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Magnetic granule chemiluminescence kit for detecting melamine and application of magnetic granule chemiluminescence kit

A chemiluminescence reagent, melamine technology, applied to a magnetic particle chemiluminescence kit for melamine detection and its application field, can solve the problems of complicated operation, high detection cost, low false positive rate and the like, and achieves low detection time, fast detection, Sensitive effect

Inactive Publication Date: 2012-07-11
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The main disadvantages of the HPLC method are that the instrument is expensive, the operation is cumbersome, time-consuming, the detection cost is expensive, the professional requirements for the detection personnel are high, and the sample pre-treatment is complicated
[0004] 2. Gas chromatography-mass spectrometry (GC-MS) has high sensitivity, low false positive rate, and the minimum detection limit for detecting melamine residues is 0.05mg / Kg
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 granule chemiluminescence kit for detecting melamine and application of magnetic granule chemiluminescence kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 kit specific components

[0036] 1. Preparation of luminescent markers

[0037] a) Synthesis of haptens

[0038] Take 0.5g (3.97mmol) of melamine in a dry 50ml round bottom flask, add 35ml of DMF and stir to dissolve, take 0.44g (7.93mmol) of KOH and 0.12g of anhydrous NaI into the above solution, add dropwise 0.646ml of tertiary bromoacetic acid Butyl ester, after being fully mixed, heated to 65°C for 10h. After the reaction, cool to room temperature, dilute with water, extract with ethyl acetate, dry, and rotary evaporate to obtain a yellow oil, ethyl acetate:petroleum ether=2:1, pass through the column to obtain the melamine hapten.

[0039] b) Preparation of luminescent markers

[0040] Take 4.5mmol / L ABEI, dissolve it in 4ml distilled water, dissolve 5.0mmol / L N-hydroxysuccinimide in 0.5ml N,N-dimethylformamide, mix the two thoroughly and react at room temperature for 3-4h . Take 15 mg of the above-prepared melamine hapten, adju...

Embodiment 2

[0056] The formation of embodiment two kits

[0057] A magnetic particle chemiluminescent detection kit for detecting melamine was assembled so that it contained the following components:

[0058] Fluorescent marker of FITC-labeled melamine monoclonal antibody

[0059] ABEI-labeled luminescent marker of melamine hapten

[0060] Separation reagent of paramagnetic nanospheres coated with anti-goat FITC monoclonal antibody

[0061] Melamine standard solution (0ng / ml, 0.01ng / ml, 0.03ng / m, 0.09ng / ml, 0.27ng / ml, 0.81ng / ml), the standard diluent is pH7.2, 0.3%NaN 3 , 0.3mol / L borate buffer. The percentage content is a mass percentage content.

[0062] The concentration of melamine quality control solution is 0.02ng / ml, 0.5ng / ml respectively, and the standard diluent is pH7.5, 0.3%NaN 3 , 0.3mol / L borate buffer. The percentage content is a mass percentage content.

[0063] Concentrated lotion is PH7.4, 0.3% Tween-20, 0.4% NaN 3 , 0.2mol / L PBS buffer solution. The percentage c...

Embodiment 3

[0064] The detection of melamine in embodiment three actual samples

[0065] 1. Sample pretreatment

[0066] (1) Milk sample pretreatment method:

[0067] Take 400 μl fresh milk sample; add 800 μl acetonitrile, vortex for 1 min, take 100 μl supernatant and add 500 μl reconstitution working solution, mix well; take 50 μl for analysis, sample dilution factor: 20.

[0068] (2) Pretreatment method of milk powder sample:

[0069] Weigh 1.0±0.05g milk powder sample into a 10ml centrifuge tube, add 2.5ml deionized water, vortex for 1min; pipette 400μl into a 2ml centrifuge tube, add 400μl acetonitrile, then add 400μl methanol, vortex for 1min, above 3000g, Centrifuge at room temperature (20-25°C) for 3-5min; take 100μl supernatant plus 500μl reconstitution working solution, mix well; take 50μl for analysis, sample dilution factor: 20

[0070] (3) Feed sample pretreatment method:

[0071] Weigh 2.0±0.05g of crushed feed sample into a 50ml polystyrene centrifuge tube; add 9ml of me...

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Abstract

The invention relates to a magnetic granule chemiluminescence kit for detecting melamine, which comprises the following reagents: a luminous marker, a fluorescein marker, a standard substance, a quality control substance and a separation reagent. The luminous marker is melamine hapten marked by an isoluminol luminous marker; the fluorescein marker is a melamine monoclonal antibody marked by fluorescein or a fluorescein derivative; and the separation reagent is paramagnetic nano micro-beads enveloped in an anti-goat FITC (fluorescein isothiocyanate) monoclonal antibody. The invention further relates to a method adopting the kit to detect the melamine in animal derived food; and the method has higher sensitivity, specificity and detecting speed for melamine.

Description

technical field [0001] The invention relates to a chemiluminescence detection kit and a testing method thereof, in particular to a magnetic particle chemiluminescence detection kit for detecting melamine content in samples such as dairy products and feed. technical background [0002] At present, the routine detection methods of melamine residue mainly include high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunoassay (ELISA), etc. . [0003] 1. High-performance liquid chromatography (HPLC) has the characteristics of high detection accuracy and low false positive rate, and the minimum detection limit for detecting melamine residues is 2 mg / kg. The main disadvantages of the HPLC method are that the instrument is expensive, the operation is cumbersome, time-consuming, the detection cost is expensive, the professional requirements of the detection personnel are high,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/52
Inventor 何方洋万宇平罗晓琴徐念琴陈炜玲刘玉梅郝士元何丽霞
Owner BEIJING KWINBON BIOTECH
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