Light induced chemiluminescent immunoassay kit of aflatoxin B1 and detecting method thereof
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A light-induced chemiluminescence and aflatoxin technology, applied in the field of light-induced chemiluminescence immunoassay, can solve the problems of expensive equipment, time-consuming, low sensitivity, etc., and achieve the effect of short detection time, simple structure and high sensitivity
Inactive Publication Date: 2009-03-25
JIANGSU INST OF NUCLEAR MEDICINE
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[0004] Currently AFB 1 There are multiple assay methods, such as: thin layer chromatography TLC (sensitivity is 5 μg/kg), high performance liquid chromatography HPLC (sensitivity is 0.0...
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Embodiment 1
[0019] Embodiment 1: Preparation kit and detection corn sample
[0020] Luminescent particles and photosensitive particles coated with streptavidin were purchased from Boyang Biotechnology (Shanghai) Co., Ltd.
[0021] Coated with AFB 1 Preparation of luminescent microparticles for artificial antigens:
[0022] Add 1 mg of luminescent microparticles to a centrifuge tube, add 12.5 μL of 1% Tween-20, 0.05 mg of AFB 1 - BSA artificial antigen, 10 μL of sodium borohydride, supplement the volume to 200 μL with 0.1M, pH 6.0 2-(N-morpholine)ethanesulfonic acid (MES) buffer solution, shake and react at 37°C for 48 hours in the dark. Add 10 μL of 0.3M, pH 5.0 carboxymethoxylamine hemihydrochloride (CMO) solution to block unbound sites, incubate at 37°C in the dark for 1 hour, and then centrifuge to separate the connected AFB 1 - BSA luminescent particles, diluted for later use.
[0042] The reagents provided by the kit are the same as in Example 1, and are used to detect barley samples.
[0043] The specific detection steps are as follows:
[0044] First process the barley sample: crush the barley sample to 20 mesh, take 5 grams of the sample and put it in a test tube, add 12.5 mL of extract (methanol:water=7:3). Stopper and vibrate for 3 minutes, filter, and use Xinhua No. 1 paper as the filter paper. Take 1mL of the filtrate and dilute it with 1mL of distilled or deionized water for later use.
[0045] Take 20μL coated with AFB 1 -BSA luminescent particles added to white opaque microwell plate; add 20 μL of AFB 1 Standard or processed samples into respective wells; add 20 μL rabbit anti-AFB 1 Antibody; continue to add 20 μL of biotinylated goat anti-rabbit antibody, incubate at 37°C for 15 minutes; add 175 μL of photosensitive particles coated with streptavidin in the dark, incubate at 37°C for...
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Abstract
The invention discloses a light induced chemiluminescent immunoassay reagent kit for aflatoxin B1and a detection method thereof, which belong to the technical field of light induced chemiluminescent immunoassay. A luminescent particle enveloped with AFB1-BSA is added into a microporosity plate, a AFB1 standard or sample, a rabbit anti-AFB1 antibody, a biotin goat anti-rabbit antibody are sequentially added for a photophobic reaction, then a photosensitive particle enveloped with streptavidin is photophobically added, and the mixture is incubated and then is detected. The AFB1-BSA enveloped on the luminescent particle competes with the AFB1 in the standard or sample for connecting to the AFB1 antibody to form a complex with the biotin goat anti-rabbit antibody and the photosensitive particle enveloped with the streptavidin, the energy is transferred to the luminescent particle to produce fluorescence by producing and transferring singlet ionic oxygen under optical excitation, a light induced chemiluminescent detector is used to detect, the intensity of optical signals is inversely proportional to the concentration of the AFB1, and the content of the AFB1 in the measured sample is determined by contrasting with the standard curve. The invention is used to detect the content of the AFB1 in foodstuff, feedstuff and products of the foodstuff and the feedstuff; and the reagent kit has the advantages of simple structure, simple and convenient operation, low cost, short detection time, and high sensitivity.
Description
technical field [0001] aflatoxin B 1 The light-induced chemiluminescence immunoassay kit and detection method thereof belong to the technical field of light-induced chemiluminescence immunoassay (LICLIA), and are used for detecting AFB in feed, grains and their products. 1 content detection. Background technique [0002] Aflatoxin B 1 (AFB 1 ) is a group of highly toxic secondary metabolites produced by the fungi Hspergillus flavus and A. parasilicus strains, especially AFB 1 It is a strong pollutant with a wide distribution range. It can cause acute poisoning death of humans and various feed animals, and can also cause carcinogenicity, teratogenicity, and mutagenicity. Even if the content of tens of μg / kg is still very toxic. More than 1mg / kg of aflatoxin in food and feed is highly toxic. Its toxicity is 10 times that of potassium chloride and 68 times that of arsenic. After eating food seriously contaminated by aflatoxin, fever, abdominal pain, vomiting, and loss of ...
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