for preparing ZEN (Zearalenone) monoclonal antibody by utilizing rabbit fetal fibroblast
  • Method for preparing ZEN (Zearalenone) monoclonal antibody by utilizing rabbit fetal fibroblast

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 animal immunization

[0051] 1.1 Choose female BaLb / C mice aged 6-8 weeks, and immunize 4 mice in each group.

[0052] 1.2 Processing of immunogen

[0053] Dilute the immunogen with sterile saline, add an equal volume of adjuvant to mix, the adjuvant must be shaken before use to make it fully mixed (soluble antigen, use Freund's complete adjuvant for the first time, use Freund's complete adjuvant for subsequent immunizations) Incomplete adjuvant, pulse immunization without adjuvant), subcutaneous injection of 200 μL / rat, of which 100 μL is adjuvant.

[0054] 1.3 Immunization of mice:

[0055] Mice were immunized according to the procedure in Table 1.

[0056] Table 1 Immunization program for mice

[0057]

Embodiment 2

[0058] The acquisition of embodiment 2 feeder layer cells

[0059] Artificial assisted mating of rabbits, two weeks of pregnancy, subcutaneous injection of atropine 1mg / kg body weight, 15 minutes later intravenous injection of ZoLetiL-50 (zoLetiL-50) 7.5 mg / kg body weight, cesarean section to take out the fetus, D-Hank's washing twice , after removing the head, limbs and viscera, cut the remaining part of the tissue into about 1mm3 size, wash 3 times with D-Hank's; add 5mL of digestive solution (0.05% trypsin + 0.04% EDTA), and pipette repeatedly for 15 minutes to digest; After the solution was turbid, let it stand for 1 min, take the upper turbid solution into another clean centrifuge tube, centrifuge and wash twice with D-Hank's solution, count the cells and adjust the density to 5×10 with culture medium (containing DMEM / F12+10% FBS). 5 cells / mL, inoculated in six-well plates and flasks, placed in CO 2 In an incubator, 37°C, 5% CO 2 , cultured statically under saturated hu...

Embodiment 3

[0061] Example 3 Cell Fusion

[0062] 3.1 Cell recovery

[0063] (1) Take out the myeloma cell cryopreservation tube from the liquid nitrogen tank, immediately immerse it in warm water at 39°C, and shake it from time to time to melt it as soon as possible;

[0064] (2) Take out the cryopreservation tube from the water bath, open the lid under aseptic conditions, suck out the cell suspension with a straw, add it to a 15mL sterile centrifuge tube, add 10mL RPMI 1640 incomplete culture medium, and mix well;

[0065] (3) Centrifuge at room temperature at 1100rpm for 5min; discard the supernatant, add RPMI 1640 complete culture solution I to resuspend the cells as a homogeneous suspension, adjust the cell density, inoculate the culture dish, and culture in a 37°C, 5% CO2 incubator;

[0066] (4) According to the growth state of the cells, replace the RPMI 1640 complete culture medium I once, and continue the culture.

[0067] 3.2 Preparation of fresh myeloma cells

[0068] (1) Cu...

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Abstract

The invention provides new application of rabbit fetal fibroblast, and particularly relates to a method for preparing a ZEN (Zearalenone) monoclonal antibody by utilizing the rabbit fetal fibroblast.The method is characterized in that mouse hybridoma is cultured by using rabbit fetal fibroblast as feeder cells of murine hybridoma, the murine feeder cells can be completely replaced, growth of monoclonal hybridoma can be promoted, and a monoclonal antibody with high valence, good stability and strong specificity can be generated; the hybridoma bred by different animal cells as the feeder cellsis used for preparing an antibody, and very high valence and very high specificity are obtained; meanwhile, the rabbit fetal fibroblast can be revived by many times for use after being cryopreserved,animal tissue isolating culture is not required to be carried out every time. The method disclosed by the invention is simple, the cost is low, the monoclonal antibody which is used for fast, sensitive, economic and efficient ZEN immunological detection can be prepared, and a wide application prospect and huge market value are obtained.

Description

technical field [0001] The invention relates to the technical field of biological immunity, and relates to a new application of rabbit fetal fibroblasts, in particular to a method for preparing zearalenone monoclonal antibody using rabbit fetal fibroblasts. Background technique [0002] Zearalenone (ZearaLenone, ZEN), also known as F2 toxin, is a mycotoxin with estrogen effect produced by Fusarium graminearum and other species. ZEN was first isolated from gibberella corn, so it was named "zearalenone", which is a metabolite of Gibberella zeara. ) β-rapenolide, which is widely present in moldy corn, sorghum, wheat, oats, barley and other cereal crops and milk, the most common fungus producing zearalenone is Fusarium graminearum, and the third line Fusarium, Fusarium equisetum, Fusarium nivale, Fusarium pink, etc., can produce various toxic and side effects on humans and animals. ZEN has estrogen-like effects, reproductive and developmental toxicity, immunotoxicity, cytotoxi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577
CPCC07K16/44C12N5/163G01N33/577
Inventor 宋绍征周丽伍燕华邹晓兰罗长财吴建彬李林
Owner WUXI ZODOLABS BIOTECH
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