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Preparation and enzyme-linked immunosorbent assay method for heavy metal mercury polyclonal antibody

An ELISA and polyclonal antibody technology, applied in the field of heavy metal detection, to achieve high specificity

Active Publication Date: 2010-11-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Penicillin G salt is used as a bifunctional chelating agent to connect heavy metal mercury and macromolecular proteins, and use it as an immunogen to prepare polyclonal antibodies and establish IC-ELISA. There is no reported precedent

Method used

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  • Preparation and enzyme-linked immunosorbent assay method for heavy metal mercury polyclonal antibody
  • Preparation and enzyme-linked immunosorbent assay method for heavy metal mercury polyclonal antibody
  • Preparation and enzyme-linked immunosorbent assay method for heavy metal mercury polyclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Preparation of polyclonal antibodies

[0022] Step 1: Synthesis of complete antigen

[0023] Dissolve 50 mg of penicillin G sodium salt or potassium salt in 10 ml of ultrapure water, add 1 g of imidazole, stir to dissolve completely, add 1M HCl drop by drop to adjust the pH to 6.8; HgCl 2 , in a water bath at 60°C for 2 hours, take it out and cool it at room temperature, and the reaction produces a yellow powdery precipitate.

[0024] Add excess 1M HCl to the resulting solution, and gently shake it while dripping. A large amount of yellow flocculent precipitates are produced in the solution. The solution is divided into 4ml EP tubes, centrifuged at 10000r / m for 5 minutes, and the supernatant is discarded; Add ultrapure water to wash the precipitate, shake for 2 minutes, then centrifuge at 10,000 r / m for 5 minutes, and repeat the washing twice to obtain penicramic acid thiol mercury salt.

[0025] Using the carbodiimide method (Harlow and Lane, 1988) and the carrier p...

Embodiment 2

[0034] ELISA method

[0035] Step 1: Potency determination

[0036] Optimize the optimal working concentration of antigen and antibody by chessboard method: Dilute the coated antigen MMPA-OVA with carbonate buffer (pH9.6) to a certain concentration (1, 1.3, 2, 2.5, 3, 4, 5, 10 μg / mL), respectively coated in rows A to H of a 96-well ELISA plate, and antibodies were also diluted to a certain number of times (4000, 8000, 10000, 12000, 16000, 32000) and added to columns 1 to 6, and the remaining columns were used as controls; indirect non-competitive OD was determined by ELISA 450Value, the value of 0.8 ~ 1.2 is the optimal value, that is, the value corresponds to the optimal working concentration. The optimal coating concentration was determined to be 2.5 μg / mL of MMPA-OVA, and the optimal antibody dilution was 1:8000.

[0037] Step 2: Establishment of IC-ELISA method for determination of heavy metal mercury

[0038] The detection method of heavy metal mercury by antibody wa...

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Abstract

The invention discloses preparation and an enzyme-linked immunosorbent assay method for heavy metal mercury polyclonal antibody in the technical field of heavy metal detection. The polyclonal antibody is prepared for the enzyme-linked immunosorbent assay by the following steps of: bonding the heavy metal mercury at one end of penicillin G sodium serving as a dual-functional chelating agent; coupling the other end with high molecular carrier proteins, namely bovine serum albumin and ovalbumin, to form immunogen MMPA-BSA and envelope antigen MMPA-OVA of mercury-chelating agent-protein; and finally emulsifying and immunizing the immunogen MMPA-BSA to prepare the polyclonal antibody. The prepared antibody has high specificity for the mercury ions, a cross reaction ratio with all other metals of less than 0.001 percent apart from the cross reaction ratio with cadmium of 7.9 percent and an adding recovery ratio of between 91.4 and 120 percent; and thus the antibody can be used for the assayof the heavy metal mercury in a water sample and can also be developed into rapid immunological detection technology for rapidly detecting the heavy metal mercury in other samples such as agricultural products and the like by combining certain pre-processing technology.

Description

technical field [0001] The invention relates to a method in the technical field of heavy metal detection, in particular to the preparation of a heavy metal mercury polyclonal antibody and its enzyme-linked immunosorbent assay method. Background technique [0002] In recent years, with the rapid development of industry and agriculture, heavy metal pollution has become increasingly serious, causing environmental and food safety problems to become increasingly prominent. Heavy metals mainly enter the human body through the water cycle, drinking water and agricultural products, threatening people's health. The heavy metals that pollute water bodies mainly include Hg, Cd, Cr, Cu, Co, Ni, etc. Among them, Hg is the most toxic. Therefore, it is particularly important to quickly detect the status of heavy metal mercury pollution. At present, the detection and analysis methods of heavy metals mainly include: atomic absorption spectrometry (AAS), inductively coupled plasma emission s...

Claims

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Application Information

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IPC IPC(8): C07K16/44G01N33/53G01N33/543
Inventor 周培奚涛邢海波曹成喜
Owner SHANGHAI JIAO TONG UNIV
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