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Method for preparing complete antigen from trimethoprim hapten T2 and application of complete antigen

A technology of trimethoprim and complete antigen, which is applied in the field of preparing complete antigen from trimethoprim hapten T2, and achieves the effects of high specificity and high sensitivity

Active Publication Date: 2015-02-11
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 T2 for 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 hapten T2 and application of complete antigen
  • Method for preparing complete antigen from trimethoprim hapten T2 and application of complete antigen
  • Method for preparing complete antigen from trimethoprim hapten T2 and application of complete antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation of Trimethoprim Hapten T2

[0031] Add 10mL of pyridine, 3.2g of p-aminobenzoic acid, 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. Dissolve 2.9g of trimethoprim in 10mL of pyridine and add it dropwise into a three-neck flask, continue to stir for 3 days, and use thin-layer chromatography with a volume ratio of methanol: tetrahydrofuran: chloroform of 1:1:5 as the developer Monitor the progress of the reaction. Distill at 60°C for 10 minutes under reduced pressure of 0.092 MPa to evaporate all the solvent to obtain a yellow oil. Add 10% (m / v) NaHCO dropwise 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 7500 r / min for 8 min, and the filter cake was dried at 60°C to obtain the trimethoprim hapten T2.

Embodiment 2

[0032] Example 2 Preparation of Trimethoprim Complete Antigen T2-ZD-BSA

[0033] Take 8 mg of trimethoprim hapten T2, dissolve it in 1 mL of DMF and mix it with 1N hydrochloric acid (the molar ratio of T2 and hydrochloric acid is 1:2.5). % sodium nitrite solution, with starch-potassium iodide test paper, it was blue-black, and the stirring reaction was continued for 2 hours to obtain the activated trimethoprim hapten T2 solution. Take BSA (the molar ratio of hapten T2 to BSA is 50:1 and 100:1, respectively), and dissolve it in 0.1M pH9.6 carbonate buffer solution, wherein the dissolved protein concentration is greater than 3mg / mL, and the carbonate The ratio of buffer to DMF was 5:1. Slowly add 14.5 mg / mL activated trimethoprim hapten T2 solution dropwise to 22 mg / mL BSA solution 3 mL, react at room temperature for 12 h, dialyze with PBS buffer for 3 days, change the water 8 times during the period, and obtain 50 ︰1 and 100︰1 two complete trimethoprim antigens (T2-ZD-BSA). ...

Embodiment 3

[0034]Example 3 Preparation of immunogen (T2-GA-BSA) and coating agent (T2-GA-OVA)

[0035] Take 8 mg of trimethoprim hapten T2, dissolve it in 1 mL of DMF, react with glutaraldehyde (the molar ratio of T2:glutaraldehyde is 1:1.5), stir and react for 2 h at 4 °C, and then react at room temperature at 25 °C Reaction 12h. Take BSA / OVA (the molar ratio of hapten T2 to BSA and OVA is 100:1) and dissolve it in 0.1M pH9.6 carbonate buffer, where the dissolved protein concentration is greater than 3mg / mL, and the carbonate The volume ratio of buffer to DMF is 5:1. Slowly add 14.5 mg / mL activated trimethoprim hapten T2 solution dropwise to 3 mL of protein 22 mg / mL BSA / OVA solution, react at room temperature for 24 hours, dialyze with PBS buffer for 3 days, change the water 8 times during the period, That is, trimethoprim immunogen (T2-GA-BSA) and coating agent (T2-GA-OVA) were obtained.

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Abstract

The invention relates to a method for preparing a complete antigen from a trimethoprim hapten T2 and application of the complete antigen, belonging to the technical field of biochemical industry. The method comprises the following steps: deriving active carboxyl group by utilizing amino group on one end of pyrimidine nucleus, purifying to obtain a single specific-structure trimethoprim carboxyl derivative (TMP-HS) which is trimethoprim hapten T2, and coupling the hapten T2 with a carrier protein to form the trimethoprim complete antigen. The antigen or antibody can be used for establishing an enzyme linked immunosorbent assay analysis method or colloidal gold test paper quick detection method, thereby being used for quickly detecting trimethoprim residues in food.

Description

technical field [0001] The invention relates to a method and application for preparing a complete antigen from a trimethoprim hapten T2, 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 animal f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D239/49C07K14/765C07K14/77C07K14/795C07K1/107C07K16/44G01N33/531
CPCC07D239/49C07K1/1077C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/531
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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