One-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk

A technology of neomycin and detection method, which is applied in the field of immunoanalysis chemistry, can solve the problems of insufficient detection limit and long detection time, and achieve the effect of high sensitivity, sensitive detection, and extensive and rapid on-site detection

Inactive Publication Date: 2011-07-20
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the detection time of the current neomycin immunoassay method is still relatively long, and the detection limit is not high enough

Method used

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  • One-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk
  • One-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk
  • One-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk

Examples

Experimental program
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Embodiment 1

[0056] (1) Synthesis of neomycin antigen by glutaraldehyde method

[0057] ① Weigh 80 mg neomycin, 40 mg bovine serum albumin (BSA) or ovalbumin (OVA) and dissolve in 10 mL, 0.01 mol / L PBS;

[0058] ② After fully dissolving, add 1.5mL 1% glutaraldehyde solution dropwise, and stir at room temperature for 30 minutes;

[0059] ③ Add 112 mg NaBH 4 , put it in a refrigerator at 4°C for 1 hour;

[0060] ④ The reaction solution was dialyzed with 0.01mol / L phosphate buffer for 3 days, and the solution was changed every 6 hours;

[0061] The obtained NEO-BSA conjugate is an immunogen, and the obtained NEO-OVA conjugate is a coating agent.

[0062] (2) Infrared spectroscopy (IR method) to identify neomycin immunogen (NEO-BSA)

[0063] ① Weigh 2 mg of NEO standard, BSA and NEO-BSA samples respectively;

[0064] ② Add 200 mg of dry potassium bromide (KBr) respectively, place the mixture in an agate mortar, mix and grind thoroughly under the irradiation of infrared light;

[0065] ③P...

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Abstract

The invention relates to a one-step ELISA (Enzyme Linked Immunosorbent Assay) method for neomycin (NEO) residues in milk, which belongs to the technical field of immunoassay chemicals. The method comprises the following steps of: coupling NEO with bovine serum albumin (BSA) or ovalbumin (OVA) to synthetize a NEO antigen (immunogen NEO-BSA or coating antigen NEO-OVA) by a glutaraldehyde method; authenticating the NEO-BSA by utilizing an infrared spectroscopy and an SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis) method; obtaining NEO antiserum by immunization and detectingthe valence of the NEO antiserum by an indirect ELISA method; and detecting the specificity of the NEO antiserum by the one-step ELISA method. Compared with the indirect competition ELISA method, theinvention greatly shortens the detecting time of the specificity of the NEO antibody, has higher sensitivity, achieves the aim of simply, rapidly and sensitively detecting NEO residues in milk and can be widely used for rapid field detection.

Description

technical field [0001] A one-step ELISA detection method for neomycin residue in milk belongs to the technical field of immunoanalysis chemistry. Background technique [0002] Neomycin (neomycin, NEO) is a multi-component antibiotic produced by Streptomyces genus Streptomyces freundii. It contains two isomers of neomycin B and C (the structural formula is shown below), but only neomycin Mycin B has antibacterial activity. Neomycin is a water-soluble alkaline antibiotic. The commonly used preparation on the market is white or off-white powder neomycin sulfate, which has strong hygroscopicity and will deliquesce due to long-term exposure to the air due to the absorption of water. Neomycin itself has extremely strong stability, and its tolerance to environmental factors such as acid, alkali, and heat is obviously stronger than other antibiotics. Neomycin is very soluble in water, but almost insoluble in organic solvents such as ethanol, ether, acetone or chloroform, and has o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N21/17G01N27/447
Inventor 匡华胥传来徐乃丰屈昌龙马伟徐丽广王利兵
Owner JIANGNAN UNIV
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