Chemiluminescent kit for aflatoxin M1 and application thereof

An aflatoxin and chemiluminescence technology, applied in the field of immunological detection, can solve the problems of long time, cumbersome and time-consuming, limited samples for detection, etc., and achieve the effect of high sensitivity and improved sensitivity

Active Publication Date: 2015-09-09
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The instrument method is a very effective, accurate and sensitive method, but the sample to be tested needs to undergo a series of pretreatments, which is tedious and time-consuming. It takes a long time from sample pretreatment to the test result, and the test cost is very high; on the other hand, this This detection method must also

Method used

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  • Chemiluminescent kit for aflatoxin M1 and application thereof
  • Chemiluminescent kit for aflatoxin M1 and application thereof
  • Chemiluminescent kit for aflatoxin M1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of hapten, antigen and monoclonal antibody

[0040] (1) Synthesis of aflatoxin M1 hapten

[0041] Reaction a: Take 15 mg of aflatoxin M, add 10 ml of dry acetonitrile to dissolve, add dropwise 1 ml of a solution containing 3 mg of boron tribromide, stir at room temperature for 4 hours, stop the reaction, detect, and complete the reaction of all raw materials. Stop the reaction, evaporate to dryness, add water and an appropriate amount of ethyl acetate, extract, stand still, separate layers, separate the water phase, dry over anhydrous sodium sulfate, and evaporate to dryness to obtain product 1.

[0042] Reaction b: Dissolve the product 1 in DMF, add 20 mg of sodium carbonate, stir, add 10 mg of bromobutyric acid, react at 60°C for 8 hours, and detect that all raw materials have been reacted, add water, extract with ethyl acetate, wash with water, dry and evaporate to dryness, and pass through silica gel for a short time. column to obtain ...

Embodiment 2

[0052] Example 2: Preparation of enzyme-labeled antigen

[0053] A, weigh 2mg HRP and dissolve in 0.5mL double distilled water; add 0.5mL newly prepared 0.06mol / L NaIO 4 Solution, 4 ℃ protected from light for 30 minutes;

[0054] B, add 0.5mL of 160mmol / L ethylene glycol, and react at room temperature for 30min;

[0055] C. Add 2 mg of aflatoxin M1-OVA, mix well, put it into a treated dialysis bag, dialyze in 1000 mL of 0.05 mmol / L sodium carbonate buffer, and overnight at 4°C;

[0056] D. Aspirate the dialysate into a 10mL centrifuge tube, add 0.25mL of newly prepared 5g / L NaBH 4 After mixing, place at 4°C for 2h; add an equal volume of saturated ammonium sulfate solution, act at 4°C for 30min, centrifuge at 3000rpm for 25min at 4°C, and discard the supernatant;

[0057] E, dissolve the precipitate in 1.5mL0.02mol / L pH7.4PBS, inhale into the dialysis bag, dialyze in 0.02mol / L pH7.4PBS, overnight at 4°C (replace PBS 3 times in the middle);

[0058] F, suck the liquid in th...

Embodiment 3

[0059] Embodiment 3: the establishment of CLEIA detection method

[0060] (1) Optimal concentration of coating antibody and enzyme-labeled antigen (square array method)

[0061] Coat the chemiluminescence plate longitudinally with serial dilutions of each coating antibody at 80.0, 40.0, 20.0, 10.0, 5.0, 2.5, 1.25, 0.625 μg / mL, 100 μL / well, place in a 37°C incubator for 2 hours, and shoot Dry; block with 150 μL / well blocking solution, place in a 37°C incubator for 2 hours, wash the plate once, and pat dry; add 50 μL / well of a series of diluted enzyme-labeled aflatoxin M1 antigen (1:1000 to 1:512000), Incubate at room temperature (20-25°C) for 15 minutes, wash the plate five times, and pat dry for the last time; add 50 μL / well of chemiluminescence A and B solutions respectively, and measure the luminescence intensity. Specificity determination was carried out with the coating antibody concentration and enzyme-labeled antigen dilution having obvious gradient changes in luminous ...

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Abstract

The invention discloses a chemiluminescent enzyme immunoassay (CLEIA) detection kit for aflatoxin M1. The kit comprises a kit body, a chemiluminescent plate arranged in the kit body and a reagent arranged in the kit body. The kit is characterized in that each hole in the chemiluminescent plate is coated with an anti-aflatoxin M1 antibody and the reagent comprises an enzyme-labeled aflatoxin M1 antigen concentrated solution, an enzyme-labeled aflatoxin M1 antigen diluent solution, aflatoxin M1 series standard solutions, a chemiluminescent substrate liquid A, a chemiluminescent substrate liquid B, a concentrated washing solution and a concentrated complex solution. The chemiluminescent enzyme immunoassay detection kit has the characteristics of high sensitivity, simplicity, rapidness and high accuracy. Compared with traditional ELISA methods, the kit provided by the invention enables operation time to be greatly reduced. The chemiluminescent enzyme immunoassay detection kit can be used for detecting aflatoxin M1 residue in milk and milk powder.

Description

technical field [0001] The invention relates to a chemiluminescent immunoassay kit for detecting aflatoxin M1, which is used for detecting the content or residual amount of aflatoxin M1 in milk and milk powder. It belongs to the field of immunological detection. Background technique [0002] Aflatoxin M1 is one of the structurally similar compounds of aflatoxin, and the probability of aflatoxin appearing in food and feed in hot and humid areas is the highest. The physical and chemical properties are quite stable and will not be destroyed by pasteurization. After mammals ingest feed or food contaminated with aflatoxin B1, it is converted into aflatoxin M1 through hydroxylation. The hazards of aflatoxin M1 are mainly carcinogenic and mutagenic, which can damage the liver tissue of humans and animals, and can lead to liver cancer and even death. Therefore, most government agencies have strict regulations on the amount of aflatoxin that can be ingested by humans and animals. ...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/577G01N33/531
Inventor 万宇平冯月君崔海峰冯静宋灏贾芳芳吴小胜
Owner BEIJING KWINBON BIOTECH
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