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Amikacin semi-antigen, and preparation method and application thereof

A technology of amikacin and hapten, which is applied in the field of hapten and its preparation, can solve the problems of complex and expensive chromatography, unsuitability for screening, cumbersome processing, etc., and achieve accurate detection method, good affinity, and detection low cost effect

Active Publication Date: 2014-01-15
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chromatography requires complex and expensive instruments, and the sample pretreatment process is cumbersome, which is not suitable for the screening of a large number of samples on site

Method used

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  • Amikacin semi-antigen, and preparation method and application thereof
  • Amikacin semi-antigen, and preparation method and application thereof
  • Amikacin semi-antigen, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Synthesis and identification of amikacin hapten (synthetic route such as figure 1 )

[0019] The mixture of 0.59 g amikacin, 0.18 g p-carboxybenzaldehyde and 1 ml pyridine in 20 ml DMSO was stirred and reacted at 60°C for 20 hours, the solvent was evaporated, purified by column chromatography in ethanol-water system The condensation product of amikacin and p-carboxybenzaldehyde was obtained by medium recrystallization with a yield of 70%.

[0020] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown, the nuclear magnetic spectrum shows that: the newly added carboxyl signal peak at about 12.7ppm and the aromatic ring signal peak between 8-9ppm in the spectrum indicate that the hapten is successfully synthesized.

Embodiment 2

[0021] Embodiment 2: Amikacin antigen

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

[0023] 1. Immunogen preparation - synthesis of amikacin hapten-bovine serum albumin (BSA) conjugate

[0024] Dissolve 15 mg of amikacin hapten in 1.5 ml of water to obtain solution I; take 10 μl of 50% glutaraldehyde (GA) and add it to solution I, stir and react at room temperature for 18 hours to obtain solution II; take 60 mg of BSA in 4.5 ml Dilute with water and add to solution II; add 24mg NaBH after reacting overnight 4 React for 3 hours; dialyze with triple distilled water for 48 hours to obtain the immunogen.

[0025] 2. Coating source preparation - synthesis of amikacin hapten-ovalbumin (OVA) conjugate

[0026] Dissolve 50mg of carbodiimide (EDC) in 2ml of water to obtain solution III; dissolve 18mg of amikacin hapten in 2ml of water to obtain solution IV; dissolve 30mg of OVA in 1ml of 0.01mol / L PBS (pH =8.0) solution to obtain ...

Embodiment 3

[0029] Embodiment 3: Amikacin monoclonal antibody

[0030] 1. Preparation of Amikacin Monoclonal Antibody

[0031] Animal immunization: Inject the immunogen into the body of Balb / c mice with an immunization dose of 150 μg / mouse to make them produce polyclonal antibodies.

[0032] Cell fusion and cloning: After the measurement result of mouse serum was higher, the splenocytes were taken and fused with SP2 / 0 myeloma cells at a ratio of 8:1, and the cell supernatant was measured by indirect competitive ELISA, and the positive wells were screened. Use the limiting dilution method to clone the positive wells until hybridoma cell lines secreting monoclonal antibodies are obtained. It is found that the titer of one of the hybridoma cell lines is significantly higher than that of the other hybridoma cell lines, and the amikacin monoclonal antibody is hybridized. The tumor cell line was named D-4-3, and the cell line was preserved on May 21, 2012 in the General Microbiology Center of ...

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Abstract

The invention discloses a semi-antigen and a preparation method and application thereof and specifically relates to an amikacin semi-antigen. The invention further discloses a preparation method and application of the amikacin semi-antigen. A rapid detection kit product established on the basis of the amikacin semi-antigen has the advantages of convenient usage, low detection cost, usage of highly efficient, accurate and rapid detection methods and capacity of simultaneous on-site monitoring of amikacin residue and screening of considerable samples.

Description

technical field [0001] The invention relates to a hapten and its preparation method and application, in particular to an amikacin hapten and its preparation method and application. Background technique [0002] Aminoglycoside antibiotics (AGs) are glycosides formed from aminosugar and aminocycloalcohol. The structure and physicochemical properties of AGs are similar. Because the structure contains multiple amino groups and hydroxyl groups, it is alkaline, polar and water-soluble. AGs have strong antibacterial effect and are commonly used antibiotics. Commonly used aminoglycosides contain 1 1, 3 or 1, 4 diaminocyclitol and 1 or 2 amino sugars. According to the structure of aminocyclitols, AGs can be divided into two categories: streptomycin and 2-deoxystreptamine. According to their sources, AGs can be divided into: antibiotics produced by Streptomyces, including streptomycin, kanamycin and neomycin; antibiotics produced by Micromonospora, mainly including gentamicin . ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H15/23C07K14/765C07K14/77C07K14/795C07K14/75C07K14/47C07K1/113C07K16/44G01N33/577G01N33/08
Inventor 何方洋万宇平杨昌松孙震蒲小容刘琳靳开颜韩雪倩
Owner BEIJING KWINBON BIOTECH
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