Hybridoma cell strain capable of secreting T-2 toxin monoclonal antibody

A technology of hybridoma cell line and monoclonal antibody, which is applied in the field of T-2 toxin monoclonal antibody, extraction and purification of T-2 toxin and HT-2 toxin, and can solve inaccurate detection results and insufficient binding ability of T-2 toxin Strong, unable to effectively extract HT-2 toxin and other issues

Active Publication Date: 2016-01-20
INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the mAb has no cross-reactivity with aflatoxin, its cross-reactivity with HT-2 toxin is 65.9%
Therefore, when used in immunoaffinity chromatography, this antibody can effectively extract T-2 toxin but not HT-2 toxin
At the same time, the titer of the monoclonal antibody is only 1:12000, the binding force to T-2 toxin is not strong, and it is easy to dissociate, resulting in inaccurate final test results

Method used

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  • Hybridoma cell strain capable of secreting T-2 toxin monoclonal antibody
  • Hybridoma cell strain capable of secreting T-2 toxin monoclonal antibody
  • Hybridoma cell strain capable of secreting T-2 toxin monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of T-2 toxin monoclonal antibody

[0040] 1. Animal immunity

[0041] The immunized animals are about 8 weeks old female BALB / c mice. The antigen T-2-BSA (made by Beijing Zhongjian Weikang Biotechnology Co., Ltd.) was used to immunize 10 mice respectively. Take an appropriate amount of immunogen (100 mg / cause) and add an equal amount of Freund's complete adjuvant to make an emulsified agent for immunization. A total of 4-6 times of immunization are performed, with an interval of 2 weeks between each time. Except for the last immunization which was intraperitoneal injection, the rest were injected subcutaneously at multiple points on the back of the neck.

[0042] 2. Preparation of hybridoma cells

[0043] ①Cultivation of myeloma cells

[0044]SP2 / 0 myeloma cells were cultured in complete medium containing 10% fetal bovine serum. When the cells are in the logarithmic growth phase, they should be diluted at a ratio of 1:3-1:10 and subcultured. U...

Embodiment 2

[0081] Example 2 Preparation of T-2 toxin, 3-acetyl deoxynivalenol, aflatoxin, ochratoxin, zearalenone immunoaffinity column

[0082] 1. Substrate Preparation

[0083] Weigh required 1 g of Sepharose matrix powder (each gram of lyophilized matrix powder can form a swelling matrix with a final volume of 3.5 ml), and dissolve it in 1 mM HCl. The matrix will immediately swell and then placed on a sintered glass filter and washed with 1 mM HCl for 15 min.

[0084] 2. Ligand (antibody) conjugation

[0085] a using coupling buffer 0.2M NaHCO 3 pH8.3 dissolves 3-acetyldeoxynivalenol, aflatoxin, ochratoxin, zearalenone, T-2 secreted by hybridoma cells 9204, 5506, 5505, 5504 and 11181 to be coupled Toxin antibody (antibody prepared in Example 1), the antibody concentration is 12.5 mg / ml, and the dissolved antibody is temporarily stored in an ice bath. Add the antibody-containing conjugation buffer described above to a fully sealable container with a lid. Quickly transfer the CNBr-...

Embodiment 3

[0093] Example 3 Detection of T-2 toxin, HT-2 toxin, 3-acetyldeoxynivalenol, aflatoxin B1, B2, G1, G2, ochratoxin A, and zearalenone in feed

[0094] 1.0 Detection of T-2 toxin, HT-2 toxin, 3-acetyldeoxynivalenol, aflatoxin B1, B2, G1, G2, ochratoxin, zearalenone in feed

[0095] In feed recovery experiment, three concentration gradients of 20μg / kg, 50μg / kg and 100μg / kg were added respectively. Five sets of parallel experiments were done for each experiment.

[0096] 1.1 Extraction of T-2 toxin, HT-2 toxin, 3-acetyl deoxynivalenol, aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone in feed:

[0097] Accurately weigh 50.0g of the sample that has been ground (particle size less than 2mm) into a 250mL conical flask with a stopper, add 5.0g of sodium chloride and accurately add 100.0mL of methanol-water (8+2), and use a homogenizer at high speed Stir and extract for 2 minutes, or shake on a shaker for 30 minutes. Quantitative filter paper filtration, accurately pipette 5.0m...

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Abstract

The invention provides a hybridoma cell strain capable of secreting a T-2 toxin monoclonal antibody and also provides the T-2 toxin monoclonal antibody secreted by the cell. The monoclonal antibody has the ascite potency ratio of 1:50000 which is superior to that of the existing monoclonal antibody. Meanwhile, the cross reaction rate of the monoclonal antibody and HT-2 toxin reaches up to 92.8%, and the cross reaction rate of the monoclonal antibody and various other antigens is lower than 0.1%. An immunoaffinity column-liquid chromatography containing the monoclonal antibody can be used for simultaneously and efficiently separating and extracting T-2 toxin and HT-2 toxin in samples and is accurate, economic and effective.

Description

technical field [0001] The present invention relates to a hybridoma cell line secreting T-2 toxin monoclonal antibody, also relates to the T-2 toxin monoclonal antibody secreted by the cell line, an immunoadsorbent, an immunoaffinity column, and a kit containing the antibody, And the method for extracting and purifying T-2 toxin and HT-2 toxin by using the immunoaffinity column. Background technique [0002] T-2 toxin and HT-2 toxin are trichothecenes (TS) produced by various fungi, mainly Fusarium tritinarum. HT-2 toxin is a metabolite of T-2 toxin. They are widely distributed in nature, and are the main toxins that commonly pollute field crops and stored grains, and are more harmful to humans and livestock. T-2 and HT-2 toxins mainly act on tissues and organs with vigorous cell division, such as thymus, bone marrow, liver, spleen, lymph nodes, gonads and gastrointestinal mucosa, etc., inhibiting the synthesis of protein and DNA in these organs. In addition, the toxin was...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/14B01J20/24B01J20/281B01D15/38C07D493/10G01N33/577C12R1/91
Inventor 李蓉王勇吴振兴冯雪雅张静王雄果旗
Owner INSPECTION AND QUARANTINE TECHNOLOGY CENTER ZHONGSHAN ENTRY EXIT INSPECTION AND QUARANTINE
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