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Enzyme-linked immunoassay kit for detecting hexavalent molybdenum ions and its composition and detection method

An enzyme-linked immunosorbent reagent and molybdenum ion technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that hexavalent molybdenum ions are in the laboratory stage, have high technical requirements for operators, and are difficult to apply widely, so as to save The effect of detection time, strong timeliness, and few detection steps

Inactive Publication Date: 2015-09-23
HENAN INST OF SCI & TECH
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Problems solved by technology

These methods have their own advantages and disadvantages. Ultraviolet spectrophotometry is simple, fast, and has little interference, but its sensitivity is not high; electrochemical analysis is sensitive and simple, and requires high technical requirements for operators; atomic absorption spectroscopy, inductively coupled plasma mass spectrometry method, inductively coupled plasma atomic emission spectrometry, hydride generation-atomic fluorescence spectrometry have the advantages of good selectivity, high measurement accuracy, simplicity, and rapidity, but the instruments used are relatively expensive and can only be detected in laboratories, and the The technical requirements for operators are relatively strict, which limits its wide use; neutron activation analysis is an analytical method based on nuclear reactions, which has the advantages of trace, fast, accurate and non-destructive analysis of multiple elements at the same time, but the It requires equipment that is not available in general laboratories, and it is difficult to be widely used
Since Reardan et al first prepared heavy metal indium (In) monoclonal antibody (mAb) through metal-chelating agent artificial antigen in 1985 and established an immunoassay method, the research on immunoassay technology for heavy metal pollution at home and abroad has been very active, but for hexavalent molybdenum Ion immunoassay technology is still in the laboratory stage

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  • Enzyme-linked immunoassay kit for detecting hexavalent molybdenum ions and its composition and detection method
  • Enzyme-linked immunoassay kit for detecting hexavalent molybdenum ions and its composition and detection method
  • Enzyme-linked immunoassay kit for detecting hexavalent molybdenum ions and its composition and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1, preparation of hexavalent molybdenum ion artificial immunization antigen and coated antigen

[0036] Preparation of artificial immune antigen Mo by isothiocyanate method 6+ -ITCBE-BSA. Take 20mL molybdic acid and 1mL HEPES buffer solution (10 mM / L) at pH 8.0, mix well to form Mo 6+ solution; weigh 10 mg isothiocyanate benzylethylenediaminetetraacetic acid (ITCBE) and dissolve it in 1 mL dimethyl sulfoxide (DMSO) to form a metal chelating agent solution; 6+ After mixing the solution with the metal chelating agent solution, adjust the pH value of the solution to 7.4, and stir at 25 °C for 24 h at a speed of 1000 r / min to form Mo 6+ - Hapten stock solution of ITCBE chelate;

[0037] Weigh 20 mg BSA and dissolve in 1 mL HEPES buffer (10mM / L) at pH 8.0 to form a carrier protein solution with a concentration of 20 mg / mL; take 1 mL Mo 6+ -ITCBE chelate hapten solution was added to 1 mL of carrier protein solution, adjusted to pH 9.0, stirred at room tempera...

Embodiment 2

[0038] Embodiment two, identification of hexavalent molybdenum ion artificial immunization antigen

[0039] Determination of Mo by the biquinoline carboxylic acid method 6+ - Carrier protein BSA concentration in ITCBE-BSA. BSA was used as the standard protein, and the concentration detection standard curve was constructed by the bisquinolinic acid method. The linear equation of the BSA concentration standard curve is: y=0.0004x+0.0072, R 2 =0.9987, where y is the absorbance of the sample at a wavelength of 562 nm, and x is the protein concentration of the sample.

[0040] Determination of Mo by ICP-AES 6+ -Mo in ITCBE-BSA 6+ concentration. 100 μg / mL of Mo 6+ The standard stock solution was diluted with 2% nitric acid to form a concentration gradient of 0 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, and the instrument software automatically draws a standard curve. A linear regression equation was obtained; the sample solution was diluted 50 times, and m...

Embodiment 3

[0041] Example 3. Preparation of anti-hexavalent molybdenum ion monoclonal antibody

[0042] Preparation of qualified antibodies is the key to assembling immunoassay kits. Because the monoclonal antibody has the advantages of strong specificity, high affinity, homogeneity, stable source, and convenient standardized production, the present invention uses a monoclonal antibody against hexavalent molybdenum ions.

[0043] First, Balb / c mice were immunized and spare mice were selected for cell fusion. use Mo 6+ -ITCBE-BSA was used to immunize 5 6-week-old female Balb / C mice, 50 μg·0.2 mL / mouse, and injected subcutaneously at multiple points on the back. For the first immunization, dilute Mo with sterile PBS 6+ - ITCBE-BSA, emulsified by mixing with an equal amount of CFA; for booster immunization, dilute Mo with sterilized PBS 6+ -ITCBE-BSA, emulsified with the same amount of IFA, the second immunization was performed 3 weeks after the first immunization, and the second immu...

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Abstract

The invention relates to an enzyme linked immunosorbent assay (ELISA) kit for detecting hexavalent molybdenum ions as well as establishment and detection methods thereof. The kit is internally provided with an ELISA plate covered by a hexavalent molybdenum ion coating antigen, a hexavalent molybdenum ion resisting monoclonal anti-antibody, an enzyme-labeled second antibody, a primer developing solution, a stopping solution, a hexavalent molybdenum ion standard solution, a concentrated washing solution and a sample treating solution. Before the detection, 0.1mol / L EDTA (Ethylene Diamine Tetraacetic Acid) chelating agent is used for treating a sample, the hexavalent molybdenum ions and the EDTA are sufficiently chelated to obtain a sample solution to be detected, and then the solution is detected. The kit provided by the invention can detect the trace hexavalent molybdenum ions and the detection sensitivity is 1ng / mL; detection steps are few so that the time is saved, the operation error is reduced and the detection cost is not more than 1 / 20 of that of a physical and chemical analysis method; the timeliness is strong and field detection can be carried out; the kit has the properties of rapidness, simplicity, convenience, sensitivity, specificity, economical efficiency and the like, and is mainly used for sieving large-batch samples polluted by residual molybdenum ions in the environment, soil, water and foods.

Description

technical field [0001] The invention relates to the fields of ELISA and hexavalent molybdenum ion detection, in particular to an ELISA kit for detecting hexavalent molybdenum ions and its composition and detection method. Background technique [0002] my country is rich in molybdenum ore resources, with a total reserve of 8.4 million tons. Molybdenum is a transition metal element, and its compounds present five valence states, of which hexavalent molybdenum is the most stable and is most closely related to human life activities. Overdose will affect the quality of life. Molybdenum is one of the essential trace elements for organisms. Various tissues of the human body contain molybdenum. The total amount in the adult body is 9 mg, and the content in the liver and kidney is the highest. Molybdenum itself has no biological activity. Only when hexavalent molybdenum ions combine with purine to form a coenzyme can it have biological activity. Its physiological functions mainly inc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/531
Inventor 张海棠王自良姜金庆范国英黄华国
Owner HENAN INST OF SCI & TECH
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