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Preparation method of methylene blue hapten and methylene blue immunogen

A methylene blue and hapten technology, applied in chemical instruments and methods, animal/human proteins, albumin peptides, etc., can solve the problems of short exposed groups and difficulty in producing polyclonal antibodies, and achieve the effect of easy availability of raw materials and competitive inhibition Excellent, easy-to-operate effect

Inactive Publication Date: 2020-06-19
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation of methylene blue immunogen usually needs to implement the process of synthesizing methylene blue immunogen. In the process of synthesizing cyclomethylene blue immunogen, the methylene blue metabolite azure A or azure C can be directly used as a hapten for synthesis. However, when using azure A or azure When the methylene blue immunogen synthesized by Qing C is used to immunize mice, there is a problem that the exposed group is short, which makes it difficult to produce polyclonal antibodies that have a competitive inhibitory effect on methylene blue

Method used

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  • Preparation method of methylene blue hapten and methylene blue immunogen
  • Preparation method of methylene blue hapten and methylene blue immunogen
  • Preparation method of methylene blue hapten and methylene blue immunogen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of methylene blue hapten

[0036] 1, Synthesis of 2-amino-5-dimethylaminophenylthiosulfuric acid (intermediate 1)

[0037]

[0038] Add aluminum sulfate 18 hydrate, sodium thiosulfate, zinc chloride and N,N-dimethyl-p-phenylenediamine in a molar ratio of 1:2:1:1 until the number of moles is equal to the number of moles of the above-mentioned reaction raw materials 3 to 4 times the total amount of water, the reaction temperature is 0°C, and the stirring time is 5 to 10 minutes.

[0039] Then, according to the molar ratio of potassium dichromate to aluminum sulfate 18 hydrate of 1:3.5-4.0, drop 0.5-1.0mol / L potassium dichromate solution, react at 0°C for 2 hours, and obtain the product; slowly rise to room temperature , suction filtration, the filter cake was successively washed with an equal volume of the reaction solution with water and half the volume of acetone, drained by an oil pump, and beaten with methyl tert-butyl ether with an equal vo...

Embodiment 2

[0048] Embodiment 2, the preparation of methylene blue immunogen

[0049] The methylene blue hapten prepared in Example 1, dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were put into a beaker according to a molar ratio of 1:1:1, and Add dimethylformamide (DMF) solution to the beaker, dissolve it completely, and react under magnetic stirring for 8 hours at room temperature, then let it stand overnight at 4°C; after the reaction, centrifuge at 4°C, 4000r.p.m 10min, take the supernatant. Add the above supernatant to 5 mL of BSA solution with a concentration of 10-20 mg / mL, and react in an ice-water bath for 6 h. After the reaction, the solution was transferred to the treated dialysis bag, and dialyzed at 4° C. for 3 days with phosphate buffer solution as the dialysate, and the dialysate was changed 3-4 times a day. After dialysis, at 4°C,

[0050] Centrifuge at 4000r.p.m for 10min, and take the supernatant, which is the methylene blue immunogen. Through UV sca...

Embodiment 3

[0051] Embodiment 3, the result verification of methylene blue immunogenicity

[0052] Dilute the methylene blue immunogen with phosphate buffer solution to obtain a 2 mg / mL immunogen solution, add 100 μL of the immunogen solution to 100 μL of Freund’s complete adjuvant, mix well, take three 4-week-old Balb / c mice, and use Abdominal injection for immunization; two weeks after immunization, take 100 μL of immunogen solution and add 100 μL of Freund’s incomplete adjuvant, mix well, and then use abdominal injection for immunization; one week later, take 100 μL of immunogen solution for tail vein booster immunization Ten days later, 50 μL of the immunogen solution was injected into the tail vein for booster immunization. On the third day of the last immunization, blood was collected from the orbit, bathed in water at 37°C for 1 hour, the supernatant was taken, and the serum titer and sensitivity were determined by ELISA method.

[0053] according to image 3 , the immune antibod...

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Abstract

The invention relates to the technical field of medicines, and provides a preparation method of methylene blue hapten and immunogen. The preparation method comprises the following steps: carrying outchemical reaction on N, N-dimethyl p-phenylenediamine, aluminum sulfate hydrate, sodium thiosulfate, zinc chloride and potassium dichromate to obtain an intermediate 1; secondly, carrying out chemicalreaction on N-methylaniline, dimethyl pyridine, ethyl 5-bromovalerate and sodium hydroxide to obtain an intermediate 2, and carrying out chemical reaction on the intermediate 1 and the intermediate 2under the action of a catalyst silver carbonate to obtain the methylene blue hapten; and finally, after the methylene blue hapten reacts with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), bovine serum albumin (BSA) is added for a reaction, and the methylene blue immunogen is prepared. According to the methylene blue immunogen prepared by the method, the coupling ratio is about 28: 1, the mouse can be stimulated to generate an antibody, the antibody titer is about 64000, the methylene blue concentration is 39.0 ng / mL when the inhibition rate is 50% and the competitive inhibition effect on methylene blue is excellent.

Description

technical field [0001] The invention belongs to the technical field of antigen preparation, and in particular relates to a preparation method of a methylene blue hapten and a methylene blue immunogen. Background technique [0002] Methylene blue (chemical formula C 16 h 18 ClN 3 S, molecular weight: 319.86) is a phenothiazine salt, the appearance is dark green bronze luster crystal (trihydrate), soluble in water / ethanol, insoluble in ether, relatively stable in the air, the aqueous solution is alkaline, poisonous. Methylene blue is widely used in chemical indicators, dyes, biological dyes and medicines. It has certain therapeutic effects in bacillary dysentery, cancer, bacterial and viral infections, and central nervous diseases. It is also used in aquaculture. It is used to treat certain fish diseases or as a disinfectant. [0003] However, methylene blue is somewhat toxic, and high concentrations of methylene blue can poison animals and cause death. The U.S. Food and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D279/20C07K14/765
CPCC07D279/20C07K14/765
Inventor 黄宣运杨光昕沈晓盛黄冬梅史永富王媛蔡友琼
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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