Method and test paper for rapidly detecting microcystins

A technology of microcystin and test paper, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of low quality and no sales, etc., and achieve the effect of simple detection method, intuitive results and easy use

Inactive Publication Date: 2012-06-13
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the main enzyme-linked immunoassay technology (Enzyme-linked immunosorbent assay, ELISA) for the detection of microcystins at home and abroad, my country has sold ELISA detection kits for MC-LR produced in the United States, an

Method used

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  • Method and test paper for rapidly detecting microcystins
  • Method and test paper for rapidly detecting microcystins
  • Method and test paper for rapidly detecting microcystins

Examples

Experimental program
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Embodiment 1

[0027] Embodiment 1 complete antigen preparation

[0028] 3 mg of microcystin was dissolved in 1.2 ml of DMF, 100 microliters of activation solution (12 mg of EDC, HCL and 7 mg of NHS dissolved in 1 ml of DMF) was added, and the reaction was activated overnight at 4°C. Take 20 mg of KLH keyhole limpet protein and dissolve in 5 ml of carbonic acid buffer solution with pH=9.6 for 1 hour, and overnight at 4°C.

[0029] After mixing, dialyze with 0.01M PBS for 3 days, centrifuge to get the supernatant, use lorry method to measure the concentration of MCLR-KLH conjugate to 1.4 mg / ml, and then dilute to different concentrations as needed.

Embodiment 2

[0030] Preparation of embodiment 2 monoclonal antibody (mouse anti-MC-LR)

[0031] Animal immunization: Mix an appropriate amount of MCLR-KLH with an equal volume of complete Freund's adjuvant, fully hatch, take five 8-week-old female BALB / C mice, subcutaneously at 3 points on the back of the neck, subcutaneously in the armpit (popliteal) of the limbs, and intraperitoneally. Each mouse was injected with 500 μl of post-hatch antigen (equivalent to 100 μg of immune antigen per mouse). After 14 days, an appropriate amount of MCLH-KLH was mixed with an equal volume of incomplete Freund's adjuvant, and after complete hatching, the antigen was injected subcutaneously at multiple points and intraperitoneally, and immunized once every 14 days by this method. Seven days after the third basic immunization, blood was collected from the tail vein to detect the titer.

[0032] MCLH-K coated enzyme samples, the indirect method to determine the potency. Mouse serum serum was serially dilut...

Embodiment 3

[0038] The preparation of embodiment 3 colloidal gold

[0039] With reference to the preparation method of colloidal gold particles introduced by Frens1973, get 1ml 1% chloroauric acid (HAuCl 4 ) solution, added to 100ml of water, heated to boiling, then added 0.5-4ml of 1% trisodium citrate, mixed and boiled for 5min, until the color did not change. The method can prepare gold particles with different diameters ranging from 15 to 60 nm, and the particle size depends on the amount of trisodium citrate added.

[0040] 0.01% chloroauric acid (HAuCl 4 ) aqueous solution 100ml is heated to boiling, adds 1.5ml of 1% trisodium citrate, mixes evenly, and boils for 5 minutes, after the color of colloidal gold solution changes from blue to purple, it is cooled for later use to obtain a colloid with a particle size of 30nm (± 2nm). gold.

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Abstract

The invention relates to the detection of a water sample and establishes a rapid colloidal gold immunochromatography detection method and test paper of microcystins MC-LR. The method comprises the following steps of: firstly, preparing a colloidal gold with the average diameter of about 30 nm and utilizing the colloidal gold to mark an anti-microcystins MC-LR monoclonal antibody; covering a conjugate of a microcystins MC-LR hapten and keyhole limpet hemocyanin on a nitrocellulose membrane as a detection belt; taking a goat anti-mouse second antibody as a control belt; and establishing an immunochromatography test paper method for rapidly detecting the microcystins MC-LR. According to the method, the sensitivity can reach to 3 ng/mL and a judged result can be observed by naked eyes only after 3-5 min; and the method has the characteristics of rapidness, direct observation, simplicity in operation, convenience for use and the like, so that the method can be used as an effective way for screening whether a lot of field freshwater and freshwater products are infected by the microcystins MC-LR.

Description

technical field [0001] The invention relates to a water sample detection method and a reagent, in particular to a method and a test paper for rapid detection of microcystin. Background technique [0002] With the development of industrialization and urbanization and the large-scale use of chemical fertilizers in agricultural production, a large amount of nutrients are injected into the water body, and the eutrophication of the lake is becoming more and more serious, resulting in the rapid growth of algae and the excessive reproduction of blue-green algae, which will cause water odor, transparency, and water consumption. Dissolved oxygen, among which Microcystis, Oscillatoria, Anabaena, Nostoc and other algae and their metabolites are toxic and carcinogenic, endangering human health. Surveys show that 80% of the blooms in freshwater lakes in my country are toxic. Among the toxins produced by toxic cyanobacteria blooms, Microcystin (MC) is one of the most widely distributed an...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 何培民胡乐琴蔡春尔缪辉南汪卿
Owner SHANGHAI OCEAN UNIV
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