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Microcystin-LR enzyme-linked immune detection kit

A technology for enzyme-linked immunosorbent assay and microcystin, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems that the accuracy is difficult to meet the requirements, limit the number of routine detections, and the price is high, and achieve the effect of high sensitivity

Inactive Publication Date: 2017-07-28
TIANJIN AGRICULTURE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, HPLC-MS or LC-MS are mostly used for the detection of algal toxins in water bodies. Such detection techniques are often complex in operation, time-consuming, high in price, and difficult to meet the requirements of precision, which limits the number of routine detections. The detection technology has shown a good application prospect in the detection field of algal toxins, and has attracted the attention of researchers at home and abroad. In view of this, an effective, fast, simple, sensitive and low-cost detection of microcystin-LR is prepared. The kit is of great significance

Method used

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  • Microcystin-LR enzyme-linked immune detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of anti-microcystin-LR antibody

[0038] The antibody of the present invention is a monoclonal antibody secreted by a hybridoma cell (CGMCC NO.13802), and the preparation method of the hybridoma cell line (CGMCC NO.13802) and the antibody is a conventional preparation method in the art. That is: firstly, the microcystin-LR hapten is coupled with the carrier protein, prepared as an immunogen, and then the immunogen is immunized to mice, and the splenocytes and myeloma cells are subjected to cell fusion and hybridoma cell cloning The desired hybridoma cell line and the monoclonal antibody secreted therefrom can be obtained through EL screening, wherein the carrier protein can be bovine serum albumin (BSA) or chicken ovalbumin (OVA). Specifically, the method may include the following steps:

[0039] Preparation of immune antigen Microcystin-LR-BSA

[0040] 1. Activation of Microcystin-LR: Dissolve 1mg of Microcystin-LR in 0.05mL DMSO solution, measu...

Embodiment 2

[0054] Example 2 Preparation of microcystin-LR enzyme marker

[0055] Activation of Microcystin-LR: Dissolve 1mg of Microcystin-LR in 0.05mL DMSO solution, measure NHS and EDC respectively and add them to the above solution (the molar ratio of Microcystin-LR:NSH:EDC is 1 : 1.5: 2.5), shake vigorously at room temperature for 50 min in the dark. Coupling of microcystin-LR: Slowly add the above solution dropwise to horseradish peroxidase solution (10mg protein dissolved in 1mL 0.1mol / L sodium bicarbonate solution), shake at room temperature for 2h in the dark, 4°C Overnight; the reaction product was dialyzed in 0.01mol / L PBS buffer solution at 4°C for 72 hours in the dark, and the dialysate was changed every 6 hours to obtain enzyme-labeled microcystin-LR and horseradish peroxidase.

Embodiment 3

[0056] Preferred (dot matrix method) of embodiment 3 antibody and enzyme marker concentration

[0057] Coat longitudinally with anti-microcystin-LR antibody at dilutions of 20 μg / mL, 15 μg / mL, 10 μg / mL, 7.5 μg / mL, 5 μg / mL, 2.5 μg / mL, 1 μg / mL, 0.5 μg / mL ELISA plate, 100 μL / well, place overnight at 2-8°C, wash three times with washing solution, and pat dry each time; 300 μL / well blocking solution is blocked, place at 37°C for 2 hours, wash the plate three times, and pat dry each time; at the same time, add 50 μL 0ng / mL microcystin-LR standard solution and 50 μL serially diluted enzyme standard substance (1:100 to 50000), place at room temperature for 30 minutes, wash the plate three times, pat dry each time, add 100 μL of substrate chromogenic solution, and place at room temperature After 15 minutes, 50 μL of stop solution was added to measure the absorbance value. The specificity test was carried out with the absorbance value having obvious gradient changes with the concentra...

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Abstract

The invention discloses a microcystin-LR enzyme-linked immune detection kit, and belongs to the technical field of enzyme-linked immune analysis. The kit comprises a box, an enzyme label plate and a reagent, the enzyme label plate and the reagent are arranged in the box, and microcystin-LR is detected by an ELISA (enzyme-linked immunosorbent assay) direct competition method. The kit is characterized in that the enzyme label plate wraps microcystin-LR antibodies, and the reagent comprises microcystin-LR horse radish peroxidase labeled antigen solution, microcystin-LR standard solution, condensed scrubbing solution, substrate developing solution and reaction stop solution. The enzyme-linked immune detection kit has the advantages that the kit is simple, convenient, rapid, sensitive, accurate and low in cost, the sensitivity of the kit can reach 0.05ng / mL, and the kit is particularly application to residue detection of microcystin-LR in water and algae food.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay kit, in particular to a microcystin-LR enzyme-linked immunoassay kit. Background technique [0002] Microcystin (MC) is a secondary toxic metabolite produced by the explosive reproduction of cyanobacteria in water bodies. MC is a liver toxin that can accumulate in the digestive glands of mussels and scallops and enter fish, birds, and mammals along the food chain. Animals and humans and other advanced nutritional organisms, causing poisoning and even death. MC is a kind of biologically active cyclic heptapeptide compound. Due to the difference in the composition of two variable amino acids in the polypeptide, there are more than 60 isomers. Among them, MC-LR, MC-RR, and MC-YR are the most common and most abundant microcystins (L, R, and Y represent leucine, arginine, and tyrosine, respectively). The toxicity of MC is related to its structure. Adda is an essential group to express the toxicity of M...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/535
CPCG01N33/577G01N33/535G01N2333/405
Inventor 齐红莉王睿睿王茜徐海龙郭立杨广
Owner TIANJIN AGRICULTURE COLLEGE
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