ELISA test box for detecting zearalenone and preparing and detecting method thereof

A technology of zearalenone and zearalenone alone, which is applied to the zearalenone enzyme-linked immunoassay test kit and the field of preparation and detection, which can solve the problem of requiring specialized technical personnel, unfavorable promotion and application, detection Expensive and other problems, to achieve the effect of simple operation, convenient use and fast detection

Active Publication Date: 2009-04-22
BEOSON JIANGSU FOOD SAFETY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the physical and chemical analysis techniques of ZEN mainly include thin-layer chromatography, gas-liquid chromatography, mass spectrometry and nuclear magnetic resonance and other methods. Due to the complicated sample pretreatment of the above analysis methods, specialized technicians and detection are required for operation. Expensive, not conducive to the promotion of applications

Method used

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  • ELISA test box for detecting zearalenone and preparing and detecting method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The coated plate is coated with solid-phase antigen, which can be 96 or 48 or 24-well microplate, with 10mmol / LpH9.5 Na 2 CO 3 -NaHCO 3 Dilute the vomitoxin-ovalbumin (ZEN-OVA) to 1.0 μg / mL, add 100 μl to each well of a 96-, 48-, or 24-well microplate, place at 5°C overnight, discard the coating solution, washed twice, followed by adding phosphate buffer solution containing 5% bovine serum albumin, pH 7.5, blocking overnight at 8°C, discarding the blocking solution, blowing dry, sealing the strips and storing them in a freezer at -20°C;

[0021] The standard zearalenone is obtained by diluting the pure zearalenone, the diluent is deionized water, and the concentration of ZEN is: 25ng / mL;

[0022] Preparation of zearalenone-bovine serum albumin (ZEN-BSA) conjugate: Dissolve ZEN in 100mL pyridine, add carboxymethylhydroxylamine hydrochloride, stir overnight at room temperature, blow dry with nitrogen, add 55ml distilled water , use sodium hydroxide solution to adjust t...

Embodiment 2

[0026] The coated plate is coated with solid-phase antigen, which can be 96 or 48 or 24-well microplate, with 55mmol / L pH9Na 2 CO 3 -NaHCO 3 Dilute the vomitoxin-ovalbumin (ZEN-OVA) to 0.55 μg / mL, add 100 μl to each well of a 96 or 48 or 24-well microplate, place it overnight at 2°C, discard the coating solution, washed 5 times, added phosphate buffer solution containing 1% bovine serum albumin, pH 7, blocked overnight at 5°C, discarded the blocking solution, dried, sealed the strips and stored at -40°C;

[0027] The standard zearalenone is obtained by diluting the pure zearalenone, the diluent is deionized water, and the concentration of ZEN is: 50ng / mL;

[0028] Preparation of zearalenone-bovine serum albumin (ZEN-BSA) conjugate: Dissolve ZEN in 3 mL of pyridine, add carboxymethylhydroxylamine hydrochloride, stir overnight at room temperature, blow dry with nitrogen, add 100 mL of distilled water , adjust the pH to about 8.5 with sodium hydroxide solution, then extract 5 ...

Embodiment 3

[0032] The coated plate is coated with solid-phase antigen, which can be 96 or 48 or 24 well microwell plate, with 100mmol / LpH10 Na 2 CO 3 -NaHCO 3 Dilute the vomitoxin-ovalbumin (ZEN-OVA) to 0.1 μg / mL, add 100 μl to each well of a 96-, 48-, or 24-well microplate, place at 8°C overnight, discard the coating solution, washed 3 times, added phosphate buffer solution containing 3% bovine serum albumin, pH 8, blocked overnight at 2°C, discarded the blocking solution, dried, sealed the strips and stored at -30°C;

[0033] The standard zearalenone is obtained by diluting the pure zearalenone, the diluent is deionized water, and the concentration of ZEN is: 0.001ng / mL;

[0034] Preparation of zearalenone-bovine serum albumin (ZEN-BSA) conjugate: Dissolve ZEN in 51.5mL pyridine, add carboxymethylhydroxylamine hydrochloride, stir overnight at room temperature, blow dry with nitrogen, add 10ml Distilled water, adjust the pH to about 9.5 with sodium hydroxide solution, then extract 4 ...

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Abstract

The invention relates to an ELISA kit for detecting zearalenone, the detection is rapid, sensitive, accurate, quantitative, simple in operation, low in requirements on sample purity and strong in specificity, thereby being particularly applicable to the detection of large quantities of samples; and the invention also provides a preparation of the kit and a detection method. The kit comprises washing liquid, color developing liquid A, color developing liquid B and stop solution, and the kit is characterized in that: the kit also comprises a coated plate, a zearalenone standard product, a zearalenone monoclonal antibody freeze-dried product and an enzyme-labeled goat anti-mouse antibody free-dried product; when in detection, the coated plate is taken, 50mu1-100mu1 of the ZEN standard product or a well processed sample is added into the respective micropores, 50mul-100mul of the anti-ZEN antibody is added, the incubation is carried out at 35 DEG C-45 DEG C for about 0.5 hour-1 hour, the washing liquid is used for washing for 3 times-5 times, 50mu1-100mu1 of the horseradish peroxidase (HRP)-goat anti-mouse antibody is added, the incubation is carried out at about 35 DEG C-45 DEG C for about 0.5 hour-1 hour, the washing liquid is used for washing for 3 times-5 times, 50mu1-100mu1 of the color developing liquid A and 50mu1-100mu1 of the color developing liquid B are added, the mixture is placed still in the dark for 10 minutes-20 minutes, then the stop solution is added, the absorbance value is measured at 450nm, and the ZEN content in the sample is calculated from a standard curve.

Description

(1) Technical field [0001] The invention relates to the field of biotechnology, in particular to a test box for enzyme-linked immunoassay of zearalenone and a preparation and detection method. (2) Background technology [0002] Zearalenone (ZEN) can be produced by Fusarium graminearum, Fusarium yellow, Fusarium pink, Fusarium moniliforme, Fusarium three-line, Fusarium solani, Fusarium equisetum, Fusarium oxysporum and other Fusarium Bacteria produce. In the natural state these Fusarium fungi mainly infect maize, causing virus and infection of maize. Especially when the corn harvest season encounters cloudy and rainy days, it is more susceptible to infection. In addition to corn, wheat, barley, and oats can also be infected with these Fusarium fungi. ZEN is a kind of estrogen-like toxin. Animals will produce estrogen hypertoxic toxic reaction after ingestion. Zearalenone has strong reproductive toxicity and teratogenic effect, which can cause estrogen poisoning in animals....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543
Inventor 徐祖奇张建中毛丽华
Owner BEOSON JIANGSU FOOD SAFETY TECH CO LTD
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