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Antigen mimotope that mimics norfloxacin and its application

A mimetic epitope, norfloxacin technology, applied in the direction of material inspection products, instruments, analytical materials, etc., can solve the health and environmental threats of testing personnel, restrict the application and promotion of immunological detection methods, and norfloxacin is expensive Carcinogenicity and other issues, to achieve good results, save costs, and reduce the effects of human health hazards

Inactive Publication Date: 2016-10-26
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of establishing an immunological detection method, standard norfloxacin must be used as a raw material to prepare a competing antigen or a solid-phase coating antigen. It poses a great threat to the health of personnel and the environment, thus restricting the application and promotion of immunological detection methods to a certain extent.

Method used

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  • Antigen mimotope that mimics norfloxacin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Affinity panning and identification of norfloxacin antigen mimotope

[0024] 1) Affinity panning of norfloxacin antigen mimic epitopes: the specific method is: dilute anti-norfloxacin monoclonal antibody with 10 mM PBS (pH 7.4), and coat 96 wells with a final concentration of 100 μg / mL ELISA plates were incubated overnight at 4°C. The next day, after washing 10 times with TBST (50 mM NaCl, pH 7.5 containing 0.1% Tween-20 (v / v) ), add 300 μl of blocking solution (3% BSA-PBS) and incubate at 4°C for 2 hours. After 2 hours, discard the blocking solution, wash 5 times with TBST, add 100 μl phage peptide library (phage display ring heptapeptide library or dodecapeptide library, purchased from NEB Company, dilute the phage stock solution 10 times with TBS, about 1.0× 10 11 pfu), shake and react for 1 hour at 22-26°C. Unbound phages were discarded, washed 10 times with TBST, bound phages were eluted with 0.2 M Glycine-HCl (pH 2.2), and immediately neutralized wi...

Embodiment 2

[0028] Example 2. The sequencing of the mimotope encoding gene of norfloxacin antigen and the determination of its amino acid sequence

[0029] The phages identified by indirect competition ELISA displaying mimotopes of the norfloxacin antigen were amplified, and the DNA sequencing templates of the phages were extracted. The brief process is as follows: For phage amplification, after the first step of centrifugation, transfer 800 µl of the phage-containing supernatant to a new centrifuge tube. Add 200 µl PEG / NaCl to precipitate the phage. After centrifugation, resuspend the pellet in 100 µl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), add 250 µl absolute ethanol to precipitate DNA, centrifuge and wash with 70% ethanol Precipitation (DNA sequencing template). The pellet was finally resuspended in 20 µl of sterilized water, and 2 µl was taken for agarose gel electrophoresis analysis; 5 µL of phage template was used for DNA sequencing, and its -96 gIII sequencin...

Embodiment 3

[0030] Example 3. The application of mimotopes of norfloxacin antigens as competitive antigens in ELISA

[0031] (1) Sample extraction

[0032] Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS ( Phosphate buffer, pH=7.2)

[0033] After mixing, the sample extract is ready for use.

[0034] (2) Coating and sealing

[0035] Dilute anti-norfloxacin monoclonal antibody with 10 mM PBS (pH 7.4), coat the microtiter plate with 10 µg / mL, and incubate overnight at 4°C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v / v)), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, then wash the plate 6 times with PBST stand-by.

[0036] (3) Establishment of standard curve

[0037] Take out the strips treated in step (2), and put 50 µl of phage displaying mimotopes of norfloxac...

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Abstract

The invention belongs to the field of biotechnology and relates to an antigen mimic epitope of norfloxacin, the amino acid sequence of which is DHAYWPKFWGKA. Norfloxacin antigen mimetic epitope of the present invention can replace expensive and highly toxic norfloxacin standard substance, and be used as competition antigen or solid-phase coating antigen to the immunological detection of norfloxacin, this antigen mimetic table The bit has similar immune response properties to the natural norfloxacin molecule and works very well. The harm of norfloxacin to human health is reduced, the cost is saved, and the invention has high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to norfloxacin antigen mimic epitopes and applications thereof. Background technique [0002] Norfloxacin (Norfloxacin, NOR) is a broad-spectrum antibiotic, which can absorb water in the air, and its color will gradually become darker when exposed to light. It is slightly soluble in dimethylformamide, very slightly soluble in water or ethanol, and is Soluble in hydrochloric acid or sodium hydroxide solution, it has high antibacterial activity against aerobic gram-negative bacilli, and has good antibacterial effect on most bacteria in vitro, such as most bacteria of Enterobacteriaceae, including Citrobacter, cloacae Enterobacter, Enterobacter aerogenes, Escherichia coli, Klebsiella, Proteus, Salmonella, Shigella, Vibrio, Yersinia, etc. Norfloxacin also has antibacterial activity against multidrug-resistant bacteria in vitro. It also has good antibacterial effect on penicill...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K7/06C12N15/11G01N33/68G01N33/577
Inventor 何庆华许杨熊争平
Owner NANCHANG UNIV
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