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Enzyme linked immunosorbent assay kit for detecting ribavirin and application thereof

A ribavirin and kit technology, applied in the field of enzyme-linked immunosorbent assay kits for ribavirin detection, can solve the problems of high capital and personnel investment costs, and achieve simple pre-treatment process, high accuracy, and low price Effect

Active Publication Date: 2016-07-20
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The instrument method has the advantages of high sensitivity and accu

Method used

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  • Enzyme linked immunosorbent assay kit for detecting ribavirin and application thereof
  • Enzyme linked immunosorbent assay kit for detecting ribavirin and application thereof
  • Enzyme linked immunosorbent assay kit for detecting ribavirin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The preparation of embodiment 1 kit components

[0028] 1. Preparation of ribavirin hapten

[0029] a) Take (9ci)-5-nitro-1H-1,2,4-thiazole-3-carboxamide 5g, add β-D-ribofuranose-1,2,3,5-tetraacetate 3.26g , add DMSO to dissolve, add bis(p-nitrophenyl) phosphate 1.2g, heat in an oil bath, and react at 170°C for 2h. Stop the reaction, add water, extract with ethyl acetate, evaporate to dryness, apply to a silica gel column, elute and separate with petroleum ether / ethyl acetate (v / v, 2 / 1), and obtain intermediate a4.15g.

[0030] b) Dissolve intermediate a in aqueous sodium hydroxide solution, react at 60°C for 2 hours, stop the reaction, neutralize, rotary evaporate, evaporate to dryness, add ethanol for recrystallization, and obtain 2.11 g of nitroribavirin.

[0031] c) Take 2.1 g of nitroribavirin, add methanol to dissolve, add a catalytic amount of palladium carbon, add hydrogen, stir at room temperature for 6 h, detect that the raw material is completely reacted, r...

Embodiment 2

[0046] Embodiment 2 detects the formation of the ELISA kit of ribavirin

[0047] An enzyme-linked immunosorbent assay kit for detecting ribavirin was set up to include the following components:

[0048] (1) A microtiter plate coated with a ribavirin-coupled antigen;

[0049] (2) 6 bottles of ribavirin standard solution, the concentrations are 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, 81 μg / L;

[0050] (3) Ribavirin antibody labeled with horseradish peroxidase;

[0051] (4) Substrate chromogenic solution is made up of A liquid and B liquid, and A liquid is carbamide peroxide, and B liquid is tetramethylbenzidine;

[0052] (5) The stop solution is 2mol / L sulfuric acid;

[0053] (6) The washing solution has a pH value of 7.4, contains 0.5% to 1.0% Tween-20, 0.01‰ to 0.03‰ sodium azide preservative, and 0.1 to 0.3mol / L phosphate buffer, and the percentage is weight volume percentage;

[0054] (7) The complex solution is a phosphate buffer solution with a pH value of 7.0 and 0....

Embodiment 3

[0055] The detection of ribavirin in embodiment 3 animal tissue

[0056] 1. Sample pretreatment

[0057] Weigh 1.0±0.05g tissue sample into a 50ml polystyrene centrifuge tube, add 10ml methanol, shake with a shaker for 2min, mix well, centrifuge at 3000g room temperature (20-25℃ / 68-77℉) for 5min; pipette 1ml of the upper layer Put the organic phase into a clean and dry glass tube of 10ml, and dry it in a water bath at 50-60°C (68-86°F) under nitrogen flow; add 1ml of reconstitution working solution, vortex for 3min; take 50μl for analysis.

[0058] 2. Detection with kit

[0059] Add 50 μl of standard solution / sample to the corresponding microwells, then add 50 μl / well of antibody working solution, shake and mix gently, cover the plate with a cover film and place it in a dark environment at 25°C for 30 minutes. Carefully uncover the cover plate membrane, shake off the liquid in the well, wash with 250 μl / well of working washing solution for 4-5 times, with an interval of 10 s...

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Abstract

The invention provides an enzyme linked immunosorbent assay kit for detecting ribavirin. The kit comprises an elisa plate coated with a coating antigen, a ribavirin standard substance solution, an enzyme-labeled antibody, substrate developing liquid, a stop solution, a scrubbing solution and reconstitution fluid. The coating antigen is a ribavirin coupled antigen, and the enzyme-labeled antibody is an enzyme-labeled ribavirin antibody. The invention further discloses a method for detecting ribavirin through the enzyme linked immunosorbent assay kit. The method comprises the steps that firstly, sample pretreatment is conducted; then, detection is conducted through the kit; finally, a detection result is analyzed. The enzyme linked immunosorbent assay kit can be used for detecting the content of ribavirin in animal tissue samples, and is easy and convenient to operate, low in expenditure, high in sensitivity and suitable for screening a large number of samples, and on-site monitoring is achieved.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunoassay kit for detecting ribavirin, which can qualitatively and quantitatively detect the residual amount of ribavirin in animal tissues. Background technique [0002] Ribavirin (RBV, ribavirin) is a highly efficient broad-spectrum antiviral nucleoside drug, which has a good inhibitory effect on a variety of DNA and RNA viruses, and has multiple action sites, high efficacy, and low toxicity. , It is not easy to produce drug resistance and has few side effects. It has been used in the treatment of many viruses. Clinically, it has a high curative effect on the treatment of influenza, various respiratory syncytial viruses, viral hepatitis, human immunodeficiency virus and epidemic hemorrhagic fever. However, such drugs are prone to drug resistance, causing virus mutations, and long-term use can cause toxic effects on body cells. The most pro...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535
CPCG01N33/535G01N33/577G01N2430/00
Inventor 冯才伟罗晓琴冯静赵正苗吴小胜曹东山王建霞王琳琛何方洋刘玉梅
Owner BEIJING KWINBON BIOTECH
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