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Chlordecone antigen and antibody and preparation method thereof

A technology of chlordecone and monohydroxy chlordecone, applied in the fields of antibodies and their preparation, and chlordecone antigens, can solve the problems of long sample pretreatment process, increasing the difficulty of separation and extraction, and instrumental analysis, etc.

Inactive Publication Date: 2011-09-21
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] For the detection and analysis techniques of chlordecone and mirex, etc., there are only instrumental analysis techniques at present, such as: high performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography Chromatography-Electron Capture Detector (GC-ECD) and Gas Chromatography-Secondary Mass Spectrometry (GC-MS-MS). However, instrumental analysis technology requires expensive and sophisticated instruments, specialized technicians, and very long In addition, there are many derivatives of mirex and chlordecone, and the derivatives have little difference in structure and polarity, and the derivative of chlordecone, hydroxychlordecone ( Chlordecone Hydrate ) and Cleaver ( Kelevan ) is easy to pyrolyze and reduce, so it increases the difficulty of separation, extraction and instrumental analysis to a large extent

Method used

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  • Chlordecone antigen and antibody and preparation method thereof
  • Chlordecone antigen and antibody and preparation method thereof
  • Chlordecone antigen and antibody and preparation method thereof

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Embodiment 1

[0035] Example 1: Preparation of Chlordecone Immunization Antigen and Coating Antigen

[0036] 1. Synthesis of hapten: Under the catalysis of sodium borohydride, chlordecone can be converted into hydroxyl-containing monohydroxychlordecone. The operation steps are as follows:

[0037] 1) Weigh 0.05g of chlordecone and dissolve it in n-propanol, dissolve 0.012g of sodium borohydride in n-propanol, and slowly add it dropwise to the n-propanol solution of chlordecone, and stir the reaction at 37°C 24h;

[0038] 2) Spin-dry n-propanol with a rotary evaporator, and the resulting substance is washed with 5ml of 10% H 2 SO 4 The solution was washed with water, and the resulting acidic solution was extracted with benzene;

[0039] 3) After the benzene liquid was evaporated to dryness, it was recrystallized with n-hexane, and the obtained crystal was monohydroxychlordecone, which was stored in a refrigerator at 4°C until use.

[0040] 2. Preparation of immune antigen and coated anti...

Embodiment 2

[0046] Embodiment two: the preparation of chlordecone antibody:

[0047] One, the chlordecone antigen that embodiment two obtains is prepared into PBS solution, and Freund's complete adjuvant ( CFA, Complete Freund's Adjuvant ) mixed in equal volume at 1:1; five 8-week-old female Balb / c mice were given subcutaneous multi-point immunization injections on the back (0.05mg / 0.5ml / mouse to 0.2mg / 0.5ml / mouse) immune complex and adjuvant After three weeks, the thigh muscle was intravenously immunized with the same dose of compound and Freund's incomplete adjuvant ( IFA, Incomplete Freund's Adjuvant) mixture; two weeks later, the thigh muscle vein was boosted again with the mixture of the same dose of compound and Freund's incomplete adjuvant, the tail was docked to take blood, and the serum titer of the mouse was determined, and the mouse with the highest titer was selected for cell fusion experiment; Three days before the fusion, the pure PBS solution of the immune complex was in...

Embodiment 3

[0052] 1. Coating: Dilute the coating antigen (chlordecone-bovine serum albumin complex) with 0.1M pH9.5 carbonate coating buffer to a protein content of 10 μg / ml. Add 0.1ml to the reaction well of each ELISA microplate plate, overnight at 4°C. The next day, the solution in the well was discarded and washed 3 times with PBST washing buffer. (referred to as washing, the same below).

[0053] 2. Blocking: add 0.3ml PBST+0.3%Gelatin to each well, and incubate at 37°C for 1 hour. Then wash.

[0054] 3. Adding samples: Add 0.1ml of the antiserum to be tested at a certain dilution to the above-mentioned coated reaction wells, and incubate at 37°C for 1 hour. Then wash. (Make blank wells, negative control wells and positive control wells at the same time).

[0055] 4. Add enzyme-labeled antibody: Add 0.1ml of fresh 1 / 4000 diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 0.5-1 hour and wash.

[0056] 5. Add substrate solution for color development: Ad...

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Abstract

The invention relates to a chlordecone antigen, a chlordecone antibody and a preparation method thereof. The antigen is an immune antigen obtained by connecting hydroxyl of single hydroxyl chlordecone with amino in keyhole limpet hemocyanin or bovine serum albumin through N,N-carbonyldiimidazole serving as a coupling agent; and hybridoma prepared from the antigen is subjected to specificity screening, in vitro culture and purification to form the antibody. By a characteristic of high-efficiency specificity competitive combination of the antigen and the antibody, the complicated pretreatment process is not needed, the whole detection process can be completed in 5-8 hours, and a detection threshold for detecting the chlordecone is 6.25 to 0.312mu g / ml. Compared with the conventional instrument detection, the invention is quicker and cheaper, and can be used for analytical detection, quantitative determination, quality standard and purification of chlordecone samples and blockage of bioeffect of the chlordecone. The materials are efficiently, quickly and cheaply detected and analyzed.

Description

technical field [0001] The invention relates to a chlordecone antigen, an antibody and a preparation method thereof. Background technique [0002] Chlordecone ( Chlordecone ) is the Stockholm Convention ( Stockholm Convention ) is a persistent organic pollutant that is generally banned ( POPs, Persistent Organic Pollutants ), they were widely used in agriculture as organochlorine pesticides in the 1950s and 1970s. Due to the high toxicity, high stability and bioaccumulation of these substances, chlordecone and its analogues have caused huge damage to the environment and crops. , and even threaten the normal living conditions of human beings. Due to the great harm of this substance, it is particularly urgent to improve the detection and monitoring of this substance and improve the detection method of this substance. [0003] For the detection and analysis techniques of chlordecone and mirex, etc., there are only instrumental analysis techniques at present, such as: high ...

Claims

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Application Information

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IPC IPC(8): C07K14/795C07K14/765C07K16/44
Inventor 邓湘波方法
Owner SHANGHAI UNIV
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