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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 the problems of increasing detection costs, increasing operation steps, and prolonging detection time, so as to reduce detection costs, operation steps, and samples. Handling simple effects

Inactive Publication Date: 2009-08-05
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

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Preparation of chlortetracycline acetic acid, artificial immunogen, coating antigen, and anti-chlortetracycline antibody

[0038] 1.1 Preparation of chlortetracycline acetic acid

[0039] Weigh 300 mg of chlortetracycline hydrochloride (chlortetracycline and chlortetracycline base can be used in other embodiments, and the usage amount is the same as chlortetracycline hydrochloride), dissolve it with 25ml pH10 phosphate buffer, and add 58.5mg chlorine Sodium acetate was fully reacted at room temperature for 10 hours, then filtered, diluted hydrochloric acid to adjust the pH value to 4.0, allowed to stand for 2 hours, suction filtered and dried at 50°C to obtain chlortetracycline acetic acid.

[0040] 1.2 Preparation of artificial immunogen

[0041] Dissolve 118.8mg of chlortetracycline acetic acid with 2ml of N,N-dimethylformamide (DMF), add 55mg of N,N dicyclohexylcarbimide (DCC) and N-hydroxysuccinimide (NHS) while stirring 28.8mg, stirred at 4°C for more than 10...

Embodiment 2

[0060] Example 2 Establishment of chlortetracycline enzyme-linked immunoassay indirect competitive detection method (ELISA)

[0061] 2.1 Determination of the original coating concentration and the working concentration (dilution factor) of the chlortetracycline antibody

[0062] Three reference points of high, medium, and low are determined by the square matrix titration test: the point with the absorbance value of about 1.0 and the largest difference in absorbance between the left and right neighbors is the reference point. Operation steps: Coat the first row of 96-well microtiter plate with 8μg / kg coating source, and then coat the second to seventh rows with 4, 2, 1, 0.5, 0.25, 0.125μg / kg coating original. 4 ℃ overnight, 1% ovalbumin in 37 ℃ for 1 hour, wash twice, pat dry, add 100μl in the first column to the seventh column of the microtiter plate, the dilution multiples of 1000, 2000, 4000, 8000, 16600, 32000, 64000 chlortetracycline antibody, incubate at 37°C for 30 minutes, ...

Embodiment 3

[0098] Example 3 Accuracy and precision test of the present invention

[0099] 3.1 Now take pig muscle as an example to illustrate the method and results of meat tissue sample processing (the same test can use animal muscle tissue such as chicken, duck, cattle, or sheep).

[0100] A standard of chlortetracycline at a concentration of 100μg / kg was added to the pig muscle tissue to be tested, and pig muscle tissue without chlortetracycline was set as a blank as a control, and each concentration was repeated 5 times. Operation steps: Weigh 5 grams of homogenized pig muscle tissue for each sample, prepare test samples and blank samples according to the aforementioned method and dosage, and then add 5 ml of pH4.0 Mcllvain buffer, shake evenly, 4℃, Centrifuge at 4000 rpm, take the supernatant, add 0.3ml of 20% trichloroacetic acid, shake well, centrifuge again, 4℃, 8000 rpm, take the supernatant, dilute 10 times and adjust the pH to 7.4 (add 1N NaOH About 100 microliters is enough), tak...

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Abstract

The invention discloses an enzyme-linked immunoassay kit suitable for the analysis of aureomycin residues and an application thereof. The kit provided by the present invention includes a box body, an ELISA plate and reagents arranged in the box body, wherein the reagents include washing solution, sample diluent, horseradish peroxidase-labeled goat anti-rabbit antibody, substrate solution, color development solution and stop solution. The core of the present invention lies in the coated antigen that can specifically react with the aureomycin antibody that is coated with the coating solution, the aureomycin standard solution and the aureomycin antibody, and the antibody is the serum obtained by immunizing rabbits with an artificial immunogen . The coating antigen is a complex of aureomycin acetic acid and ovalbumin. The artificial immunogen is a complex of aureomycin acetic acid and bovine serum albumin. The kit of the invention has strong specificity and high sensitivity.

Description

Technical field [0001] The invention belongs to the technical field of enzyme-linked immunoassay for residual analysis of chlortetracycline in animal source foods. Specifically, it belongs to an enzyme-linked immunoassay kit suitable for chlortetracycline residue analysis and its application. Background technique [0002] Chlortetracycline is one of the main members of tetracycline drugs. It is a derivative of polycyclic tetracenecarboxamide core and has antibacterial effect. Its similar drugs mainly include tetracycline, oxytetracycline, desmethylchlortetracycline and doxycycline Wait. [0003] Since chlortetracycline has an inhibitory effect on gram-positive bacteria, gram-negative bacteria, spirochetes, rickettsiae, mycoplasma, chlamydia, protozoa, etc., its application is extremely wide. However, irrational use of drugs can cause chlortetracycline to accumulate residues in the edible tissues of animals, and then endanger human health. In view of this, it is imperative to moni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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