Kit for detecting cadmium ions based on direct competition enzyme-linked immunoadsordent assay and application thereof

A technology of enzyme-linked immunoassay and detection kit, which is applied in the field of environmental detection and can solve problems such as long detection time

Inactive Publication Date: 2017-06-20
HENAN INST OF SCI & TECH
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on the immunological detection technology of heavy metal cadmium ion pollution at home and abroad is very active. Among them, the patent with the notification number CN104155424A discloses a cadmium ion blocking ELISA kit with a detection range of 5.0-512 μg / L and a long detection time

Method used

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  • Kit for detecting cadmium ions based on direct competition enzyme-linked immunoadsordent assay and application thereof
  • Kit for detecting cadmium ions based on direct competition enzyme-linked immunoadsordent assay and application thereof
  • Kit for detecting cadmium ions based on direct competition enzyme-linked immunoadsordent assay and application thereof

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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 Cd-ITCBE-cBSA immunogen ( figure 1 )

[0106] (1) Weigh 10 mg of ITCBE and dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to form a metal chelating agent solution; weigh 4.2 mg of cadmium chloride and dissolve it in 1 mL of HEPES buffer (no toxicity to cells. It is A hydrogen ion buffer that can control a constant pH range for a long time) (10mmol / L) to form Cd 2+ solution; the metal chelating agent solution and Cd 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 Cd-ITCBE chelate hapten.

[0107] (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 as cBSA.

[010...

Embodiment 2

[0110] Preparation of anti-cadmium ion monoclonal antibody

[0111] The anti-cadmium ion-specific monoclonal antibody is prepared by immunizing Balb / C mice with Cd-ITCBE-cBSA immunogen, and is realized by the following steps:

[0112] (1) Mice immunization: 5 female Balb / C mice aged 6-8 weeks were immunized with Cd-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 fu...

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Abstract

The invention provides a kit for detecting cadmium 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, a Cd-ITCBE chelate hapten with the mass concentration of 20mu g / mL to 50mu g / mL, an anti-cadmium 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 cadmium 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-cadmium ion monoclonal antibody solution is prepared from Cd-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 cadmium ion detection kit based on a direct competitive enzyme-linked immunosorbent assay and a preparation method and application thereof. Background technique [0002] Heavy metal cadmium (cadmium, Cd) plays an important role in electroplating, batteries, automobiles, aviation, pigments, paints, printing, plastics and other industries due to its good performance, but it has also become one of the public hazards of environmental pollution. Cd is mainly in ionic form (Cd 2+ ) exists, it can endanger human health through food chain enrichment, and has toxic effects on the human body such as kidney, bone, lung, and cardiovascular, as well as carcinogenic, teratogenic, and mutagenic effects. The Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) 2+ Listed as the third priority food pollution source, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58
CPCG01N33/581
Inventor 姜金庆刘长忠王磊贺永惠杨雪峰王自良范国英胡建和雷壮牛琳琳王亚楠刘耀东
Owner HENAN INST OF SCI & TECH
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