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A heavy metal, enzyme-labeled technology, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of inability to use rapid detection, high detection cost, expensive, etc., and achieves low HRP activity loss, low synthesis cost, Simple preparation method
Inactive Publication Date: 2014-03-12
CHINA AGRI UNIV
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Problems solved by technology
However, the detection of copper ions by these methods requires expensive equipment, high detection costs, time-consuming, and cannot be used for rapid on-site detection.
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Embodiment 1
[0056] Embodiment 1, test kit for detecting whether heavy metal copper is contained in the sample
[0057] 1. The composition of the kit
[0058] (1) The composition of kit of the present invention
[0059] 1. Coating agent: anti-copper monoclonal antibody, which is secreted by the hybridoma cell line Cu-EDTA 6A9 with the deposit number CGMCC No.3987;
[0060] 2. Enzyme label: enzyme-labeled heavy metal copper, which is a cross-linked product formed by a complex and horseradish peroxidase through a covalent bond; the complex is p-aminobenzylethylenediaminetetraacetic acid and BSA The conjugate formed by the covalent bond is then connected with the heavy metal copper through a coordination bond to form a complex.
[0062] 4. Washing solution: Prepare each 1 liter of washing solution as follows: mix 8.0g NaCl, 0.2g KH 2 PO 4 , 2.96gNa 2 HPO 4 12H 2 O. Dissolve 1 mL of Tween-20 in water and make up to 1 L with wat...
Embodiment 2
[0118] Embodiment 2, the application of kit
[0119] Dilute the EDTA-Cu chelate standard solution prepared above with the sample diluent to the following different Cu ion concentrations: 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL, 0.31 ng / mL, 0.156 ng / mL, 0.078 ng / mL, and 0.039 ng / mL.
[0120] (1) Antibody coating: Dilute 1 mg / mL monoclonal antibody at 1:2000 and add to the microtiter plate, 100 μL per well, and incubate at 37°C for 3 hours; pour off the solution in the microtiter plate, wash with Wash the plate 4 times with liquid and shake dry;
[0121] (2) Add the above-mentioned different concentrations of EDTA-Cu chelate standard solution (experimental well) to the microtiter plate of step (1), 50 μL per well, and do not add EDTA-Cu chelate standard in the control well Solution and add 50μL sample diluent;
[0122] (3) Add 0.5 μg / mL enzyme-labeled substance to the above-mentioned experimental wells and control wells, 50 μL per well; incubate at 37°C for 30 minute...
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Abstract
The invention discloses an enzyme label and application thereof. The heavy metal enzyme label is a conjugate consisting of a complex and an enzyme, wherein the complex is a complex consisting of a conjugate of a chelant and a carrier protein and heavy metal. A method for preparing a copper-ion enzyme label has the advantages of simpleness, low synthesis cost and low HRP activity loss. The ELISA detection method constructed by the enzyme label has extremely high sensitivity. The method for preparing the enzyme label for copper ion ELISA detection has important reference value in successfully preparing an enzyme label for ELISA detection of other heavy metals, and the copper-ion rapid immunological detection method constructed by the method has wide application prospect.
Description
technical field [0001] The invention relates to a heavy metal enzyme marker and its application. Background technique [0002] Heavy metals are important pollutants in the environment and agricultural products. They can be enriched in animals and humans through the food chain, especially in the liver, which will cause serious harm to human health. Copper is one of the trace elements necessary for the growth of living organisms, but high concentrations of copper will have a toxic effect on living organisms. Aquatic organisms are particularly sensitive to the toxic effects of copper ions. [0003] At present, the analysis methods of heavy metal copper mainly include atomic absorption spectrometry, inductively coupled plasma emission / mass spectrometry, X-ray fluorescence spectrometry, potential stripping analysis and biosensor detection developed in recent years. However, the detection of copper ions by these methods requires expensive instruments and equipment, high detectio...
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