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Hydrochlorothiazide semi-antigen and complete antigen as well as preparation method thereof

A technology of hydrochlorothiazide and complete antigen, applied in chemical instruments and methods, animal/human protein, serum albumin, etc., can solve the problems of high detection cost, long cycle, complicated preparation, etc., and achieve high sensitivity and high specificity

Inactive Publication Date: 2015-03-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The instrumental analysis method has the disadvantages that the sample must be diluted, filtered, and extracted in multiple steps, and the preparation is complicated and cumbersome.
Although the instrument method is the confirmatory method for the detection of hydrochlorothiazide, due to its cumbersome operation and long sample pretreatment process, the detection cost is high and the cycle is long, which cannot meet the requirements of rapid screening of large batches of samples and rapid on-site detection

Method used

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  • Hydrochlorothiazide semi-antigen and complete antigen as well as preparation method thereof

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Effect test

Embodiment 1

[0028] The preparation of embodiment 1 hydrochlorothiazide hapten

[0029] Hydrochlorothiazide 71mg (about 0.24mmol), dissolved in 20mL dimethylformamide (DMF), add 66mg potassium carbonate (about 0.48mmol), mix well, then add 107μL 6-bromohexanoic acid ethyl ester (about 0.48mmol) 60℃ Reflux for 12 hours, add 20 mL of ultrapure water and 20 mL of ethyl acetate, and separate the organic phase. Ethyl acetate was removed with a rotary evaporator to obtain the hapten of hydrochlorothiazide.

Embodiment 2

[0030] Example 2 Preparation of Hydrochlorothiazide Complete Antigen

[0031] Take 15 mg of hydrochlorothiazide hapten, add 2 mL of DMF to dissolve, then add NHS and EDC respectively (the molar ratio of hydrochlorothiazide hapten, NHS, and EDC is 1:1.5:1.5), stir and react at 4°C for 6 hours, and then react at room temperature 12h. Take 30mg of bovine serum albumin (the molar ratio of hydrochlorothiazide hapten to bovine serum albumin is 80:1), add 6mL of 0.1M pH9.6 carbonate buffer to dissolve, and the dissolved protein concentration is greater than 3mg / mL, and the carbonic acid The volume ratio of salt buffer solution to DMF is 5:1. The activated hydrochlorothiazide hapten solution was slowly added dropwise to the carrier protein solution, and reacted at room temperature for 24 hours. Dialyze with PBS buffer for 2 days, during which the water is changed 4 times, and the complete antigen of hydrochlorothiazide can be obtained.

Embodiment 3

[0032] Example 3 Preparation of Hydrochlorothiazide Complete Antigen

[0033] Take 15 mg of hydrochlorothiazide hapten, add 2 mL of DMF to dissolve, and pre-cool at 0°C for 30 min. At 0°C, add tri-n-butylamine and isobutyl chloroformate (the molar ratio of hydrochlorothiazide hapten, tri-n-butylamine, and isobutyl chloroformate is 1:2:2), and stir for 1 hour at 0°C. Take 30mg of bovine serum albumin (the molar ratio of hydrochlorothiazide hapten to bovine serum albumin is 80:1), add 6mL of 0.1M pH7.4 phosphate buffer to dissolve, pre-cool at 0°C for 30min, and the dissolved protein concentration is greater than 3mg / mL. At 0°C, the activated hydrochlorothiazide hapten solution was slowly added dropwise to the carrier protein solution, reacted at 0°C for 1 hour, and then reacted at room temperature for 24 hours. Dialyze with PBS buffer for 2 days, during which the water is changed 4 times, and the complete antigen of hydrochlorothiazide can be obtained.

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Abstract

The invention discloses hydrochlorothiazide semi-antigen and complete antigen as well as a preparation method thereof, and belongs to the technical field of biochemical industry. The hydrochlorothiazide semi-antigen is obtained by dissolving hydrochlorothiazide in methanol, adding potassium carbonate, then reacting with 6-bromine ethyl caproate, and performing extractive separation. The hydrochlorothiazide complete antigen is obtained by coupling carboxyl groups on the hydrochlorothiazide semi-antigen with amino groups on carrier protein. Experimental results show that the antiserum titer obtained by using the hydrochlorothiazide complete antigen disclosed by the invention to immunize animals can reach 81000, the limit of detection is 0.01ng / mL, and the half inhibiting concentration is 0.1ng / mL. Generated antibodies are high in specificity and sensitivity. The antigens or the antibodies disclosed by the invention have a wide application prospect.

Description

technical field [0001] The invention relates to a hydrochlorothiazide hapten and a complete antigen and a preparation method thereof, belonging to the technical field of biochemical industry. Background technique [0002] Hydrochlorothiazide is a diuretic and an antihypertensive drug, and its structure is shown in chemical formula Ⅰ. Mainly applicable to cardiogenic edema, hepatic edema and renal edema: such as nephrotic syndrome, acute glomerulonephritis, chronic renal failure and edema caused by excessive adrenal cortex hormone and estrogen; hypertension; diabetes insipidus disease. Illegal addition of hydrochlorothiazide in antihypertensive drugs and health foods can lead to uncontrollable usage and dosage of chemicals, which may easily cause adverse reactions caused by drug abuse, and even cause drug injury. The state explicitly prohibits the addition of chemical ingredients in Chinese patent medicines and health foods . Taking it without the patient's knowledge will ...

Claims

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Application Information

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IPC IPC(8): C07D285/28C07K14/765C07K14/795C07K14/77G01N33/577G01N33/543
CPCC07D285/28C07K14/765C07K14/77C07K14/795G01N33/543G01N33/577
Inventor 匡华彭双胥传来徐丽广马伟刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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