Immunofluorescence quenching detecting method for microcystin-LR
A technology of microcystin and a detection method, applied in the field of immunoassay chemistry, can solve the problems such as the inability to meet the requirements of rapidity, convenience and accuracy, the lack of commercial supply of specific protein phosphatase, poor specificity and the like, and the convenience of cleaning and separation is achieved. , Simplified reaction conditions, easy operation effect
Inactive Publication Date: 2012-07-11
JIANGNAN UNIV
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Problems solved by technology
Instrumental analysis methods mainly include high-performance liquid chromatography (HPLC), mass spectrometry, liquid chromatography-mass spectrometry, etc. Although these methods are sensitive, they require expensive instruments and equipment, professional operators, and relatively high requirements for inspection materials. Further sample pretreatment can be carried out, which can no longer meet the requirements of modern detection for fast, convenient and accurate
The biochemical method is mainly the protein phosphatase inhibition test method, which has the advantage of being fast and can detect a large number of samples in a few hours, but its biggest disadvantage is that specific protein phosphatases are not commercially available and have poor specificity
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Embodiment 1
[0044] (1) Preparation of immunogen:
[0045] ① Take 0.25 mL of ethanol solution dissolved with microcystin-LR (MC-LR), add 0.75 mL of deionized water, and add 150 μL of carbodiimide (EDC) to obtain liquid A.
[0046] ② Dissolve 2 mg of bovine serum albumin (BSA) in 1 mL of 25% ethanol solution to obtain liquid B.
[0047] ③ Add liquid A to liquid B dropwise, then add 150 μL of carbodiimide (EDC), mix well, and react at 4°C for 12 hours. A mixed liquid of microcystin-LR-BSA conjugate was obtained.
[0048] ④ Transfer the mixture of microcystin-LR-BSA conjugates into a dialysis bag, and dialyze with 6×1 L deionized water for 4-6 days. Finally, the liquid in the dialysis bag was made into powder by freeze-drying method, and the artificial antigen: microcystin-LR-BSA was obtained as the immunogen.
[0049] Dialysis bag pre-treatment: Take a 10cm dialysis bag, boil it for 5 minutes, rinse it with 60°C deionized water for 3 minutes, and store it in 4°C deionized water for later ...
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The invention discloses a detection method for immunofluorescence quenching of microcystin-LR, which pertains to the field of immunological analysis chemical technology and comprises the preparation of immunogen, primordial covering and antibodies, the modification of magnetic nanoparticles, the coupling of the magnetic nanoparticles and the primordial covering, the preparation of gold nanoparticles, the preparation of a gold nano-probe, the fixing of the primordial covering, the immunoreaction in a flow injection system, the covering of a fluorescent enzyme target and the detection of the flow injection fluorescence quenching. The invention greatly improves the detection sensitivity of the microcystin-LR, has simpler and more convenient operation, achieves the aim of high pass, has the advantages of convenient cleaning and separation and simple operation for taking the magnetic nanoparticles as the solid phase carrier, and simplifies the reaction conditions for taking the gold nanoparticles, modified by DNA, instead of the traditional HRP as the probe to carry out immunoreaction.
Description
technical field [0001] The invention relates to an immunofluorescence quenching detection method for microcystin-LR, which belongs to the technical field of immunoanalysis chemistry. Background technique [0002] With the development of the economy, sewage containing a large amount of nitrogen and phosphorus nutrients enters lakes and reservoirs, causing eutrophication of water bodies, causing abnormal proliferation of algae, releasing secondary metabolites algal toxins (Algae Toxins), threatening the safety of human drinking water. safety and the safety of other organisms in the water body. Among them, Microcystins (Microcystins, MCs) are widely distributed and more harmful. They are a family of cyclic heptapeptides with similar structures produced by certain species of freshwater cyanobacteria, mainly Microcystis aeruginosa. There are more than 60 isomers known. Its structure is as follows: [0003] [0004] It contains five fixed amino acids and two variable amino a...
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IPC IPC(8): G01N33/543G01N33/532
Inventor 胥传来徐丽广彭池方陈伟马伟李灼坤
Owner JIANGNAN UNIV