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ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection

An enzyme-linked immunosorbent reagent and substrate technology, which is used in measurement devices, special data processing applications, color/spectral property measurement, etc. High requirements are required to achieve the effect of low cost, easy separation and improved sensitivity

Active Publication Date: 2006-12-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object detected by ELISA kits is generally animal urine or tissue. The treatment of urine is relatively simple, or no treatment is required. However, for animal tissue samples, the current commercial kits need to take complicated extraction and separation steps, first acidification and then alkali and then centrifuged. Some of them need to be purified by C18 column, ranging from more than ten steps to dozens of steps. Reagent fee
In addition, searching related patents found that the Chinese patent "A Clenbuterol Enzyme Immunoassay Kit and Its Detection Method" (Application No. 02137941.6) mentioned a clenbuterol extraction method. In this method, animal tissue samples were first Acidify and then freeze and thaw twice, centrifuge to absorb the supernatant, then adjust it to alkaline with alkali, and then extract it with isobutanol.
[0006] Therefore, the existing products at home and abroad are generally poor in stability, complicated in tissue sample pretreatment and detection steps, high in equipment requirements, and expensive in price, which seriously affects the detection and monitoring of clenbuterol residues, so the development stability is high , simple operation, low equipment requirements, cheap clenbuterol ELISA kit and simple and fast tissue sample pretreatment method have very important economic and social significance

Method used

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  • ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection
  • ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection
  • ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of ELISA Kit Samples

[0034] (1) Preparation of buffer: buffer (pH7.4PBST) KH 2 PO 4 0.4g, Na 2 HPO 4 12H 2 O5.8g, NaCl 16g, KCl 0.4g, Tween-20 0.05% 1mL, add distilled water to 2000mL.

[0035] (2) Preparation of blocking solution: 1.0-5.0 g of skimmed milk powder was dissolved in 100 mL of distilled water.

[0036] (3) Preparation of substrate solution: 30 μL of 30% hydrogen peroxide was dissolved in 19 mL of chromogenic solution (pH5.0 phosphoric acid-citric acid buffer 0.2M Na 2 HPO 4 25.7mL, 0.1M citric acid 24.3mL, add distilled water 50mL), store at 4°C.

[0037] (4) Preparation of substrate buffer solution: Dissolve 80 mg o-phenylenediamine OPD in 10 mL substrate solution and store at 4°C.

[0038] (5) Coating of ELISA microplate: Coating antigen with pH9.6, 0.05mol / L carbonate buffer solution (containing 1~2g sodium carbonate and 2~4g sodium bicarbonate, double distilled water 1L ) diluted to 0.1-5ug / mL, add 100uL to each well o...

Embodiment 2

[0049] The detection method of embodiment 2 kit

[0050] (1) Take the kit out of the refrigerated environment, place it at room temperature (20-24°C) to equilibrate for more than 30 minutes, fix enough strips for standards and samples on the bracket, and do two parallel experiments for standards and samples, in order serial number.

[0051] (2) Add 50 μL of standard substance to the standard well, and add 50 μL of the sample to be tested into the sample well. Then add 50 μL of enzyme markers to each well, and tap to mix well. Incubate at room temperature for 60 min.

[0052] (3) Pour out the liquid in the well, turn the microwell frame upside down on the absorbent paper and pat (3 times for each round of plate washing) to ensure that the liquid in the well is completely removed. Fill the wells with 250 μL distilled water, pour off the liquid in the microwells again, and repeat the operation 3 times.

[0053] (4) Add 100 μL of chromogenic solution (mix equal volumes of subs...

Embodiment 3

[0059] Example 3 Detection of Liver

[0060] Mince the negative and positive pig liver samples (concentration confirmed by GC-MS), weigh 5g and put it into a 25mL beaker, add 5mL distilled water, put it in a 95°C water bath, pour it into a 5mL centrifuge tube after 5min, and place it at 4000r / Centrifuge for about 5 minutes at a rotational speed of 1 min, and the supernatant is the sample extract, and then detected by using the ELISA kit of the present invention. The experimental results are shown in Table 1.

[0061]

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Abstract

The invention relates to an enzyme couple immune detecting field, especially disclosing a clenobuterol enzyme immune agent box, relative detecting method, and a method for preparing the sample of animal organism, wherein said box comprises a box body, a 96 / 40 porous enzyme mark plate, a horse peroxide enzyme mark antibody, a clenobuterol standard solution, a substrate liquid, a substrate buffer liquid and a reaction ending liquid; the holes of said mark plate packs the packing antibody that combined with the clenobuterol antibody. The invention uses direct competitive enzyme couple immune adsorption analysis technique, to simplify the operation and reaction time, to reduce error. The inventive method has simple operation, low cost, and short time, while the extracted sample can be directly used in enzyme couple immune method and golden mark immune laminated analysis, to be sample of prior analysis.

Description

technical field [0001] The invention relates to the field of enzyme-linked immunoassay detection, in particular to a clenbuterol detection ELISA kit, a detection method thereof, and a sample preparation method of animal tissue before detection. Background technique [0002] Clenbuterol, commonly known as clenbuterol, belongs to beta stimulant, chemical name is 2-[(tert-butylamino)methyl]-4-ammonia-3,5-dichlorobenzyl alcohol hydrochloride, medicine It is called "Cetansu", which has been illegally added to the feed in recent years to increase the lean meat rate of fat animals and accelerate the growth of animals. Because the additive dose is 5 to 10 times the therapeutic dose, it has high residues in the animal body and is given to the animal. harm to consumers. Since 1997, my country's Ministry of Agriculture and other relevant departments have issued documents many times, listing clenbuterol as a prohibited drug that seriously endangers people's health, and prohibiting its ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/547G01N33/577G01N21/31G06F19/00G06F19/10
Inventor 孙远明杨金易潘科肖治理雷红涛王弘谌国莲吴青沈玉栋黄丽
Owner SOUTH CHINA AGRI UNIV
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