Test kit special for enzyme linked immunosorbent assay adsorption of vomitus toxin and preparing and detecting method thereof

An enzyme-linked immunosorbent and deoxynivalenolase technology, applied in the biological field, can solve problems such as the need for specialized technicians, high detection costs, and unfavorable promotion and application, and achieve the effects of simple operation, rapid detection, and convenient use

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

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods of vomitoxin include high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), etc., because the sample pretreatment of the above-mentioned analytical methods is relatively complicated, and special operations are required during operation. High technical personnel and high testing costs are not conducive to popularization and application

Method used

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  • Test kit special for enzyme linked immunosorbent assay adsorption of vomitus toxin and preparing and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The coated plate is coated with vomitoxin solid-phase antigen, which adopts 96 or 48 or 24 well microwell plate, with 10mmol / L pH9 Na 2 CO3-NaHCO 3 Dilute the vomitoxin-ovalbumin (DON-OVA) to 1.0 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, wash 4, add salt buffer solution containing 5% bovine serum albumin, pH7.5, block overnight at 2°C, discard the blocking solution, blow dry, seal the strips and store in a freezer at -20°C;

[0021] The deionized toxin (DON) standard substance is obtained by diluting the pure product of deoxynivalenol (DON), the diluent is deionized water, and the concentration of DON is: 0.001ng / mL;

[0022] Preparation of vomitoxin-bovine serum albumin (DON-BSA) conjugate: DON was dissolved in pyridine, and 1-butylboronic acid was added to the reaction flask; the mixture was stirred overnight at room temperature to generate 7,15-oxy- (Butylboron)-deoxynivalenol; add suc...

Embodiment 2

[0027] The coated plate is coated with vomitoxin solid-phase antigen, which adopts 96 or 48 or 24 well microwell plate, with 100mmol / L pH10 Na 2 CO 3 -NaHCO 3 Dilute the vomitoxin-ovalbumin (DON-OVA) to 0.1 μg / mL, add 100 μl to each well of a 96-, 48-, or 24-well microplate, place at 5°C overnight, discard the coating Wash twice, add phosphate buffer solution containing 1% bovine serum albumin, pH 7, block overnight at 8°C, discard the blocking solution, blow dry, seal the strips and store in a freezer at -40°C;

[0028] Described deoxynivalenol (DON) standard substance is obtained by diluting from the pure product of deoxynivalenol (DON), the diluent is deionized water, and the concentration of DON is: 1000ng / mL;

[0029] Preparation of vomitoxin-bovine serum albumin (DON-BSA) conjugate: DON was dissolved in pyridine, and 1-butylboronic acid was added to the reaction flask; the mixture was stirred overnight at room temperature to generate 7,15-oxy- (Butylboron)-deoxynivale...

Embodiment 3

[0034] The coated plate is coated with vomitoxin solid-phase antigen, which adopts 96 or 48 or 24 well microwell plate, with 55mmol / L pH9.5 Na 2 CO 3 -NaHCO 3 Dilute the vomitoxin-ovalbumin (DON-OVA) to 4.95 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, followed by adding phosphate buffer solution containing 3% bovine serum albumin, pH 8, blocked overnight at 5°C, discarded the blocking solution, dried, sealed the strips and stored at -30°C;

[0035] Described deoxynivalenol (DON) standard substance is obtained by diluting from deoxynivalenol (DON) pure product, diluent is deionized water, DON concentration is: 500ng / mL;

[0036] Preparation of vomitoxin-bovine serum albumin (DON-BSA) conjugate: DON was dissolved in pyridine, and 1-butylboronic acid was added to the reaction flask; the mixture was stirred overnight at room temperature to generate 7,15-oxy- (Butylboron)-deoxynivale...

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Abstract

The invention relates to a special enzyme-linked immunosorbent assay kit for emetic toxin. 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 special 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 which is coated by emetic toxin solid-phase antigen, an emetic toxin (DON) standard product, an emetic toxin (DON) monoclonal antibody freeze-dried product and an enzyme-labeled goat anti-mouse antibody free-dried product. When in detection, the coated plate is taken, 50muL-100muL of the DON standard product or a well processed sample is added into the respective micropores, 50mul-100mul of the anti-DON 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, 50muL-100muL 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, 50muL-100muL of the color developing liquid A and 50muL-100muL 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 DON 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 special test box for ELISA detection of vomitoxin and a preparation and detection method. (2) Background technology [0002] Vomitoxin, chemically called deoxynivalenol (DON), belongs to the trichothecene family of fungi, and is mostly distributed in grain seeds such as wheat, barley, and corn, and its pollution to grain Fusarium toxins rank first in the rate and contamination level, have high cytotoxicity and immunosuppressive properties, and are produced by several Fusarium pink fungi. DON is usually present in mg / kg levels in wheat, corn and rice. [0003] At present, the detection methods of vomitoxin include high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), etc., because the sample pretreatment of the above-mentioned analytical methods is relatively complicated, and special operations are required during operation. T...

Claims

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Application Information

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