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Rifaximin hapten and rifaximin artificial antigen, and preparation methods and application thereof

A rifaximin and antigen technology, applied in the field of rifaximin artificial antigen and its preparation, can solve the problems of expensive instruments, unsuitable for large-scale sample screening and detection, cumbersome operation, etc., and achieve simple synthesis method and high yield , the effect of high purity

Inactive Publication Date: 2020-08-21
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry (MS) for the detection of rifaximin residues. Although these methods have strong specificity and high sensitivity, they are cumbersome to operate and expensive, and are not suitable for large quantities. Sample screening test

Method used

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  • Rifaximin hapten and rifaximin artificial antigen, and preparation methods and application thereof
  • Rifaximin hapten and rifaximin artificial antigen, and preparation methods and application thereof
  • Rifaximin hapten and rifaximin artificial antigen, and preparation methods and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, preparation and identification of rifaximin hapten

[0034] 1. Preparation of rifaximin hapten

[0035] Rinse the 50ml round bottom flask, dry it with ethanol, fix it on the stirrer, and add a stirring bar. Weigh 1g of rifamycin S, dissolve it with 6ml DMF, stir, and keep the sample after it is completely dissolved. At this time, the solution is purple. Weigh 300mg NBS, add it, stir, when it is found that the reaction is complete through TLC, process it, and add it to ice In water, a precipitate precipitated out, and the solid was obtained by filtration and dried to obtain 670 mg of Intermediate 1.

[0036] Weigh 500mg of intermediate 1, dissolve it with 5ml of methanol, stir, add 2ml of triethylamine, then add 100mg of 2-aminoisonicotinic acid, stir, spot plate monitoring, when the raw materials do not change, process the reaction, spin dry the reaction solution, The residue was purified by column chromatography, the ratio of developing solvent: (dichlo...

Embodiment 2

[0043] Embodiment 2, the preparation of rifaximin artificial antigen

[0044] 1. Synthesis of immunogen

[0045] (1) Dissolve 30.4mg of rifaximin hapten in 1.5ml of DMF, stir at 200rpm for 10min, add 25.75mg of EDC and 15.46mg of NHS to dissolve, stir at room temperature (500rpm) and activate for 2-3h.

[0046] (2) Weigh 50mg of BSA and dissolve it in 3.5ml of 0.1M sodium bicarbonate solution, stir at 200rpm for 10min to fully dissolve, cool down in an ice bath to 0-4°C, stir at 1000rpm, add the reaction solution in step 1 dropwise (1ml / min), stirred at 500rpm for 24h.

[0047] (3) Put the reaction product into distilled water and rinse the dialysis bag (10cm), dialysis with 1L 0.01M PBS (1×, pH7.2) at 4°C (100rpm) for 3 days, and change the solution 3 times a day (once in the morning, once in the middle of the day and once in the evening). , changing the medium 9 times in total, centrifuge the dialysis product at 5000rpm for 6min, aliquot 1.5ml / tube, number the antigen, an...

Embodiment 3

[0052] Example 3, Rifaximin Artificial Antigen Immunized Animals to Prepare Monoclonal Antibodies

[0053] 1. Animal immunity

[0054] Use the immunogen (rifaximin-BSA) prepared in Example 2 at 100 μg per mouse, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in an equal volume, and inject subcutaneously on the back of the neck to immunize 6-8-week-old Balb / c female mice, on the 7th, 14th, and 28th day after the initial immunization, the immunogen and Freund's incomplete adjuvant were mixed in equal volumes, and each additional immunization was given once, and the immune complex was 100 μg / rat 3 days before fusion, without Freund's adjuvant. Adjuvant was used for booster immunization once again.

[0055] 2. Cell fusion and cloning

[0056] According to the conventional method, the splenocytes of the immunized mice were mixed with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then the preheated fusion agent (PEG4000) ...

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Abstract

The invention discloses a rifaximin hapten and a rifaximin artificial antigen, and a preparation method and application of the rifaximin hapten and the rifaximin artificial antigen. The rifaximin artificial antigen provided by the invention is an antigen obtained by coupling the rifaximin hapten shown as a formula I which is described in the specification with a carrier protein. The rifaximin artificial antigen provided by the invention is simple in synthesis method, high in purity and high in yield, and has important value for preparation of a rifaximin antibody and detection of rifaximin drug residues.

Description

technical field [0001] The invention belongs to the field of rapid detection of drug residues, and relates to a rifaximin artificial antigen and a preparation method and application thereof. Background technique [0002] Rifaximin belongs to the rifamycin class of antibacterial drugs and is a semi-synthetic derivative of rifamycin. It mainly inhibits the synthesis of bacterial RNA by irreversibly binding to the β-subunit of the bacterial DNA-dependent RNA polymerase, thereby achieving purpose of sterilization. It has good antibacterial activity against Gram-positive bacteria (such as Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Corynebacterium, etc.) and Gram-negative bacteria (such as Escherichia coli, etc.) . [0003] In veterinary medicine, rifaximin is mainly used as a udder injection agent during the dry period of dairy cows to prevent and treat mastitis during the dry period of dairy cows. It is widely used due to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D498/22C07K14/435C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53G01N33/577
CPCC07D498/22C07K14/435C07K14/765C07K14/77C07K14/795C07K16/44G01N33/5308G01N33/577
Inventor 马立才聂靖东丁亚芳邢维维刘薇李蓉蓉
Owner 北京维德维康生物技术有限公司
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