Method for quickly detecting lead poisoning based on magnetic separation and quantum dots marks and kit thereof

A kit and rapid technology, applied in measurement devices, analytical materials, instruments, etc., to achieve the effects of high photochemical stability, easy operation, and not easy to photolysis

Inactive Publication Date: 2014-04-02
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods mainly focus on the rapid detection of environmental water samples,

Method used

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  • Method for quickly detecting lead poisoning based on magnetic separation and quantum dots marks and kit thereof
  • Method for quickly detecting lead poisoning based on magnetic separation and quantum dots marks and kit thereof
  • Method for quickly detecting lead poisoning based on magnetic separation and quantum dots marks and kit thereof

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Example Embodiment

[0046] Example 1

[0047] 1) Pb immune antigen synthesis: Pb (National Center for Analysis and Testing of Non-ferrous Metals and Electronic Materials, GSB04-1742-2004), bifunctional chelating agent [(R)-2-thiocyano-3-(4-aminophenyl) )Propyl]-(SS)cyclohexane-1,2 diethylenetriaminepentaacetic acid (p-SCN-Bn-CHX-A"-DTPA) (Macroyclics, USA) and hemocyanin (KLH) (Sigma, United States, H7017) and triethylamine (TEA) (domestic analytical grade) according to Pb:p-SCN-Bn-CHX-A"-DTPA:KLH:TEA=9:12:200:13(W / W / W / W), the reaction was shaken at 25°C for 22h; the reaction product was washed with PBS buffer, and unreacted small molecules were filtered out through an ultrafiltration tube (30KD); the obtained Pb immune antigen was diluted with PBS buffer.

[0048] 2) Pb detection antigen synthesis: Pb, p-SCN-Bn-CHX-A"-DTPA, bovine serum albumin (BSA) (Sigma, USA, A1933) and TEA are combined according to Pb: p-SCN-Bn-CHX- A"-DTPA:BSA:TEA=9:12:200:13 (W / W / W / W) ratio of synthetic detection antigen. ...

Example Embodiment

[0049] Example 2

[0050] Preparation and purification of anti-Pb monoclonal antibody (PbMAb):

[0051] The 1.0 mg / ml Pb immune antigen and an equal volume of Freund's complete adjuvant (FCA) are thoroughly mixed by the syringe double push method to form a white water-in-oil emulsion, that is, an antigen emulsifier. Multipoint immunization was used to immunize 6-week-old female Balb / C mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 211), immunized once every 2 weeks, and immunized 5 times in total. In the second, third, and fourth immunizations, the Pb immune antigen was mixed with an equal volume of Freund’s incomplete adjuvant (FIA), and the same dose was used to boost the immunization. In the fifth immunization, only Pb was used to immunize the antigen without Add adjuvant. The titer and specificity of mouse serum after mouse immunization are as follows Figure 3-4 Shown. Spleen cells were prepared from the immunized mice with the highest antiserum tite...

Example Embodiment

[0052] Example 3

[0053] Anti-Pb immunomagnetic beads (PbMAb-Fe 3 O 4 ) Preparation: Because nano magnetic beads ( M-280Tosylactivated, lifetechnologies, USA, 14203) has a low background value, and the antibody is covalently attached to the surface of the beads, which is an excellent choice for immunoprecipitation of protein complexes. The magnetic aggregation of the beads is gentle and fast and the incubation time is very short. Therefore, the choice of nano magnetic beads M-280Tosylactivated, its particle size is 280nm. The synthesis steps of anti-Pb immunomagnetic beads are as follows:

[0054] (1) Take 165 μL of nano magnetic beads, magnetically separate for 1 min, and remove the supernatant;

[0055] (2) Add 100μg of anti-lead monoclonal antibody and 0.1M pH7.4 phosphate buffer to make the volume become 150μL, vortex and shake;

[0056] (3) Add 100μL 3M(NH 4 ) 2 SO 4 Vortex the 0.1M pH7.4 phosphate buffer.

[0057] (4) Incubate with shaking at 37°C for 12-18h;

[0058] (5) Mag...

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Abstract

The invention provides a method for quickly detecting lead in liquid biological samples based on magnetic separation and quantum dots (QDs) marks. The method comprises the steps of (1), preparing a lead-resisting monoclonal antibody; (2), coupling the lead-resisting monoclonal antibody with nano magnetic beads through covalent bonds to prepare lead-resisting immune nano magnetic beads; and (3), adding bifunctional chelating agents, BSA (Bovine Serum Albumin) and triethylamine (TEA) into the liquid biological samples, performing incubation and adding the lead-resisting immune nano magnetic beads to mix fully, then performing magnetic separation, adding biotinylation BSA antibodies and streptavidin quantum dots (QDs) into sediments obtained by magnetic separation, and performing fluorescence detection by using a fluorescence microplate. The invention also provides a kit for quickly detecting lead in liquid biological samples.

Description

technical field [0001] The invention belongs to the technical field of biology and new medicine. specifically. The invention provides a method for rapidly detecting lead content in blood based on magnetic separation and quantum dot labeling technology and a kit for rapidly detecting lead content in blood. Background technique [0002] Lead (Pb) pollution has attracted much attention because of its persistence, bioaccumulation and toxicity, and has been one of the hot research topics in the international scientific community. In recent years, with the acceleration of industrialization and urbanization in my country, lead pollution is on the rise. The heavy metal lead that enters the environment through the discharge of industrial "three wastes" has even directly affected the food chain and the quality of agricultural products. In many places, the lead content in foods such as grains, vegetables, and fruits seriously exceeds the standard or is close to the critical value, wh...

Claims

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Application Information

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IPC IPC(8): G01N33/577
CPCG01N33/577G01N33/542G01N33/54326
Inventor 孙志伟黄沛力孙湖泊王萌萌王吉龙王晖郝凤桐李惠玲
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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