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Amantadine artificial antigen and preparation method and application thereof

A technology of amantadine and amantadine hydrochloride, which is applied in the field of antigen preparation and amantadine hapten, can solve the problems that the accuracy, sensitivity, and specificity of the kit cannot fully meet the detection requirements, and achieve the preparation method Simple and feasible, low cost and high yield

Inactive Publication Date: 2016-08-31
北京维德维康生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Enzyme-linked immunosorbent assay kits for the detection of amantadine have been developed abroad, but domestically produced kits cannot fully meet the detection requirements in terms of accuracy, sensitivity, and specificity.

Method used

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  • Amantadine artificial antigen and preparation method and application thereof
  • Amantadine artificial antigen and preparation method and application thereof
  • Amantadine artificial antigen and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the preparation of amantadine hapten

[0028] 1. Preparation of amantadine hapten

[0029] Dissolve 0.94g of amantadine hydrochloride in 30ml of anhydrous pyridine, add 0.49g of maleic anhydride, after dissolving, add 10mg of 4-dimethylaminopyridine, reflux at 70°C for 3h, remove pyridine by rotary evaporation, add 10ml of deionized water , adjust the pH to 5.0 with glacial acetic acid, precipitate a large amount of precipitate, filter, wash the precipitate with water, and dry to obtain the hapten.

[0030] 2. Identification of amantadine hapten

[0031] The resulting product was identified by mass spectrometry, -MS: M-1=248.7 ( figure 1 ).

[0032] The results showed that its chemical structure was as shown in Formula 1, which was the amantadine hapten.

[0033]

[0034] Formula 1.

Embodiment 2

[0035] Example 2, Preparation and Identification of Amantadine Artificial Antigen

[0036] 1. Synthesis of amantadine immune antigen

[0037] Weigh 5.57mg of hapten and dissolve it in 1ml DMF, add 4.3mg of EDC, 5.2mg of NHS, and stir at room temperature for 2h; After stirring overnight, dialyze in PBS at 4°C for 72 hours, during which time the dialysate was changed 6 times. The dialysate was passed through a filter membrane with a pore size of 0.22 μm under sterile conditions, distributed into ampoule bottles, and stored at -20°C.

[0038] Coating original was prepared in the same way.

[0039] 2. Identification of amantadine artificial antigen

[0040] The immunogen MALDI-TOF-MS identification results showed that the coupling ratio was: R=(AMD-MH-BSA)-(BSA) / 249.7=(73828.009-67485.901) / 249.7=25.40( figure 2 and image 3 ). That is, in the immunogen, the molar ratio of the amantadine hapten (formula I) coupled to bovine serum albumin (BSA) is 25.40:1.

Embodiment 3

[0041] Example 3, Preparation and Specific Identification of Enzyme-labeled Monoclonal Antibody

[0042] 1. Preparation of amantadine monoclonal antibody

[0043] 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.

[0044] 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 ...

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Abstract

The invention discloses an amantadine hapten, a corresponding artificial antigen and a preparation method and application of the amantadine hapten and the corresponding artificial antigen. The amantadine hapten is a product as shown in formula 1, and the amantadine antigen can be obtained by connecting the product with carrier protein. The amantadine antigen can be used for preparing an amantadine specific antibody. The preparation method is simple and practicable, low in cost and high in hapten yield. The amantadine artificial antigen has the advantages that the specific antibody aiming at amantadine can be produced by immunizing animals, the amantadine artificial antigen can be used for preparing an enzyme-linked immunosorbent assay kit for detecting amantadine residues, and the amantadine artificial antigen is simple, fast, large in sample processing amount, high in sensitivity, high in specificity, and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to an amantadine hapten, an antigen preparation method and an application thereof. Background technique [0002] Amantadine (1-adamantanamine) was synthesized in 1960, also known as amantadine and tricyclodecanylamine, it is an organic compound of 1-adamantine or 1-aminoadamantane, which means that its structure is an adamantine There is one substituted amino group in the alkane backbone and four methine positions. It is white crystalline powder with bitter taste, easily soluble in water or ethanol. It is mainly used for the prevention of respiratory tract infection caused by influenza A virus and the treatment of patients with influenza A virus infection. It was approved as a preventive drug in 1966 when the Asian cold was popular in the United States, and it was established on the basis of preventive medicine in 1976. It is confirmed on the Internet tha...

Claims

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Application Information

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IPC IPC(8): C07C231/02C07C233/09C07K14/765C07K14/77C07K14/795C07K14/00C07K16/44G01N33/53G01N33/577
CPCC07C231/02C07C233/09C07K14/00C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53G01N33/577
Inventor 秦誉丁亚芳李延山张秀芹吴雨洋王照鹏杨柳李向梅聂丽贾良曦
Owner 北京维德维康生物技术有限公司
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