Method and application for preparing complete antigen from trimethoprim hapten t2

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

Active Publication Date: 2016-04-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 and application for preparing complete antigen from trimethoprim hapten t2
  • Method and application for preparing complete antigen from trimethoprim hapten t2
  • Method and application for preparing complete antigen from trimethoprim hapten t2

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Embodiment 1

[0030] The preparation of embodiment 1 trimethoprim hapten T2

[0031] Add 10 mL of pyridine, 3.2 g of p-aminobenzoic acid, and 5 mg of 4-dimethylaminopyridine in sequence into a 100 mL 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 at 0.092 MPa to evaporate all the solvent to obtain a yellow oil. Add 10% (m / v) NaHCO dropwise 3 When the solution is no longer turbid, filter and adjust the supernatant to recrystallize under acidic pH 5.0 to precipitate a white solid, centrifuge at 7500r / min for 8min, and dry the filter cake at 60°C to obtain trimethoprim hapten T2.

Embodiment 2

[0032] The preparation of embodiment 2 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). The sodium nitrite solution was blue-black when detected by starch-potassium iodide test paper, and the stirring reaction was continued for 2 hours to obtain 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, where the dissolved protein concentration is greater than 3mg / mL, and carbonate buffer The ratio of solution to DMF is 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 hours, 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-Z...

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 at 4°C for 2 hours, and then react at room temperature at 25°C 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 carbonate buffer The volume ratio of liquid 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 Patents(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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