Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen

A technology of trimethoprim and complete antigen, applied in the field of biochemical industry, to achieve the effect of high specificity and high sensitivity

Inactive Publication Date: 2015-02-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a method and application for preparing trimethoprim complete antigen from trimethoprim hapten compound T1 aiming at the deficiencies and defects of existing trimethoprim detection technology and antigen synthesis and corresponding antibody preparation

Method used

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  • Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen
  • Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen
  • Method for preparing complete antigen from trimethoprim semiantigen compound T1 and use of complete antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Preparation of Trimethoprim Hapten T1

[0027] Add 10mL of pyridine, 2g of succinic anhydride, and 5mg of 4-dimethylaminopyridine in sequence to a 100mL three-necked flask, and heat in a water bath at 60°C with magnetic stirring until the solution is clear. Then 2.9 g of trimethoprim was dissolved in 10 mL of pyridine and added dropwise to the three-necked flask, and the stirring reaction was continued for 3 days. TLC was used to monitor the progress of the reaction using methanol: chloroform with a volume ratio of 1:5 as the developing solvent. Distill at 50°C for 10 minutes under reduced pressure at 0.090 MPa to evaporate all the solvent to obtain a pale yellow oil. Add dropwise mass volume ratio 10% NaHCO 3 The solution was no longer turbid, filtered and the supernatant was recrystallized under acidic conditions of pH 5.0 to precipitate a white solid, centrifuged at 6000 r / min for 10 min, and the filter cake was dried at 50°C to obtain the trimethoprim ha...

Embodiment 2

[0028] Example 2 Preparation of Trimethoprim Complete Antigen

[0029] Take 8 mg of trimethoprim hapten T1, dissolve it in 1 mL of DMF, and mix it with tri-n-butylamine and isobutyl chloroformate (the molar ratio of T1: tri-n-butylamine: isobutyl chloroformate is 1: 1.2: 1.2) 0 The reaction was carried out at ℃ for 1 h to obtain the activated trimethoprim hapten compound T1 solution. Take BSA / OVA (the molar ratio of hapten T1 to BSA and OVA is 100:1), dissolve it in 0.01M pH6.5 phosphate buffer, pre-cool at 0°C for 30 minutes, and the dissolved protein concentration is greater than 3mg / mL , and the volume ratio of phosphate buffer saline to DMF was 5:1 to obtain a protein solution. At 0°C, the activated hapten T1 solution was slowly added dropwise to the protein solution, reacted at 0°C for 2 hours, and then reacted at room temperature for 12 hours. Dialyze with PBS buffer for 3 days, during which the water was changed 8 times to obtain the complete trimethoprim antigen (T1-...

Embodiment 3

[0030] Example 3 Preparation of Trimethoprim Antiserum

[0031] Using the antigen (T1-MA-BSA) prepared in Example 2 as the immunogen, 8-week-old female BALB / C mice were selected as immunized animals, and Freund's adjuvant was used for immunization, and 5 mice were immunized. 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; Inject immunization at multiple points, and boost immunization every 3 weeks.

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Abstract

The invention discloses a method for preparing a complete antigen from a trimethoprim semiantigen compound T1 and a use of the complete antigen, and belongs to the technical field of biochemical engineering. An amino group at one end of a pyrimidine nucleus is derived to form active carboxyl, a trimethoprim carboxyl derivative (TMP-HS) with a single specific structure is obtained by purification, the derivative is the trimethoprim semiantigen compound T1, and the trimethoprim semiantigen compound T1 is coupled to a carrier protein to form the trimethoprim complete antigen T1-MA-BSA. The antigen or antibody can be used for building an enzyme-linked immunosorbent assay adsorption analysis method and a colloidal gold test paper rapid-detection method so that trimethoprim residue in foods can be fast detected.

Description

technical field [0001] The invention relates to a method and application for preparing a complete antigen from a trimethoprim hapten compound T1, belonging to the technical field of biochemical engineering. Background technique [0002] Trimethoprim (TMP), also known as trimethoprim, and dimethoprim (Diaveridine, DVD) are dihydrofolate reductase inhibitors of diaminopyrimidines, white crystals Powder, insoluble in water, slightly soluble in chloroform, ethanol, acetone, but easily soluble in organic acids such as glacial acetic acid. Its antibacterial ability is weak, so it is often not used alone, but it can be used in combination with sulfonamides, furans, quinolones and other antibiotics, which can greatly enhance the application effect of antibiotics, and can treat chicken pullorum, fowl cholera, bacterial infections and other livestock and poultry disease. However, the overuse and irrational use of antibiotics and synergists have resulted in excessive drug residues in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D239/49C07K14/765C07K14/77C07K14/795C07K1/107C07K16/44G01N33/543
CPCC07D239/49C07K1/1077C07K14/765C07K14/77C07K14/795C07K16/06C07K16/44C07K19/00
Inventor 匡华徐乃丰胥传来徐丽广马伟刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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