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Indirect competitive ELISA kit for detecting mercury ions and preparation method thereof

An enzyme-linked immunosorbent reagent and mercury ion technology, which is applied in the field of enzyme-linked immunosorbent immunoassay and ion detection, can solve the problems of poor selectivity, high price, and low sensitivity

Inactive Publication Date: 2015-11-18
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 competitive ELISA kit for detecting mercury ions and preparation method thereof
  • Indirect competitive ELISA kit for detecting mercury ions and preparation method thereof
  • Indirect competitive ELISA kit for detecting mercury ions and preparation 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 of ovalbumin OVA and dissolve in 1 mL of 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 for...

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 docking the tail, 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 taken and stored at -20°C for later use.

[0052] (...

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Abstract

The invention relates to an indirect competitive ELISA kit for detecting mercury ions and a preparation method thereof. The kit contains an ELISA plate coated with mercury ion-coated antigen, anti-mercury ion monoclonal antibody, enzyme-labeled secondary antibody, substrate chromogenic solution, stop solution, mercury ion standard solution, washing liquid concentrate and sample processing liquid. The kit of the present invention can detect mercury ions in trace amounts, and is used for detecting mercury ions in the environment, soil, water, food, medicine, cosmetics, etc., and has the characteristics of fast, simple, sensitive, specific and economical, and has few detection steps, saving The detection time is short, the operation error is reduced, the timeliness is strong, and on-site detection can be carried out. The kit has low requirements on sample pre-treatment and simple processing process. It can be used not only for the screening of large batches of samples, but also for the rapid detection of small batches of samples. , Food inspection, environmental pollution monitoring and evaluation, etc. provide effective technical means.

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 Patents(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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