Copper ion detection kit based on direct competitive ELISA (enzyme-linked immunosorbent assay) and application of kit

An enzyme-linked immunoassay, copper ion technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problem of long detection time

Active Publication Date: 2017-06-13
HENAN INST OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on heavy metal copper ion pollution immunoassay technology is very active at home and abroad. Among them, the patent with the notification number CN103558387A di

Method used

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  • Copper ion detection kit based on direct competitive ELISA (enzyme-linked immunosorbent assay) and application of kit
  • Copper ion detection kit based on direct competitive ELISA (enzyme-linked immunosorbent assay) and application of kit
  • Copper ion detection kit based on direct competitive ELISA (enzyme-linked immunosorbent assay) and application of kit

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Experimental program
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preparation example Construction

[0042] In the present invention, the preparation method of the detection plate coated with goat anti-mouse IgG secondary antibody preferably comprises the following steps:

[0043] 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;

[0044]Ⅱ. Add 200-300 μL / well of pig negative serum with a mass concentration of 3-6% to the detection plate obtained in the above step 1, 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 second...

Embodiment 1

[0105] Synthesis of Cu-ITCBE-cBSA immunogen ( figure 1 )

[0106] (1) Weigh 10mg ITCBE and dissolve it in 1mL dimethyl sulfoxide (DMSO) to form a metal chelating agent solution; weigh 9.0mg copper nitrate Cu(NO 3 ) 2 Dissolve in 1mL of HEPES buffer at pH 8.0 (has no toxic effect on cells. It is a hydrogen ion buffer that can control a constant pH range for a long time) (10mM / L) to form Cu 2+ solution; the metal chelating agent solution and Cu 2 + The solutions were mixed, and the pH was adjusted to 7.0 with NaOH, and then reacted on a shaker at room temperature for 12 hours to form Cu-ITCBE chelate hapten.

[0107] (2) Weigh 66 mg of BSA and 11.6 mg of EDC and dissolve them 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 as cBSA....

Embodiment 2

[0110] Preparation of anti-copper ion monoclonal antibody

[0111] The anti-copper ion-specific monoclonal antibody is prepared from Cu-ITCBE-cBSA immunogen immunized Balb / C mice, and is realized by the following steps:

[0112] (1) Mice immunization: five female Balb / C mice aged 6-8 weeks were immunized with Cu-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.

[0113] (2) Cell fusio...

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Abstract

The invention provides a copper ion detection kit based on direct competitive ELISA (enzyme-linked immunosorbent assay). The kit comprises a detection plate coated with a goat anti-mouse IgG secondary antibody, an enzyme-labeled Cu-ITCBE chelate hapten with the mass concentration being 20-50 mu g/mL, an anti-copper-ion monoclonal antibody solution with the mass concentration being 10-20 mu g/mL, 1.0-3.0 mol/L of a stop buffer, a sample diluent, a substrate color developing solution, a scrubbing solution, a copper ion standard solution and an EDTA (ethylene diamine tetraacetic acid) treatment solution with the mass concentration being 10-20 mg/mL, wherein the detection plate is subjected to vacuum sealed packaging; the coating concentration of the goat anti-mouse IgG secondary antibody is 50-200 mu g/mL; the anti-copper-ion monoclonal antibody solution is prepared from a Cu-ITCBE-cBSA immunogen immune Balb/C mouse. The kit has the characteristics of high sensitivity and high specificity and 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 test kit for detecting copper ions based on a direct competitive enzyme-linked immunosorbent assay and a preparation method and application thereof. Background technique [0002] Heavy metal copper is an important metal element that exists in nature and is also a trace element necessary for the human body. A large amount of copper is a nutrient for crops or per capita. Anemia, diarrhea and other symptoms will occur due to copper deficiency, but excessive intake of copper can also cause harm. With the rapid development of industry and agriculture, the use of copper has become more and more extensive, and the amount of copper consumption has continued to increase. The discharge of copper-containing pollutants has also increased, and the pollution of the soil and other environments has gradually emerged. The main pollution sources of copper are wastewa...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/531G01N33/577
Inventor 王秋霞欧长波马景周余燕魏小兵张艳红刘兴友姜金庆孙勇
Owner HENAN INST OF SCI & TECH
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