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Hapten spe-ede and its corresponding artificial antigen and its application

A compound, diglycidyl ether technology, applied in animal/human protein, anti-bacterial immunoglobulin, serum albumin, etc., can solve the problems of unsatisfactory rapid detection, expensive instruments, high detection cost, etc., and achieve low cost, High potency, simple instrument requirements

Active Publication Date: 2017-01-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional detection methods of spectinomycin include gas chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry, microbial methods, etc., but they all require expensive instruments and high detection costs, which cannot meet the needs of on-site rapid detection

Method used

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  • Hapten spe-ede and its corresponding artificial antigen and its application
  • Hapten spe-ede and its corresponding artificial antigen and its application
  • Hapten spe-ede and its corresponding artificial antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the preparation of immunogen and coating former

[0032] The schematic diagram of the synthetic route of SPE-EDE-BSA is shown in figure 1 .

[0033] 1. Preparation of hapten

[0034] Dissolve 37.5mg spectinomycin hydrochloride in 5mL3g / 100ml NaHCO 3 Aqueous solution, add 0.5mL EDE-DMF solution dropwise while stirring under ice bath conditions (20mg EDE is dissolved in 0.5mL DMF to obtain 0.5mL EDE-DMF solution), and then stir at 4°C for 15min to obtain a hapten solution (the hapten is SPE-EDE).

[0035] The hapten is represented by formula (I).

[0036] Formula (Ⅰ).

[0037] 2. Preparation of immunogen

[0038] 1. Dissolve 50mg BSA in 5mL0.1mol / L Na 2 CO 3 aqueous solution.

[0039] 2. Add the hapten solution obtained in step 1 dropwise into the BSA solution obtained in step 1 while stirring, and then stir at room temperature for 16 hours.

[0040] 3. Take the solution obtained in step 2, put it into a dialysis bag, and dialyze it in PBS buffer...

Embodiment 2

[0043] Embodiment 2, the preparation of polyclonal antibody

[0044] Dilute the immunogen solution prepared in Example 1 with sterilized physiological saline to a solution of 2 mg / mL (based on protein content), add an equal volume of Freund's complete adjuvant for emulsification, and obtain the first immune preparation. Dilute the immunogen solution prepared in Example 1 with sterilized physiological saline to a solution of 2 mg / mL (based on protein content), add an equal volume of Freund's incomplete adjuvant for emulsification, and obtain a booster immune preparation.

[0045] Take 4-month-old female New Zealand white rabbits: the first immunization is carried out by multi-point intradermal injection on the back of the neck. , 4 weeks later, 6 weeks later, 8 weeks later and 10 weeks later each booster immunization once, using multi-point subcutaneous injection on the back of the neck. ); 10 days after the last booster immunization, blood was collected from the heart, serum ...

Embodiment 3

[0046] Example 3, Determination of Optimal Coating Concentration and Optimal Antibody Working Concentration

[0047]Using the square method. The original coating solution prepared in Example 1 was serially diluted 1:1000 to 1:50000 times (volume ratio) with carbonate buffer solution (pH 9.5, 0.05 mol / L). The serum (antibody) prepared in Example 2 was serially diluted from 1:1000 to 1:100000 times (volume ratio) with PBS buffer (pH7.4, 0.01mol / L). The best antigen coating concentration and the best antibody working concentration were determined by indirect ELISA method. by OD 490nm When the value is close to 1.5, the corresponding antigen coating concentration and antibody concentration are the optimal antigen coating concentration and the optimal antibody working concentration.

[0048] The optimal coating concentration of the coating source (SPE-EDE-OVA) is 0.3 μg / mL (based on protein amount), and the optimal working concentration of the antibody is 1:1500.

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Abstract

The invention discloses a half antigen SPE-EDE and a corresponding artificial antigen and application of the half antigen. A protection formula (I) shows a compound. A method that a compound is shown in a preparation formula (I) is provided, and the method comprises the following steps: spectinomycin hydrochloride is made to react with ethylene glycol bis(2,3-epoxypropyl) ether, and the compound shown in the formula (I) is generated; the compound shown in the formula (I) and conjugate (the artificial antigen) of carrier protein are further provided. The antibody obtained by serving the any conjugate as the immunogen is provided. The invention further provides a kit. The kit comprises the conjugate and the antibody. The invention further provides any conjugate or the antibody or the application of the knit to detection of spectinomycin or spectinomycin derivatives (like spectinomycin hydrochloride). The application has the great practical value in detection of the spectinomycin and the spectinomycin derivatives, operation is easy, the cost is low, and the requirement of a detection limit can be met. The formula (I) is shown in the description.

Description

technical field [0001] The invention relates to a hapten SPE-EDE and its corresponding artificial antigen and application. Background technique [0002] Spectinomycin (SPE) is an aminoglycoside broad-spectrum antibiotic produced by Streptomyces spectiformis. The drug can effectively treat and prevent a variety of livestock and poultry diseases caused by streptococcus, E. Therefore, it is widely used in animal husbandry production. [0003] Because spectinomycin will cause potential harm to human body, especially nephrotoxicity and ototoxicity, the maximum residue limit of spectinomycin residues in animal food has been clearly defined at home and abroad. In 2002, the European Union promulgated the maximum residue limits of spectinomycin in muscle, fat, liver, and kidney of all food animals as 300, 500, 1000, and 1500 μg / kg, respectively. The Ministry of Agriculture of China also promulgated the maximum residue limits of spectinomycin in animal tissues in 2003. The maximum ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D493/04C07K14/765C07K14/77C07K16/12G01N33/53
CPCC07D493/04C07K14/765C07K14/77C07K16/12C07K16/1232C07K16/1235
Inventor 沈建忠江海洋王战辉温凯丁双阳张素霞史为民曹兴元夏曦李建成
Owner CHINA AGRI UNIV
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