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Enzyme-linked immunosorbent assay kit for detecting ceftiofur, and applications thereof

An enzyme-linked immunosorbent reagent, ceftiofur technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of limited application range, complicated operation process, difficult to popularize and apply, etc., and achieve convenient use, high sensitivity, and detection method Efficient effect

Inactive Publication Date: 2017-04-26
JIANGSU WISE SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commonly used physical and chemical analysis methods require expensive instruments and equipment, skilled professionals and other conditions, and the operation process is complicated and the detection time is long, so the scope of application is limited, and it is difficult to popularize and apply in actual production

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting ceftiofur, and applications thereof
  • Enzyme-linked immunosorbent assay kit for detecting ceftiofur, and applications thereof
  • Enzyme-linked immunosorbent assay kit for detecting ceftiofur, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The preparation of embodiment 1 kit components

[0031] 1. Preparation of ceftiofur hapten

[0032] A mixture of 440mg ceftiofur, 360mg phthalic acid and 2mL Abitha in 25m1 DMSO was stirred and reacted at 80°C for 15h, the solvent was evaporated, and purified by column chromatography to obtain phthalic acid monoceftiofur amine.

[0033] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown, the two groups of aromatic ring signal peaks increased around 7.8 and 8.3ppm, and the shuttle base signal peak increased around 13.3ppm, indicating that the hapten was successfully synthesized.

[0034] 2. Antigen preparation

[0035] Immunogen preparation—ceftiofur hapten was conjugated with bovine serum albumin (BSA) to obtain immunogen.

[0036] Take 15 mg of the ceftiofur hapten prepared by the above method and dissolve it in 1 mL of DMF to obtain solution (1); take 30 mg of EDC and NHS and dissolve them fully i...

Embodiment 2

[0048] Embodiment 2 detects the formation of the ELISA kit of ceftiofur

[0049] The enzyme-linked immunosorbent assay kit that group law detects ceftiofur, it comprises following component:

[0050] (1) Enzyme plate coated with ceftiofur-conjugated antigen;

[0051] (2) 5 bottles of ceftiofur standard solution, the concentrations are 0μg / L, 0.75μg / L, 3μg / L, 9μg / L, 27μg / L;

[0052] (3) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0053] (4) Ceftiofur-specific antibody;

[0054] (5) The substrate chromogenic solution is composed of A liquid and B liquid, A liquid is urea peroxide, and B liquid is tetramethyl bismuth

[0055] (6) The stop solution is 2mol / L sulfuric acid;

[0056] (7) The washing solution has a pH value of 7.4, contains 0.5%~1.0% Tween-20, 0.01%~0.03%o sodium azide preservative, and 0.1~0.3mol / L phosphate buffer, the Percentages are weight volume percentages;

[0057] (8) The complex solution is a phosphate buffer solution with a pH...

Embodiment 3

[0059] The detection of ceftiofur in the pork tissue of embodiment 3

[0060] 1. Sample pretreatment

[0061] Homogenize the sample with a homogenizer; weigh 3.0±0.05g of the homogenized tissue sample into a 50m1 polystyrene centrifuge tube, add 100μL sample extractant and 900μL deionized water, vortex to mix, and then add 5m1 acetonitrile , use a vortex instrument to vortex for 2min, mix well, centrifuge at 3000g room temperature (20-250℃) for 5min; pipette 3m1 supernatant into a 50m1 polystyrene centrifuge tube, add 1m12M sodium hydroxide solution to mix, and then add 6m1 For n-hexane, vortex for 3min with a vortexer, centrifuge at 3000g room temperature (20-25°C) for 5min; pipette 2m1 of the supernatant into a 10ml clean and dry glass test tube, dry it under nitrogen or air flow at 50-60°C, add 1ml Vortex the reconstituted working solution for 2 min with a vortex instrument; take 50 μL for analysis.

[0062] 2. Detection with kit

[0063] Add 50 μL of standard working so...

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Abstract

The present invention provides an enzyme-linked immunosorbent assay kit for detecting ceftiofur, wherein the kit comprises an enzyme label plate coated with a coating antigen, a ceftiofur standard substance solution, an enzyme-labeled secondary antibody, a ceftiofur specific antibody, a substrate coloring solution, a stop solution, a washing solution, a reconstitution solution, and a sample extraction agent, wherein the coating antigen is a ceftiofur conjugated antigen, and the enzyme-labeled secondary antibody is an enzyme-labeled goat anti-mouse anti-antibody. The invention further discloses a method for detecting the ceftiofur by using the kit, wherein the method comprises: pre-treating a sample, detecting with the kit, and finally analyzing the detection result. According to the present invention, the kit can be used for detecting the ceftiofur content in pork, has advantages of simple operation, low cost, high sensitivity and on-site monitoring, and is suitable for screening of a large number of samples.

Description

technical field [0001] The invention relates to enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting ceftiofur, which can qualitatively and quantitatively detect the residual amount of ceftiofur in pork tissue samples. Background technique [0002] Ceftiofur (ceftiofur), also known as Saidefu, is the first third-generation cephalosporin antibiotic exclusively for animals. In 1988, the FDA approved ceftiofur sodium for the treatment of bovine respiratory bacterial diseases, when the drug was first marketed in the United States. Since then, FDA has successively approved ceftiofur sodium, ceftiofur hydrochloride and ceftiofur crystalline free acid for the treatment of respiratory diseases in 1-day-old chickens, turkeys, pigs, beef cattle, dairy cows, horses, goats and sheep. Canada, Japan and some European countries have also officially approved it for the treatment of respiratory diseases in pigs, beef cattl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535
CPCG01N33/535
Inventor 洪霞刘静立雯馨
Owner JIANGSU WISE SCI & TECH DEV
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