Synthetic method of general artificial antigen of phthalate plasticizers for immunodetection

A phthalate and phthalic acid technology, applied in the field of biochemical industry, can solve the problems that antibodies cannot be universally detected by phthalates, and the cross-reaction rate is small, and achieves good antibody versatility and sensitivity. high effect

Active Publication Date: 2013-07-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current common method is to introduce active amino groups on the benzene ring of phthalates and then couple them with carrier proteins to expose the side chain groups of phthalates as antigenic determinants. After the antigen is immunized, it only has a good inhibitory effect on specific phthalates, and the cross-reaction rate is small. The obtained antibodies cannot be used for the general detection of phthalates

Method used

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  • Synthetic method of general artificial antigen of phthalate plasticizers for immunodetection
  • Synthetic method of general artificial antigen of phthalate plasticizers for immunodetection
  • Synthetic method of general artificial antigen of phthalate plasticizers for immunodetection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Preparation of phthalate plasticizer universal hapten

[0032] Take 0.5g of phthalic acid, 1mL of 6-(fluorenylmethoxycarbonyl-amino)-1-hexanol, 10μL of tetrabutyl titanate, 10mg of dicyclohexylcarbodiimide (DCC), react at 95°C for 12h, add 1M NaOH aqueous solution, neutralize the reaction solution to pH 7.4-8, discard the aqueous phase after separation, wash the organic phase once more, spin evaporate at 35°C under reduced pressure, and dealcoholize. Add 10 mL of N,N-dimethylformamide (DMF) solution containing 20% ​​piperidine to the product, react at room temperature for 20 minutes, remove the fluorenyl moxycarbonyl protecting group, and obtain a phthalate plasticizer Universal hapten.

Embodiment 2

[0033] Embodiment 2, the preparation of phthalate ester plasticizer universal artificial antigen

[0034] Take 25mg (0.0686mmol) phthalate plasticizer universal hapten, add 2mL N,N-dimethylformamide (DMF) to dissolve, add 0.5mL glutaraldehyde solution with a mass concentration of 2.5% at 4°C (0.1mmol), keep away from light and mix well, stir and react for 1h, take 58.3mg (0.000857mmol) bovine serum albumin (the molar ratio of hapten and bovine serum albumin is 80:1), add 10mL0.1MpH9.6 carbonic acid Dissolve in salt buffer solution, wherein the concentration of dissolved bovine serum albumin is greater than 3mg / mL, and the volume ratio of carbonate buffer solution to N,N-dimethylformamide (DMF) is 5:1. At 0°C, the activated hapten solution was slowly added dropwise to the bovine serum albumin solution, reacted for 1 hour at 0°C, and reacted for 6 hours at room temperature. Dialyze with PBS buffer solution for 2 days, during which the PBS buffer solution is changed 4 times to o...

Embodiment 3

[0035] Embodiment 3, preparation of phthalate plasticizer antiserum

[0036] The general artificial antigen of phthalate plasticizers obtained in Example 2 was used as the immunogen, and female BALB / C mice aged 6-8 weeks were used as immunized animals, and Freund's adjuvant was used for immunization. 5 rats. The Freund's adjuvant immunization method is as follows: Firstly, take an appropriate amount of immunogen and mix it with an equal volume of Freund's complete adjuvant, emulsify it, and inject it subcutaneously at multiple points on the back of the neck, and boost the immunization every 3 weeks.

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Abstract

A synthetic method of a general artificial antigen of phthalate plasticizers for immunodetection belongs to the field of bio-chemical engineering technology. The synthetic method of the present invention comprises the following steps of carrying out an esterification reaction between phthalic acid and 6-(fluorenyl methoxy carbonyl acyl-amino)-1-hexanol, removing fluorene methoxy carbonyl acyl protecting groups to form a hapten, and coupling with amino from carrier protein to obtain the general artificial antigen of phthalate plasticizers. Experimental results disclose that titer of antiserum obtained by immunizing animals by the antigen of the invention is up to 160000;the 50% inhibiting concentration IC50 for DBP, DEHP and DINP is less than 500 ng / ml; and the generated antibodies have good generality and high sensitivity. The antigen or antibodies of the invention can be used for the establishment of enzyme-linked immunosorbent assay and colloidal gold test paper rapid detection methods, thus can be used for rapid detection of residues of phthalic acid ester plasticizers in food, and have broad application prospects.

Description

technical field [0001] The invention discloses a method for synthesizing a general artificial antigen of a phthalate plasticizer for immunodetection, which belongs to the technical field of biochemistry. Background technique [0002] A plasticizer is an additive that increases the softness of a material or liquefies a material. The most common plasticizers are Phthalate Esters (PAEs), which are esterified derivatives of phthalic acid. [0003] Phthalate plasticizers are colorless liquids with a little aromatic smell or no smell, medium viscosity, high stability, low volatility, low cost, low water solubility, but easily soluble in most organic solvents. Phthalates are widely used in daily life and industry, with di(2-ethylhexyl) phthalate (DEHP) as the largest, accounting for three-quarters of plasticizer production, followed by ortho Dibutyl phthalate (DBP). Phthalate ester plasticizers are classified as suspected environmental hormones. Their biological toxicity is main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K14/795C07K16/44C07C219/14C07C213/02G01N33/577G01N33/02
CPCY02P20/55
Inventor 胥传来刘丽强王利兵匡华徐利广马伟胡拥明
Owner JIANGNAN UNIV
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