Cyproheptadine hapten, antigen, preparation method and application thereof

A technology of cyproheptadine and hapten, which is applied in the field of monoclonal antibody preparation, cyproheptadine hapten, and antigen. It can solve the problems of cumbersome operation steps, high cost, and inapplicability to large-scale sample screening and detection, and achieve the processing sample volume. Large size, low cost, simple and feasible preparation method

Active Publication Date: 2016-09-14
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, high-performance liquid chromatography is mostly used at home and abroad. These methods have strong specificity and high sensitivity, but the sample pretreatment steps are cumbersome and costly, and they are not suitable for screening and testing of large batches of samples.

Method used

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  • Cyproheptadine hapten, antigen, preparation method and application thereof
  • Cyproheptadine hapten, antigen, preparation method and application thereof
  • Cyproheptadine hapten, antigen, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1. Preparation of Cyproheptadine Hapten

[0028] A mixture of 3.3 g of cyproheptadine, 7.49 g of ethyl chlorocarbonate and 17 mL of toluene was refluxed for 3.5 hours. After cooling, the reaction mixture was washed with hydrochloric acid and water, then dried and concentrated. Treat with n-hexane to obtain pale yellow crystals, recrystallize, solidify the residue from ethanol to obtain pale yellow needle crystals. Add 50ml of water to the product, add 8.36g of lithium hydroxide monohydrate, and test pH=10 or more with a pH test paper. Hydrolysis reaction at room temperature for more than 3h. Extract with 100ml of ethyl acetate, extract the aqueous phase of the hydrolysis reaction once or twice to remove impurities, then adjust the pH to 4 with 5mol / L hydrochloric acid, extract with ethyl acetate, and spin dry to obtain the cyproheptadine hapten CHD-OCL .

[0029] The structural formula of cyproheptadine hapten is shown in formula I.

Embodiment 2

[0030] Example 2. Preparation and identification of cyproheptadine artificial antigen

[0031] 1. Synthesis of cyproheptadine immune antigen

[0032] (1) Dissolve 31.7mg CHD-OCL in 3ml DMF;

[0033] (2) Add 1ml 0.01MPBS to help dissolve, add EDC38mg, NHS38mg, activate at room temperature for 2h;

[0034] (3) Dissolve 100mg of BSA in 10ml of 0.1M sodium bicarbonate solution, add the activated substance to the protein in an ice-water bath at 4°C, and stir overnight at 4°C;

[0035] (4) Put in 1L0.01MPBS and dialyze 8 times to obtain the whole antigen CHD-OCL-BSA;

[0036] (5) Aliquot and store at -20℃ for later use.

[0037] The structural formula of cyproheptadine immune antigen is shown in formula II.

[0038] 2. Synthesis of Cyproheptadine Coating Antigen

[0039] (1) Dissolve 31.7mg CHD-OCL in 3ml DMF;

[0040] (2) Add 1ml 0.01MPBS to help dissolve, add EDC38mg, NHS38mg, activate at room temperature for 2h;

[0041] (3) Dissolve 100mg of OVA in 10ml of 0.1M sodium bicarbonate solution, add ...

Embodiment 3

[0047] Example 3. Preparation and specific identification of enzyme-labeled monoclonal antibody

[0048] 1. Preparation of Cyproheptadimumab

[0049] 1. Use the above-prepared immunogen at 100μg / mouse, dissolve the immunogen in saline and mix the same volume with Freund's complete adjuvant, and immunize 6-8 week old Balb / c female mice by subcutaneous injection on the back of the neck. After the first immunization On 7, 14, and 28 days, the immunogen and Freund's incomplete adjuvant were mixed in equal volume, and each additional immunization was added once. Three days before fusion, the immune complex was 100μg / mouse without Freund's adjuvant and another additional immunization was performed.

[0050] 2. According to the conventional method, take the spleen cells of the immunized mouse and mix with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add the preheated fusion agent (PEG4000) for fusion within 45s. Suspend evenly in HAT medium, add appropri...

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Abstract

The present invention discloses a cyproheptadine hapten, a corresponding artificial antigen and a corresponding monoclonal antibody, and further discloses preparation method and applications of the cyproheptadine hapten, the corresponding artificial antigen and the corresponding monoclonal antibody. According to the present invention, the cyproheptadine hapten is the product represented by a formula I, and the product represented by the formula I and carrier protein are connected to obtain the cyproheptadine antigen, wherein the cyproheptadine antigen can be used for preparing the cyproheptadine specific antibody; the preparation method has characteristics of simpleness, feasibility, low cost, and high hapten yield; and after the cyproheptadine artificial antigen of the present invention is used to immunize animals, the cyproheptadine-specific antibodies can be produced, wherein the antibodies can be used for preparing the enzyme-linked immunosorbent assay kit for detecting the cyproheptadine residue, and the kit has advantages of simpleness, rapidness, large sample treatment amount, high sensitivity, strong specificity, and the like.

Description

Technical field [0001] The invention belongs to the technical field of food safety detection, and specifically relates to a preparation method and application of cyproheptadine hapten, antigen, and monoclonal antibody. Background technique [0002] Cyproheptadine (Cyproheptadine) chemical name is 1-methyl-4-(5H-dibenzo[a,d]cycloheptatriene-5-ylidene)piperidine hydrochloride sesquihydrate, alias Cyproheptadine Din, diphenylcycloheptadidine, and partial pain, the English alias is 2,6-dichloro-N-(2-imidazolin-2-yl)aniline. Molecular formula C21H21N·HCl·3 / 2H2O; molecular weight: 350.89. Cyproheptadine is white or light yellow crystalline powder, odorless and slightly bitter in taste. Melting point 113°C. It is easily soluble in methanol; dissolved in chloroform, the solution is emulsified (if it is dissolved in a dry product, the solution is clear); slightly soluble in ethanol; slightly soluble in water, and the aqueous solution is acidic; almost insoluble in ether. Cyproheptadin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D211/70
CPCC07D211/70C07K14/765C07K16/44C07K19/00G01N33/531G01N33/577
Inventor 温凯王照鹏王战辉吴小平贾良曦秦誉王进苏丽芳覃丹凤曾江龙殷缓缓陈银辉
Owner 北京维德维康生物技术有限公司
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