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Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin

An enzyme-linked immunosorbent reagent, amikacin technology, applied in microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as sample processing time and reagents are not optimized, and achieve detection time. The effect of short, high detection efficiency and simple processing method

Inactive Publication Date: 2012-07-18
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention patent with the application number of 200710064347 discloses an enzyme-linked immunosorbent assay kit and method for detecting neomycin drug. The patent adopts the method of directly activating protein to couple neomycin and bovine serum albumin (bovine serum albumin, BSA) To obtain the immunogen, the same method is used to couple neomycin and thyroid protein to obtain the coating agent. The prepared monoclonal antibody can only specifically recognize neomycin, and the sample processing time and reagents have not been optimized.

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin
  • Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin
  • Monoclonal antibody, enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 immunogen and coating former

[0033] 1.1 Synthesis of neomycin-BSA

[0034] Weigh 10.0 mg of neomycin and 100.0 mg of BSA and dissolve in 20 mL of PBS solution (pH 7.4), stir evenly, add 50.0 mg of EDC dissolved in 1 mL of pure water dropwise, and stir and react at room temperature for 8 hours. Finally, the reaction solution was transferred into a dialysis bag, dialyzed in PBS (pH7.4) solution at 4°C for 4 days, centrifuged and freeze-dried. like figure 1 , 2 As shown, the coupling was identified by matrix-assisted laser desorption (MALDI-TOF-MS), and stored at -20°C for future use.

[0035] 1.2 Synthesis of neomycin-OVA

[0036] Weigh 20.0 mg of neomycin and 200.0 mg of OVA and dissolve in 20 mL of PBS solution (pH 7.4), stir evenly, and then slowly add 80.00 mg of EDC dissolved in 1 mL of pure water. The reaction was stirred at room temperature for 2 hours. Finally, the reaction solution was transferred into a dialysis bag, and d...

Embodiment 2

[0037] The preparation of embodiment 2 monoclonal antibody

[0038] Preparation of hybridoma cells: With reference to Yang Hanchun's "Animal Immunology", the immunogen neomycin-BSA prepared in Example 1 was used to immunize Balb / C mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention), and the immunization procedure was as follows: : For basic immunization, the immunogen was emulsified with an equal volume of complete Freund's adjuvant, and then injected subcutaneously at multiple points on the back of the mouse, and then boosted immunization once every 2 weeks, and emulsified with incomplete adjuvant. Finally, intraperitoneal injection three days before fusion (preferably resting for one month after the end of immunization) for booster immunization, doubling the amount of antigen without adding adjuvant.

[0039]At the time of fusion, a Balb / C mouse that had undergone the final booster immunization was taken, sacrifi...

Embodiment 3

[0044] The establishment of embodiment 3 neomycin indirect competition ELISA detection method

[0045] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0046] Carbonate buffer (pH9.6): Accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, dissolved in a small amount of ultrapure water, and adjusted to 1000mL.

[0047] Washing solution (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O 2.90g, KCl 0.20g, dissolved in a small amount of ultrapure water, Tween 20 0.50mL was added, and the volume was adjusted to 1000mL.

[0048] Phosphate buffer (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 Dissolve 2.90g of O, 0.20g of KCl in a small amount of ultrapure water, and dilute to 1000mL.

[0049] Blocking solution: Accurately weigh 10.00 g of ovalbumin, add 1000 mL of phosphate buffer, stir and mix until the protein is completely dissolved...

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Abstract

The invention discloses a specific monoclonal antibody capable of identifying neomycin, amikacin and paromomycin and an enzyme-linked immunosorbent assay (ELISA) method and kit for detecting neomycin, amikacin and paromomycin. The monoclonal antibody is secreted by a hybridoma cell EDC / 5G04 and the hybridoma cell is collected in the China Center for Type Culture Collection, with collection number being CCTCC NO:C201144. The ELISA method comprises the steps of preparation of immunogen, coating antigen and the antibody, treatment and detection of samples and the like. Compared with the prior art, the monoclonal antibody, the ELISA method and the kit have the following main advantages that the prepared monoclonal antibody can simultaneously identify neomycin, amikacin and paromomycin, thus improving the detection efficiency of the prior art; the animal tissue sample treatment method is simple and short in time; and the ELISA method and the kit also have the characteristics of high detection sensitivity, good precision and good accuracy.

Description

technical field [0001] The present invention relates to a monoclonal antibody capable of recognizing neomycin, amikacin and paromomycin, and an ELISA method and reagents for detecting neomycin, amikacin and paromomycin box. Background technique [0002] Aminoglycoside (AMGs) antibiotics are a class of water-soluble alkaline antibiotics that are extracted from the culture fluid of Streptomyces or Micromonospora, or semi-synthesized from natural products. Currently commonly used in veterinary clinics are: streptomycin, gentamicin, neomycin, kanamycin, amikacin and so on. It shows a strong bactericidal effect on most Gram-negative bacilli, and it also has an effect on Gram-positive bacteria. The main sensitive bacteria are Enterobacter sensitive strains, and it is effective against Streptococcus, Hepatitis, Clostridium, and Rickettsia. However, because AMGs are easy to accumulate in the renal cortex and inner ear perilymph, causing ototoxicity and nephrotoxicity, and the micr...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N5/20G01N33/577C12R1/91
Inventor 袁宗辉王玉莲闫彩霞彭大鹏潘源虎黄玲利陈冬梅陶燕飞戴梦红刘振利廖峰
Owner HUAZHONG AGRI UNIV
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