Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit

A technology of chloramphenicol and hapten, which is applied in the preparation of chloramphenicol hapten and antigen, and the application field of quantum dot immunofluorescence kit, which can solve the problem of human body producing drug-resistant bacteria beads, destroying the balance state, allergic reaction, etc. problem, to achieve the effect of simple and feasible preparation method, low cost and high sensitivity

Active Publication Date: 2016-03-02
CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, chloramphenicol residues in animal-derived foods are ingested by the human body for a long time along the food chain, which can cause various diseases
In mild cases, the balance of normal flora in the human body will be destroyed, and the flora

Method used

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  • Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit
  • Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit
  • Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0033] Embodiment 1, the preparation of chloramphenicol hapten

[0034] The preparation method of chloramphenicol hapten, the specific operation steps include:

[0035] Put 323mg of chloramphenicol bulk drug in a 10ml single-neck bottle, dissolve 5ml of pyridine, cool down to 0-5 degrees in an ice bath, add 230mg of p-carboxybenzenesulfonyl chloride in batches, heat up to the outside after 1 hour, stir and react for 18 hours, TLC detects, The crude drug chloramphenicol was completely reacted and the reaction solution was poured into 25ml of ice water to precipitate a white precipitate, filtered, washed with water, and dried to obtain 450 mg of hapten, which was detected by mass.

[0036] The structural formula of the hapten is shown in formula I:

[0037]

Example Embodiment

[0038] Embodiment 2, the preparation of chloramphenicol artificial antigen

[0039] 1. Synthesis of Chloramphenicol Immune Antigen (CAP-BSA)

[0040] ①Weigh BSA50mg, dissolve in 3.5ml CB solution, weigh hapten 50 / 67000*60*507=22.7mg and dissolve in 1.5ml DMF, add NHS50 / 67000*120*115.09=10.3mg, EDC50 / 67000*120* 191.7=17.2 mg, activated at room temperature for 2 hours;

[0041] ② The activation was added dropwise to the CB solution of BSA under ice bath, reacted overnight at 4°C, and dialyzed with PBS.

[0042] The structural formula of the immunogen is as follows.

[0043]

[0044] 2. Synthesis of Chloramphenicol Coated Antigen (CAP-OVA)

[0045] ①Weigh OVA33.5mg, dissolve in 3.5ml CB solution, weigh hapten 33.5 / 45000*60*507=22.5mg and dissolve in 1.5ml DMF, add NHS33.5 / 45000*120*115.09=10.3mg, EDC33. 5 / 45000*120*191.7=17.17mg, activated at room temperature for 2 hours;

[0046] ② The activation was added dropwise to the CB solution of OVA under ice bath, reacted overni...

Example Embodiment

[0049] Example 3. Preparation of chloramphenicol-specific antibody

[0050] 1. Preparation of polyclonal antibodies to chloramphenicol

[0051] Take the immunogen (CAP-BSA) solution prepared in Example 1, and dilute it with pH 7.4, 0.01M PBS buffer to obtain an immunogen dilution, which is used for the preparation of polyclonal antibodies. New Zealand white rabbits were used as immunized animals.

[0052] The immunization process is as follows:

[0053] The first immunization: The immunogen dilution is mixed with an equal volume of Freund's complete adjuvant to make an emulsifier, and it is injected subcutaneously on the back of the neck at multiple points, and the immunization dose is 2.5 mg per animal;

[0054] Booster immunization: 4 weeks after the first immunization, 8 weeks after 8 weeks and 12 weeks after the first immunization, a booster immunization was performed, and the immunogen dilution was mixed with an equal volume of Freund's incomplete adjuvant to emulsify. T...

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Abstract

The invention discloses chloramphenicol hapten, corresponding artificial antigen and a monoclonal antibody marked by a quantum dot and further discloses a preparing method and application of the chloramphenicol hapten, the corresponding artificial antigen and the monoclonal antibody marked by the quantum dot. The chloramphenicol antigen can be obtained when the chloramphenicol hapten and carrier protein are connected. The chloramphenicol antigen can be applied to preparing a chloramphenicol specific antibody. The preparing method is easy and convenient to implement and low in cost, and the yield of the hapten is high. The artificial antigen and the chloramphenicol specific antibody marked by the quantum dot can be used for preparing a quantum dot immunofluorescence reagent kit for detecting chloramphenicol residues and have the advantages of being simple, fast, large in quantity of treated samples, high in sensitivity, high in specificity and the like.

Description

technical field [0001] The invention relates to a hapten, an antigen and a preparation method thereof, in particular to a preparation method of a chloramphenicol hapten and an antigen and their application in a quantum dot immunofluorescence kit. Background technique [0002] Chloramphenicol (Chloramphenicol, CAP) is a cheap and highly effective broad-spectrum antibiotic that has a good inhibitory effect on both Gram-positive and Gram-negative bacteria. It was once widely used in agriculture and animal husbandry. However, the residual chloramphenicol in food of animal origin is ingested by the human body for a long time along the food chain, which can cause various diseases. In mild cases, the balance of normal flora in the human body will be destroyed, and flora imbalance will cause the human body to produce drug-resistant bacteria beads, which will bring adverse effects on the use of antibiotics for future illnesses; people with antibiotic allergies will have allergic reac...

Claims

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Application Information

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IPC IPC(8): G01N33/558
Inventor 江海洋沈建忠吴小平王战辉丁双阳李建成史为民温凯王照鹏王世恩王文珺韩京朋
Owner CHINA AGRI UNIV
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