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Enzyme immunoassay method for heavy metal chromium detection and enzyme immunoassay detection reagent kit

An immune analysis and heavy metal technology, applied in the field of immune analysis, can solve problems such as DNA damage, high requirements for detection samples, and chromosomal abnormalities

Inactive Publication Date: 2015-08-19
深圳市三方圆生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mechanism of hexavalent chromium leading to cancer is: in the presence of low concentration of hexavalent chromium, serious DNA damage is formed, and through the mismatch repair (mismatch repair, MMR) pathway, resulting in chromosomal abnormalities; and under high doses of hexavalent chromium, The generation of microsatellite instability leads to the complete loss of functional mismatch repair (mismatch repair, MMR), which increases the frequency of gene mutations and eventually leads to cancer
However, the detection results of the instrument method are accurate, but there are generally problems such as expensive instruments, high requirements for the detection samples, professional and technical personnel are required to operate, and it takes a long time to detect a large number of samples, so it is difficult to realize the needs of on-site rapid detection.
However, the sensitivity of chemical chromogenic method is not high enough, it is more susceptible to the interference of other substances in the sample, and when a large number of samples are detected, more consumables are required and it takes a long time, etc.

Method used

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  • Enzyme immunoassay method for heavy metal chromium detection and enzyme immunoassay detection reagent kit
  • Enzyme immunoassay method for heavy metal chromium detection and enzyme immunoassay detection reagent kit
  • Enzyme immunoassay method for heavy metal chromium detection and enzyme immunoassay detection reagent kit

Examples

Experimental program
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Embodiment 1

[0047] A preparation method for chromium complete antigen, comprising the following steps:

[0048] Weigh 0.5mg iEDTA and dissolve it with 100μL DMSO to prepare iEDTA solution, that is, figure 1 Ingredient I as indicated; subsequently 100 μL containing 1.0 μM Cr 3+ Add the solution of iEDTA dropwise to the iEDTA solution, and react for 3 hours at 25°C and 125rpm to obtain Cr 3+ -iEDTA complex, i.e. as figure 1 Complex II shown. 2.5 mg carrier protein (BSA or OVA) was dissolved in 800 μL, 0.1 M, pH value of 7.4 Tris-HCl buffer to prepare a buffer system containing carrier protein. Then Cr 3+ - The iEDTA complex was added dropwise to the buffer system containing the carrier protein to prepare a reaction mixture. Add triethylamine solution to adjust the pH of the reaction mixture to 9.0-9.5, and react at 25° C. and 125 rpm for 24 hours. After the reaction, add the reaction liquid to the ultrafiltration tube that has been pretreated with 0.1M EDTA·2Na, and perform centrifuga...

Embodiment 2

[0050] The preparation method of colloidal gold particle-coupled horseradish peroxidase and anti-chromium monoclonal antibody complex comprises the following steps:

[0051] (1) Measure 100mL of three-distilled water and add it to a clean and dry flask, then add 2mL of 1% chloroauric acid solution; place it on a heating stirrer until the solution boils, then quickly add 4mL of 1% trisodium citrate solution , stop heating after the color of the reaction solution is stable, let it cool at room temperature, and prepare the following figure 2 Colloidal gold particles (AuNP) shown;

[0052] (2) Take 5 mL of colloidal gold particle (AuNP) solution with a diameter of 20 nm and place it in a clean and dry low-adsorption glass bottle, and adjust its pH to 8.2-8.5 with 0.25 M potassium carbonate solution. Then, add dropwise the mixed solution according to the amount ratio of substances [horseradish peroxidase (HRP) / anti-chromium monoclonal antibody (Mab) is 12:1], after stirring slowl...

Embodiment 3

[0054] Checkerboard titration method to determine the optimal reaction concentration of antigen and antibody, including the following steps:

[0055] (1) The detection antigen was serially diluted with coating buffer (0.05M, pH 9.6 carbonate buffer), and 100 μL per well was added to a 96-microwell plate;

[0056] (2) Place the plate in a sealed bag to prevent the liquid in the well from volatilizing, and coat at 4°C for 16 hours;

[0057] (3) Pour off the liquid in the well, add PBS-T (0.015M, pH 7.4 phosphate buffer solution, containing 0.05% Tween 20), let it stand for 10s, then discard the liquid in the well and place it on clean absorbent paper to clear the well. Blot up the inner liquid and repeat 3 times;

[0058] (4) Add 200 μL of blocking buffer (5% skimmed milk powder) to each well, seal the plate, and place it at 37° C. for 1 hour;

[0059] (5) Repeat plate washing operation (3);

[0060] (6) Take out the required number of microwells, add the gradiently diluted a...

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Abstract

The invention provides an enzyme immunoassay method for heavy metal chromium detection and an enzyme immunoassay detection reagent kit. Colloidal gold particles are coupled with horse radish peroxidase and anti-chromium monoclonal antibodies for forming enzyme marker compounds, and a detection method is built; through preprocessing chromium in samples and detecting original competitive enzyme-linked labelled antibody compounds, the detection on the chromium content in the samples to be tested is realized through chromogenic reaction amplification signals. The enzyme immunoassay method for chromium detection belongs to a one-step process competitive enzyme-linked immunoassay method, and belongs to a simple convenient fast and high-flux detection method.

Description

technical field [0001] The invention relates to the technical field of immunoanalysis, in particular to an immunoanalysis method for detecting chromium (including trivalent chromium, hexavalent chromium and total chromium) and an immunoanalysis detection kit for detecting chromium. Background technique [0002] Chromium is a silver-white metal with a shiny surface, hard and brittle texture, and good ductility. There is no free chromium in nature, and the main mineral is chromite. Common valences are trivalent and hexavalent. Trivalent chromium has thermodynamic stability and is an essential trace element for the human body. It has biological functions. For humans, adequate chromium supplementation can effectively improve the body's tolerance to glucose and effectively enhance the activity of insulin; chromium deficiency can increase the risk of cardiovascular disease. However, excessive chromium will cause harm to human health. In vitro experiments have proved that high co...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/577
CPCG01N33/535G01N33/53
Inventor 钟松清江天久谭攀唐勇邹军辉谢冬霞刘家飞
Owner 深圳市三方圆生物科技股份有限公司
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