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Method for detecting dihydroartemisinin, and used hapten, antigen, antiserum and antibody

A dihydroartemisinin and detection method technology, applied in the biological field, can solve problems such as high technical requirements for operation, long operation time, and poor reaction rate, and achieve the effects of low cross-reaction rate, high sensitivity, and low cost

Inactive Publication Date: 2017-11-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods require expensive instruments and equipment, high detection costs, long operation time, high operational technical requirements and cannot be used for on-site detection
In addition, the predecessors have also produced antibodies corresponding to dihydroartemisinin, but the sensitivity is generally 100-500ng / mL. At the same time, there is a high and poor reaction rate to other derivatives of artemisinin such as artesunate, so it cannot be done to the specific detection of dihydroartemisinin

Method used

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  • Method for detecting dihydroartemisinin, and used hapten, antigen, antiserum and antibody
  • Method for detecting dihydroartemisinin, and used hapten, antigen, antiserum and antibody
  • Method for detecting dihydroartemisinin, and used hapten, antigen, antiserum and antibody

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1, the preparation of hapten

[0047] 1. Preparation of compounds

[0048] 1, the preparation of the compound shown in formula (Ⅶ)

[0049] The preparation method of compound shown in formula (VII) is as follows:

[0050] Materials: Cunninghamella elegans (Cunninghamella elegans, ATCC9245, American Culture Collection), PDA medium (Beijing Aoboxing Biotechnology Co., Ltd.), silica gel column (Qingdao Ocean Chemical Co., Ltd.).

[0051] Inoculate silvery mildew (ATCC9245) in PDA medium at 28°C and 160rpm for 3 days, then inoculate in fermentation medium (20g glucose, 15g sucrose, 10g peptone, 1L distilled water) and culture at 28°C at 160rpm. One day later, artemisinin was added to make the concentration 1 mg / mL, and cultured continuously for 14 days under the same conditions to obtain a fermentation broth.

[0052] Centrifuge the fermentation broth, discard the precipitated bacteria, collect the supernatant, extract the supernatant with ethyl acetate three ...

Embodiment 2

[0064] Embodiment 2, the preparation of antigen

[0065] 1. Preparation of dihydroartemisinin-ovalbumin conjugates

[0066] 1. Take 0.036 mmol of the compound represented by formula (II), 0.047 mmol of NHS and 0.040 mmol of EDC, dissolve it in 1 mL of DMSO, stir and react at 25°C for 6 hours to obtain the compound represented by formula (Ⅷ).

[0067]

[0068]

[0069] 2. Take the entire reaction solution after completing step 1 (according to the compound shown in formula (Ⅷ) has been generated according to all the compounds shown in formula (II), the reaction solution contains 0.036mmol of the compound shown in formula (Ⅷ)), slowly add dropwise In the carrier protein solution (the carrier protein solution is obtained by dissolving 0.0024mmol OVA in 5mL pH7.5, 0.1M PBS buffer), stir at 4°C for 12 hours.

[0070] 3, get the whole reaction solution that finishes step 2 (according to all carrier proteins are all coupled with the compound shown in formula (Ⅷ)), dialyze with ...

Embodiment 3

[0075] Example 3, Preparation of Antibody and Its Application in Detection of Dihydroartemisinin

[0076] 1. Using dihydroartemisinin-BSA as an antigen to prepare antibodies

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Abstract

The invention discloses a method for detecting dihydroartemisinin and the used hapten, antigen, antiserum and antibody. The detection method of dihydroartemisinin provided by the present invention comprises the use of antiserum or antibody to realize the detection of dihydroartemisinin; the antiserum and antibody are the conjugates of the compound represented by formula (II) and carrier protein respectively The antiserum and antibody prepared as immunogen; the carrier protein is bovine serum albumin or ovalbumin; the detection method of dihydroartemisinin is carried out by enzyme-linked immunosorbent method. Experiments have proved that the compound represented by the formula (II) of the present invention can be used as a hapten, and the antigen obtained after coupling with the carrier protein can be used to immunize animals, and the antibody specific for the dihydroartemisinin antigen can be obtained, and the sensitivity is high. The detection IC50 value of artemisinin can reach 1.16ng / mL.

Description

technical field [0001] The invention relates to a method for detecting dihydroartemisinin and the used hapten, antigen, antiserum and antibody in the field of biotechnology. Background technique [0002] Artemisinin derivatives such as artesunate, artemether, and dihydroartemisinin have significant curative effects on the treatment of cerebral malaria and chloroquine-resistant falciparum malaria with high efficiency, low toxicity, quick action, and low relapse rate. Dihydroartemisinin (DHA) is an antimalarial drug widely used in Southeast Asia and Africa. [0003] Counterfeit, inferior and substandard artemisinin drugs have posed a major threat to the prevention and control of malaria, and this phenomenon of counterfeiting is particularly prominent in backward regions such as Southeast Asia and Africa. Therefore, it is urgent to establish a fast and reliable qualitative and quantitative analysis method for artemisinin and its derivatives. [0004] The chemical name of dihy...

Claims

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Application Information

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IPC IPC(8): G01N33/543C07K14/765C07K14/77C07K1/107C07K16/44C07K16/06
CPCG01N33/94C07K1/1077C07K14/765C07K14/77C07K16/06C07K16/44C07K19/00G01N33/54353
Inventor 王保民宁香雪王冕谭桂玉
Owner CHINA AGRI UNIV
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