Antigen mimotope ph15 of bisphenol A and its application

A mimetic epitope, ph15 technology, applied in recombinant DNA technology, DNA/RNA fragments, material testing products, etc., can solve the problems of increasing testing costs, easily causing harm to testing personnel and the environment, and restricting the application and promotion of immunological methods. , to achieve the effect of reducing the harm to human health, good effect and cost saving

Inactive Publication Date: 2016-11-02
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method must use standard products, which not only increases the cost of detection, but also easily causes harm to the detection personnel and the environment, which restricts the application and promotion of immunological methods to a certain extent.

Method used

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  • Antigen mimotope ph15 of bisphenol A and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Affinity panning and identification of bisphenol A antigen mimotope

[0018] Affinity panning of mimotopes of bisphenol A antigen: the specific method is: dilute anti-bisphenol A monoclonal antibody with 10mM PBS (pH 7.4), and coat a 96-well microtiter plate with a final concentration of 20 µg / mL, 4 Incubate overnight at °C. The next day, wash 10 times with TBST (50mM NaCl, pH 7.5 containing 0.1% Tween-20 (v / v)), add 300 μL blocking solution (3% BSA-PBS) and incubate at 4°C for 2 hours. Discard the blocking solution after two hours, and dilute the phage stock solution 10 times with TBS, about 1.0×10 11 pfu). Shaking reaction at 35°C for 1 hour. 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 with 15 μL 1M Tris-HCl (pH 9.1). Take 10 μL of the eluted phage to measure the titer, and the rest is used to infect 20 mL of E. Coli ER2738 strain that has grown to the p...

Embodiment example 2

[0022] Implementation Case 2. Sequencing of the Bisphenol A Antigen Mimotope Encoding Gene and Determination of its Amino Acid Sequence

[0023] The phages displaying bisphenol A antigen mimic epitopes identified by indirect competition ELISA in Example 1 were amplified, and the DNA sequencing templates of the phages were extracted. The brief process is as follows: For phage amplification, transfer 800 μL of phage-containing supernatant to a new centrifuge tube after the first centrifugation. 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, 4M NaCl), add 250 μL absolute ethanol to precipitate DNA, centrifuge and wash the pellet with 70% ethanol (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 taken for DNA sequencing, and the -96 gⅢ sequencing pri...

Embodiment example 3

[0024] Example 3 Application of bisphenol A antigen mimotope as a competitive antigen in ELISA

[0025] (1) Sample extraction

[0026] Weigh 5g samples (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200rpm for 5min; filter the extract with Whatman No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS (phosphate Buffer, pH=7.2) After mixing, it is the sample extract, ready for use.

[0027] (2) Coating and sealing

[0028] Dilute the anti-bisphenol A 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.

[0029] (3) Establishment of standard curve

[0030] Take out the plates that have been treated in step (2), and put 50 μL of phage (1.0×10 11 pfu) and a series of 50...

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Abstract

The invention belongs to the field of biotechnologies and relates to an antigenic mimic epitope of bisphenol A. The amino acid sequence of the antigenic mimic epitope is YPERDAYVHFLA. The antigenic mimic epitope of bisphenol A, disclosed by the invention, can replace a bisphenol A standard which is expensive in price and strong in toxicity, is applied to the immunological detection of bisphenol A as a competitive antigen or solid-phase coating antigen, has the immunoreaction characteristics similar to those of natural bisphenol A molecules and has a very good effect, so that the injury to the health of a human body caused by bisphenol A is reduced, the cost is reduced, and the application value is very high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a bisphenol A antigen mimic epitope and an application thereof. Background technique [0002] Bisphenol A (BPA) is a chemical material used to produce shatter-resistant plastics. Data show that bisphenol A is a toxic chemical. Animal experiments have found that bisphenol A has the effect of simulating estrogen, and even a very low dose can cause animals to have precocious puberty, decreased sperm count, and prostate growth. In addition, according to literature reports, bisphenol A has certain embryotoxicity and teratogenicity. Bisphenol A can lead to endocrine disorders and threaten the health of fetuses and children. Cancer and obesity caused by metabolic disorders are also considered to be related to this. In the human living environment, bisphenol A is ubiquitous. From beverage bottles, medical equipment to food packaging, many plastic products contain bisphenol A. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/11G01N33/68G01N33/577
Inventor 许杨何庆华孙澄浩陈静
Owner NANCHANG UNIV
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