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Time-resolved fluoroimmunoassy kit for detecting race-quantity mebendazole residual

A time-resolved fluorescence and mebendazole technology, which is applied in the field of immunological detection, can solve the problems of low sensitivity and narrow emission spectrum band, and achieve the effects of high sensitivity, reduced operation steps and high accuracy

Inactive Publication Date: 2016-05-11
JIANGSU WISE SCI & TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2) The emission spectrum band is very narrow
[0007] 3) The Stokes shift (the difference between the maximum excitation wavelength and the maximum emission wavelength) is large
The main advantage of this method is that it is not sensitive to contamination, it can be applied to various immunoassays by using general immunological reagents, and this method can also give spatial positioning information; the disadvantage of this method is its low sensitivity

Method used

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  • Time-resolved fluoroimmunoassy kit for detecting race-quantity mebendazole residual

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The preparation of embodiment 1 kit components

[0058] 1. Antigen synthesis

[0059] a. Acylate the alcoholic hydroxyl group in the molecular structure of mebendazole with glutaraldehyde anhydride to obtain the hapten of mebendazole

[0060] b. Dissolve 1 g of the hapten of the mebendazole drug in 10 ml of 0.5 M sodium hydroxide solution.

[0061] c. Dissolve 1 g of carbodiimide in 5 ml of pure water, then add it to the hapten solution and stir at room temperature for 3 hours.

[0062] d. Dissolve 10 g of the carrier protein ovalbumin (OVA) or bovine serum albumin (BSA) in 35 mL of pH9.6 carbonate buffer solution, and add the carrier protein dropwise to the hapten and react overnight at 4°C.

[0063] e. The artificial antigen obtained after the reaction was dialyzed against 0.1M PBS buffer for 5 days, and the buffer was changed 4 times a day; the obtained purified artificial antigen was concentrated by ultrafiltration or lyophilized for storage.

[0064] 2. Preparat...

Embodiment 2

[0077] Example 2 Formation of Time-Resolved Fluorescence Immunoassay Kit for Detection of Trace Mebendazole Residues

[0078] A time-resolved immunoassay kit for detecting mebendazole was constructed to include the following components:

[0079] (1) ELISA plate coated with Mebendazole drug antigen

[0080] (2) Eu 3+ Labeled mebendizumab working solution

[0081] (3) Mebendazole standard with a concentration of 1 μg / mL

[0082] (4) Washing buffer: 0.05mol / LTris-HCl, pH8.0, 0.9%NaCl, 0.04%Tween20.

[0083] (5) Standard diluent: 0.05mol / LTris-HCl, pH 8.0, 0.9%NaCl buffer.

[0084] (6) Fluorescence enhancement solution: 0.1mol / L potassium hydrogen phthalate-acetic acid buffer solution, containing 15umol / L β-NTA, 0.1% TritonX-100.

Embodiment 3

[0085] The detection of mebendazole drug residue in the sample of embodiment 3

[0086] 1. Sample pretreatment

[0087] a. Tissue samples: Homogenize lean meat or visceral tissue samples with a homogenizer, accurately weigh 4g of the sample, add 4mL of ethyl acetate, shake for 10 minutes, centrifuge at room temperature for 15 minutes at 4000rpm / min, take out 2mL of the upper layer, and place it under nitrogen flow Dry at 50-60°C, add 1mL of n-hexane to dissolve the dried residue, add 1mL of standard diluent, shake vigorously for 1min, centrifuge at room temperature at 4000rpm / min for 5min, remove the upper oil phase, and take 50μl of the lower aqueous phase for measurement.

[0088] b. Urine: Take 2mL of urine into a centrifuge tube, add 4mL of ethyl acetate, shake for 5 minutes, centrifuge at 4000rpm / min at room temperature for 5 minutes, take out 2mL of the upper layer, dry it at 50-60°C under nitrogen flow, and add 1mL of standard The diluent was shaken and dissolved for 1...

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Abstract

The invention provides a time-resolved fluoroimmunoassy kit for detecting race-quantity mebendazole residual, and relates to a time-resolved fluorescence technology and an enzyme-linked immunoassay technology. The kit comprises a mebendazole drug antigen coated ELISA plate, an Eu<3+>-labeled mebendazole monoclonal antibody, a mebendazole standard substance, a washing buffer solution, a standard dilution solution and a fluorescence enhancement solution. The time-resolved fluoroimmunoassy kit for detecting race-quantity mebendazole residual is produced through comprehensively adopting the time-resolved fluorescence technology, a protein coupling technology and a biochemical preparation technology. A fluorescence antibody is prepared through coupling an anti-mebendazole monoclonal antibody with Eu<3+>, and the race-quantity mebendazole residual is detected through an enzyme linked immunosorbent assay completing method. The time-resolved fluoroimmunoassy kit has the advantages of simple structure, use convenience, low price, portability, high sensitivity, and suitableness for onsite massive screening. Compared with kits adopting the ELISA method, the kit provided by the invention is sensitive and efficient, and avoids interferences of some matrix substances.

Description

technical field [0001] The invention belongs to the field of immunological detection, and in particular relates to a time-resolved fluorescent immunoassay kit for detecting trace mebendazole residues and a preparation method thereof. It is applied to the detection of trace amounts of mebendazole residues in food of animal origin, especially in meat products. Background technique [0002] Mebendazole is a broad-spectrum anthelmintic drug. Both in vivo and in vitro tests have proved that it can directly inhibit the intake of glucose by nematodes, leading to the depletion of glycogen and making it unable to survive. It has a significant effect of killing larvae and inhibiting the development of eggs. It can be used to prevent and control intestinal parasitic diseases such as hookworms, roundworms, pinworms, whipworms, and sterculoid nematodes. Animal experiments have shown that this product can cause teratogenicity, so the residues of mebendazole in animal food pose a huge thre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531G01N21/64
Inventor 洪霞戴蔚蔚杜霞
Owner JIANGSU WISE SCI & TECH DEV
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