Antigenic mimic epitope of vardenafil and application thereof

A mimetic epitope and vardenafil technology, which is applied in the direction of material inspection products, biological testing, peptides, etc., can solve the problems of increasing testing costs, testing personnel and the environment are easy to cause harm, and restricting the application and promotion of immunological methods. Achieve the effect of reducing the harm to human health, good effect and cost saving

Inactive Publication Date: 2013-04-17
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method must use vard standard products, which not only increases the detection cost, but also easily causes harm to the

Method used

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  • Antigenic mimic epitope of vardenafil and application thereof

Examples

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Example Embodiment

[0019] Example 1. Affinity panning and identification of vardenafil antigen mimotopes

[0020] 1) Affinity panning of vardenafil antigen mimotope: the specific method is: dilute anti-vardenafil monoclonal antibody with 10 mM PBS (pH 7.4) and coat 96 wells with a final concentration of 100 μg / mL Incubate the ELISA plate overnight at 4°C. After washing 10 times with TBST (50 mM NaCl, pH 7.5 containing 0.1% Tween-20 (v / v)) the next day, add 300 μl 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 of phage peptide library (phage display cycloheptapeptide library or dodecapeptide library, purchased from NEB company, 10 times diluted phage stock solution with TBS, about 1.0× 10 11 pfu), shaking at 22-26°C for 1 hour. The unbound phage was discarded and washed 10 times with TBST. The bound phage was eluted with 0.2 M Glycine-HCl (pH 2.2) and immediately neutralized with 15 μl 1 M Tris-HCl (...

Example Embodiment

[0024] Example 2. Sequencing of vardenafil antigen mimotope coding gene and determination of its amino acid sequence

[0025] The phage displaying vardenafil antigen mimic epitope identified by indirect competitive ELISA was amplified, and the DNA sequencing template of the phage was extracted. The brief process is as follows: carry out phage amplification, after the first centrifugation, transfer 800 μl of 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 the DNA, and then wash with 70% ethanol after centrifugation Precipitation (DNA sequencing template). The pellet was finally resuspended in 20 μl sterile water, and 2 μl was taken for agarose gel electrophoresis analysis; 5 μL phage template was taken for DNA sequencing, and the -96 gIII sequencing primer was: 5’- HO CC...

Example Embodiment

[0026] Example 3. Application of vardenafil antigen mimotope as a competing antigen in ELISA

[0027] (1) Sample extraction

[0028] Weigh 5g of the sample to be tested, add 25 ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with Whatman No. 1 filter paper, take 1 ml of the filtrate and add 4 ml of PBS (phosphate buffer, pH=7.2)

[0029] After mixing, it is the sample extract, ready for use.

[0030] (2) Coating and sealing

[0031] Dilute the anti-vardenafil monoclonal antibody with 10 mM PBS (pH 7.4), coat the plate with 10 μg / ml, and incubate overnight at 4°C. After washing with PBST (10 mM PBS, 0.05% Tween-20 (v / v)) 3 times on the second day, blocking with PBS containing 3% skimmed milk powder, incubating at 37°C for 1 hour, washing the plate 6 times with PBST stand-by.

[0032] (3) Establishment of standard curve

[0033] Take out the slats processed in step (2), and put 50 μl of phage displaying mimotopes of vardenafil antigen into each...

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Abstract

The invention belongs to the field of biotechnology, and relates to an antigenic mimic epitope of vardenafil. The amino acid sequence of the antigenic mimic epitope is CKFPSHPYC. The antigenic mimic epitope of the vardenafil, provided by the invention, can replace expensive vardenafil non-standard products, can be applied to the vardenafil immunological detection as a competitive antigen or solid-phase envelope antigen, has similar immune response characteristics to natural vardenafil non-molecular, can reduce the harm to human health due to vardenafil and save the cost, and has an excellent effect and very high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to vardenafil antigen mimic epitopes and applications thereof. Background technique [0002] Vardenafil (vardenafil, vard) is a kind of phosphodiesterase-5 inhibitors (PDE-5 inhibitors) produced by Bayer AG in Germany, which is used for the treatment of male erectile dysfunction. Natural aphrodisiac drugs are considered safe and have no side effects, and are favored by consumers all over the world. However, illegal addition of PDE-5 inhibitors to natural aphrodisiac drugs will cause a series of side effects on public health, such as headaches, facial flushing, Indigestion, blurred vision and muscle aches. PDE-5 inhibitors interact with nitrate drugs and can cause severe hypotension. Therefore, it is of great significance to human health to strengthen the detection of illegally added PDE-5 inhibitors in natural aphrodisiac drugs. [0003] Currently, detection methods for s...

Claims

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Application Information

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IPC IPC(8): C07K7/06C12N15/11G01N33/68
Inventor 何庆华许杨刘星
Owner NANCHANG UNIV
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