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Monoclonal antibody for kitasamycin residual detection and preparation method and application thereof

A monoclonal antibody and guitarmycin technology, applied in the field of veterinary drug residue analysis and immunology, can solve the problems of high instrument cost, complexity and time-consuming, and achieve the effect of simple sample processing route, few organic reagents, and wide application range.

Inactive Publication Date: 2013-09-11
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, instrumental method sample processing is generally more complicated, time-consuming, and the cost of the instruments used is high, so it is difficult to popularize at the grassroots level
No reports on kitasamycin-specific antigens, antibodies, enzyme-linked immunoassays and kits have been retrieved so far

Method used

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  • Monoclonal antibody for kitasamycin residual detection and preparation method and application thereof
  • Monoclonal antibody for kitasamycin residual detection and preparation method and application thereof
  • Monoclonal antibody for kitasamycin residual detection and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of Immunogen and Coating Original

[0047] Accurately weigh 20 mg of kitasamycin standard substance and dissolve in 5 mL of methanol. Weigh 10 mg of oxycarboxymethylhydroxylamine (AOAA) and dissolve in 1 mL of water. The AOAA solution was slowly added to the kitasarmycin methanol solution, and the reaction was stirred overnight. The reaction solution was rotary evaporated to dryness at 60°C. Accurately draw 1mL DMF to dissolve the evaporated matter, add DCC 100mg and stir to react overnight.

[0048] Accurately weigh 100 mg of bovine serum albumin (BSA), and 10 mL of dissolved protein in PBS. Aspirate 1mL of DCC activation drug solution, slowly drop it into the protein solution, and stir the reaction overnight. The reaction solution was centrifuged at 8000r / min for 10min, dialyzed in saline solution at 4°C for 3 days, and the dialysate was changed every 12h. After dialysis, the reaction solution was divided into vials and freeze-dried. Store i...

Embodiment 2

[0050] Embodiment 2: Preparation of monoclonal antibody

[0051] Preparation of hybridoma cell lines: referring to Zhu Liping and Chen Xueqing's "Common Immunology Experimental Methods", the immunogen kitasamycin-AOAA-BSA prepared in Example 1 was used to immunize Balb / C mice. Equal volume of complete Freund's adjuvant was emulsified, and injected subcutaneously at multiple points on the back of the mouse. After that, boosted immunization once every 2 weeks, replaced with incomplete adjuvant emulsified, and finally intraperitoneally injected three days before fusion, boosted immunization, antigen Double the amount without adjuvant. At the time of fusion, take a Balb / C mouse that has undergone the final booster immunization, sacrifice it by bleeding from the eye socket (collect the serum, it is positive serum), soak it in 75% (volume ratio) alcohol for 5 minutes for disinfection. 3~5×10 7 The suspension of SP2 / 0 myeloma cells and spleen cells of immunized mice was added to a ...

Embodiment 3

[0054] Example 3: Establishment and Method Evaluation of Kitamycin Indirect Competition ELISA Detection Method

[0055] 3.1 ELISA related reagent preparation

[0056] Carbonate buffer preparation: Accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, Proclin300 300μL in a 500mL beaker, add 300mL of ultrapure water and stir until completely dissolved, transfer to a 1000mL volumetric flask, rinse the beaker twice with a small amount of ultrapure water (both poured into the volumetric flask), and make up to volume with ultrapure water to 1000mL. Invert up and down to mix well, that is pH 9.5-9.7 carbonate buffer solution.

[0057] PBS preparation: Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 .12H 2 Put 2.90g of O and 0.20g of KCl in a 500mL beaker, add 300mL of ultrapure water and stir to dissolve completely, then transfer to a 1000mL volumetric flask, dilute the volume of ultrapure water to 1000mL, mix upside down, that is, pH7.3~7.5PBS.

[0058] PBST prep...

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Abstract

The invention discloses a monoclonal antibody for kitasamycin residual detection and a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma KA / 2A9, which is collected by CCTCC (China Center for Type Culture Collection) and has the collection number of C201184. The preparation method comprises the steps of: (A) coupling hapten kitasamycin with bovine serum albumin so as to obtain an immunogen; (B) coupling hapten kitasamycin with ovalbumin so as to obtain a coating antigen; (C) preparing the monoclonal antibody secreted by the hybridoma KA / 2A9 by using the immunogen; (D) coating a solid phase carrier by using the coating antigen; (E) extracting a sample to be detected by using metaphosphoric acid in the presence of ethanol, carrying out back extraction by using ethyl acetate, blow-drying by using nitrogen gas, and diluting the sample so as to obtain a substance to be detected; and (F) carrying out enzyme-linked immunoassay on the substance to be detected. A kit for kitasamycin residual detection comprises an ELISA (Enzyme-Linked Immunosorbent Assay) plate coated by the coating antigen kitasamycin-AOOA-OVA, a kitasamycin standard solution, a goat anti-mouse IgG (Immunoglobulin G) antibody working solution labeled by a horseradish peroxidase, a concentrated phosphate buffer, a concentrated washing solution, a substrate mixture and a stop solution. The method has the characteristics of simplicity, convenience, rapidness, sensitiveness and accuracy.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and specifically relates to a monoclonal antibody for kitasamycin residue detection, and also relates to a preparation method of a monoclonal antibody for kitasamycin residue detection, and also relates to a kitasarmycin residue detection method. The use of monoclonal antibody for the detection of protein residues. Background technique [0002] Kitamycin is a macrolide antibiotic widely used in pig and poultry production. Improper use will lead to its residue in animals, leading to a series of adverse reactions after use by consumers+. There are mainly mild gastrointestinal reactions such as diarrhea, nausea, vomiting, allergies, liver toxicity, tinnitus, hearing impairment and cardiotoxicity (Shu Gang et al., 2006). Kitamycin residues can also cause bacterial resistance in the human body, directly affecting the treatment of human diseases (Deguchi et al....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44G01N33/577
Inventor 袁宗辉王小清彭大鹏王玉莲黄玲利陈冬梅陶燕飞戴梦红刘振利
Owner HUAZHONG AGRI UNIV
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