2, 4-dichlorophen artificial antigen, and its preparing method and use

A technology of artificial antigen and dichlorophenol, which is applied in the preparation, fluorescence immunoassay, 2, can solve the problems of unseen application environment detection, etc., and achieve the effect of simple and feasible preparation technology, strong practicability and good antibody stability

Inactive Publication Date: 2007-05-23
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hiroyuki Tanaka et al. obtained antibodies capable of simultaneously detecting 2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid through ELISA, but they were not used in specific environmental detection [Hiroyuki Tanaka, Shayu Yan, Norio Miura and Yukihiro Shoyam! Preparation of anti-2, 4-dichlorophenol and2, 4-dichlorophenoxyacetic acid monoclonal antibodies, Cytotechnology, Volume 42, Number 2, 2003: 101-107]
For the hapten prepared by the diazo method, the indirect competitive fluorescence immunoassay (FIA) detection of 2,4-dichlorophenol has not been reported at home and abroad

Method used

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  • 2, 4-dichlorophen artificial antigen, and its preparing method and use
  • 2, 4-dichlorophen artificial antigen, and its preparing method and use
  • 2, 4-dichlorophen artificial antigen, and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Synthesis and identification of 2,4-dichlorophenol hapten

[0053] Synthesis of 12,4-Dichlorophenol Hapten

[0054] 5.5mmol 0.75g of p-aminobenzoic acid was dissolved in 5ml of 5% sodium hydroxide, after cooling, 0.4g of sodium nitrite (dissolved in 3ml of water) was added dropwise in an ice bath, and 1.5ml of concentrated HCl (dissolved in 5ml of water) was added under magnetic stirring , keep the reaction temperature at 0-4°C for 30 minutes (until the KI-starch test paper turns blue, add a little urea)

[0055] Dissolve 1g of 2,4-dichlorophenol in 2ml of 10% NaOH, add the solution dropwise, and react for 20min. After standing overnight, a yellow-brown lumpy solid was obtained. Recrystallized with water and ethanol (1:1). Dry to obtain a solid powder, namely the hapten 2,4-dichloro-6-(4-carboxyphenylazo)phenol, with a yield of 84%.

[0056] Identification of 22,4-dichlorophenol hapten

[0057] Get the above-mentioned synthetic product throu...

Embodiment 2

[0058] Example 2 Synthesis and Identification of 2,4-Dichlorophenol Artificial Antigen

[0059] The hapten 2,4-dichloro-6-(4-carboxyphenylazo)phenol was linked to BSA and OVA by activated ester method and mixed anhydride method respectively to synthesize artificial antigen (immunogen and coating agent) , the specific method is:

[0060] 2.1 Synthesis of immunogen

[0061] Weigh 0.2mmol 62mg hapten 2,4-dichloro-6-(4-carboxyphenylazo)phenol and dissolve in 200μl DMF, dissolve 19.4mg NHS and 34.2mg DCC in 300μl DMF, add dropwise to the hapten solution , the reaction was stirred at room temperature for 4 h, overnight at 4 °C, and the supernatant was separated by centrifugation. The activated ester solution in the upper layer was added to 60mg BSA dissolved in 5ml carbonate buffer solution (0.05M, pH9.6), and reacted at 4°C for 4h. After the reaction was completed, the solution was put into a dialysis bag, dialyzed with 0.9% normal saline for 5 days, and the solution...

Embodiment 3

[0068] Example 3 Preparation and purification of 2,4-dichlorophenol antibody

[0069] 3.1 Preparation of antiserum

[0070] Select three adult male New Zealand white rabbits from each group (No. 1, II and III), weighing about 2-2.5Kg, and raise them in a standard experimental animal room. After one week of observation, they will be immunized (raised by the animal room of the Agricultural College of Shanghai Jiaotong University).

[0071]Slowly thaw the immunogen, dilute it to 1mg / ml with 0.9% normal saline (or PBS), inhale it into a 5ml sterile syringe according to the immunization dose, add an equal amount of complete Freund's adjuvant (CFA) for the first immunization, and use polyvinyl fluoride plastic Connect the tube to another 5ml syringe, push it until it is fully emulsified, and form a water-in-oil (W / O) state, and inject immunization according to the immunization plan.

[0072] For the first immunization, the immunogen emulsified with the same amount of Fr...

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Abstract

The invention relates to a preparation method and use for 2, 4-di-chlorophenol artificial antigen. It is synthesized by hapten 2, 4-dichlor-6-(4-hydroxyl phenyl azo) phenyl hydroxide through activating fat or admixing acid anhydride. The antigen can be used to make 2, 4-di-chlorophenol antibody, set 2, 4-di-chlorophenol indirect competing fluorescence immunity method, or testing its residuum in the aquatic environment. The advantages of the invention are that the antibody has great practicability and good stability; the preparation method is easy and feasible; the cost is low; and it is easy to produce in industrial scale.

Description

technical field [0001] The invention belongs to the field of immunochemistry and residue analysis biotechnology of small molecular organic pollutants. It involves organic synthesis, immunochemistry, biochemistry and physical and chemical testing techniques, etc. Specifically, it is a 2,4-dichlorophenol artificial antigen, its preparation method and its use. In particular, it involves the design and synthesis of 2,4-dichlorophenol hapten and artificial whole antigen of environmental hormones, the preparation method of specific antibody for immunized animals, and the establishment of fluorescence that can be applied to the detection of residual 2,4-dichlorophenol in water. Immunoassay techniques. Background technique [0002] Environmental hormones are hormone analogs in the environment, which can interfere with normal physiological metabolism, endocrine and reproductive functions by binding to hormone receptors, causing various negative biological effects. In the past 70 y...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765C07K14/77C07K16/00C07C245/08G01N33/533
Inventor 庄惠生余宇燕
Owner DONGHUA UNIV
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