Time-resolved fluorescence immunoassay kit for aflatoxin M1 in milk

Abstract

The invention discloses a time fluorescence resolved fluorescence immunoassay kit for detecting aflatoxin M1 in milk. The time-resolved fluorescence immunoassay kit for aflatoxin M1 in milk is composed of a porous coated plate, buffer solution, aflatoxin M1 standard substance, antibody freeze-dried substance of the aflatoxin M1, europium labelled Anti-MIgG, washing liquid and enhancement solution.A detection method of the time fluorescence immunoassay kit for detecting the aflatoxin M1 comprises the following steps: (1) preparation of immunogen; (2) preparation of coating antigen; (3) preparation of monoclonal antibody; (4) pretreatment and detection of a sample. The kit provided by the invention needs short detection time, has high average recovery rate, is simple in pretreatment of thesample, can be operated on site for detection, can be applied widely, needs low detection cost, and simultaneously has the advantages of strong detection specificity, small within-run and between-rundifference, high sensitivity, being simple and fast to operate, and particularly being suitable for detection of the samples in large scale.

Description

technical field

[0001] The invention belongs to the field of biological detection, and in particular relates to a time-resolved fluorescent immunoassay kit for detecting aflatoxin M1 in milk. Background technique

[0002] Aflatoxins (AFT) are secondary metabolites of fungi, mainly produced by some strains of Aspergillus flavus, A. parasiticus and A. nonius; The toxin-producing ability of Aspergillus parasiticus varies greatly from strain to strain; all strains of Aspergillus parasiticus can produce aflatoxin. Aflatoxin is a highly toxic hepatotoxin that can cause acute or chronic damage to the liver. It and hepatitis B virus are considered to be the two main causes of liver cancer; A variety of other tissues and organs can also cause serious damage. What is more serious is that aflatoxin has been proven to have the "three causes" of carcinogenicity, teratogenicity, and cell mutation. Aflatoxins are widely found in grain agricultural products such as peanuts, corn, wheat, a...

Examples

Embodiment

[0022] 1. Preparation of immunogen and coating agent

[0023] Synthesis of the immunogen (aflatoxin M1-BSA) of the present invention: Accurately weigh 324 mg of aflatoxin M1 and dissolve in 2mL N,N-dimethylformamide, add γ-aminobutyric acid solution drop by drop under stirring, and stir for 3 Hours, adjust the pH of the reaction solution to about 10. The precipitate was removed by centrifugation. Add the above reaction dropwise to the BSA solution (320mg BSA dissolved in 5mL physiological saline), then add 23mg of N-hydroxysuccinimide (NHS), 45.4mg of N,N-dicyclohexylcarbodiimide (DCC), React overnight at 4°C, remove the precipitate by centrifugation, take the supernatant and dialyze it with phosphate buffered solution (PBS) for 3 days, change the dialysate every 6 hours, freeze-dry the obtained product, and store it at -20°C for later use;

[0024] Synthesis of the original coating (aflatoxin M1-OVA): In the above reaction, after replacing BSA with OVA, the reaction conjuga...

technical field

[0001] The invention belongs to the field of biological detection, and in particular relates to a time-resolved fluorescent immunoassay kit for detecting aflatoxin M1 in milk. Background technique

[0002] Aflatoxins (AFT) are secondary metabolites of fungi, mainly produced by some strains of Aspergillus flavus, A. parasiticus and A. nonius; The toxin-producing ability of Aspergillus parasiticus varies greatly from strain to strain; all strains of Aspergillus parasiticus can produce aflatoxin. Aflatoxin is a highly toxic hepatotoxin that can cause acute or chronic damage to the liver. It and hepatitis B virus are considered to be the two main causes of liver cancer; A variety of other tissues and organs can also cause serious damage. What is more serious is that aflatoxin has been proven to have the "three causes" of carcinogenicity, teratogenicity, and cell mutation. Aflatoxins are widely found in grain agricultural products such as peanuts, corn, wheat, a...

Examples

Embodiment

[0022] 1. Preparation of immunogen and coating agent

[0023] Synthesis of the immunogen (aflatoxin M1-BSA) of the present invention: Accurately weigh 324 mg of aflatoxin M1 and dissolve in 2mL N,N-dimethylformamide, add γ-aminobutyric acid solution drop by drop under stirring, and stir for 3 Hours, adjust the pH of the reaction solution to about 10. The precipitate was removed by centrifugation. Add the above reaction dropwise to the BSA solution (320mg BSA dissolved in 5mL physiological saline), then add 23mg of N-hydroxysuccinimide (NHS), 45.4mg of N,N-dicyclohexylcarbodiimide (DCC), React overnight at 4°C, remove the precipitate by centrifugation, take the supernatant and dialyze it with phosphate buffered solution (PBS) for 3 days, change the dialysate every 6 hours, freeze-dry the obtained product, and store it at -20°C for later use;

[0024] Synthesis of the original coating (aflatoxin M1-OVA): In the above reaction, after replacing BSA with OVA, the reaction conjuga...