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Monoclonal antibody and enzyme-linked immunoassay method and reagent kit for detecting tylosin and tilmicosin residue

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, which is applied in the field of veterinary drug residue analysis and immunology, can solve problems such as difficult implementation and undisclosed core technology, and achieve less health hazards, less analysis times, and simple processing methods Effect

Inactive Publication Date: 2011-05-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The prepared monoclonal antibody can only recognize tylosin, and at the same time, the application does not specifically disclose the core technology of its invention, that is, the detailed preparation steps of the main core reagents such as the immunogen and the coating agent as the kit, so that the technical field It is difficult for skilled personnel to implement the invention and achieve the claimed effect of the invention

Method used

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  • Monoclonal antibody and enzyme-linked immunoassay method and reagent kit for detecting tylosin and tilmicosin residue
  • Monoclonal antibody and enzyme-linked immunoassay method and reagent kit for detecting tylosin and tilmicosin residue
  • Monoclonal antibody and enzyme-linked immunoassay method and reagent kit for detecting tylosin and tilmicosin residue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1 immunogen and coating former

[0038]1.1 Synthesis of hapten descarbamicose tylosin (DES)

[0039] Weigh 10 g of tylosin tartrate, dissolve it in 200 mL of sulfuric acid solution (pH 2), and reflux for 2 h. After the reaction was completed, adjust the pH to 8.8 with saturated sodium carbonate solution, transfer to a separatory funnel, add 60 mL of dichloromethane for extraction, and repeat the above operation once. Combine the dichloromethane layers, add 5 g of anhydrous sodium sulfate for shaking, and then let stand overnight. After filtration, the dichloromethane layer was taken and evaporated to dryness under reduced pressure at 60° C. to obtain the product descarbamicose tylosin.

[0040] 1.2 Preparation of mycaminosyl tylosin-p-hydrazinobenzoic acid-bovine serum albumin / ovalbumin conjugate (DES-HBA-BSA / OVA)

[0041] Weigh 770 mg of mycaminosyl tylosin (DES) and 150 mg of p-hydrazinobenzoic acid (HBA) into 12 mL of absolute ethanol...

Embodiment 2

[0050] Example 2 Preparation of Monoclonal Antibody

[0051] 2.1 Preparation of anti-tylosin and tilmicosin monoclonal antibodies:

[0052] With reference to the method in the 2001 edition of Xue Qingshan's "Principles and Techniques of In Vitro Culture" Science Press: Utilize the decarbamicose tylosin-O-carboxymethylhydroxylamine-bovine serum albumin prepared by the inventor's veterinary pharmacology laboratory (DES-AOAA-BSA) conjugates were used to immunize Balb / C mice (purchased from the Experimental Animal Center of Hubei Academy of Medical Sciences). The immunization procedure was to emulsify a protein solution containing 50-100 μg of DES-AOAA-BSA conjugate with an equal volume of Freund's complete adjuvant (purchased from sigma), and then inject it subcutaneously at multiple points on the back of the mouse. Afterwards, it was strengthened every 2 weeks, and emulsified with incomplete adjuvant (purchased from sigma company). Finally, 3 days before the fusion (preferably...

Embodiment 3

[0055] Example 3 Establishment of Tylosin Indirect Competition ELISA Detection Method

[0056] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0057] Phosphate buffer: NaCl8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O2.9g, KCl0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0058] Coating solution: Take Na 2 CO 3 1.5g, NaHCO 3 2.9g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0059] Washing solution: NaCl8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O2.9g, KCl0.2g, Tween200.5mL, thimerosal 0.1g, add double distilled water to 1000mL, adjust pH to 7.4;

[0060] Blocking solution: Ovalbumin 0.1g dissolved in 100mL phosphate buffer;

[0061] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0062] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen perox...

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Abstract

The invention belongs to the veterinary medicine residual analysis and immunity analysis technique field and specifically relates to an enzyme-linked immunity method and the reagent kit thereof which can discern specificity monoclonal antibodies of tylosin and tilmicosin and detect the residuals of tylosin and tilmicosin at the same time; the monoclonal antibodies in the invention is secreted from hybridoma cell strain P3C4 established by the applicant; the hybridoma cell strain is preserved in China Center of Type Culture Collection; the number of preservation of the hybridoma cell strain isCCTCC No: C200719; the invention discloses the preparation method and enzyme-linked immunity detection method of the monoclonal antibody, coating antigen and immunogen. Compared with the prior art, the monoclonal antibody prepared in the invention can discern tylosin and tilmicosin at the same time and add the detecting object in the prior art. The reagent kit and the method in the invention has the advantages of simple, convenience, swiftness, sensitivity and accuracy; furthermore, the reagent kit and method in the invention can detect the residuals of tylosin and tilmicosin in an animal edibility tissue at the same time.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology. It specifically relates to the preparation of a monoclonal antibody capable of simultaneously recognizing tylosin and tilmicosin, the establishment of a multi-residue enzyme-linked immunoassay method for simultaneous detection of tylosin and tilmicosin, and the development of a kit. Background technique [0002] Tylosin and tilmicosin belong to macrolide animal-specific antibiotics, and are widely used in veterinary clinics to treat pneumonia, mastitis, and systemic sepsis caused by Gram-positive bacteria, especially for the treatment of livestock and poultry Mycoplasma infection. In addition, tylosin is also widely used as a feed additive. [0003] The residues of tylosin and tilmicosin in animal tissues will threaten food safety and cause harm to human health. Tylosin and tilmicosin can easily induce bacterial resistance, and the resistance can be tran...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53G01N33/577C12N5/12
Inventor 袁宗辉叶胜强彭大鹏王玉莲黄玲利陈冬梅陶燕飞戴梦红刘振利谢长清刘宇杨波赵春保胡晓芬王小清
Owner HUAZHONG AGRI UNIV
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