Polyclonal antibody for detecting moxidectin as well as preparation method and application thereof

A technology of polyclonal antibody and moxidectin, which is applied in the preparation methods of peptides, chemical instruments and methods, and biological tests, etc., can solve the problems of moxidectin residues, threats to human health, and stable and unreasonable use of the ecological environment. Even abuse and other problems, to achieve the effect of high sensitivity, good application prospects, and high practical value

Active Publication Date: 2017-08-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] While promoting the development of animal husbandry, due to unreasonable use or even abuse, moxidectin residues in animal foods and even the emergence of drug-resistant insects seriously threaten human health and the stability of the ecological environment.

Method used

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  • Polyclonal antibody for detecting moxidectin as well as preparation method and application thereof
  • Polyclonal antibody for detecting moxidectin as well as preparation method and application thereof
  • Polyclonal antibody for detecting moxidectin as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Synthesis and identification of embodiment 1, hapten

[0068] 1. Synthesis of haptens

[0069] 1. Take a 50mL Erlenmeyer flask, add 1.012g moxidectin, 20mL anhydrous pyridine and 0.5g succinic anhydride in sequence, seal the bottle mouth with a parafilm and place it at 48°C, 240rpm, in the dark for 9 hours.

[0070] 2. After step 1 is completed, pyridine is removed by rotary evaporation to obtain the product.

[0071] 3. Dissolve the product of step 2 in 50mL of ethyl acetate, then add 15mL of 1g / 100mL sodium hydroxide aqueous solution, mix thoroughly and let stand for stratification, discard the organic phase, collect the water phase, and adjust the pH to 4-5.

[0072] 4. Take the liquid phase obtained in step 3 and extract it three times with ethyl acetate (each time adding ethyl acetate equal to the volume of the liquid, and collect the organic phase for each extraction), and combine the organic phases.

[0073] 5. Take the organic phase obtained in step 4, add ex...

Embodiment 2

[0084] Embodiment 2, preparation of immunogen and coated antigen (activated ester method)

[0085] The conjugate of hapten and BSA was named MXD-BSA as the immunogen. The conjugate of hapten and OVA was named as MXD-OVA as the coating source.

[0086] 1. Preparation of immunogen

[0087] 1. Weigh 20 mg of hapten and dissolve it in 1 mL of DMF to obtain hapten solution.

[0088] 2. Take the hapten solution prepared in step 1, add 20 mg EDC and 20 mg NHS, place it on a magnetic stirrer, and react at room temperature at 400 rpm for 2 hours.

[0089] 3. Take 50 mg of BSA and dissolve it in 3 mL of PBS buffer solution containing 10% (volume percentage) DMF to obtain a protein solution.

[0090] 4. Add the liquid phase completed in step 2 dropwise to the protein solution prepared in step 3, then transfer to a dialysis bag with a molecular weight cut-off of 7KDa, and then place the dialysis bag in PBS buffer solution for dialysis at 4°C for 72h (every 12 Change the solution every...

Embodiment 3

[0098] Embodiment 3, the preparation of polyclonal antibody

[0099] The amount of MXD-BSA added is based on the total protein amount, that is, the total protein concentration of the MXD-BSA solution is detected by the Bradford method, and the total protein concentration multiplied by the added volume is the added amount of MXD-BSA.

[0100] 1. Preparation of polyclonal antibodies

[0101] Take 2 New Zealand white rabbits, numbered MXD-BSA-3 and MXD-BSA-5 respectively. The preparation method of the first immunization preparation: take the MXD-BSA solution prepared in Example 2, dilute it with PBS buffer, then mix and emulsify it with an equal volume of Freund's complete adjuvant. The preparation method of the booster preparation: take the MXD-BSA solution prepared in Example 2, dilute it with PBS buffer, then mix and emulsify it with an equal volume of Freund's incomplete adjuvant.

[0102] Carry out eight times of immunization to New Zealand white rabbits, the specific step...

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Abstract

The invention discloses a polyclonal antibody for detecting moxidectin as well as a preparation method and an application thereof. Firstly, a compound (a hapten) is protected, and a structural formula is shown in a formula (I). The invention also provides a conjugate obtained by conjugation of the compound shown in the formula (I) and a carrier protein. The invention also provides an antibody obtained by using the conjugate as an immunogen. The antibody is concretely a polyclonal antibody. The invention also provides a kit for detecting moxidectin, and the kit comprises the polyclonal antibody. The structure of moxidectin is reconstructed for the first time, the hapten is obtained, the polyclonal antibody of moxidectin is prepared, and home and abroad blanks are filled. The conjugate of the hapten and the carrier protein is used for preparing the moxidectin antibody, and the preparation process has the advantages of simplicity, economy, high sensitivity and high practical value. The product has good application prospects in the residual detection of veterinary drug.

Description

technical field [0001] The invention relates to a polyclonal antibody for detecting moxidectin and its preparation method and application. Background technique [0002] Moxidectin (Moxidectin, MXD) is a semi-synthetic new macrolide drug produced by fermentation of Streptomyces. Moxidectin has broad-spectrum anthelmintic activity and is highly active against nematodes and arthropod parasites in dogs, cattle, sheep, and horses. Due to its high anthelmintic activity and long duration of efficacy, moxidectin is used more and more widely at home and abroad, and has a tendency to replace abamectin. [0003] While promoting the development of animal husbandry, due to unreasonable use or even abuse, moxidectin residues in animal foods and even the emergence of drug-resistant insects seriously threaten human health and the stability of the ecological environment. [0004] In 1990, EU 2377 / 90 stipulated that the maximum content of moxidectin in animal (cattle, sheep, horse) liver wa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D493/22C07K14/765C07K14/77C07K1/107C07K16/44C07K16/06G01N33/53
CPCC07D493/22C07K14/765C07K14/77C07K16/44G01N33/53G01N2333/36
Inventor 王战辉沈建忠李红芳张素霞史为民温凯杨慧娟
Owner CHINA AGRI UNIV
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