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Enzyme-linked immune assay kit adapted to biomycin residue analysis and application

A detection kit and residue analysis technology, applied in analytical materials, measuring devices, instruments, etc., can solve problems such as unfavorable work efficiency, increase operation steps, increase detection cost, etc., to shorten detection time, reduce operation steps, reduce The effect of testing costs

Inactive Publication Date: 2006-08-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main disadvantages of using microbiological methods are: long detection time (2 to 3 days), lack of specificity, and even inability to characterize a certain class of drugs (NikolaosA.Botsoglou, DimitriosJ.Fletouris.Drug residues in foods pharmacology, food safety and analysis [M]. New York, 2001)
[0006] The main disadvantages of the application of high performance liquid chromatography (HPLC) are: long detection time, low sensitivity, expensive equipment, and difficulty in popularization and application at the grassroots level
[0009] The existing ELISA method for detecting certain veterinary drug residues, due to the differences in the specificity of the prepared antibodies, requires column purification to eliminate interference when processing tissue samples, which will inevitably increase operating steps, increase detection costs, and prolong Detection time (generally extended by about 2 hours), is not conducive to improving work efficiency

Method used

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  • Enzyme-linked immune assay kit adapted to biomycin residue analysis and application
  • Enzyme-linked immune assay kit adapted to biomycin residue analysis and application
  • Enzyme-linked immune assay kit adapted to biomycin residue analysis and application

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1 prepares aureomycin acetic acid, artificial immunogen, coating antigen, anti-aureomycin antibody

[0038] 1.1 Preparation of aureomycin acetic acid

[0039] Take by weighing 300mg aureomycin hydrochloride (can replace with aureomycin, aureomycin alkali in another embodiment, its usage amount is identical with aureomycin hydrochloride), dissolve with the phosphate buffer saline solution of 25mlpH10, add 58.5mg chlorine Sodium acetate, fully react at room temperature for 10 hours, filter, adjust the pH value to 4.0 with dilute hydrochloric acid, let stand for 2 hours, and dry at 50°C after suction filtration to obtain aureomycin acetic acid.

[0040] 1.2 Preparation of artificial immunogen

[0041] Dissolve 118.8 mg of aureomycin acetic acid with 2 ml of N, N-dimethylformamide (DMF), add N, N dicyclohexyl carboximide (DCC) 55 mg, N-hydroxysuccinimide (NHS) while stirring 28.8 mg, stirred at 4°C for more than 10 hours, centrifuged at 4°C (10000r / min), added ...

Embodiment 2

[0058] Embodiment 2 establishes aureomycin ELISA indirect competitive detection method (ELISA)

[0059] 2.1 Determination of the coating concentration of the original coating and the working concentration (dilution factor) of the aureomycin antibody

[0060] Determine the three reference points of high, middle and low by the square matrix titration test: the point with the absorbance value of about 1.0 and the difference between the left and right adjacent absorbance values ​​is the largest as the reference point. Operation steps: Coat the first row of the 96-well ELISA plate with 8 μg / kg of coating source, and the second to seventh rows are coated with 4, 2, 1, 0.5, 0.25, 0.125 μg / kg of coating Original. Overnight at 4°C, block with 1% ovalbumin at 37°C for 1 hour, wash twice, pat dry, add 100 μl to the first column to the seventh column of the microtiter plate, and the dilution factor is 1000, 2000, 4000, 8000, 16000, 32000, 64000 chlortetracycline antibody, incubate at 37...

Embodiment 3

[0090] Embodiment 3 The accuracy and precision test of the present invention

[0091] 3.1 Now take pig muscle as an example to illustrate the processing method and results of meat tissue samples (the same test can use animal muscle tissue such as chicken or duck or cattle or sheep).

[0092]Add chlortetracycline standard substance with a concentration of 100 μg / kg into the porcine muscle tissue to be tested, and another porcine muscle tissue without chlortetracycline was used as a blank as a control, and each concentration was repeated 5 times. Operation steps: Weigh 5 grams of homogenized porcine muscle tissue for each sample, prepare test samples and blank samples according to the aforementioned method and dosage, then add 5 ml of pH4. Centrifuge at 4000 rpm, take the supernatant, add 0.3ml of 20% trichloroacetic acid, shake well and then centrifuge at 4°C, 8000 rpm, take the supernatant, adjust the pH value to 7.4 after 10-fold dilution (add 1N NaOH About 100 microliters),...

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Abstract

The present invention discloses an ELIA kit applicable to aureomycin recidue analysis and its application. Said kit includes kit body, enzyme-labelled plate and reagent, in which said reagent includes washing solution, sample diluent, horseradish peroxidase labeled antibody, substrate solution, color developing solution and stop buffer. The invented kernel lies in that it has the coated antigen, which is coated by coating solution and can be specifically reacted with aureomycin antibody, aureomycin standard solution and aureomycin antibody. Said antibody is the blood serum that is obtained by immunizing rabbit by utilizing artificial immune source, the coated antigen is the compound formed from aureomycin acetic acid and egg-white albumin, and the artificial immune source is the compound composed of aureomycin acetic acid and bovine serum albumin.

Description

technical field [0001] The invention belongs to the technical field of enzyme-linked immunoassay detection for aureomycin residue analysis in animal source food. Specifically, it belongs to an enzyme-linked immunoassay kit suitable for analysis of aureomycin residues and its application. Background technique [0002] Chlortetracycline is one of the main members of tetracyclines. It is a derivative of the core of polycyclic tetracenecarboxamide and has antibacterial effects. wait. [0003] Because aureomycin has inhibitory effect on gram-positive bacteria, gram-negative bacteria, spirochetes, rickettsia, mycoplasma, chlamydia, protozoa, etc., it is widely used. However, irrational drug use can lead to the accumulation and residue of aureomycin in the edible tissues of animals, and then endanger human health. In view of this, it is imperative to monitor the residues of aureomycin in meat, eggs, milk and other animal source foods. [00...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/531
Inventor 袁宗辉赵春保王玉莲常超陈冬梅陶燕飞彭大鹏王帅兵
Owner HUAZHONG AGRI UNIV
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