Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof

An enzyme-linked immunosorbent reagent and mercury ion technology, applied in the field of enzyme-linked immunosorbent assay and ion detection, can solve the problems of high price, low sensitivity, and failure to reach the limit value.

Inactive Publication Date: 2013-12-25
ZHENGZHOU UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, the ELISA method requires a matching microplate reader and supporting reagents, and the operation process is more complicated. Moreover, the country is currently in the research and development stage, and imported foreign products are expensive, so the application of the ELISA method is greatly restricted.
However, commercialized rapid detection instruments have low sensitivity (the lower detection limit is 0.1mg / L), which cannot reach the limits stipulated by the national standard (0.05mg / L for sewage, 0.001mg / L for drinking water), and poor selectivity, which cannot meet the requirements Sensitive and accurate detection requirements

Method used

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  • Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof
  • Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof
  • Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1, the preparation of mercury ion artificial immune antigen

[0041] Preparation of artificial complete antigens, including immune antigen Hg, by the isothiocyanate method 2+ -ITCBE-BSA and coating antigen Hg 2+ -ITCBE-OVA. Hg 2+ -ITCBE-OVA as an example, its preparation method is as follows.

[0042] (1) Weigh 20 mg ovalbumin OVA and dissolve it in 1 mL HEPES buffer solution with a concentration of 0.01 mol / L and a pH value of 9.0 to form a carrier protein solution;

[0043] (2) Weigh 10mg of isothiocyanate benzylethylenediaminetetraacetic acid chelating agent (ITCBE) and dissolve it in 1mL dimethyl sulfoxide (DMSO) to form a metal chelating agent solution;

[0044] (3) Add 0.5mL metal chelator solution dropwise to the carrier protein solution, mix well, add 100uL, 1.5moL / L tri-n-butylamine, react at room temperature on a shaker for 24h, and make OVA-ITCBE solution;

[0045] (4) Dissolve 11.25mg of mercury nitrate in 100uL of distilled water to form a...

Embodiment 2

[0047] Embodiment two, mercury ion artificial immune antigen identification

[0048] Determination of Hg by the biquinoline carboxylic acid method 2+ - Carrier protein BSA concentration in ITCBE-BSA. BSA was used as the standard protein, and the concentration gradients of 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, and 80 μg / mL were prepared, and the concentration detection standard curve was constructed by the biquinolinic acid method. The linear equation of the BSA concentration standard curve is: y=0.0035x+0.0065, R 2 =0.9981, where y is the absorbance of the sample at a wavelength of 562 nm, and x is the protein concentration of the sample.

[0049] Determination of Hg by ICP-AES 2+ -Hg in ITCBE-BSA 2+ concentration. 100 μg / mL of Hg 2+ -Hg in ITCBE-BSA 2+ The standard stock solution was diluted with 2% nitric acid to a concentration gradient of 0 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, 0.6 μg / mL, and 0.8 μg / mL. The instrument software automatically draws a st...

Embodiment 3

[0050] Embodiment three, the preparation of anti-mercury ion monoclonal antibody

[0051] (1) Immunization of animals. with Hg 2+ -ITCBE-BSA was used to immunize 5 Balb / c mice at a dose of 50 μg·0.2 mL / mouse, and injected subcutaneously at multiple points on the back. For the first dose, dilute Hg with sterile phosphate buffered saline (PBS)2+ -ITCBE-BSA, emulsified with an equal amount of complete Freund's adjuvant (CFA); for booster immunization, dilute Hg with sterilized PBS 2+ -ITCBE-BSA, emulsified with an equal amount of Freund's incomplete adjuvant (IFA), the second immunization was performed 3 weeks after the first immunization, and then immunized once every 3 weeks, a total of 5 immunizations, 10 days after the third immunization Blood was collected by tail docking, placed in a water bath at 37°C for 30 minutes, placed overnight at 4°C, centrifuged at 800 r / min at 4°C for 5 minutes, and the supernatant was collected and stored at -20°C for later use.

[0052] (2)...

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Abstract

The invention relates to an indirect competition enzyme linked immunoreagent kit for detecting mercury ions and a manufacturing method of the kit. An elisa plate enveloped by mercury ion envelope antigens, mercury-ion-resisting monoclonal antibodies, elisa second antibodies, a substrate color developing solution, a stop solution, a mercury ion standard solution, a washing liquor concentrated solution and a sample treating liquid are arranged in the kit. The kit can detect the mercury irons in a trace mode, is used for detecting the mercury irons in the environment, the soil, water, foods, medicines, cosmetics and the like, and has the advantages of being rapid, easy and convenient to use, sensitive, peculiar, economical and the like. The kit is few in detection step and high in timeliness, saves detection time, reduces operation errors and can perform field detection. The kit is low in requirement for pretreating samples, simple in treatment process, not only can be used for screening the samples in large batch, but also can perform rapid detection on the samples in small batch, and not only provides technical support for environment and food safety, but also provides effective technological means for food import and export inspection, food inspection, monitoring and evaluation of environmental pollution and the like.

Description

technical field [0001] The invention relates to ELISA and ion detection, in particular to an indirect competitive ELISA kit for detecting mercury ions and a preparation method thereof. Background technique [0002] Mercury, commonly known as mercury, is the only metal that exists in a liquid state at room temperature and pressure. It is chemically stable and can evaporate at room temperature. Mercury vapor and mercury compounds are often highly toxic. Mercury is ubiquitous in nature. my country is a large country in the production, use and emission of mercury, and the situation of mercury pollution prevention and control is very severe. Heavy metal mercury is a highly toxic substance that pollutes the environment for a long time. It is highly toxic and harmful to human health. Mercury pollution has become a major global environmental problem, and it has also attracted great attention from the Chinese government. The prevention and control of mercury pollution has been listed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
Inventor 王爱萍周景明张改平祁元明张守涛祁艳华李永欣席宇栗宁段倩倩郑伟王新洲何文博
Owner ZHENGZHOU UNIV
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