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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

AI Technical Summary

Problems solved by technology

The above methods mainly focus on the rapid detection of environmental water samples, but there are few reports on the rapid detection of heavy metals in biological 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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Experimental program
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Effect test

Embodiment 1

[0047] 1) Synthesis of Pb immune antigen: Pb (National Center for Analysis and Testing of Nonferrous Metals and Electronic Materials, GSB04-1742-2004), bifunctional chelating agent [(R)-2-thiocyano-3-(4-aminophenyl )propyl]-(S-S)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 / The ratio of W) was shaken at 25°C for 22 hours; 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 were synthesized according to Pb:p-SCN-Bn-CHX- The ratio of A"-DTPA:BSA:TEA=9:12:200:13 (W / W / W / W) was used to synthesize the detec...

Embodiment 2

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

[0051] Mix 1.0 mg / ml of Pb immune antigen with an equal volume of Freund's complete adjuvant (FCA) through the double-push method of the syringe to form a white water-in-oil emulsion, that is, the antigen emulsifier. Using the multi-point immunization method, 6-week-old female Balb / C mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 211) were immunized once every 2 weeks for a total of 5 immunizations. During the 2nd, 3rd, and 4th immunization, the Pb immunization antigen was mixed with an equal volume of Freund's incomplete adjuvant (FIA), and the same dose was used to boost immunization. At the 5th immunization, only the Pb immunization antigen was used without Add adjuvant. The titer and specificity of mouse serum after mouse immunization are as follows: Figure 3-4 shown. Take the immunized mice with the highest antiserum titer to prepare splenocytes, and mix splenocytes wi...

Embodiment 3

[0053] Anti-Pb immunomagnetic beads (PbMAb-Fe 3 o 4 ) preparation: because nano magnetic beads ( M-280Tosylactivated, lifetechnologies, USA, 14203) has low background and the antibody is covalently attached to the bead surface, making it an excellent choice for immunoprecipitation of protein complexes. Magnetic aggregation of microbeads is gentle and rapid with minimal incubation time. Therefore, the nano magnetic beads M-280Tosylactivated, its particle size is 280nm. Anti-Pb immunomagnetic beads synthesis steps are as follows:

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

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

[0056] (3) Add 100 μL 3M (NH 4 ) 2 SO 4 Vortex 0.1M pH 7.4 phosphate buffer.

[0057] (4) Shake and incubate at 37°C for 12-18 hours;

[0058] (5) Magnetic separation for 2 minutes, remove the super...

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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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