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A kind of lomefloxacin immunogen and its preparation method and detection test paper card

A technology of lomefloxacin and test strips, which is applied in the fields of biomedicine and medical testing, can solve the problems of low detection limit and inability to estimate the specificity of monoclonal antibodies, and achieve quick results, easy large-scale application, and simple operation

Inactive Publication Date: 2019-01-29
HENAN INST OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, according to the prior art, Du Fubin et al. reported the synthesis and identification method of the artificial antigen of lomefloxacin, specifically using the carbodiimide method and the mixed acid anhydride method to synthesize the lomefloxacin immunogen and the coating agent, but the prepared lomefloxacin The immunogen has not been immunized to mice, therefore, the specificity of the monoclonal antibody cannot be estimated; another example, the patent with the notification number CN100418982C discloses a conjugate of lomefloxacin and its preparation method, using the carbodiimide method The prepared conjugate of lomefloxacin was used to immunize white rabbits, and the minimum detection limit of antiserum was 1ppb (1μg / L)
Therefore, prior art needs lower detection kit of lomefloxacin detection limit

Method used

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  • A kind of lomefloxacin immunogen and its preparation method and detection test paper card
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  • A kind of lomefloxacin immunogen and its preparation method and detection test paper card

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preparation example Construction

[0039] The present invention also provides a preparation method of lomefloxacin immunogen, comprising the following steps:

[0040] 1) Carrying out dehydration condensation reaction of aminovaleric acid solution and lomefloxacin solution, extracting the obtained reaction solution, and then carrying out back extraction to obtain the LOM hapten with carbon chain extension;

[0041] 2) dissolving the carbon chain extended LOM hapten obtained in step 1) in N,N-dimethylformamide to obtain a carbon chain extended LOM hapten solution; N-hydroxysuccinimide and 1 - Ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is mixed with the LOM hapten solution extended by the carbon chain, and a displacement reaction is carried out under dark conditions to obtain an activated LOM hapten;

[0042] 3) mixing bovine serum albumin, glucose and phosphate buffer, adjusting the pH value of the mixture to 9.0-10, and reacting under shaking conditions to obtain activated cBSA;

[0043] 4) Dissol...

Embodiment 1

[0114] The synthetic method of lomefloxacin immunogen is as attached figure 1 .

[0115] (1) LOM 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, and stir in an ice bath; PBS (0.01mol / L, pH 7.4) dissolves Lomesal hydrochloride Star (lomefloxacinhydrochloride), under stirring state, was added dropwise into valeric acid-sodium hydroxide solution, and reacted with magnetic stirring for 2h; extracted with ethyl acetate, washed with dilute hydrochloric acid, and back-extracted with sodium bicarbonate solution to obtain the LOM hapten .

[0116] (2) BSA activation: Glycosylation modification method, in which the aldehyde group on the glucose is coupled to the amino group on the protein in an alkaline environment, the amino group is blocked, and the number of coupling-COOH on the BSA is increased. The specific process is as follows: Weigh 66 mg of bovine serum albumin (BS...

Embodiment 2

[0119] Preparation method of anti-lomefloxacin monoclonal antibody

[0120] 1. The anti-lomefloxacin-specific monoclonal antibody is prepared by immunizing Balb / C mice with a lomefloxacin-bovine serum albumin (LOM-cBSA) conjugate, which is achieved by the following steps:

[0121] (1) Mouse immunization: 5 8-10 week old female Balb / C mice were immunized with LOM-cBSA at a dose of 60 μg / mouse and a volume of 0.2 ml. The immunogen diluted in PBS was completely emulsified with an equal volume of FCA for the first immunization, and then boosted every 3 weeks, and emulsified with FIA. After 5 times of immunization, the blood was collected by docking the tail to separate the serum, and the mice with high titer and good inhibitory effect were screened by indirect ELISA and indirect competitive ELISA (ciELISA) as spare mice for fusion. Three days before the fusion, the hyperimmunized mice were injected with 60 μg of immunogen in the tail vein and intraperitoneally.

[0122] (2) Cell...

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Abstract

The invention provides a lomefloxacin immunogen which has the structure represented by the formula I. A preparation method of the lomefloxacin immunogen is provided and comprises the steps of carrying out C chain derivatization of carboxyl on lomefloxacin, and activating BSA by using a glycosylation modification method, then synthesizing and immunizing by using a carbodiimide two-step method, and thus obtaining the immunogen having the characteristics of high coupling rate and strong immunity. The invention also provides a test paper card for detecting the lomefloxacin, wherein the test paper card has the advantages of fast detection speed and high sensitivity; and the detection limit of the test paper card can be reach 0.3 ng / ml, and the requirement of rapid detection can be met.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and medical testing, and in particular relates to a lomefloxacin immunogen, a preparation method thereof and a detection test paper card. Background technique [0002] Lomefloxacin (LOM) is a new type of long-acting fluoroquinolone antibacterial drug, its chemical name is 1-ethyl-6,8-difluoro-1,4-dihydro-7-(3-methyl-1 -piperazinyl)-4-oxo-3-quinolinecarboxylate hydrochloride. Fluoroquinolones have strong antibacterial effects on a variety of Gram-positive and Gram-negative bacteria, so they are widely used in animal husbandry and aquaculture to treat and prevent bacterial infections, and as feed additives to promote animal growth. However, long-term consumption of food contaminated by such drugs will cause drug resistance and drug resistance in humans, and in severe cases, it will cause toxic side effects such as neurotoxicity, renal failure, and potential carcinogenicity. Therefore, countrie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/765G01N33/577G01N33/531
CPCG01N33/531G01N33/558
Inventor 姜金庆刘长忠赵坤杨雪峰王自良范国英胡建和雷壮牛琳琳王靖文
Owner HENAN INST OF SCI & TECH
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