Indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury

An enzyme-linked immunosorbent assay technology, which is applied in the field of indirect competitive enzyme-linked immunosorbent assay based on heavy metal mercury monoclonal antibodies, can solve problems such as the lack of breakthroughs in heavy metal mercury, and achieve easy remedial and recovery work, high-throughput Quantitative measurement, the effect of great practical significance

Inactive Publication Date: 2010-02-24
NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury
  • Indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury
  • Indirect competitive enzyme-linked immunosorbent assay for measuring heavy metal mercury

Examples

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Embodiment 1

[0033] Example 1: Preparation of Monoclonal Antibody for Detection of Heavy Metal Mercury

[0034] The complete antigen of heavy metal mercury was synthesized by the isothiocyanate method, and the complete antigen was generated by combining the thiocyanate on the bifunctional chelating agent in the hapten complex with the free amino group on the macromolecular carrier protein. Specifically: use isothiocyanate-benzyl-ethylenediaminetetraacetic acid (ITCBE) as a bifunctional chelating agent to form a mercury-chelating agent (Hg-ITCBE) complex with heavy metal cadmium, and then use bovine serum whitening Protein (BSA) and keyhole limpet hemocyanin (KLH) are used as carrier proteins to couple with the Hg-ITCBE complex, and finally form the complete antigens of Hg-ITCBE-BSA and Hg-ITCBE-KLH, that is, the coating and the immunogen.

[0035]Four 6-week-old female BALB / c mice were taken, and their tails were docked one week before immunization to collect blood as negative serum. One ...

Embodiment 2

[0038] Example 2: Identification of Mercury Monoclonal Antibody Specificity

[0039] In this example, some common heavy metal ions in the environment were selected for cross-reactivity determination to investigate the specificity of mercury monoclonal antibodies. Selected metal ions are: Zn 2+ , Pb 2+ , Cd 2+ , Al 3+ , Ni 2+ , Mg 2+ , Ca 2+ ,Co 2+ , Cu 2+ And so on 9 kinds. Operate according to the specific experimental steps of the indirect competitive ELISA method in the summary of the invention. With concentration of 0.1, 1, 10, 20, 50, 100, 500, 1000μg / LHg 2+ Standard solution and Zn with concentration of 0.01, 0.1, 1, 5, 10, 50mg / L 2+ , Pb 2+ , Cd 2+ , Al 3+ , Ni 2+ , Mg 2+ , Ca 2+ ,Co 2+ , Cu 2+ A series of standard solutions are added to the microtiter plate as samples, and the OD of each well in the plate is determined 450 value. The concentration IC of each competitor that produces 50% inhibition or binding according to the inhibition curve 50 (m...

Embodiment 3

[0042] Embodiment 3: heavy metal mercury enzyme-linked immunosorbent assay method

[0043] 1: Determination of the optimal working concentration of antigen and antibody

[0044] Optimize the optimal working concentration of the coated antigen and antibody by square array titration: use CBS to serially dilute the coated antigen Hg(II)-ITCBE-BSA to 1000 times, 2000 times, 4000 times, 8000 times the coated enzyme Columns 1-3, 4-5, 6-9, 10-12 of the target plate; use 1% gelatin as the blocking solution; the antibody is diluted to 5000 times, 10000 times, 20000 times, 40000 times, 80000 times, 160000 times Times, 320000 times and 640000 times, on the A-H line of the ELISA plate; add goat anti-mouse secondary antibody IgG-HRP (1:5000, diluted in PBS); TMB color development, 2M sulfuric acid stop, measure OD 450 value. by OD 450 The best working concentration is when the value is slightly greater than but close to 1 and the combination of antigen and antibody concentration is the ...

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Abstract

The invention discloses an indirect competitive enzyme-linked immunosorbent assay (ELISA) for measuring heavy metal mercury, belonging to a method for measuring heavy metal mercury in an environment water sample. The method comprises the following steps: taking a monoclonal antibody for specially identifying mercury-chelating agent EDTA compound Hg-EDTA as the base, coating the coated antigen mercury-chelating agent-albumen on a 96-hole ELISA plate; incubating at 4 DEG C over night and then sealing by phosphate buffer solution PBS including 1% of gelatin, after washing the ELISA plate, addinga mixing solution of the specificity mercury monoclonal antibody and samples to be measured, incubating at 37 DEG C and then washing the plate, adding an ELISA secondary antibody, incubating at 37 DEGC, adding an enzyme reaction substrate after washing the plate, and adding a reaction stop solution for stopping the reaction after the incubation, and then measuring the absorbance value of each hole by an ELISA reader; obtaining a standard competitive inhibit curve, then executing Logit transition on the curve, and drawing the standard curve of the mercury in the sample to carry out the quantitative analysis. The method is suitable for the measurement of trace mercury in an environment water sample, simultaneously provides the technical support for the fast scene measurement of heavy metalpollution emergency in order to remedy the pollution accident and go back to work in time.

Description

technical field [0001] The invention relates to a fast, accurate and high-throughput assay method for heavy metal mercury, in particular to an indirect competitive enzyme-linked immunosorbent assay method (ELISA method) based on heavy metal mercury monoclonal antibody. Background technique [0002] Heavy metals generally exist in nature at natural concentrations, and the mercury content in natural water bodies is very small, generally not exceeding 0.1 μg / L. The standard limit for drinking water in my country is 0.001 mg / L. However, due to the increasing mining, smelting, processing and commercial manufacturing activities of heavy metals, many heavy metals such as cadmium and mercury enter the atmosphere, water and soil environment, causing serious environmental pollution, and exist in various chemical states or chemical forms. After entering the environment or ecosystem, heavy metals will persist, accumulate and migrate, causing harm, and can eventually endanger human healt...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/543
Inventor 孙成何欢喻恺杨绍贵徐敏杰
Owner NANJING UNIV
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