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Cyproheptadine hapten, artificial antigen and antibody and preparation method and application thereof

A technology of artificial antigen and cyproheptadine is applied in the field of preparation of cyproheptadine hapten, artificial antigen and antibody, and can solve the problems of insufficient specificity and recognition power of cyproheptadine antibody, lack of cyproheptadine immunological analysis method, etc. , to achieve the effect of high accuracy, high sensitivity and high specificity

Active Publication Date: 2017-02-08
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Immunological analysis technology has been widely used in the field of drug residue detection due to its advantages of sensitivity, specificity, rapidity and simplicity. The synthesis of artificial antigen is the key to the establishment of hapten substance immunoassay method. For cyproheptadine, because of hapten design and The reason for the synthesis is that the specificity and recognition ability of the cyproheptadine antibody obtained by the prior art is not enough, and there is a lack of relevant technical reports on the immunological analysis method of cyproheptadine

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Hapten Synthesis and Identification

[0033] 1. Synthesis of cyproheptadine hapten

[0034] The synthetic route of cyproheptadine hapten is as attached figure 1 shown.

[0035] (1) Dissolve 0.55g desmethylcyproheptadine and 1mL pyridine in 10mL dimethyl sulfoxide (DMSO);

[0036] (2) Weigh 0.39g tert-butyl bromoacetate and dissolve it in 5mL DMSO, then slowly drop it into the solution described in step (1) at 40°C, continue the reaction for 4 hours, evaporate the solvent, and separate by column chromatography to obtain cyproheptadine Hapten intermediates;

[0037] (3) Add 20 mL DMSO and 5 mL formic acid to the cyproheptadine hapten intermediate prepared in step (2), react at room temperature for 20 hours, evaporate the solvent, and recrystallize in the ethanol-water system to obtain the target hapten (I). The total yield of the two-step reaction was 62%.

[0038] 2. Identification of cyproheptadine hapten

[0039] Get above-mentioned object product and ...

Embodiment 2

[0040] Example 2: Artificial Antigen Synthesis and Identification

[0041] 1. Synthesis of Cyproheptadine Antigen (Ⅱ)

[0042] Active ester synthesis of cyproheptadine antigen (Ⅱ):

[0043] (1) Weigh 20 mg of cyproheptadine hapten and dissolve it in 1 mL of N,N-dimethylformamide (DMF);

[0044] (2) Dissolve 30 mg of carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in 0.2 mL of water, add them to the hapten solution, and stir at room temperature for 24 hours;

[0045] (3) Weigh 50 mg of BSA and dissolve it in 3.8 mL of CB (pH 9.6) solution, slowly add the solution after step (2) into the BSA-CB solution drop by drop, and stir and react at room temperature for 24 hours;

[0046] (4) Put the solution reacted in step (3) into a dialysis bag, and dialyze with 0.01mol / L PBS solution at 4°C for 72 hours, during which the dialysate was changed 3 times a day to obtain the target product. Store at 20°C.

[0047] In the above method, human serum albumin, hemocyanin or ovalbumin can ...

Embodiment 3

[0050] Example 3: Preparation of Monoclonal Antibody

[0051] (1) Animal immunization: Inject the immunogen into Balb / c mice at an immunization dose of 150 μg / mouse to make them produce antiserum.

[0052] (2) Cell fusion and cloning: Splenocytes from Balb / c mice that produce specific antibodies were fused with myeloma cell SP20, and the cell supernatant was measured by indirect competitive enzyme-linked immunoassay method, and positive wells were screened. Positive wells were cloned by limiting dilution method to obtain and establish monoclonal antibody-producing hybridoma cell lines.

[0053](3) Cell cryopreservation and recovery: take the hybridoma cells in the logarithmic growth phase and make a cell suspension with the cryopreservation medium, aliquot them into cryopreservation tubes, and store them in liquid nitrogen for a long time. When recovering, take out the cryopreservation tube, put it into a 37°C water bath to thaw quickly, remove the cryopreservation solution b...

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Abstract

The invention provides a cyproheptadine semiantigen, an artificial antigen, an antibody, preparation methods of cyproheptadine semiantigen and the artificial antigen, and application of the artificial antigen and the antibody. The cyproheptadine semiantigen has a molecular structure shown as a formula (I), and the cyproheptadine artificial antigen has a molecular structure shown as a formula (II). The titer of a monoclonal antibody obtained from the antigen reaches 1:300000, and the 50% inhibiting concentration is 0.20 mu g / L. The generated antibody is high in specificity, high in sensitivity and high in accuracy. The antigen and the antibody have wide application prospect on detection of cyproheptadine residue in food.

Description

technical field [0001] The invention belongs to the technical field of food safety, and in particular relates to a preparation method and application of a cyproheptadine hapten, an artificial antigen and an antibody. Background technique [0002] Cyproheptadine (Cyproheptadine, molecular formula: C 21 h 21 N), also known as diphenylcycloheptidine, is an antihistamine with anti-5-HT, blocking H 1 Receptor and anticholinergic effect, it is mainly used to treat allergic diseases such as urticaria, eczema, contact dermatitis, skin itching, allergic rhinitis, etc. It can also be used to improve the appetite of patients. [0003] Cyproheptadine is a prescription drug for humans, but it is currently found to be added in some domestic feed products. The reason may be to increase the animal's appetite to achieve the purpose of weight gain. Consumption of animal-derived products containing cyproheptadine residues is harmful to human health, and the effect on children is more obviou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D211/70C07K14/765C07K14/77C07K14/795C07K16/44G01N33/531G01N33/577
CPCC07D211/70C07K1/1077C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/531G01N33/577
Inventor 何方洋冯才伟孙震陶光灿顾蓉蓉崔廷婷白莉齐向武
Owner BEIJING KWINBON BIOTECH
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