Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Reagent box and detection for ochracin A

An ochratoxin and kit technology, which is applied in the field of time-resolved fluorescence immunoassay, can solve the problems of unpractical application, unsatisfactory sensitivity and reagent stability, etc., and achieves the effects of simple structure, high sensitivity and convenient use.

Inactive Publication Date: 2005-05-11
JIANGNAN UNIV
View PDF0 Cites 13 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, the detection sensitivity and the stability of the reagents cannot meet the requirements, so that it cannot be practically applied, so so far there is no domestic kit available on the market

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Reagent box and detection for ochracin A
  • Reagent box and detection for ochracin A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1 prepares kit and detects corn sample

[0015] OTA is a typical hapten, so it has reactogenicity in the immune reaction, but it needs to be combined with macromolecular substances to be immunogenic. The carboxyl group in OTA is an active group that can combine with the amino group of the protein. Therefore, the carbodiimide method can be used to combine OTA with the macromolecular protein KLH, and use the synthetic OTA-KLH as an artificially synthesized immune antigen for animal immunization.

[0016] Preparation of OTA-KLH antigen:

[0017] 1. Dissolve 1-2mg OTA with 1ml dimethylformamide (DMF);

[0018] 2. Use 1ml of 0.13mol / L NaHCO 3 Dissolve 1-2mg KLH carrier protein in coupling buffer;

[0019] 3. Add 0.4-0.8mg OTA solution to KLH solution;

[0020] 4. Dissolve 2-4mg carbodiimide (EDC) in 1mL double-distilled water, and add 50-100μL to the above mixture, and let it work at room temperature for 2 hours (protect from light);

[0021] 5. After centrif...

Embodiment 2

[0060] Embodiment 2 prepares kit

[0061] Preparation of OTA-KLH antigen: same as Example 1.

[0062] Preparation of polyclonal ochratoxin A antibody: same as Example 1.

[0063] Eu 3+ - Preparation of goat anti-rabbit antibody:

[0064] Take 1ml of 5g / L goat anti-rabbit antibody dissolved in 50mmol / L PBS pH7.0, change the buffer condition through PD-10 column, and the eluent is 50mmol / L NaCl containing 0.155mol / L NaCl 2 CO 3 -NaHCO 3 pH9.0 buffer. The protein peaks were collected and quantified by UV absorption analysis (1.46A 280 -0.74A 260 ), dilute the goat anti-rabbit antibody to 2g / L with the eluent above. Take 500μl and add Eu containing 0.2mg 3+ -N 2 -[p-isocyanate-benzyl]-diethylenetriaminetetraacetic acid (Eu 3+ -DTTA) in a vial with magnetic stirring at 28°C for 16 hours. The reaction solution was chromatographed on a Sephadex-G50 column (1×40 cm) equilibrated with 80 mmol / LTris-HCl pH 7.8 buffer solution to collect protein peaks, and diluted for use. ...

Embodiment 3

[0080] Embodiment 3 prepares kit

[0081] Preparation of OTA-KLH antigen

[0082] 1. Dissolve 2mg OTA with dimethylformamide (DMF);

[0083] 2. Use 0.13mol / L NaHCO 3 Dissolve 2mg of KLH carrier protein in coupling buffer;

[0084] 3. Add 0.8mg OTA solution to the KLH solution;

[0085] 4. Dissolve 4 mg of carbodiimide (EDC) in double distilled water, and add 100 μL to the above mixture, and let it act for 2 hours at room temperature (protect from light);

[0086] 5. After centrifugation, take the supernatant and separate it on a column (Sephadex G-25) for UV scanning detection.

[0087] The preparation of the polyclonal ochratoxin A antibody was the same as that in Example 1, with a few omissions.

[0088] Eu 3+ - Preparation of goat anti-rabbit antibody:

[0089] Take 2ml of 5g / L goat anti-rabbit antibody dissolved in 50mmol / L PBS pH7.0, change the buffer condition through PD-10 column, and the eluent is 50mmol / L NaCl containing 0.155mol / L NaCl 2 CO 3 -NaHCO 3 pH 8...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

A detection method includes encapsulating microhole with OTA-BSA, adding OTA standard or sample and OTA antibody, washing out unconnected OTA antibody and EU3+ yangkangtu antiody, adding intensifier to determine fluorescent intensity CPS by time resolution fluorometer, presenting inverse ratio of fluorescent intensity to OTA concentration, comparing to standard curve for obtaining OTA concentration in sample. The kit applying TRFIA to detect OTA based on labelling immune reaction is also disclosed.

Description

technical field [0001] A kit for detecting ochratoxin A and a detection method thereof belong to the technical field of time-resolved fluorescent immunoassay (TRFIA), and are used for detecting the content of ochratoxin A (abbreviated as OTA) in grain, feed and food. Background technique [0002] Ochratoxin A (OTA) is a toxin produced by the fungus Aspergillus ochraceous and several Penicillium fungi. OTA has been proven to cause damage to the kidneys of animals and humans, and is also a carcinogen. Most of the poisoning caused by mycotoxins is caused by mold-contaminated grains, oil crops, and fermented foods, and mycotoxin poisoning is often manifested in obvious places The clinical manifestations are more complex, including acute poisoning, chronic poisoning, carcinogenicity, teratogenicity and mutagenicity. OTA can be isolated from most grains, including barley, wheat, oats, corn, coffee beans, etc. Poultry fed with these grains will also be contaminated. Therefore, in ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N21/64G01N33/533G01N33/547G01N33/569
Inventor 陶文沂金坚黄飚张莲芬时瑾
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com