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ELISA kit for rapidly detecting monensin

An enzyme-linked immunosorbent reagent, monensin technology, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of cumbersome pretreatment steps, long analysis time, and inappropriate determination of a large number of samples, and achieves reduction of operation steps, time saving effect

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

[0003] At present, HPLC and LC/MS/MS are the main methods for determining residues in animal-derived foods. Due to the shortcomings of such methods, such as lo

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0043] Example 1 Preparation of kit components

[0044] 1. Antigen synthesis

[0045] a. Take 10 g of carrier bovine serum albumin (BSA) and dissolve it in 35 mL of pH 9.6 carbonate buffer, and add 10 g of ethylenediamine for activation;

[0046] b. Dissolve 1g of monensin in 10ml of 0.5M sodium hydroxide solution;

[0047] c. Dissolve 1g carbodiimide in 5ml pure water, then add it to the monensin solution and stir for 3 hours at room temperature;

[0048] d. Add the activated carrier protein BSA dropwise to the monensin solution and stir overnight at 4°C;

[0049] e. Dialysis the obtained artificial antigen against 0.1M PBS buffer for 5 days, changing the buffer 4 times a day; concentrating the obtained purified artificial antigen by ultrafiltration or freeze-drying for storage.

[0050] 2. Preparation of monoclonal antibodies for monensin drugs

[0051] a. Animal immunization procedure: use monensin artificial antigen as immunogen to immunize BALB / C mice with an immunization dose of 50 ...

Example Embodiment

[0066] Example 2 Establishment of an enzyme-linked immunoassay kit for rapid detection of monensin

[0067] This enzyme-linked immunoassay kit consists of the following components:

[0068] (1) Enzyme-labeled plate coated with monensin antigen;

[0069] (2) Monensin monoclonal antibody working solution with a protein concentration of 0.5ug / L;

[0070] (3) The concentration is 1μg / mL monensin standard product;

[0071] (4) Standard dilution: 0.05mol / LTris-HCl, pH8.0, 0.9% NaCl buffer.

[0072] (5) Goat anti-mouse enzyme-labeled secondary antibody working solution labeled with horseradish peroxidase;

[0073] (6) The level of the substrate color-developing solution A is hydrogen peroxide, and the level of the substrate color-developing solution B is o-phenylenediamine;

[0074] (7) Washing buffer is 0.05mol / LTris-HCl, pH8.0, 0.9% NaCl, 0.04% Tween20.

[0075] (8) The stop solution is 2mol / L sulfuric acid.

Example Embodiment

[0076] Example 3 Detection of monensin residues in samples

[0077] 1. Sample pretreatment

[0078] a. Tissue sample: Use a homogenizer to homogenize the sperm or visceral tissue sample, accurately weigh 4g of the sample, add 4mL ethyl acetate, shake for 10 minutes, centrifuge at 4000rpm / min at room temperature for 15 minutes, take out 2mL of upper liquid, and 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 4000rpm / min at room temperature for 5 minutes, remove the upper oil phase, and take 50μl of the lower water phase for determination.

[0079] b. Urine: Take 2mL urine into the centrifuge tube, add 4mL ethyl acetate, shake for 5 minutes, centrifuge at 4000rpm / min at room temperature for 5 minutes, take out 2mL upper liquid, dry at 50-60℃ under nitrogen flow, add 1mL standard product The diluent was shaken and dissolved for 1 min, and 50 μl was taken for measurement.

[008...

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Abstract

The invention provides an ELISA kit for detecting the residual quantity of monensin in an animal-derived food, and relates to an immunoassay detection technology. The kit comprises an enzyme labeled plate coated with a monensin antigen, a monensin antibody, a monensin standard substance, a standard substance dilution solution, an enzyme labeled secondary antibody, a substrate coloring solution, a washing buffer solution and a stopping solution. A monensin monoclonal antibody is prepared in the invention. The kit adopts the highly specific monensin monoclonal antibody, and a main reagent is provided with as an operating fluid, so the operating steps are reduced, the time is saved, and the residual quantity of monensin in a meat product especially is detected.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunoassay detection, and in particular relates to the preparation of a monensin monoclonal antibody and an enzyme-linked immunoassay kit. The monoclonal antibody of the present invention is secreted by the hybridoma cell line C2F3C2 established by the applicant. The invention discloses a preparation method of a specific monoclonal antibody, a coating agent and an immunogen and an enzyme-linked immunoassay method. Compared with the prior art, the present invention broadens the detection objects of the prior art. The kit and method of the present invention have the advantages of simplicity, rapidity, sensitivity, accuracy, etc., and can simultaneously detect animal-derived foods, especially meat products. Residues of phenomycin. Background technique [0002] Monensin belongs to the class of antibiotics and is used for coccidiosis in chickens and growth promotion of b...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 洪霞戴蔚蔚
Owner JIANGSU WISE SCI & TECH DEV
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