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ELISA kit for detecting fluoromethylquinoline and detection method thereof

An enzyme-linked immunosorbent reagent, the technology of flumequine, is applied in the direction of measuring devices, material analysis and instruments through observation of the influence on chemical indicators, and can solve complex instruments and equipment, which are not suitable for on-site monitoring and screening of a large number of samples, Solve problems such as cumbersome process, achieve the effect of improving sensitivity, saving operation time, and being easy to carry

Inactive Publication Date: 2006-10-11
贵州勤邦食品安全科学技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The chemical methods for detecting flumequine residue mainly include thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), gas-mass spectrometry (GC / MS), liquid-mass spectrometry ( HPLC / MS), capillary electrophoresis (CE), etc., are not suitable for on-site monitoring and screening of a large number of samples due to complex equipment and cumbersome processes

Method used

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  • ELISA kit for detecting fluoromethylquinoline and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 Preparation of kit components

[0063] 1. Antigen Synthesis

[0064] a. Synthesis of hapten

[0065] Flumequine and 6-aminocaproic acid are obtained by acylation.

[0066] Specific steps for the hapten: take 200 mg of flumequine, 100 mg of 6-aminocaproic acid and 50 mg of NHS (activated ester) and stir with water overnight.

[0067] b. Immunogen synthesis

[0068] The immunogen was obtained by coupling the flumequine hapten with rabbit serum albumin by the carbodiimide method.

[0069] c. Preparation of coated pro-flumequine-conjugated antigen

[0070] The immunogen was obtained by coupling the flumequine hapten with human serum albumin by a carbodiimide method.

[0071] The preparation process of the immunogen: take 100 mg of EDC, dissolve it fully with 2.5 ml of 10 mmol / LPBS solution of pH 8.0 (I liquid); take 15 g of the flumequine hapten, dissolve it with 2 ml of 0.2 oml / L NaOH solution (II liquid); Take 30mg of rabbit serum albumin, dissolve it i...

Embodiment 2

[0090] Example 2 The formation of an enzyme-linked immunosorbent assay kit for detecting flumequine

[0091] An ELISA kit for detecting flumequine was set up to include the following components:

[0092] (1) a microtiter plate coated with flumequine-coupled antigen;

[0093] (2) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0094] (3) Flumequine monoclonal antibody working solution;

[0095] (4) 6 bottles of flumequine standard solution, the concentrations are 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, 81 μg / L;

[0096] (5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A liquid is carbamide peroxide, and the substrate chromogenic liquid B liquid is tetramethylbenzidine;

[0097] (6) The stop solution is 2mol / L hydrochloric acid;

[0098] (7) The concentrated washing solution is 0.02M, pH 7.4, and contains 0.8%-1.2% Tween 80 and 0.5% thimerosal preservative phosphate buffer;

[0099] (8) The c...

Embodiment 3

[0100] The detection of flumequine residue in the sample of embodiment 3

[0101] 1. Sample pretreatment

[0102]Animal tissue (chicken, shrimp): Weigh 2.0g of homogenized tissue sample into a 50ml centrifuge tube, add 8ml of PB buffer. Fully mix up and down for 30 minutes, add 10ml of dichloromethane, mix well for 10 minutes, over 3000g, centrifuge at 15°C for 10 minutes, take 5ml of the upper organic phase into a dry bottle (clear and free of impurities), and rotary evaporate at 50°C until dry / dry with nitrogen , vortexed with 1ml of the diluted complex solution for 30s, dissolved the dry residue, added 1ml of n-hexane and mixed for 30s, above 3000g, centrifuged at 15°C for 10min (if the lower layer is obviously turbid, please repeat this step), gently suck off the upper organic layer For the phase and the middle part of the liquid, take the lower layer 100μl + 100μl PB buffer and mix well for analysis. (the sample was diluted 2 times)

[0103] 2. Detection with kit

[0...

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Abstract

The invention discloses an enzyme immunity test agent box for testing the apurone drug, which comprises: an enzyme mark plate that coated by coat source; enzyme mark matter; apurone specific antibody working solution (when the enzyme mark plate is coated by antibody and the enzyme mark matter is the enzyme mark antibody or the enzyme mark plate is coated by the anti antibody and the enzyme mark matter is the enzyme mark antigen); apurone standard solution; base color display solution; stop solution; concentrated washing solution; and concentrated compound solution. The invention also discloses a method for using said agent box to test apurone, which comprises: processing pretreatment on the sample; then using test box to test; at last analyzing the test result. The inventive agent box can be used to test the left amount of apurone in chicken and shrimp, etc, with simple operation, lower cost, and high sensitivity.

Description

technical field [0001] The invention relates to an ELISA kit and method for detecting flumequine in animal tissues. Background technique [0002] Flumequine is the second-generation fluoroquinone antibacterial drug, which has good curative effect on livestock, poultry and aquatic animal diseases caused by Escherichia coli, mycoplasma and Aeromonas hydrophila, because of its high safety and tolerance , high biological activity and unique high permeability in tissues are therefore widely used, and are widely used for treatment, prevention and growth promotion. Due to its drug resistance and potential carcinogenicity, it has received the attention of residue monitoring at the same time as its application. [0003] The chemical methods for detecting flumequine residue mainly include thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), gas-mass spectrometry (GC / MS), liquid-mass spectrometry ( HPLC / MS), capillary electrophoresi...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/78G01N33/577
Inventor 沈建忠何方洋万宇平冯才伟吴小平罗晓琴
Owner 贵州勤邦食品安全科学技术有限公司
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