Magnetic-particle chemiluminescence kit used for detecting monensin and applications of the kit

A chemiluminescence reagent, the technology of monensin, which is applied to a magnetic particle chemiluminescence kit for detecting monensin and its application field, can solve the problem of low false positive rate, influence of reaction time and temperature, poor reagent stability, etc. problem, to achieve the effect of low detection time, fast detection and high sensitivity

Inactive Publication Date: 2014-02-19
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 2. Gas chromatography-mass spectrometry (GC-MS) has high sensitivity and low false positive rate
The react

Method used

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  • Magnetic-particle chemiluminescence kit used for detecting monensin and applications of the kit

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0035] Example 1 Preparation of specific components of the kit

[0036] 1) Preparation of monensin artificial antigen

[0037] The immunogen was obtained by coupling monensin with bovine serum albumin (BSA) by activated ester method (NHS-DCC).

[0038] 3) Preparation of monoclonal antibodies

[0039] Animal immunization: Balb / c mice were immunized with immunogen at a dose of 100 μg / mice to produce antiserum.

[0040] Cell fusion and cloning: take the spleen cells of immunized Balb / c mice and fuse with SP2 / 0 myeloma cells in a ratio of 9:1 to obtain a hybridoma cell line of monoclonal antibody.

[0041] Cell cryopreservation and recovery: make 1×10 hybridoma cells with cryopreservation solution 9 cells / ml, and stored in liquid nitrogen for a long time. When resuscitated, take out the cryopreservation tube, immediately put it into a 37°C water bath to thaw quickly, remove the cryopreservation solution by centrifugation, and transfer it to a culture bottle for culture.

[00...

Example Embodiment

[0052] Example 2 Construction of the kit

[0053] A magnetic particle direct competition chemiluminescence detection kit for the detection of monensin was constructed to contain the following components:

[0054] Fluorescent label for FITC-labeled monensin monoclonal antibody

[0055] Luminescent label for ABEI-labeled monensin hapten

[0056] Separation reagent for paramagnetic nanobeads coated with goat anti-FITC monoclonal antibody

[0057] Monensin standard solution (0ng / ml, 0.02ng / ml, 0.06ng / ml, 0.18ng / ml, 0.54ng / ml, 1.6ng / ml), the standard dilution solution is pH7.2, 0.03%NaN 3 , 0.1mol / L PBS buffer. The percentage content is the mass percentage content.

[0058] The concentration of monens quality control solution is 0.05ng / ml, 1.0ng / ml, and the dilution of quality control solution is pH7.2, 0.03%NaN 3 , 0.1mol / L PBS buffer. The percentage content is the mass percentage content.

[0059] The concentrated wash solution is pH7.2, 0.2-0.4% Tween-20, 0.02-0.04% NaN ...

Example Embodiment

[0060] Embodiment 3 Detection of monensin in actual samples

[0061] 1. Sample pretreatment

[0062] (1) Pork / liver pretreatment method

[0063] Homogenize the sample with a homogenizer; weigh 3.0±0.05g of the homogenate into a 50ml polystyrene centrifuge tube, add 5ml methanol, then add 1ml deionized water, shake for 5min, above 3000g, room temperature (20-25℃) Centrifuge for 5min; take 2ml of supernatant into a 50ml plastic centrifuge tube, add 2ml of 1M sodium hydroxide solution and mix, then add 6ml of n-hexane, vortex for 3min, centrifuge at 3000g or more at room temperature (20-25℃) for 5min; take 3ml Transfer the supernatant to a 10ml glass test tube, blow dry under nitrogen flow at 50-60°C, add 1ml of reconstituted working solution and vortex for 2-3min; take 50μl for analysis, sample dilution ratio: 2.

[0064] (2) Pre-treatment method of chicken / liver

[0065] Homogenize the sample with a homogenizer; weigh 3.0±0.05g of the homogenate into a 50ml polystyrene centr...

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Abstract

The invention relates to a magnetic-particle chemiluminescence kit used for detecting monensin. Reagents of the kit comprise a luminescent marker, a fluorescein marker, a standard substance, a quality control substance and a separating reagent. The luminescent marker relates to monensin semiantigen marked by an isoluminol luminescent maker. The fluorescein marker relates to a monensin monoclonal antibody marked by fluorescein or derivatives thereof. The separating reagent relates to paramagnetic nano beads coated with antigoat FITC monoclonal antibody. The invention also relates to a method of detecting the monensin in animal-source food by using the kit. The method has high sensitivity, high specificity and rapid detection speed for monensin detection.

Description

technical field [0001] The invention relates to a direct chemiluminescence detection kit and a detection method thereof. In particular, it is a magnetic particle competition direct chemiluminescent detection kit for the detection of monensin drug residues in animal tissues, feed and other samples. Background technique [0002] Monensin is a polyether antibiotic widely used in the treatment of animal diseases. Its main mechanism of action is to interfere with the normal exchange of sodium and potassium ions in coccidian cells, and a large amount of sodium ions enter the cells, causing sodium The concentration of ions in the cell is high, the osmotic pressure rises, and more water enters the coccidian. In order to remove the excess sodium in the cell, the coccidia must use energy to pump out the excess sodium, and eventually the limited energy is used up. ; Then the sodium and potassium ion exchange was interrupted and stopped, and the cells swelled, deformed, ruptured, and d...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/54326G01N33/577G01N2446/00G01N2446/80
Inventor 何方洋周德刚冯静岳欣荣韩京朋余厚美
Owner BEIJING KWINBON BIOTECH
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