Method for preparing zeranol monoclonal antibody based on cross reactivity of structural analogue

A zearatol, monoclonal antibody technology, applied in biochemical equipment and methods, chemical instruments and methods, and analysis by chemical reaction of materials, etc., can solve the problem of low antibody sensitivity and uncertain quality of ZAL artificial antigens and other problems to achieve the effect of strong affinity

Inactive Publication Date: 2017-11-14
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The key to determining the sensitivity of immunological detection methods is the quality of the antibody. At present, the sensitivity of ZAL immunological detection antibodies is reported to be at the ng level, and some are even higher, mainly because the quality of the

Method used

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  • Method for preparing zeranol monoclonal antibody based on cross reactivity of structural analogue
  • Method for preparing zeranol monoclonal antibody based on cross reactivity of structural analogue
  • Method for preparing zeranol monoclonal antibody based on cross reactivity of structural analogue

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, ZAN artificial antigen

[0044] 1. Preparation of ZAN artificial antigen

[0045] (1) Weigh 2 mg of ZAN and dissolve it in 2 mL of anhydrous pyridine, then add 4 mg of CMO, reflux on a rotary evaporator at 115 ° C for 2 h, and then dry by rotary evaporation; wash the dried product twice with methanol, each time 1 mL, dried by rotary evaporation; then re-dissolve the dried product (spin-dried product after washing with methanol) with 1 mL double-distilled water, adjust the pH value of the solution to 8.0 with 2M NaOH; extract 3 times with an equal volume of toluene, discard the toluene layer, and keep Aqueous phase layer; finally, the obtained aqueous phase was extracted 3 times with an equal volume of ethyl acetate, the aqueous phase was discarded, and the ethyl acetate phase was left, and dried by rotary evaporation to obtain ZAN-O;

[0046] (2) Dissolve ZAN-O with 1mL N,N-dimethylformamide (DMF), stir magnetically, add 2mgEDC and 1.2mg NHS while stirrin...

Embodiment 2

[0062] Embodiment 2, monoclonal antibody

[0063] 1. Animal immunity and immune effect determination

[0064] 1.1. Animal immunity

[0065] Select 6 healthy SPF grade 6-week-old female BALB / c mice, and use ZAN-O-BSA to immunize, and the immunization dose is 50 μg protein (ZAN-O-BSA) per mouse; adopt the back subcutaneous immunization method, 4 -Injection at 6 points, immunization four times in total, immunization interval is 3-4 weeks. For the first immunization, dilute ZAN-O-BSA with PBS to 100 μg protein / 200 μL, then add an equal amount of FCA, and fully emulsify (take a drop of the emulsified emulsion, put it on the water surface, and the milk drop will float on the water surface, and no diffusion occurs, which proves that the emulsification is complete), inject 200 μL subcutaneously into BALB / c mice. For the next three booster immunizations, FIA can be used instead of FCA for emulsification, and other reagents and procedures remain unchanged.

[0066] 1.2. Determinatio...

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Abstract

The invention discloses a method for preparing a zeranol monoclonal antibody based on the cross reactivity of a structural analogue. The method comprises the steps of firstly preparing an artificial antigen ZAN-O-BSA by using a structural analogue ZAN of ZAL, immunizing animals by using the artificial antigen ZAN-O-BSA to generate antiserum; and by adopting the ZAL as a detection target object, screening out a hybridoma cell capable of secretion of an anti-ZAL monoclonal antibody by blocking ELISA screening and preparing the ZAL monoclonal antibody. Practice proves that the monoclonal antibody prepared through the method is high in sensitivity and high in affinity, IC50 is 577pg/mL, immunological detection methods, such as zeranol enzyme-linked immunosorbent assay, chemiluminiscence, time resolution and fluorescence polarization can be built by using the monoclonal antibody prepared through the method, and fast detection products, such as a zeranol colloidal gold test strip and a Raman-enhanced colloidal gold test strip, can be prepared.

Description

technical field [0001] The invention relates to a preparation method of a monoclonal antibody, in particular to a method for preparing a high-sensitivity monoclonal antibody to zearalenol based on the cross-reactivity of structural analogs. Background technique [0002] Alpha-zearalenol, also known as Zearalanol (ZAL), chemical name right cyclotetradecone phenol, is the reduction product of mycotoxin zearalenone (ZEARALENONE, ZEN), is a plant Estrogen can improve the ability of plants to resist cold and frost. [0003] In the 1960s, it was found that ZAL has the function of promoting protein deposition in ruminants, which can significantly improve the feed conversion efficiency of cattle and sheep, and shorten the fattening cycle of cattle and sheep. Therefore, it is widely used as a growth-promoting agent for ruminants. form. Subsequent studies have shown that ZAL is harmful to monogastric animals such as rats, dogs and monkeys, especially harmful to reproduction. Althou...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K14/77C07K16/16C12N5/20G01N21/64G01N21/65G01N21/76G01N33/577
CPCC07K14/765C07K14/77C07K16/16G01N21/6445G01N21/658G01N21/76G01N33/577G01N2333/415
Inventor 张改平胡骁飞邓瑞广李青梅王方雨姚静静孙亚宁柴书军杨艳艳滕蔓赵东
Owner HENAN ACAD OF AGRI SCI
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