Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of unoptimized reagents and long sample processing time, and achieve short detection time, high detection efficiency and excellent processing methods. simple effect

Inactive Publication Date: 2012-07-25
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 200920246849.1 discloses a gentamicin ELISA detection kit, which uses the direct activation protein method to couple gentamicin and bovine serum albumin (bovine serum albumin, BSA) to obtain the immunogen, Using the same method to couple gentamicin and thyroid protein to obtain the coating agent, the prepared monoclonal antibody can only recognize gentamicin, and the sample processing time is long, and the reagents have not been optimized.

Method used

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  • Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin
  • Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin
  • Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting gentamicin and sisomicin

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Experimental program
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Effect test

Embodiment 1

[0031] The preparation of embodiment 1 immunogen and coating former

[0032] 1.1 Synthesis of gentamicin-BSA

[0033] Weigh 164.0mg of gentamicin and 200.0mg of BSA and dissolve them in 15mL of pure water (pH7.4), stir evenly, then add 430.00mg of carbodiimide (EDC) dissolved in 5mL of pure water dropwise, at room temperature The reaction was stirred for 8 hours. Finally, the reaction solution was transferred into a dialysis bag, dialyzed in PBS solution (pH7.4) at 4° C. for 4 days, centrifuged and freeze-dried. Such as 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.

[0034] 1.2 Synthesis of gentamicin-OVA

[0035] Weigh 148.0 mg of gentamycin and 40.0 mg of OVA and dissolve them in 12 mL of pure water (pH7.4), stir evenly, then add dropwise 30.00 mg of EDC4 dissolved in 4 mL of pure water, and stir at room temperature for 8 hours. Finally, the reaction solution was transferred into...

Embodiment 2

[0036] The preparation of embodiment 2 monoclonal antibody

[0037] Preparation of hybridoma cells: With reference to Yang Hanchun's "Animal Immunology", the immunogen gentamicin-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 For: basic immunization, the immunogen was emulsified with an equal volume of complete Freund's adjuvant, and 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.

[0038] At the time of fusion, a Balb / C mouse that had undergone the final booster immunization was taken, sacrificed by bleeding fro...

Embodiment 3

[0043] Embodiment 3 Establishment of gentamicin indirect competition ELISA detection method

[0044] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0045] 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.

[0046] 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, Tween20 0.50mL was added, and the volume was adjusted to 1000mL.

[0047] Phosphate buffer saline (PBS) (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, and the volume was adjusted to 1000mL.

[0048] Blocking solution: Accurately weigh 10.00 g of ovalbumin, add 1000 mL of phosphate buffer, stir and mix until the protein ...

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Abstract

The invention discloses a specific monoclonal antibody capable of recognizing gentamicin and sisomicin simultaneously. The monoclonal antibody of the invention is secreted by hybridoma DEC / 2D5 which is preserved in China Center for Type Culture Collection with the preservation number of CCTCC NO: C201145. The invention also discloses a preparation method of the specific monoclonal antibody, coating antigen and immunogen as well as an enzyme linked immunosorbent assay kit. Compared with the prior art, the monoclonal antibody prepared according to the invention can recognize gentamicin and sisomicin simultaneously, therefore, the detection efficiency of the prior art is improved. The kit and the method of the invention have the advantages of simplicity and convenience in operation, sensitivity, accuracy and the like.

Description

technical field [0001] The invention relates to a monoclonal antibody capable of recognizing gentamicin and sisomycin, an enzyme-linked immunoassay method and a kit for detecting gentamicin and sisomycin. Background technique [0002] Gentamicin and sisomycin belong to aminoglycoside antibiotics (AMGs), which cause ototoxicity and nephrotoxicity due to the accumulation of these antibiotics in the renal cortex and inner ear perilymph, as well as the toxicity produced by aminoglycoside inactivating enzymes. Microbial drug-resistant strains are serious, so the detection of their residues in animal-derived foods has attracted increasing attention. Enzyme-linked immunoassay (ELISA) method has been widely used in the field of rapid residue detection of AMGs due to its simplicity, rapidity, sensitivity and specificity. However, the current ELISA methods mainly focus on single residue detection, and there are few ELISA methods that use one antibody to simultaneously detect AMGs wit...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543
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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