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Preparation method and application of ofloxacin hapten

A technology of ofloxacin and hapten, applied in the field of antigen preparation and ofloxacin hapten, can solve the problems such as accuracy, sensitivity and specificity that cannot be fully detected, and achieve a simple and feasible preparation method and high yield , the effect of low cost

Inactive Publication Date: 2016-11-09
北京明日达科技发展有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] ELISA kits for the detection of ofloxacin have been developed at home and abroad, but the domestically produced kits cannot fully meet the detection requirements in terms of accuracy, sensitivity, and specificity.

Method used

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  • Preparation method and application of ofloxacin hapten
  • Preparation method and application of ofloxacin hapten
  • Preparation method and application of ofloxacin hapten

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the preparation of ofloxacin hapten

[0028] 1. Preparation of ofloxacin hapten

[0029] Add 1000mg ofloxacin raw material and 50ml DMF to a 100ml round bottom flask, stir magnetically at 20-24°C for 30-60min, add NHS 797.0mg, EDC 1327.6mg, stir magnetically at 20-24°C for 3-4 hours, then add 4-amino Ethyl butyrate hydrochloride 696.0 mg, 20-24 ° C magnetic stirring reaction overnight, the reaction solution was added dropwise to 300 ml of ice water, stirred for 30-40 min to precipitate off-white solid, filtered, the filter cake was washed with 20 ml of water, and the filter cake was collected for 50 ℃ blow drying for 5-6 hours to obtain 1052.6 mg ofloxacin 4-aminobutyric acid ethyl ester intermediate.

[0030] Add 1052mg ofloxacin 4-aminobutyric acid ethyl intermediate 1052mg into a 50ml round bottom flask, methanol 30ml, stir magnetically at 20-24°C for 30-60min, then add 6.0ml of 1M sodium hydroxide aqueous solution dropwise, stir magnetically at 20-24°...

Embodiment 2

[0035] Embodiment 2, preparation and identification of ofloxacin artificial antigen

[0036] 1. Synthesis of ofloxacin immune antigen

[0037] 1. Dissolve 20mg ofloxacin hapten in 1.5ml DMF, stir at 200rpm for 10min, add 21.5mg of EDC to dissolve, then add 13mg of NHS, stir at room temperature (500rpm) to activate for 2-3h.

[0038] 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, put the reaction solution in step 1 in the reaction box in an ice bath (<4°C) and stir (1000rpm) drop by drop Add (1ml / min), stir (500rpm) and react for 24h.

[0039] 3. Put the reaction product into the distilled water and rinse the dialysis bag (10cm), 1L of 0.01M pH7.2 PBS, stir at 100rpm at 4°C, dialyze for 3 days, change the solution 3 times a day (one in the morning, one in the middle and one in the evening), and change the solution 9 times in total , centrifuge the dialysis product at 5000rpm for 6min, aliquot 1.5m...

Embodiment 3

[0044] Example 3, Preparation and Specific Identification of Enzyme-labeled Monoclonal Antibody

[0045] One, the preparation of ofloxacin monoclonal antibody

[0046] 1. Use the immunogen prepared above at 100 μg / mouse, dissolve the immunogen in normal saline and mix it with Freund’s complete adjuvant in equal volumes, inject subcutaneously on the back of the neck and immunize 6-8 week-old Balb / c female mice, after the first immunization On the 7th, 14th, and 28th days, mix the immunogen and Freund's incomplete adjuvant in equal volumes, and each booster immunization once, and 100 μg of the immune complex per mouse 3 days before the fusion, and then booster immunization without Freund's adjuvant.

[0047] 2. Proceed according to the conventional method, take the splenocytes of the immunized mice and mix them with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add the preheated fusion agent (PEG4000) within 45s for fusion, Suspend evenly with H...

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Abstract

The invention discloses ofloxacin hapten and a corresponding artificial antigen and also discloses a preparation method and application of the ofloxacin hapten and the corresponding artificial antigen. The formula of the ofloxacin hapten is as shown in formula 1. An ofloxacin antigen can be obtained when the ofloxacin hapten is connected with carrier protein. The ofloxacin antigen can be used for preparing a specific ofloxacin antibody. The preparation method is simple, practicable, low in cost and high in hapten yield. The artificial ofloxacin antigen can be used for producing the specific antibody aiming at ofloxacin by immunizing animals, the specific antibody can be used to prepare an enzyme-linked immunosorbent assay kit for detecting residual ofloxacin, and the kit is simple, fast, large in processing sample quantity, high in sensitivity, high in specificity and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and in particular relates to a method for preparing an ofloxacin hapten, an antigen and an application thereof. Background technique [0002] Ofloxacin, alias: fleroxacin, ofloxacin, ofloxacin, is a synthetic broad-spectrum antibacterial drug. Staphylococcus, Streptococcus (including Enterococcus), Streptococcus pneumoniae, Neisseria gonorrhoeae, Escherichia coli, Citrobacter, Shigella, Klebsiella pneumoniae, Enterobacter, Serratia, Proteus, influenza Haemophilus, Acinetobacter, and Helicobacter have good antibacterial effects, and they also have certain antibacterial effects on Pseudomonas aeruginosa and Chlamydia trachomatis. It still has anti-tuberculosis effect, and can be used together with isoniazid and rifampicin to treat tuberculosis. [0003] The drug has been widely used in livestock, poultry and aquaculture. Ofloxacin drug residues are not only directly harmful to the ...

Claims

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Application Information

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IPC IPC(8): C07D498/06C07K14/795C07K14/77C07K14/765C07K14/47C07K1/107C07K16/44G01N33/68G01N33/53
CPCC07D498/06C07K14/47C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/53G01N33/68G01N2333/76G01N2333/765G01N2333/77G01N2333/795
Inventor 曹罡吴雨洋王照鹏刘薇秦誉覃丹凤
Owner 北京明日达科技发展有限责任公司
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