Streptomycin hapten and its preparation method and application

A streptomycin and hapten technology, applied in the field of hapten and its preparation, can solve the problems of high cost, expensive equipment, complicated operation and the like, and achieve the effects of convenient use, low detection cost and simple synthesis method

Active Publication Date: 2016-12-14
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of streptomycin residues mainly includes instrumental detection methods, microbiological detection methods, and immunological methods, such as high performance liquid chromatography, fluorescence detection, thin layer chromatography, mass spectrometry, cup and saucer method, paper method, enzyme method, etc. ELISA has the advantages of high sensitivity, less time-consuming, low cost, large-scale detection, etc.

Method used

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  • Streptomycin hapten and its preparation method and application
  • Streptomycin hapten and its preparation method and application
  • Streptomycin hapten and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Synthesis and identification of streptomycin hapten

[0019] The mixed solution of 0.58g streptomycin and 0.07g ethylenediamine in 50ml methanol is reacted at room temperature for 5-10 hours, the solvent is evaporated, and the streptomycin hapten is obtained quantitatively.

[0020] Take the above-mentioned product and measure by hydrogen nuclear magnetic resonance spectroscopy, such as figure 2 As shown, the newly added peaks at 1.6, 2.3, and 3.4 ppm (mg / L) in the spectrum are the signal peaks of the aminoethyl fragment, and the peaks around 7.7 ppm are the CH signal peaks on the enamine double bond, indicating the synthesis of hapten success.

Embodiment 2

[0021] Example 2: Streptomycin antigen

[0022] The streptomycin hapten is coupled with the carrier protein to obtain the streptomycin antigen.

[0023] 1. Preparation of immunogen-synthesis of streptomycin hapten-bovine serum albumin conjugate

[0024] Take 20 mg of streptomycin hapten and dissolve it in 2 ml of water to obtain solution I; take 0.5 ml of 3% glutaraldehyde and add dropwise to solution I, stir at room temperature for 24 hours to obtain solution II; take 50 mg of bovine serum albumin (BSA) Dissolve with 4ml of water to obtain solution III; slowly add solution II to solution III, stir at room temperature and react overnight; add NaBH 4 30mg reduction; dialyzed with 0.02M PBS for three days and replaced the dialysate three times a day to obtain streptomycin immunogen.

[0025] 2. Preparation of the original coating-synthesis of streptomycin hapten-ovalbumin conjugate

[0026] Take 50mg ovalbumin (OVA) and dissolve in 4ml water to obtain solution I; take 50mg each of carbod...

Embodiment 3

[0029] Example 3: Streptomycin monoclonal antibody

[0030] 1. Preparation of Streptomycin Monoclonal Antibody

[0031] Animal immunization: Inject the immunogen into Balb / c mice at a dose of 150μg / mouse to produce polyclonal antibodies.

[0032] Cell fusion and cloning: After the mouse serum assay result is high, take its spleen cells and fuse with SP2 / 0 myeloma cells at a ratio of 8:1. Use indirect competitive ELISA to measure the cell supernatant and screen positive wells. Use the limiting dilution method to clone the positive wells until a hybridoma cell line secreting monoclonal antibodies is obtained.

[0033] Cell cryopreservation and resuscitation: The monoclonal hybridoma cell line of streptomycin is made into 5×10 with cryopreservation solution 7 Cells / ml cell suspension, long-term storage in liquid nitrogen. Take out the cryopreservation tube during resuscitation, immediately put it into a 37℃ water bath for rapid thawing, centrifuge to remove the cryopreserved liquid, and...

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Abstract

The invention discloses a streptomycin hapten, as well as a preparation method and application thereof, and in particular relates to a streptomycin hapten. The invention further discloses a preparation method and an application of the streptomycin hapten. A quick detection enzyme-linked immune kit product built based on the streptomycin hapten is convenient to use, low in detection cost and efficient, accurate and quick in detection, can be used for simultaneously detecting large batches of samples, and is suitable for site monitoring and streptomycin residue in animal derived foods and screening of lots of samples.

Description

Technical field [0001] The invention relates to a hapten and a preparation method and application thereof, in particular to a streptomycin hapten and a preparation method and application thereof. Background technique [0002] Streptomycin (SM) is an important aminoglycoside antibiotic. As an antibiotic, it plays an important role in the treatment of infectious diseases in livestock, but its drug resistance is strong, and the side effects are relatively large. Excessive streptomycin residues in food may cause serious harm to people. In order to ensure the safety of animal-derived foods, the Ministry of Agriculture Document No. 235 "Maximum Residue Limits of Veterinary Drugs in Animal Foods" stipulates that streptomycin is 200μg / L in milk and 600μg / kg in muscle, fat and liver. It is 1000 μg / kg in the kidney. [0003] At present, the detection of streptomycin residues mainly includes instrumental detection methods, microbiological detection methods, and immunological methods, such a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H15/238C07H1/00C07K14/765C07K14/77C07K14/795C07K14/75C07K14/47C07K1/113C07K16/44G01N33/577
Inventor 万宇平罗晓琴孙震冯静李勇杨秀贤刘琳扶胜李晓芳
Owner BEIJING KWINBON BIOTECH
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