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6-methylmercaptopurine (6-MMP) immunogen and 6-(MMP) detection reagent

A detection reagent, methylmercapto technology, which is applied in the field of anti-6-purine specific antibody and 6-purine detection reagent, can solve the problems such as the complexity of 6-MMP method, and achieve the effect of short cycle, accurate detection result and high sensitivity

Active Publication Date: 2014-04-09
苏州博源医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention is to overcome the complex defect of the method for detecting 6-MMP existing in the prior art, and adopts the immunoassay reagent developed by 6-MMP specific antibody to make up for these shortcomings

Method used

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  • 6-methylmercaptopurine (6-MMP) immunogen and 6-(MMP) detection reagent
  • 6-methylmercaptopurine (6-MMP) immunogen and 6-(MMP) detection reagent
  • 6-methylmercaptopurine (6-MMP) immunogen and 6-(MMP) detection reagent

Examples

Experimental program
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Embodiment 1

[0046] Example 1 Synthesis of 6-MMP immunogen

[0047] 1. Synthesis of 6-MMP derivatives, the chemical structure of which is shown in formula (II).

[0048]

[0049] Formula (Ⅱ)

[0050] The synthetic approach and method of this 6-MMP derivative are as follows:

[0051]

[0052] (1) Dissolve 5.0g 6-MMP and 4.45g K with 30ml DMF 2 CO 3 , add 10ml of DMF solution containing 4.06g of ethyl 6-bromohexanoate, and stir the reaction at 40°C for 30min.

[0053] (2) The reacted solution was then neutralized with 1N HCl solution, and extracted with ethyl acetate. The organic phase was dried, filtered, dried in vacuo and purified on normal phase silica gel. Dissolve 1.5 g of the purified product with 25 ml of methanol, and then add 600 mg of LiOH.H 2 O aqueous solution, stirred at room temperature for 4 hours. After concentration, the product was diluted with water and washed with ether, and the pH of the aqueous phase solution was adjusted to 5.0 with 1N HCl solution. Fina...

Embodiment 2

[0058] Example 2 Preparation of anti-6-MMP specific antibody

[0059] (1) Dilute the synthesized 6-MMP immunogen to 1.5 mg / ml with PBS to obtain an antigen solution, then mix the antigen solution with Freund's complete adjuvant, and inject it into rabbits;

[0060] (2) After 2 to 3 weeks, inject the rabbit once with 1.0ml of the same antigen solution mixed with Freund's incomplete adjuvant, and then once every four weeks, twice in total, and extract the antiserum of the rabbit to obtain effective antibodies.

Embodiment 3

[0061] The preparation of embodiment 3 6-MMP detection reagent, this detection reagent:

[0062] 1. Contain the anti-6-MMP specific antibody in the above-mentioned embodiment 2

[0063] 2. Containing 6-MMP enzyme-labeled conjugates, taking HRP as an example, the preparation method is as follows:

[0064] (1) Weigh 20mg of HRP and dissolve it in 5ml of 0.2M, pH 8.5 phosphate buffer at room temperature;

[0065] (2) Activation of 6-MMP derivatives: Weigh 10 mg of 6-MMP derivatives into a small beaker, and add 350 μL of DMF, 350 μL of absolute ethanol, 700 μL of 10 mM potassium phosphate buffer at pH 5.0, 40 mg of EDAC and 5 mg of Sulfo-NHS, stirred and reacted at room temperature for 30 minutes;

[0066] (3) Add the activated 6-MMP derivative dropwise to the HRP solution, stir overnight at 2-8° C., and dialyze the coupled antigen to obtain the HRP-6-MMP conjugate.

[0067] 3. Containing 3,3',5,5'-tetramethylbenzidine (3,3'5,5'-Tetramethylbenzidine, TMB) detected by convention...

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Abstract

The invention discloses a 6-methylmercaptopurine (6-MMP) immunogen, a synthesis method of the 6-MMP immunogen, an anti-6-MMP specific antibody obtained from the immunogen, a detection reagent and a preparation method of the detection reagent. The immunoassay reagent developed by adopting the high-titer 6-MMP specific antibody can accurately determine the content of 6-MMP. Compared with the traditional method such as high performance liquid chromatography, the immunoassay reagent provided by the invention has the advantages of high sensitivity, high specificity, simplicity in operation, rapid speed, accurate detection result and the like, and has a great importance to guiding clinical and reasonable medication.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a 6-(methylmercapto)purine immunogen, an anti-6-(methylmercapto)purine specific antibody and a 6-(methylmercapto)purine detection reagent. Background technique [0002] 6-(methylmercapto) purine (6-Methylmercaptopurine, 6-MMP), its structural formula is shown in formula (I). [0003] [0004] 6-MMP is a metabolite of 6-(mercapto)purine (6-Mercaptopurine, 6-MP). Clinically, 6-MP is widely used in the treatment of many important diseases, including: acute leukemia, organ transplantation and some autoimmune diseases, etc., but the improper use of this drug can cause serious and even life-threatening blood toxicity. Mardini [1] It is recommended to determine the dosage of 6-MP by measuring the concentration of its metabolite 6-MMP in the patient's blood when using 6-MP. The results of the literature show that when the concentration of 6-MMP in the blood is less than 0.6 μg / mL, The do...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765C07K14/795C07K14/47C07K1/113C07K16/44G01N33/532G01N33/535G01N33/543
Inventor 虞留明田军袁红霞蔡江丽
Owner 苏州博源医疗科技有限公司
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