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Kit for detecting lead 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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on immunological detection technology of heavy metal lead ion pollution at home and abroad is very active, and the patent with the notification number CN103472230B discloses an indirect competitive enzyme-linked immunosorbent assay kit for detecting lead ions and its preparation method, and its detection sensitivity is 10.31ng / mL , and the detection time is longer

Method used

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

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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 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

[0106] Synthesis of Pb-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 10.25mg lead nitrate Pb(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) (10mmol / L) to form Pb 2+ solution; the metal chelating agent solution and Pb 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 Pb-ITCBE chelate hapten.

[0108] (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 store...

Embodiment 2

[0111] Preparation of anti-lead ion monoclonal antibody

[0112] Described anti-lead ion specific monoclonal antibody is prepared by Pb-ITCBE-cBSA immunogen immunization Balb / C mouse, realizes by following steps:

[0113] (1) Mice immunization: 5 female Balb / C mice aged 6-8 weeks were immunized with Pb-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) Cell fusion: NSO cel...

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Abstract

The invention provides a kit for detecting lead 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 Pb-ITCBE chelate hapten with the mass concentration of 20mu g / mL to 50mu g / mL, an anti-lead 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 lead 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-lead ion monoclonal antibody solution is prepared from Pb-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 lead ion detection kit based on a direct competitive enzyme-linked immunosorbent assay and a preparation method and application thereof. Background technique [0002] Trace lead is one of the essential metal trace elements for organisms. With the increase of human mining, smelting, processing and commercial manufacturing of lead, a large amount of lead enters nature in different forms, and enters the biosphere along with the transmission of the biological chain. Because lead has the characteristics of biomagnification and is not easy to be metabolized, it not only pollutes water sources, the atmosphere and soil, but also seriously threatens the safety of humans and animals. In 1938, British scholars reported for the first time that excessive lead content in pastures could cause lead poisoning in grazing sheep, which is characterized by severe diarrh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58
CPCG01N33/581
Inventor 姜金庆刘长忠张慧辉王自良杨雪峰李广领齐永华范国英张海棠李艺
Owner HENAN INST OF SCI & TECH
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