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Chemiluminescent enzyme-linked immunosorbent assay kit of tenuazonic acid and application method thereof

A technology of alternarinic acid and chemiluminescent enzyme, which is applied in the field of chemiluminescent enzyme-linked immunoassay kits for alternarinic acid, can solve the problems of rapid detection of samples, poor accuracy and sensitivity, High operational technical requirements and other issues, to achieve good coating effect, high sensitivity, high sensitivity effect

Inactive Publication Date: 2014-01-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TLC is the commonly used method for the initial detection of alternarinic acid. It is simple to operate, but the accuracy and sensitivity are poor.
The sensitivity and accuracy of the instrumental analysis method are high, but the equipment investment is large, the operation technology requirements are high, the pretreatment is cumbersome, the detection cost is high, and it is difficult to meet the rapid detection of on-site and large-scale samples, and it is not easy to promote; moreover, due to the chemical properties of TeA The above performance is strongly acidic, and it is easy to chelate with metals, and the established instrument detection methods all have certain defects
Although the ELISA method has the advantages of strong specificity, rapidity, and sensitivity, it is also suitable for the screening of large batches of samples, but its sensitivity has certain restrictions.

Method used

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  • Chemiluminescent enzyme-linked immunosorbent assay kit of tenuazonic acid and application method thereof
  • Chemiluminescent enzyme-linked immunosorbent assay kit of tenuazonic acid and application method thereof
  • Chemiluminescent enzyme-linked immunosorbent assay kit of tenuazonic acid and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The preparation of embodiment 1 alternarinic acid polyclonal antibody, coating antigen

[0049] (1) Preparation of coated antigen

[0050] The coated original TeAHGA-OVA was prepared by active ester method.

[0051] (2) Preparation of polyclonal antibody to alternarinic acid

[0052] Three rabbits were immunized with the immunogen TeAHGA-BSA as the immunogen, and the basic immunization was carried out first, and the immunization dose was 1 mg / kg body weight. After diluting the immune antigen with normal saline, adding an equal volume of Freund's complete adjuvant to fully emulsify it, adopt multi-point subcutaneous immunization on the back, and boost the immunization after 3 weeks. Booster immunization every two weeks thereafter. From the third booster immunization, blood was collected from the ear vein on the 8th day after immunization, and the titer was determined by indirect competitive ELISA method. After a high titer was obtained, the hapten was injected di...

Embodiment 2

[0053] Example 2 Establishment of chemiluminescent enzyme immunoassay

[0054] (1) Optimization of coating antigen and antibody concentration

[0055] 1) Dilute the coated antigen at 0.1 μg / L, 0.2 μg / L, 0.3 μg / L, 0.4 μg / L, 0.6 μg / L with coating solution (0.05 mol / L pH 5.0 carbonate buffer) and Coat the opaque white luminescent plate longitudinally, 100 μL / well, at 37°C for 24 h, wash twice with washing solution, and pat dry on absorbent paper.

[0056] 2) Add 150 μL / well of the prepared blocking solution for blocking, overnight at 37°C, spin dry and put in an oven to dry.

[0057] 3) Add 50 μL / well of alternarinic acid standard series solution diluted with 0.01 mol / L PBS

[0058] 4) Add 50 μL / well of alternarinic acid monoclonal antibody serially diluted with 0.01 mol / L PBST (1: 28000, 1: 30000, 1: 32000, 1: 34000, 1: 36000), at 37°C 30min, wash the plate 5 times, and pat dry on absorbent paper.

[0059] 5) Add 100 μL / well of diluted horseradish peroxidase-labeled goat ant...

Embodiment 3

[0069] Example 3 Alternaria tenone acid chemiluminescent enzyme-linked immunoassay kit

[0070] (1) Composition of the kit

[0071] 1) Chemiluminescent microtiter plate coated with alternarinic acid antigen: the microtiter plate is a 96-well detachable opaque white luminescent plate, which has been coated with alternarinic acid antigen and blocking solution; The streptosporonic acid antigen was a conjugate of TeAHGA and ovalbumin (OVA), and the coating concentration was 0.3 μg / L.

[0072] Preparation of ELISA plate: Take a 96-well detachable opaque white luminescent plate, dilute the coated antigen to 0.3 μg / L with coating solution, add 100 μL to each well, and overnight at 37°C, pour out the liquid in the well, wash with washing solution for 2 Once, pat dry on absorbent paper. Then add 150 μL of blocking solution to each well, incubate overnight at 37°C, pour off the liquid in the well, dry in an oven at 37°C, and store in a vacuum-sealed aluminum foil bag at 4°C.

[0073]...

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Abstract

The invention discloses a chemiluminescent enzyme-linked immunosorbent assay kit of a tenuazonic acid and an application method thereof, and belongs to the technical field of chemiluminescent enzyme-linked immunosorbent assay. An indirect competition method is adopted by the assay kit; the kit comprises a chemiluminescent ELISA (Enzyme-Linked Immuno-Sorbent Assay) plate coated with tenuazonic acid antigens, a tenuazonic acid standard substance, tenuazonic acid antibodies, enzyme-labeled antibodies, a chemiluminescent liquid and a washing liquid. The application method of the kit comprises the following steps: (1) pretreating a sample to be detected; (2) orderly adding the solution or sample of the tenuazonic acid standard substance and the tenuazonic acid antibodies, and adding the enzyme-labeled antibodies after a competing reaction, and finally adding the chemiluminescent liquid to carry out the quantitative determination of the tenuazonic acid through a chemiluminescent immunoassay analyzer; (3) processing and analyzing the results. The chemiluminescent enzyme-linked immunosorbent assay kit of the tenuazonic acid provided by the invention is high in sensitivity, good in stability and suitable for screening of lots of samples, and thus has enormous practical application and popularization significance.

Description

technical field [0001] The present invention relates to a method for detecting alternarinic acid, more specifically, to a chemiluminescent enzyme-linked immunoassay kit for alternarinic acid and a use method thereof. Background technique [0002] Tenuazonic acid (TeA) is a toxic metabolite produced by Alternaria, Magnaporthe oryzae, etc. The structure is shown in figure 1 . It is a colorless viscous liquid, easily soluble in methanol, ethanol, dimethyl sulfoxide and other organic solvents, but has low solubility in chloroform, benzene and acetone, and has biological activities such as anti-tumor, anti-virus, anti-bacteria and insecticide . At the same time, alternariic acid is also one of the natural pollutants that pollute agricultural food, feed, etc. It is widely found in grains, vegetables, fruits, and field crops. It is the most toxic Alternaria toxin. Long-term consumption and accumulation can cause chronic Poisoning can lead to massive bleeding in the gastrointesti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558G01N21/76
CPCG01N33/5308G01N21/76G01N33/54393G01N2333/37
Inventor 杨金易张燕王弘孙远明雷红涛沈玉栋徐振林李萍
Owner SOUTH CHINA AGRI UNIV
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