Ofloxacin-hemocyanin coating antigen and preparation method thereof and detection test card

A technology of ofloxacin and hemocyanin, which is applied in the fields of biomedicine and medical testing, can solve the problems of inability to estimate the specificity of monoclonal antibodies, etc., and achieve the effects of easy large-scale promotion and application, quick results, and reduced error rates

Inactive Publication Date: 2017-05-31
HENAN INST OF SCI & TECH
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Problems solved by technology

But according to the prior art, people such as Du Fubin report the synthesis and identification method of ofloxacin artificial antigen, specifically adopt carbodiimide method and mixed acid anhydride method to synthesize ofloxacin immunogen and coating former, but the prepared oxygen The floxacin immunogen has not been immunized to mice, therefore, the specificity of the monoclonal antibody cannot be estimated; as another example, the patent with the notification number CN100418982C discloses a conjugate of ofloxacin and its preparation method, using carbon The conjugate of ofloxacin prepared by the diimine method was used to immunize white rabbits, and the minimum detection limit of the antiserum was 1ppb (1μg / L)

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  • Ofloxacin-hemocyanin coating antigen and preparation method thereof and detection test card
  • Ofloxacin-hemocyanin coating antigen and preparation method thereof and detection test card
  • Ofloxacin-hemocyanin coating antigen and preparation method thereof and detection test card

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preparation example Construction

[0044] The invention provides a preparation method of ofloxacin-hemocyanin coating former, comprising the following steps:

[0045] 1) Carrying out dehydration condensation reaction of aminovaleric acid and ofloxacin in a buffer solution system, extracting and back-extracting the obtained reaction solution in sequence to obtain the ofloxacin hapten with carbon chain extension;

[0046]2) The carbon chain extended hapten ofloxacin hapten that described step 1) is obtained is dissolved in N, N-dimethylformamide, obtains the ofloxacin hapten solution that carbon chain extends; The ester is mixed with the carbon chain extended ofloxacin hapten solution, stirred and reacted to obtain activated ofloxacin hapten;

[0047] 3) mixing hemocyanin and phosphate buffer solution, adjusting the pH value of the mixture to 9.0-10, and reacting under shaking conditions to obtain hemocyanin;

[0048] 4) dissolving the activated hemocyanin obtained in step 3) in a phosphate buffer solution conta...

Embodiment 1

[0108] Ofloxacin detects the synthesis of antigen, and the synthesis reaction is as attached figure 1 shown.

[0109] Ofloxacin carboxyl C chain extension: Weigh 2mmol of aminovaleric acid and add it to the Erlenmeyer flask, then adjust the pH value with 2ml of sodium hydroxide solution, stir in an ice bath; dissolve ofloxacin in methanol containing 50% DMF, stir In the state, add aminovaleric acid-sodium hydroxide solution drop by drop, and react with magnetic stirring for 2 hours; extract with ethyl acetate, wash with dilute hydrochloric acid, and back-extract with sodium bicarbonate solution to obtain the OFL hapten.

[0110] The detection antigen of ofloxacin-hemocyanin (OFL-KLH) is realized by the mixed acid anhydride coupling method: Weigh 20 mg OFL hapten and dissolve it in 2 ml DMF, add 10 μL triethylamine, and put it in an ice bath at 4°C Reaction 1h. Then 30 μL of isobutyl chloroformate was added, and the stirring reaction was continued for 1 h. 30mg of KLH was di...

Embodiment 2

[0112] Synthesis of levofloxacin (LOF) immunogen; Synthetic reaction as attached figure 2 shown.

[0113] (1) BSA activation: Glycosylation modification method, in which the aldehyde group on the glucose is coupled to the carboxyl group on the protein in an alkaline environment, the carboxyl group is blocked, and the number of coupled amino groups on the BSA is increased. The specific process is as follows: Weigh 66 mg of bovine serum albumin (BSA) and 1 mg of glucose and dissolve them in PBS buffer, adjust the pH value to 9.5 with NaHCO3, and react on a shaker at 40° C. for 2 hours to obtain activated cBSA.

[0114] (3) Synthesis of immunogen: LOF immunogen was synthesized by a two-step carbodiimide method. Take 19.3mg of LOF hapten, stir and dissolve with 2.3ml N,N-dimethylformamide (DMF), then add 14.4mg of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethyl Aminopropyl) carbodiimide hydrochloride (EDC.HCl) 51.2 mg, dissolved at room temperature, protected from light, sha...

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Abstract

The invention provides an ofloxacin-hemocyanin coating antigen and a preparation method thereof. The structure of the ofloxacin-hemocyanin coating antigen is shown in the description. The preparation method of the ofloxacin-hemocyanin coating antigen includes the steps that C-chain derivatization is conducted on carboxyl of ofloxacin, activation is conducted on KLH at the same time, an acid-anhydride coupling method is adopted to synthesize the coating antigen, and the prepared coating antigen has the advantages of high coupling efficiency and reactogenicity. The invention further provides a test card for testing the ofloxacin, the test card is fast in detection and high in specificity and sensitivity, the detection limit of the test card can reach 0.06 ng/ml, and the requirements of rapid detection are met at the same time.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and medical testing, and in particular relates to an ofloxacin-hemocyanin coating agent, a preparation method thereof and a detection test paper card. Background technique [0002] Ofloxacin (OFL) is a third-generation fluoroquinolone drug, also known as ofloxacin, an antibiotic developed by Japan's Daiichi Pharmaceutical Co., Ltd. in 1980, because the basic structure of the molecule introduces hydrophobic The unique fluorine atom and the hydrophilic piperazine ring have enhanced its antibacterial activity, and compared with the early quinolones, its bacterial drug resistance has also been greatly reduced. This type of drug inhibits bacterial DNA replication, transcription and repair by inhibiting bacterial DNA gyrase and topoisomerase IV. It has the characteristics of broad antibacterial spectrum, high efficiency, low toxicity, and strong tissue penetration. After that, it was one of the mos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/795G01N33/68G01N33/577
CPCC07K14/795G01N33/577G01N33/68G01N33/9446G01N2333/795
Inventor 范国英王磊姜金庆王自良张慧辉张海棠陈金山贺永惠赵坤杨雪峰
Owner HENAN INST OF SCI & TECH
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