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Enzyme linked immunosorbent assay kit used for detecting ochratoxin A, and applications thereof

An ochratoxin and enzyme-linked immunoassay detection technology, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of unsuitable for on-site monitoring and screening, long detection cycle, and various reagents, etc., and achieves a simple pretreatment process , high accuracy and low pre-processing requirements

Active Publication Date: 2014-02-12
BEIJING KWINBON BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the analysis methods for ochratoxin A residues mainly include chemical analysis and immunochemical analysis. Chemical analysis was first applied to the detection of toxin residues. In 1973, thin-layer chromatography became the detection method of the International Association for Chemical Analysis (ACAC). The official method of ochratoxin A, thin-layer chromatography has the advantages of simple method and cheap reagents, but it has poor sensitivity, many reagents required, long detection cycle, instrument detection, such as liquid chromatography-mass spectrometry It has the characteristics of high sensitivity, but the equipment is expensive and requires complicated sample pretreatment, so it is not suitable for on-site monitoring and screening of a large number of samples

Method used

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  • Enzyme linked immunosorbent assay kit used for detecting ochratoxin A, and applications thereof
  • Enzyme linked immunosorbent assay kit used for detecting ochratoxin A, and applications thereof
  • Enzyme linked immunosorbent assay kit used for detecting ochratoxin A, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 Preparation of kit components

[0038] 1. Preparation of ochratoxin A hapten

[0039] 1) Dissolve 20 mg of ochratoxin A, 10 μl of 1,3-propanediamine, catalytic amount of 4-dimethylaminopyridine (DMAP) in 2 ml of N,N'-dimethylformamide (DMF), Get liquid I;

[0040] 2) Dissolve 20mg of N,N’-dicyclohexylcarbodiimide (DCC) in 0.5ml of DMF to obtain liquid II;

[0041] 3) Slowly add solution II to solution I dropwise at 0°C, and continue to react for 20 h after returning to room temperature;

[0042] 4) Evaporate the solvent and perform column chromatography (eluent: dichloromethane / methanol, volume ratio 20:1) to obtain the ochratoxin A hapten. The synthetic route is shown in figure 1 .

[0043] Get above-mentioned product and measure through proton nuclear magnetic resonance spectrum, such as figure 2 As shown in the NMR spectrum, the new amide characteristic peak at 8.0ppm, the disappearance of the carboxyl signal peak, and the three groups of alkane si...

Embodiment 2

[0068] Example 2 Formation of an ELISA Kit for Detecting Ochratoxin A

[0069] An enzyme-linked immunosorbent assay kit for detecting ochratoxin A was set up to include the following components:

[0070] (1) A microtiter plate coated with ochratoxin A-conjugated antigen;

[0071] (2) 6 bottles of ochratoxin A standard solution, the concentrations are 0μg / L, 1μg / L, 3μg / L, 9μg / L, 27μg / L, 81μg / L;

[0072] (3) Concentrate the enzyme conjugate;

[0073] (4) Enzyme conjugate diluent;

[0074] (5) The substrate chromogenic solution is composed of substrate solution A and substrate solution B, the substrate solution A is carbamide peroxide, and the substrate solution B is tetramethylbenzidine;

[0075] (6) The stop solution is 2mol / L sulfuric acid.

Embodiment 3

[0076] The detection of ochratoxin A in the sample of embodiment 3

[0077] 1. Sample pretreatment

[0078] Homogenize the feed sample with a homogenizer; weigh 5.0g±0.05g of the homogenized feed sample into a 50ml polystyrene centrifuge tube, add 25ml of 50% methanol, shake vigorously with a shaker for 5min, keep at room temperature (20 Centrifuge at -25°C / 68-77°F) for 5 min; take 500 μl of the supernatant to a 2ml polystyrene centrifuge tube, add 500 μl of 10% sodium chloride aqueous solution and shake with a shaker for 1 min, mix well; take 20 μl for analysis.

[0079] 2. Detection with kit

[0080] Add 20 μl of ochratoxin A standard solution / sample to the microwells of the microtiter plate coated with ochratoxin A conjugated antigen, and then add 100 μl of enzyme conjugate working solution (use enzyme conjugate diluent for concentrated enzyme conjugate Dilute according to the volume of 1:20), seal the plate with a cover plate, react in the dark at 25°C for 10 minutes, po...

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Abstract

The invention provided an enzyme linked immunosorbent assay kit used for detecting ochratoxin A. The enzyme linked immunosorbent assay kit comprises an elisa plate containing ochratoxin A coupling antigen, a standard solution of ochratoxin A, a concentrated enzyme conjugate, an enzyme conjugate working solution, a substrate color development solution, and a stopping solution. The invention also discloses a method used for detecting ochratoxin A using the enzyme linked immunosorbent assay kit, and comprises steps of pretreatment of samples, detection via the enzyme linked immunosorbent assay kit, and analysis of detection results. The enzyme linked immunosorbent assay kit can be used for detecting ochratoxin A residual quantity in forage (raw materials, auxiliary materials and concentrates); operation is simple and convenient; cost is low; sensitivity is high; and the enzyme linked immunosorbent assay kit is capable of realizing on-site monitoring, and is suitable for screening of a large amount of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting ochratoxin A, which is suitable for the determination of ochratoxin A in feed (raw materials, batch materials, concentrated materials). technical background [0002] Ochratoxin A (Ochratoxin A) is produced by two types of molds, Aspergillus and Penicillium, and often contaminates food and feed. Ochratoxin A can cause acute and chronic toxicity in animals, is a strong carcinogen, can cause irreversible lethal poisoning to the kidney, cause kidney atrophy, and can also cause fetal malformation, abortion or even death, seriously affecting the function and growth of animals. Potential hazards to humans are of concern. For this reason, the International Agency for Research on Cancer (JARC) has identified it as a Class IIB carcinogen, and strengthening the detection of ochratoxin A is an important method to pr...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/532G01N21/78
CPCG01N33/535G01N33/577G01N33/68
Inventor 何方洋万宇平冯静吴继华李勇韩雪倩何丽霞朱亮亮
Owner BEIJING KWINBON BIOTECH
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