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2, 4, 6-trichlorophen artificial antigen, and its preparing method and use

A technology of artificial antigen and trichlorophenol, which is applied in the field of fluorescence immunoassay and achieves the effects of strong practicability, good antibody stability, and simple and feasible preparation technology.

Inactive Publication Date: 2006-05-24
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Modern preparation technology has obtained 2,4,6-trichlorophenol of higher purity (>99%) [Grassacyl ether intermediate 2,4,6-trichlorophenol preparation technology, fine chemical intermediates, 1980, 2: 296], but there are few reports on the immunoassay of 2,4,6-trichlorophenol at home and abroad. Roger Glave et al obtained anti-2,4,6-trichlorophenol antiserum by immunizing rabbits, and used enzyme Linked immunoassay (ELISA) optimizes the detection conditions, but has not been applied to specific environmental detection [Roger Glave, Franciso Sanchez-Baeze, Franciso Camps, M.Pilar Marco, Indirect competitive immunoassay for trichlorophenol determination rationalevaluation of the competitor heterology effect , Analytica Chimica Acta, 2002, 452:191-206] [Patricia Noguera, a Angel Maquieira, a Rosa Puchades, * a Ernesto Brunet, bMa Mar Carramolinob and Juan Carlos Rodri guez-Ubis.Development of anenzyme-linked immunosorbent assay for screening contamination by chlorophenols inenvironmental waters.J.Environ.Monit., 2002(4):442-448]
However, there is no report on the fluorescence immunoassay (FIA) both at home and abroad.

Method used

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  • 2, 4, 6-trichlorophen artificial antigen, and its preparing method and use
  • 2, 4, 6-trichlorophen artificial antigen, and its preparing method and use
  • 2, 4, 6-trichlorophen artificial antigen, and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Synthesis and identification of 2,4,6-trichlorophenol hapten

[0050] 1 Synthesis of 2,4,6-trichlorophenol hapten

[0051] Dissolve 4.5g (47.6mmol) of chloroacetic acid in 7.5ml of water, slowly add 2.55g of sodium carbonate powder under stirring, and the pH of the solution is 7.5. Take 6.7g (38.1mmol) of 2,4,6-trichlorophenol, 10ml of water, and 5ml of 25% sodium hydroxide, and dissolve them completely. The pH of the solution is 8, and then add the above-mentioned sodium chloroacetate solution under stirring, and keep the pH at 7~ 8 (adjusted with dilute alkali), add 0.08g of catalyst polyethylene glycol-6000, continue to heat the reaction for 2h, while paying attention to controlling the pH of the reaction solution to 9~10 (adjusted with dilute alkali), keep warm at 80~90°C for 3h, the reaction is completed, Add a small amount of activated carbon for decolorization, boil for 10 minutes, filter while hot, adjust the pH of the filtrate to 1 with hyd...

Embodiment 2

[0054] Example 2 Synthesis and Identification of 2,4,6-Trichlorophenol Artificial Antigen

[0055] The hapten 2, 4, 6-trichlorophenol phenoxyacetic acid is connected to BSA and OVA by the activated ester method and the mixed anhydride method respectively, to synthesize artificial antigens (immunogen and coating former). The specific methods are:

[0056] 2.1 Synthesis of immunogen

[0057] Weigh 0.2mmol 51mg hapten 2,4,6-trichlorophenol phenoxyacetic acid and dissolve in 200μl DMF, dissolve 19.4mg NHS and 34.2mg DCC in 300μl DMF, add dropwise to the hapten solution, stir at room temperature React for 8 hours, overnight at 4°C, and centrifuge to separate the supernatant. 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 distilled water for 2 days, and dialyzed with pH7.0 ph...

Embodiment 3

[0064] Example 3 Preparation and purification of 2,4,6-trichlorophenol antibody

[0065] 3.1 Preparation of antiserum

[0066] Select two adult male New Zealand white rabbits from each group (number 1 and II), 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 Jiao Tong University).

[0067] Slowly thaw the immunogen, dilute it to 1 mg / ml with 0.9% normal saline (or phosphate buffer solution), inhale it into a 5ml sterile syringe according to the immune dose, add an equal amount of complete Freund's adjuvant (CFA) for the first immunization, and use poly The vinyl fluoride plastic tube is connected to another 5ml syringe, and pushed until it is fully emulsified to form a water-in-oil (W / O) state, and the immunization is injected according to the immunization plan.

[0068] For the first immunization, the immunogen emulsif...

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Abstract

The invention relates to a preparation method and use for 2, 4, 6-tri-chlorophenol artificial antigen. It is synthesized by hapten 2, 4, 6-trichlorobenzende oxygen acetic acid through activating fat or mixing acid anhydride. The antibody can be used to make 2, 4, 6-tri-chlorophenol antibody, set 2, 4, 6-tri-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 relates to a 2,4,6-trichlorophenol artificial antigen, a preparation method and an application thereof, and belongs to the field of immunochemistry and residual analysis biotechnology of small molecular organic pollutants. It involves organic synthesis, immunochemistry, biochemistry and physical and chemical testing techniques, etc. In particular, it involves the design and synthesis of 2, 4, 6-trichlorophenol hapten and artificial whole antigen of environmental hormones, and the preparation method of specific antibody for immunized animals, and the establishment of methods that can be applied to the detection of 2, 4, 6 residues in water and soil - Fluorescence immunoassay technique for trichlorophene. 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 n...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07C231/02C07C235/34C07K14/77C07K16/00G01N33/533
Inventor 余宇燕庄惠生
Owner DONGHUA UNIV
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