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Method of detecting monensin by immunomagnetic bead purification-enzyme-linked immunoassay and special monoclonal antibody used therein

A monoclonal antibody, monensin technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, anti-bacterial immunoglobulin, etc., can solve the problems of limited sensitivity, high degree of instrumentation, cumbersome processing, etc. Achieve the effect of improving detection sensitivity, improving work efficiency and high separation efficiency

Active Publication Date: 2014-09-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Microbiological methods are economical and simple, but have limited sensitivity
Because monensin lacks ultraviolet, fluorescent and electrochemical characteristics, it must be derivatized to introduce chromophores based on HPLC. Although the sensitivity is high, the pretreatment process is cumbersome
The emergence of liquid chromatography tandem mass spectrometry technology avoids the derivatization process in the pretreatment, and the sensitivity is further improved. It is a definitive method for the detection of various veterinary drug residues. The detection limit is 0.05-0.2 μg / kg, but the degree of instrumentation is high. It is expensive and requires professional operators, which is not conducive to the promotion at the grassroots level and the screening of a large number of samples

Method used

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  • Method of detecting monensin by immunomagnetic bead purification-enzyme-linked immunoassay and special monoclonal antibody used therein
  • Method of detecting monensin by immunomagnetic bead purification-enzyme-linked immunoassay and special monoclonal antibody used therein
  • Method of detecting monensin by immunomagnetic bead purification-enzyme-linked immunoassay and special monoclonal antibody used therein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the preparation of immunogen and coating former

[0028] 1. Preparation of immunogen

[0029] (1) Dissolve 20 mg of monensin in 2 mL of DMF, then add 20 μL of tri-n-butylamine, then add 10 μL of isobutyl chloroformate, and shake at 120 rpm for 30 minutes at room temperature to obtain reaction solution I.

[0030] (2) Take 50 mg of BSA and dissolve it in 3 mL of carbonate buffer solution with pH 9.6 and 0.05 M to obtain reaction solution II.

[0031] (3) Add the reaction solution I dropwise to the reaction solution II, then shake at room temperature for 24 hours.

[0032] (4) Put the solution obtained in step (3) into a dialysis bag, dialyze in pH7.4, 0.01M PBS buffer for 3 days (change the liquid twice a day), then centrifuge at 10000rpm for 10min, and collect the supernatant, which is Immunogen solution, aliquoted and stored at -20°C.

[0033] 2. Preparation of coating agent

[0034] (1) Dissolve 20 mg of monensin in 1.5 mL of DMF, then add 25 mg of E...

Embodiment 2

[0041] Embodiment 2, the acquisition of hybridoma cells

[0042] First immunization: fully emulsify the immunogen solution with the same amount of complete Freund's adjuvant, and inject 6-week-old Balb / c mice subcutaneously, 0.2 mL each;

[0043] Booster immunization twice: from the first immunization, booster immunization once every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the method and dosage are the same as the first immunization;

[0044] One week after the last booster immunization, the fundus vein blood was collected to measure the titer and inhibition. When there was inhibition and the titer reached 1:10000 or more, the last immunization was performed as follows: intraperitoneal injection of 0.1 mL of immunogen solution without any adjuvant, and the mice were killed three days later , whose spleen was fused with myeloma cells.

[0045] A hybridoma cell line stably secreting monoclonal antibody was named MON. Monensin monoclo...

Embodiment 3

[0046] Example 3, Preparation of monensin monoclonal antibody

[0047] The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate to RPMI-1640 medium, the final concentration of calf serum is 20% (mass percentage composition), the final concentration of sodium bicarbonate is 0.2 % (mass percentage).

[0048] The monensin monoclonal antibody hybridoma cell line MON was placed in the cell culture medium, cultured at 37°C for 2 days, and the obtained cell culture supernatant was purified by the octanoic acid-saturated ammonium sulfate method to obtain monensin monoclonal antibody Cloning antibody solution (stored at -20°C).

[0049] Protein concentration in monoclonal antibody solution (mg / ml) = 1.45×OD 280 -0.74×OD 260 .

[0050] Protein concentration in monensin monoclonal antibody solution = 3.209 mg / ml.

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Abstract

The invention discloses a method of detecting monensin by immunomagnetic bead purification-enzyme-linked immunoassay and a special monoclonal antibody used therein. The accession number of a monensin monoclonal antibody hybridoma cell strain MON (hybridoma cell strain MON for short) provided by the invention is CGMCC No.8952. The monoclonal antibody secreted by the hybridoma cell strain MON also belongs to the scope of protection of the invention. Immunomagnetic beads obtained by coupling the monoclonal antibody and magnetic beads also belong to the scope of protection of the invention. The immunomagnetic beads are used for enriching and purifying MON molecules in a sample, the concentrated sample is detected by indirect competitive ELISA finally, and the MON content to be measured is calculated. The method can detect MON samples exceeding the limit of detection (1 ng / mL) of common immunodetection methods, and effectively improves the detection sensitivity.

Description

technical field [0001] The invention relates to an immunomagnetic bead purification-enzyme-linked immunoassay method for detecting monensin and a special monoclonal antibody thereof. Background technique [0002] Monensin (monensin, MON), also known as rumenin, monensic acid, and Yukepeng, was first isolated from the fermentation broth of Streptomyces cinnamonensis by Haney et al. in 1967. Monensin resists It has a wide spectrum of coccidia and has a strong inhibitory effect on various Eimeria coccidia. It is the leading anticoccidial drug used in the late 20th century. In addition, monensin can also be used as a growth promoter for cattle and pigs to improve feed utilization and weight gain. Due to the high toxicity of monensin and its potential harm to humans and animals, the issue of its residue in animal tissues has been paid more and more attention. The maximum residue limit of monensin in animal foods approved in my country is: 0.05 μg / mL for edible tissue of cattle ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12C07K14/765C07K14/77C07D493/10G01N33/577C12R1/91
Inventor 沈建忠李建成张筱筱郭燕刘爽
Owner CHINA AGRI UNIV
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