Zeranol immunoaffinity column as well as preparation method and application thereof

A technology of zearalanol and immunoaffinity, applied in the field of immunoaffinity chromatography, can solve the problems of inability to selectively adsorb target molecules, introduce interference substances, cumbersome operation, etc., simplify the sample pretreatment process and improve the analysis quality , good purification effect

Active Publication Date: 2022-01-21
BEIJING KWINBON BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, LLE needs to use a large amount of organic solvents, which is cumbersome and time-consuming; SPE has become one of the most commonly used extraction methods because of its small amount of organic solvents, large adsorption capacity, and rapid separation.
At present, the SPE columns commonly used for the determination of zearalanol include mixed anion exchange columns (MAX columns) and hydrophilic-lipophilic balance columns (HLB columns), etc. Interfering substances may be introduced in the process

Method used

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  • Zeranol immunoaffinity column as well as preparation method and application thereof
  • Zeranol immunoaffinity column as well as preparation method and application thereof
  • Zeranol immunoaffinity column as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation of embodiment 1 zearalenol monoclonal antibody

[0021] 1. Synthesis of zearalenol hapten (synthetic route see attached figure 1 )

[0022] Take 0.32g of zearalanone, add 10mL of N,N-dimethylformamide (DMF) to dissolve, add 0.13g of potassium L-hydroxyproline, stir well, add 2.12g of trimethylsilyl cyanide, and stir at room temperature for 8h , stop the reaction, add water 20mL, 1mol / L hydrochloric acid 3mL, fully stir for 30min, stop stirring, add water 50mL, add ethyl acetate 20mL×3 for extraction three times, combine the organic phases, evaporate to dryness to obtain a red oil; Dissolve in 80% sulfuric acid, react at 90°C for 4 hours, stop the reaction, add 6mol / L NaOH to adjust the pH value to 6, add 100mL of dichloromethane for extraction, separate the water phase, evaporate the organic phase to dryness, put on a silica gel column, and use The mixed solution of dichloromethane and methanol with a volume ratio of 10:1 was eluted and separated to ob...

Embodiment 2

[0039] Example 2 Preparation of Zearallol Immunoaffinity Column

[0040] 1. Activation of solid phase carrier

[0041] Weigh 1 g of cyanogen bromide-activated agarose gel 4B, add 10 mL of 1 mmol / L HCl solution, soak for 30 min to make it swell; then centrifuge at 3000-8000 r / min for 2-10 min to separate the solid-phase carrier, Wash the swollen solid-phase carrier with pH 9.0 CB buffer 3 to 5 times, adding 10 mL of buffer solution each time, and finally centrifuge at 5000 r / min for 10 min to separate the solid-phase carrier.

[0042] 2. Antibody Conjugation

[0043] Add 30mg of zearalenol monoclonal antibody to the activated solid phase carrier, then add 10mL of 0.1mol / L pH9.0 CB buffer solution, and shake at 150r / min at 37°C for 1h; after the reaction, centrifuge Conjugates, with 0.1mol / L NaHCO pH 8.3 3 The conjugate was washed with buffer for 3 to 5 times, and 10 mL of buffer was added each time.

[0044] 3. Antibody blocking

[0045] Add 10 mL of 0.1 mol / L Tris-HCl solut...

Embodiment 3

[0048] The use of embodiment 3 zearalenol immunoaffinity columns

[0049] 1. Sample preparation

[0050] Accurately weigh 10.0g (accurate to 0.01g) homogeneous sample, add 2.0g sodium chloride; mix with 40.0mL methanol-water solution (80:20, V / V), extract by shaking for 15min, and centrifuge at 4000r / min 5min; accurately pipette 10mL supernatant, add 40mL PBS solution to dilute, mix well, filter with glass microfiber filter paper until the filtrate is clear, and set aside.

[0051] 2. Immunoaffinity column purification

[0052] Accurately pipette 10mL of the above-mentioned filtrate, inject it into a syringe, adjust the pressure of the syringe to make it slowly pass through the immunoaffinity column at a speed of 2 s / drop; wash the column with 20mL water (for the sample solution with a darker color, first use 10mL 0.1% spit Wash with warm 20-PBS, then wash with 10mL 10% methanol-water, and finally wash with 10mL water), flow rate 1s / drop, discard the effluent until 2-3mL air...

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Abstract

The invention discloses a zeranol immunoaffinity column, and the zeranol immunoaffinity column comprises a solid phase carrier coupled with a zeranol monoclonal antibody, a plastic tube loaded with the solid phase carrier, and a preserving fluid, and the zeranol monoclonal antibody is prepared by using a zeranol hapten-carrier protein conjugate as an immunogen. The zeranol hapten is obtained by taking zearalenone as an initial raw material, reacting zearalenone with trimethylsilyl cyanide under the catalysis of L-hydroxyproline salt to obtain a cyanohydrin compound, and then carrying out hydrolysis reaction on cyano groups. The zeranol immunoaffinity column provided by the invention is simple in structure, convenient to use and high in specificity, and zeranol residues in animal-derived food can be rapidly extracted.

Description

technical field [0001] The invention belongs to the technical field of immunoaffinity chromatography, in particular, the invention relates to a zearalanol immunoaffinity column and its preparation method and application. Background technique [0002] Zearalanol, also known as "R-cyclotetradecone phenol", is a kind of ideal weight-increasing agent for skin embedding, which can improve the conversion rate of livestock and poultry feed and the lean meat rate of ketone bodies. However, studies have shown that zearalanol has certain hazards to mammals, especially reproductive hazards. Although most of zearalanol will be metabolized in the body, some of it will remain at the embedding site, and after eating animal-derived foods containing zearalanol residues through the food chain, it will lead to a decrease in the level of gonadotropin in consumers. It also has adverse effects such as lowering, endocrine disorders, growth and development disorders, and affecting the development ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531B01D15/38
CPCG01N33/577G01N33/531B01D15/3823
Inventor 万宇平马玉华王兆芹李晓芳赵杨李天伟
Owner BEIJING KWINBON BIOTECH
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