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Direct Competitive ELISA Kit and Its Application in Mercury Ion Food Contamination

A technology for detection kits and enzyme-linked immunoassays, which can be used in measuring devices, instruments, scientific instruments, etc., and can solve problems such as long detection time

Active Publication Date: 2019-06-07
HENAN INST OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the immunological detection technology of heavy metal mercury ion pollution is very active at home and abroad. Among them, the patent with the notification number CN103472231B discloses a mercury ion indirect competition ELISA kit with a detection sensitivity of 10.31ng / mL and a long detection time.

Method used

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  • Direct Competitive ELISA Kit and Its Application in Mercury Ion Food Contamination
  • Direct Competitive ELISA Kit and Its Application in Mercury Ion Food Contamination
  • Direct Competitive ELISA Kit and Its Application in Mercury Ion Food Contamination

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preparation example Construction

[0043] In the present invention, the preparation method of the detection plate coated with goat anti-mouse IgG secondary antibody preferably comprises the following steps:

[0044] I. Add 100-150 μL / well of goat anti-mouse IgG secondary antibody coating solution with a coating concentration of 50-200 μg / mL to the detection wells of the detection plate, and incubate for the first time at 35-40°C for 1.5-3 hours. After incubation, Remove the coating solution, and wash the plate for the first time with the washing solution for 2 to 5 times;

[0045] II. Add 200-300 μL / well of pig negative serum with a mass concentration of 3-6% to the detection plate obtained in the step I, and incubate for the second time at 35-40° C. for 1.5-3 hours, and remove the coating solution after incubation , washing the plate for the second time with washing solution for 2-5 times, and drying the detection plate naturally at 23-27° C. to obtain a detection plate coated with goat anti-mouse IgG secondary ...

Embodiment 1

[0106] Synthesis of Hg-ITCBE-cBSA immunogen ( figure 1 )

[0107] (1) Weigh 10mg ITCBE and dissolve it in 1mL dimethyl sulfoxide (DMSO) to form a metal chelating agent solution; weigh 11.25mg mercuric nitrate Hg (NO 3 ) 2 Dissolve in 1 mL of HEPES buffer (10mmol / L) with pH 8.0 (has no toxic effect on cells. It is a hydrogen ion buffer and can control a constant pH range for a long time) (10mmol / L) 2+ solution; the metal chelating agent solution and Hg 2+ The solutions were mixed, and the pH value was adjusted to 7.0 with NaOH, and then reacted on a shaker at room temperature for 12 hours to form the Hg-ITCBE chelate hapten.

[0108] (2) Weigh 66 mg of BSA and 11.6 mg of EDC and dissolve in 5 mL of PBS buffer, slowly add 7 mg of ethylenediamine (dissolved in 3 mL of PBS+DMF solution in advance) under stirring conditions, and shake at 37°C for 2 hours. The reaction solution was dialyzed against PBS for 4 days, and the activated carrier protein BSA was lyophilized and stored ...

Embodiment 2

[0111] Preparation of anti-mercury ion monoclonal antibody

[0112] The anti-mercury ion-specific monoclonal antibody is prepared by immunizing Balb / C mice with the Hg-ITCBE-cBSA immunogen, and is realized by the following steps:

[0113] (1) Mice immunization: 5 female Balb / C mice aged 6-8 weeks were immunized with Hg-ITCBE-cBSA, the dose was 60 μg / mouse, and the volume was 0.2 mL. The immunogen diluted with PBS was completely emulsified with an equal volume of FCA for the first immunization, and then boosted every 4 weeks, and emulsified with FIA. 7 days after immunization for 5 times, the blood was collected by docking the tail to separate the serum, and the mice with high titer and good blocking effect were screened by indirect ELISA and indirect competitive ELISA (ciELISA) as spare mice for fusion. 3 days before the fusion, the hyperimmunized mice were injected with 50 μg of immunogen in the tail vein and intraperitoneally, each with a volume of 100 μL.

[0114] (2) Cel...

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Abstract

The invention provides a kit for detecting mercury ions based on a direct competition enzyme-linked immunoadsordent assay. The kit comprises a detection plate coated with a goat anti-mouse IgG secondary antibody, an Hg-ITCBE chelate hapten with the mass concentration of 20mu g / mL to 50mu g / mL, an anti-mercury ion monoclonal antibody solution with the mass concentration of 10mu g / mL to 20mu g / mL, 1.0mol / L to 3.0mol / L of a stopping solution, a sample diluting solution, a substrate color developing solution, a washing solution, a mercury ion standard product solution and an EDTA (Ethylene Diamine Tetraacetic Acid) treatment solution with the mass concentration of 10mg / mL to 20mg / mL, wherein the detection plate is sealed and packaged in vacuum; the coating concentration of the goat anti-mouse IgG secondary antibody is 50mu g / mL to 200mu g / mL; the anti-mercury ion monoclonal antibody solution is prepared from Hg-ITCBE-cBSA immunogen immunized Balb / C mice. The kit provided by the invention has the characteristics of high sensitivity and high specificity and also has the advantage of short detection time.

Description

technical field [0001] The invention belongs to the technical field of environmental detection, and in particular relates to a direct competition ELISA detection kit and application thereof in mercury ion food pollution. Background technique [0002] Mercury, commonly known as mercury, is the only metal that exists in a liquid state at normal temperature and pressure. With the rapid development of modern society, mercury is more and more widely used in agriculture, industry, pharmaceutical industry, metallurgy and dentistry. Mercury vapor and mercury compounds are highly toxic. With the application in various fields, mercury has formed a three-dimensional pollution cycle of the atmosphere, water and soil, and is enriched in plants and fish, then enters the human body through the food chain, and is enriched in liver, kidney, brain and other tissues. Inhibit human enzyme activity, disrupt normal metabolism, and even cause deformities, which seriously threaten human health and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/558G01N33/577
CPCG01N33/558G01N33/577
Inventor 王秋霞马景周欧长波张艳红魏小兵余燕刘兴友姜金庆秦保亮
Owner HENAN INST OF SCI & TECH
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