Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay and fumonisin technology, applied in the field of chemical detection, to achieve easy separation, improved sensitivity, and good specificity

Inactive Publication Date: 2010-10-20
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] But the minimum detectable concentration of this prior art is 5 μ g / L, along with the continuous improvement of people's health requirement, and the further understanding to the toxicity of mycotoxin, the maximum residue limit (RML) of fumonisin will also have the same stricter requirements

Method used

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  • Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay
  • Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay
  • Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Preparation and purification of polyclonal antibody against keyhole limpet hemocyanin (KLH)

[0028] Fully emulsify the antigen KLH with the same amount of complete Freund's adjuvant, inject 1ml of New Zealand white rabbits intradermally on the back; second immunization: two weeks later, use Freund's incomplete adjuvant, use the same method and Dosage, for immunization; for the third immunization, the operation is the same as the second immunization. Five days after the third immunization, blood was collected from the ear vein to measure the titer. When the titer reached 1:10,000 or more, booster immunization: 1 ml of antigen without adjuvant was injected into the ear vein; blood was collected from the heart after 1 week, and the collected serum was stored at 37°C. After incubating for 1 hour, move to 4°C refrigerator overnight, centrifuge at 3500 rpm for 10 minutes, take the supernatant, and purify it with an affinity chromatography column to obtain rabbit IgG against ...

Embodiment 2

[0030] Fumonisin FB 1 Preparation of complete antigen conjugates

[0031] ① Preparation of fumonisin-keyhole limpet hemocyanin conjugate: put 1ml KLH (10mg / ml) into a dialysis bag, place it in 200ml PBS (containing 0.2% glutaraldehyde) solution and dialyze at 4°C for 16h, then transfer to PBS Dialyze in medium for 8h to remove unreacted glutaraldehyde. Add 2 mg of FB to the KLH dialyzate 1 , 4°C for 16h. Add 10mg Tris and react for 2h to block unreacted protein sites. Finally, it was dialyzed with PBS for 2-3 days and stored at -20°C.

[0032] ② Preparation of ovalbumin-fumonisin conjugate: Dissolve 2.5 mg ovalbumin (OVA) in 0.1 ml 0.01 MPB buffer, add 10 ul of 50% glutaraldehyde, and stir overnight at room temperature. Dialyze against PBS overnight at 4°C to remove excess GA. 0.5mg fumonisins (FB 1 ) was dissolved in 0.2ml of 25% ethanol, added to the activated OVA dialyzate (about 0.15ml), added 0.1ml of 1M carbonic acid buffer (pH9.5), and stirred overnight at 4°C. ...

Embodiment 3

[0034] Preparation of immunomagnetic beads

[0035] Take 50 μl of magnetic beads into a 1.5ml centrifuge tube, place them in a magnetic field to separate the magnetic beads from the storage solution, wash them twice with washing buffer (PBS, pH7.4), take 5 μl of anti-KLH-IgG and use 200 μl of coupling buffer (Sodium phosphate solution, 0.01% Tween20, pH8.2) was added after dilution, and the magnetic beads were resuspended. After shaking and incubating at room temperature for 10-30 minutes, the magnetic beads were washed twice with washing buffer, and 400 μl of 5% skimmed milk powder solution was added to Block the free complexes of unbound antibodies, and wash the magnetic beads twice with washing buffer; then FB 1 - Dilute KLH with coupling buffer to 10 μg / ml, add 200 μl, shake and incubate at room temperature for 10-30 minutes, wash the magnetic beads twice with washing buffer; transfer the magnetic bead suspension to a new tube, and place it in a magnetic field for magnetic...

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Abstract

The invention relates to a method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay, belonging to the technical field of chemical detection. The method comprises the steps of: preparing fumonisins-KLH conjugate and fumonisins-OVA conjugate, combining the fumonisins-KLH conjugate with an immuno-magnetic bead, and preparing a magnetic bead for detecting the fumonisins; then, using the fumonisins-KLH conjugate to prepare fumonisins monoclonal antibody, applying a competition ELISA method for detecting the fumonisins together with the magnetic bead for detecting the fumonisins, and obtaining the magnetic bead for detecting the fumonisins by a magnetic separating method; and developing and obtaining the detection result by an enzyme linked immunosorbent assay. The method is used for the fumonisins sample which is lower than detection limit, and enlarges the combination superficial area by enrichment of the immuno-magnetic bead and the full diffusion of the magnetic bead in the liquid, thus indirectly changing the detection limit, improving the detection sensitivity and avoiding undetected error.

Description

technical field [0001] The invention relates to a method in the technical field of chemical detection, in particular to a method for detecting fumonisins based on immunomagnetic beads combined with enzyme-linked immunosorbent assay. Background technique [0002] Fumonisins are mycotoxins produced by Fusarium moniliforme, and 11 derivatives are currently known. Since the discovery of fumonisins in 1989, it has received widespread attention from all over the world, and many countries have conducted systematic research on it. Fumonisins can contaminate corn and its products, and have been detected in some grain-based products such as noodles, beer, condiments, and even in asparagus. Studies have confirmed that fumonisins can cause leukoencephalomalacia in horses, and neurotoxicity can lead to disturbance of consciousness, blindness and movement disorders, and even death. It can cause pulmonary edema syndrome in pigs, and can induce human esophageal cancer, liver cancer, gastr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/531
Inventor 严亚贤王雨晨孙建和
Owner SHANGHAI JIAO TONG UNIV
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