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Amlodipine hapten, amlodipine artificial antigen, amlodipine antibody as well as preparation method and application thereof

A technology of amlodipine and hapten, applied in chemical instruments and methods, animal/human protein, transferrin, etc., to achieve high sensitivity, high sensitivity and high specificity recognition ability, wide detection range effect

Active Publication Date: 2021-02-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key to the establishment of the above method is to design a suitable amlodipine hapten and obtain antibodies with high sensitivity and specificity, but there are no related reports about amlodipine haptens, artificial antigens, and antibodies in the prior art

Method used

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  • Amlodipine hapten, amlodipine artificial antigen, amlodipine antibody as well as preparation method and application thereof
  • Amlodipine hapten, amlodipine artificial antigen, amlodipine antibody as well as preparation method and application thereof
  • Amlodipine hapten, amlodipine artificial antigen, amlodipine antibody as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Synthesis and identification of embodiment 1 amlodipine hapten AN-SSA

[0048] 1. Test method

[0049] 450mg of amlodipine was dissolved in 10mL of anhydrous dichloromethane, then 500mg of succinic anhydride and 100mg of 4-dimethylaminopyridine were added to the above solution, heated and stirred at 60°C for 20 hours, and the mixture was obtained by TLC (thin layer chromatography) Silica gel plate) to monitor the reaction process and finally confirm the product, the developing solvent is methanol:ethyl acetate=1:5. After the reaction product is spin-dried, add an appropriate amount of ethyl acetate and saturated saline to extract three times, add 100 mg of anhydrous sodium sulfate to the ethyl acetate layer for drying, and finally concentrate to obtain the amlodipine hapten AN-SSA. The synthesis process Such as figure 2 shown.

[0050] 2. Identification

[0051] Amlodipine standard was used for NMR identification, and then the target product was purified and dissol...

Embodiment 2

[0058] Synthesis and identification of the complete antigen of embodiment 2 amlodipine

[0059] 1. Coupling of amlodipine complete antigen and carrier protein

[0060] (1) Dissolve 8 mg of amlodipine hapten, 3 mg of NHS and 4 mg of EDC in sequence in 200 μL of DMF, stir at room temperature for 4 hours, and fully activate;

[0061] (2) Weigh 10 mg of bovine serum albumin (BSA) and add it to 2 mL of PBS buffer solution (0.1M, PH=7.4);

[0062] (3) Slowly add the solution obtained in step (1) dropwise to the solution obtained in step (2), and stir for 7h:

[0063] Dialyze with 0.1M (PH=7.4) buffer solution for two days, change the liquid 4 times every day, and obtain amlodipine complete antigen (AN-SSA-BSA) after the dialysis ends, wherein the formula of PBS buffer solution is: Na2HPO4 12H2O 2.90g , NaCl 8.50g, KCl 0.20g, KH2PO4 0.20g, add distilled water to 1000mL.

[0064] Similarly, replace BSA with lactoferrin (LF) and chicken ovalbumin (OVA) as the carrier protein to obta...

Embodiment 3

[0067] The preparation of embodiment 3 amlodipine polyclonal antibody

[0068] (1) Select 2 (one male and one male) adult healthy New Zealand white rabbits for immunization. The dose of each rabbit is 500 μL of the complete antigen AN-SSA-LF (1 mg / mL). Emulsified with Freund's complete adjuvant at a volume ratio of 1:1, and immunized New Zealand white rabbits. Afterwards, AN-SSA-LF was emulsified with Freund's incomplete adjuvant at a volume ratio of 1:1, and a booster immunization was performed every three weeks, for a total of four booster immunizations;

[0069] (2) Seven days after the third immunization, blood was collected from the ear veins of rabbits, centrifuged to obtain the supernatant, and stored at -20°C for further use.

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Abstract

The invention discloses an amlodipine hapten, an amlodipine artificial antigen, an amlodipine antibody as well as a preparation method and an application thereof, amlodipine hapten ANSSA is firstly prepared, the amlodipine artificial antigen and the amlodipine polyclonal antibody are obtained by applying the hapten. The antibody has high sensitivity and high specificity recognition capability on amlodipine, the half inhibition concentration is 0.31 mu g / kg, the quantitative detection range is 0.04758-2.078 mu g / kg, the detection limit is 0.085 mu g / kg, and the cross reaction rate on felodipineand nitrendipine is lower than 1%. Besides, a colloidal gold immunochromatography technology is established by utilizing the amlodipine antibody, the colloidal gold immunochromatography technology has a wide detection range and high sensitivity, detection of amlodipine can be specifically recognized, the visual detection line is 20 ng / mL, and the colloidal gold immunochromatography technology hasa great application prospect in immune rapid detection of amlodipine in health-care food.

Description

technical field [0001] The invention relates to the technical field of food safety detection, more specifically, to an amlodipine hapten, an artificial antigen, an antibody and a preparation method and application thereof. Background technique [0002] Amlodipine is currently the most common antihypertensive drug, and amlodipine is a third-generation dihydropyridine calcium channel blocker. The mechanism of action of amlodipine is to relax the smooth muscle of the arterial wall, reducing the total peripheral resistance, thereby lowering blood pressure. In order to increase sales and make huge profits, unscrupulous merchants add amlodipine and other drugs to health food to attract consumers by lowering blood pressure to achieve good results. This practice not only violates my country's "Food security law 》, and it also seriously damages the rights and physical and mental health of consumers. In 2019, the Food Safety Office of the State Council, the Ministry of Public Securi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D211/90C07K14/765C07K14/77C07K14/79C07K16/06C07K16/44G01N33/558G01N33/58G01N33/68
CPCC07D211/90C07K14/77C07K14/765C07K14/79C07K16/065C07K16/44G01N33/94G01N33/6854G01N33/581G01N33/585G01N33/558
Inventor 雷红涛华启诚李向梅沈兴李兆栋韦晓群王锦林建浩沈玉栋孙远明
Owner SOUTH CHINA AGRI UNIV
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