Cadmium ion detection kit and application thereof

A technology of cadmium ions and kits, which is applied in the field of immunoassay, can solve the problems of being unable to detect the concentration of trace cadmium ions, being unable to use on-site detection, and being unfavorable for popularization and application, etc., achieving long storage time of reagents, short detection time, no Effects of radioactive contamination

Active Publication Date: 2018-08-03
苏州仁端生物医药科技有限公司
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  • Abstract
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Problems solved by technology

[0003] Traditional heavy metal ion detection methods include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, voltammetry, ion chromatography, and electrothermal atomization atomic absorption spectrometry, etc. However, these detection methods must use large analytical instruments and cannot be used in the field Detection, and the cost of inspection is high, the processing capacity is limited, the detection time is long, and the accuracy is limited by the instrument, which is not conducive to the promotion and application in daily life
[0004] CN 106872709 A discloses a kit for detecting cadmium ions based on direct competitive ELISA and its application. The kit includes a detection plate coated with goat anti-mouse IgG secondary antibody, enzyme-labeled Cd-ITCBE chelate half Antigen, anti-cadmium ion monoclonal antibody solution, the anti-cadmium ion monoclonal antibody solution is prepared by immunizing Balb / C mice with Cd-ITCBE-cBSA immunogen, but the lowest detection line of the kit is 0.04 μg / L, low sensitivity, cannot be used to detect the concentration of trace cadmium ions

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  • Cadmium ion detection kit and application thereof
  • Cadmium ion detection kit and application thereof
  • Cadmium ion detection kit and application thereof

Examples

Experimental program
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Embodiment 1

[0053] The preparation of embodiment 1 cadmium ion complete antigen

[0054] (1) Dissolve 10 mg of bifunctional chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-N-hydroxysuccinimide ester (DOTA-NHS) Form a metal chelator solution in 1 mL dimethylsulfoxide (DMSO), 30 mg polylysine (PLL) or 15 mg human serum albumin (HSA) in 1 mL 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer in the liquid;

[0055] (2) Slowly drop the DOTA-NHS solution into the carrier protein solution at room temperature, stir while dripping, adjust the pH to 9.0, and stir overnight at room temperature. The next day, the reaction solution is colorless or slightly yellow and transparent. Use 15mL centrifugal ultrafiltration Tube ultrafiltration, washed 3 times with HEPES buffer, concentrated to 2mL, which is the coupling compound of bifunctional chelating agent and protein;

[0056] (3) After diluting the coupling compound of bifunctional chelating agent and protein 5 times...

Embodiment 2

[0057] The preparation of embodiment 2 cadmium ion monoclonal antibody

[0058] (1) Dissolve 60 μg of cadmium ion complete immunogen Cd-DOTA-PLL in 200 μL of sterile normal saline, mix with 200 μL of complete Freund’s adjuvant, fully emulsify, and inject multipoint subcutaneously into the abdomen of Balb / c female mice; Two weeks later, the mixture of immunogen Cd-DOTA-PLL and incomplete Freund's adjuvant was used for booster immunization, and the immunization site was subcutaneous on the back of the neck. From the third immunization, blood was collected from the orbit of mice one week after immunization to detect serum efficacy. The fourth immunization did not add adjuvant, and the immunogen Cd-DOTA-PLL was directly used for intraperitoneal immunization;

[0059] (2) Three days after the end of immunization, the splenocytes of the immunized mice were fused with SP2 / 0 myeloma, and after more than three times of subcloning, the hybridoma cells capable of stably secreting anti-he...

Embodiment 3

[0062] Example 3 Preparation of magnetic particle suspension coupled with cadmium ion-coated detection agent

[0063] (1) Add 1 mL of 2-(N-morpholine) ethanesulfonic acid (MES) buffer solution with a concentration of 0.05 mol / L to 1 mg of carboxylated magnetic particles, vortex and mix well, place on a magnetic stand, let stand for 5 min, remove supernatant, washed three times;

[0064] (2) Add 100 μL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 100 μL N-hydroxysuccinimide (NHS), shake for 30 minutes and wash three times ;

[0065] (3) Add 30 μg of cadmium ions to coat the original Cd-DOTA-HSA, vortex, rotate the reaction tube, and incubate at room temperature for 2 hours;

[0066] (4) Remove the supernatant, add a certain amount of washing solution (TBS+0.05% Tween-20), and wash 3 times;

[0067] (5) Block with buffer solution containing 1% BSA for 4 times, 10 min each time, to obtain magnetic particle suspension coupled with cadmium ion-coated det...

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Abstract

The invention provides a cadmium ion detection kit as well as a preparation method and an application thereof. Complete antigen of a cadmium ion is a complex of the cadmium ion, a chelating agent andcarrier protein, wherein the chelating agent comprises any one of or a combination of any two of EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriamine pentaacetic acid), a derivative of DTPA, DOTA (1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid) and DOTA-NHS (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-N-hydroxysuccinimide eater). The heavy metal cadmium ion is coupled with the carrier protein through a bifunctional chelating agent, the complete antigen of the cadmium ion is obtained, a cadmium ion monoclonal antibody with high immunogenicity and high valence for the cadmium ion is prepared, and the formed kit has high specificity and sensitivity and can be used for detecting the cadmium ion.

Description

technical field [0001] The invention belongs to the field of immune analysis, and relates to a cadmium ion detection kit and application thereof, in particular to a cadmium ion detection kit based on magnetic particles and chemiluminescence technology, a preparation method and application thereof. Background technique [0002] Cadmium (Cd), a heavy metal, is a common environmental pollutant mainly derived from industrial activities such as mining and smelting. Cadmium released into the environment is enriched in cadmium in soil, through crops and aquatic animals and plants in water, and finally enters the human body through diet. The absorption rate of cadmium in the gastrointestinal tract is about 5-10%, while the absorption rate of cadmium in the lungs is as high as 10-50%. The biological half-life of cadmium in the human body is as long as 10-30 years, and it can damage the liver, kidney, lung, bone, cardiovascular system, immune system and nervous system. The main haza...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/577G01N33/532G01N21/76
CPCG01N21/763G01N33/532G01N33/54326G01N33/577G01N2446/90
Inventor 周小进
Owner 苏州仁端生物医药科技有限公司
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