Magnetic particle chemiluminescence kit for detecting trenbolone, and applications thereof

A chemiluminescence reagent and kit technology, applied in chemiluminescence/bioluminescence, analysis by chemical reaction of materials, measurement devices, etc., can solve the problems of poor reagent stability, reaction time and temperature effects, etc. The effect of low time, fast detection and high sensitivity

Inactive Publication Date: 2013-02-13
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reaction time and temperature have a great influence on the enzyme activity, and the stability of the reagent is poor

Method used

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  • Magnetic particle chemiluminescence kit for detecting trenbolone, and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of the concrete component of embodiment one kit

[0036] 1) Synthesis of Trenbolone Hapten

[0037] Synthesize trenbolone hapten from trenbolone by succinic anhydride method.

[0038] 2) Preparation of artificial antigen

[0039] The trenbolone hapten and bovine serum albumin were coupled to the immunogen by the carbodiimide method.

[0040] 3) Preparation of monoclonal antibodies

[0041] Animal immunization: Immunize Balb / c mice with the immunogen at a dose of 100 μg / mouse to produce antiserum.

[0042] Cell fusion and cloning: Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 9:1 to obtain hybridoma cell lines of monoclonal antibodies.

[0043] Cell cryopreservation and recovery: the hybridoma cells were made into 1×10 cryopreservation medium 9 cells / ml for long-term storage in liquid nitrogen. When recovering, take out the cryopreservation tube, put it into a 37°C water bath to thaw quickly, remove the cry...

Embodiment 2

[0054] The formation of embodiment two kits

[0055] The magnetic particle chemiluminescence kit for detection of trenbolone was assembled to contain the following components:

[0056] Fluorescent marker of FITC-labeled trenbolone monoclonal antibody

[0057] Luminescent marker of ABEI-labeled trenbolone hapten

[0058] Separation reagent of paramagnetic nanobeads coated with goat anti-FITC monoclonal antibody

[0059] Trenbolone standard solution (0ng / ml, 0.01ng / ml, 0.03ng / ml, 0.1ng / ml, 0.5ng / ml, 1.0ng / ml), the standard diluent is pH7.4, 0.03%NaN 3 , 0.1mol / LPBS buffer. The percentage content is a mass percentage content.

[0060] The concentrations of Trenbolone quality control solution are 0.02ng / ml and 0.8ng / ml respectively, and the quality control dilution solution is pH7.4, 0.03% NaN 3 , 0.1mol / LPBS buffer. The percentage content is a mass percentage content.

[0061] Concentrated lotion is PH7.2, 0.2-0.4% Tween-20, 0.2-0.4% NaN 3 , 0.1-0.2mol / L PBS buffer soluti...

Embodiment 3

[0062] The detection of trenbolone in the actual sample of embodiment three

[0063] 1. Sample pretreatment

[0064] (1) Feed, tissue (chicken / liver, pork / liver, shrimp, fish, etc.) pretreatment method

[0065] Use a homogenizer to homogenize tissue and feed samples; weigh 2.0±0.05g of homogenate into a 50ml polystyrene centrifuge tube, add 10ml of acetonitrile-0.1M sodium hydroxide solution, shake with an oscillator for 10min, over 3000g, and store at room temperature ( 20-25°C) and centrifuge for 10min; pipette 0.5ml supernatant into a 50ml polystyrene centrifuge tube, add 0.5ml 0.1M sodium hydroxide solution, add 5ml chloroform after shaking, vortex for 2min with a vortex instrument, Above 3000g, centrifuge at room temperature (20-25°C) for 10min. (If the sample is emulsified, put it in a water bath at 70°C for 2-4 minutes, and centrifuge again until the lower layer is clear); remove the upper layer, take 1ml of the lower layer of clear organic phase into a 10ml clean gla...

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Abstract

The present invention relates to a magnetic particle chemiluminescence kit for detecting trenbolone. The kit comprises a luminous marker, a fluorescein marker, a standard substance, a quality control substance and a separation reagent, wherein the luminous marker is isoluminol luminous marker labeled trenbolone hapten, the fluorescein marker is fluorescein labeled trenbolone monoclonal antibody or fluorescein derivative labeled trenbolone monoclonal antibody, and the separation reagent is sheep anti-FITC monoclonal antibody coated paramagnetic nanometer microbeads. The present invention further relates to a method for detecting trenbolone in foods of animal origin by using the kit, wherein the method provides characteristics of high sensitivity, high specificity and rapid detection for trenbolone detection.

Description

technical field [0001] The invention relates to a direct chemiluminescent detection kit and a testing method thereof. In particular, the magnetic particle competition direct chemiluminescent detection kit for the detection of trenbolone drug residues in feed, aquatic products, urine, tissue and other samples. technical background [0002] The chemical name of trenbolone is norandrostrienolone, which is a synthetic steroidal male hormone and a growth promoter for livestock. Human consumption of meat products with residues of this substance will bring harm to health. In addition, trenbolone has emerged as a possible source of doping in sports competitions. The European Union prohibits the import of food using this type of product, and it is also one of the 31 kinds of veterinary drugs prohibited by the customs in my country. [0003] Currently, the detection methods of trenbolone include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/532G01N33/533G01N21/76
Inventor 冯才伟罗晓琴冯月君扶胜聂雯莹张荃
Owner BEIJING KWINBON BIOTECH
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