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Immunofluorescent rapid test strip of zearalenone and preparation method and use thereof

A technology for detecting zearalenone and test paper, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as large impact, difficult removal, and harm to human health, and achieves a simple preparation method, convenient use, and low cost. Effect

Active Publication Date: 2014-11-26
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ZEN has strong reproductive toxicity and teratogenic effects, which can cause hyperestrogenism in animals, lead to infertility or abortion, and have a great impact on pigs, poultry, and ruminants, and bring great economic losses to animal husbandry; ZEN It also has cytotoxicity, immunotoxicity, liver toxicity, potential carcinogenicity, etc., and ZEN is stable to high temperature, not easy to remove, and seriously endangers human health

Method used

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  • Immunofluorescent rapid test strip of zearalenone and preparation method and use thereof
  • Immunofluorescent rapid test strip of zearalenone and preparation method and use thereof
  • Immunofluorescent rapid test strip of zearalenone and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of Zearalenone Monoclonal Antibody

[0039] 1. Use the active ester method to couple ZEN with OVA to prepare the immune antigen (ZEN-OVA), couple ZEN with BSA to prepare the detection antigen (ZEN-BSA), immunize BALB / c mice, cell fusion, and prepare ZEN monoclonal antibody.

[0040] Three healthy BALB / c mice aged 6-8 weeks (purchased from the Institute of Animals, Southern Medical University) were taken and numbered respectively. Each mouse was immunized with a dose of 100 μg each time. The immunogen ZEN-OVA was mixed with an equal volume of Freund’s complete Adjuvant (prime immunization) or incomplete adjuvant (boost) emulsification (0.2 mL), subcutaneous injection at multiple points on the back, immunization cycle of 14 days, 7 days after each booster immunization, blood was collected from the tail to obtain serum, and stored in Store at -20°C for later use. ZEN-BSA was used as the coating agent, the titer of antiserum was determined by indirect...

Embodiment 2

[0043] Example 2 Preparation of anti-ZEN monoclonal antibody-fluorescent microsphere probe

[0044] Carboxylation of fluorescent microspheres: Dissolve 100 μl fluorescent microspheres in 400 μl triple-distilled water, then add 100 μl 100 mg / ml EDC and 60 mg / ml NHS respectively, and incubate at room temperature for 30 min. The reaction solution was centrifuged at 4°C and 9000 rpm / min for 2 min, the supernatant was discarded, and this step was repeated 3 times to remove excess EDC and NHS. The pellet was resuspended in 500 μl PH7.2 PBS, and sonicated for 3 minutes.

[0045] Preparation of anti-ZEN monoclonal antibody-fluorescent microsphere probe: Mix 500 μl of the carboxylated fluorescent microsphere solution with 100 μl of anti-ZEN monoclonal antibody (45 μg / ml) dissolved in triple distilled water, and react at room temperature for 2 h. Then 2 μl of 10% BSA was added to block the remaining carboxyl groups of the microspheres and incubated for 1 h. Centrifuge at 4°C, 90...

Embodiment 3

[0046] Example 3 Coating of antigens and antibodies

[0047] Spray C and T lines on the nitrocellulose membrane (NC membrane) using an automatic film sprayer, C line is the quality control line, coated with goat anti-mouse IgG, the concentration of goat anti-mouse IgG is 0.8~1.2mg / ml, preferably 1mg / ml, the spray volume is 1μl / cm; the T line is the detection line, the complete antigen ZEN-BSA coated with ZEN, the concentration is 1.2~1.8mg / ml, preferably 1.5mg / ml, the spray volume is 1μl / cm; C , T line spacing is 5mm. Dry the NC film sprayed with C and T lines at 37°C for 6-12 hours.

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Abstract

The invention discloses an immunofluorescent rapid test strip of zearalenone and a preparation method and use of the immunofluorescent rapid test strip of zearalenone. The test strip disclosed by the invention comprises a sample pad, a fluorescent microsphere probe joint pad, a nitrocellulose membrane, absorbent paper and a plastic bottom plate. According to the invention, zearalenone pollution of foods, grains, feed and the like can be detected on the basis of an immune principle of an antigen-antibody. The immunofluorescent rapid test strip of zearalenone, provided by the invention, can be used for fast detection of a field of zearalenone by just 5-10 minutes, and can achieve quantitative detection if being combined with a quantitative fluorescence detector (card reader), and an operator does not need professional training. Therefore, the immunofluorescent rapid test strip has a good market prospect.

Description

technical field [0001] The invention relates to a detection technology for zearalenone pollution in foods, grains and feedstuffs, in particular to a zearalenone immunofluorescence rapid detection test strip and a preparation method and application thereof. Background technique [0002] Zearalenone (Zenralenone, ZEN) is an estrogen-like mycotoxin produced by a variety of Fusarium fungi. Many links such as storage and use may be polluted by ZEN. ZEN has strong reproductive toxicity and teratogenic effects, which can cause hyperestrogenism in animals, lead to infertility or abortion, and have a great impact on pigs, poultry, and ruminants, and bring great economic losses to animal husbandry; ZEN It also has cytotoxicity, immunotoxicity, hepatotoxicity, potential carcinogenicity, etc., and ZEN is stable to high temperature, not easy to remove, and seriously endangers human health. At present, most countries have strictly limited the content of ZEN in food, grain and feed. [...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/558
Inventor 王宏唐勇
Owner JINAN UNIVERSITY
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