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Cyproheptadine hapten, antigen, and preparation method and applications of cyproheptadine hapten and antigen

A cyproheptadine and hapten technology, which is applied in the fields of antigen, monoclonal antibody preparation, and cyproheptadine hapten, can solve the problems of unsuitability for large-scale sample screening and detection, complicated operation steps and high cost, and achieves a simple and feasible preparation method. , The effect of processing large sample volume and low cost

Active Publication Date: 2015-07-22
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, and preparation method and applications of cyproheptadine hapten and antigen
  • Cyproheptadine hapten, antigen, and preparation method and applications of cyproheptadine hapten and antigen
  • Cyproheptadine hapten, antigen, and preparation method and applications of cyproheptadine hapten and antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, preparation 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 get light yellow crystals, recrystallize, solidify the residue from ethanol to get light yellow needle crystals. 50ml of water was added to the product, and 8.36g of lithium hydroxide (monohydrate) was added, and the pH was detected to be above 10 by pH test paper. Hydrolysis at room temperature for more than 3 hours. Extract with 100ml of ethyl acetate, extract the hydrolysis reaction water phase once or twice to remove impurities, then adjust the pH value to about 4 with 5mol / L hydrochloric acid, extract with ethyl acetate, and spin dry to obtain the hapten CHD-OCL.

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

Embodiment 2

[0030] Embodiment 2, preparation and identification of cyproheptadine artificial antigen

[0031] 1. Synthesis of Cyproheptadine Immunization Antigen

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

[0033] (2) Add 1ml0.01MPBS to aid dissolution, 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 at 4°C overnight;

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

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

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

[0038] 2. Synthesis of cyproheptadine-coated antigen

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

[0040] (2) Add 1ml0.01MPBS to aid dissolution, add EDC38mg, NHS38mg, activate at room temperature for 2h;

[0041] (3) Dissolve 100mg of OVA in 10ml...

Embodiment 3

[0047] Example 3, Preparation and Specific Identification of Enzyme-labeled Monoclonal Antibody

[0048] 1. Preparation of cyproheptadinumab

[0049] 1. Use the immunogen prepared above at 100 μg / mouse, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in equal volumes, inject subcutaneously on the back of the neck and immunize 6-8 week-old Balb / c female mice, after the first immunization On the 7th, 14th, and 28th days, mix the immunogen and Freund's incomplete adjuvant in equal volumes, and each booster immunization once, and 100 μg of the immune complex per mouse 3 days before the fusion, and then booster immunization without Freund's adjuvant.

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

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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 in particular relates to a preparation method of a cyproheptadine hapten, an antigen, a monoclonal antibody and an application thereof. Background technique [0002] The chemical name of Cyproheptadine is 1-methyl-4-(5H-dibenzo[a,d]cycloheptatriene-5-ylidene)piperidine hydrochloride sesquihydrate, also known as cyproheptadine Dichloroheptidine, Bitongding, English alias 2,6-dichloro-N-(2-imidazolin-2-yl)aniline. Molecular formula C21H21N·HCl·3 / 2 H2O; molecular weight: 350.89. Cyproheptadine is white or light yellow crystalline powder, odorless and slightly bitter. The melting point is 113°C. Soluble in methanol; dissolved in chloroform, the solution will emulsify (if dissolved in dry product, the solution will be clear); slightly soluble in ethanol; slightly soluble in water, the aqueous solution is acidic; almost insoluble in ether. Cyproheptadine is an antiallergic drug fo...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531
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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