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Indirect competitive ELISA kit for detecting lead ions and preparation method thereof

A technology of enzyme-linked immunosorbent reagent and lead ion, which is applied in the field of enzyme-linked immunosorbent immunoassay and ion detection, and can solve problems such as complicated operation process

Inactive Publication Date: 2015-12-02
HENAN INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ELISA method requires a supporting microplate reader and supporting reagents, and the operation process is more complicated.

Method used

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  • Indirect competitive ELISA kit for detecting lead ions and preparation method thereof
  • Indirect competitive ELISA kit for detecting lead ions and preparation method thereof
  • Indirect competitive ELISA kit for detecting lead ions and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment one, the preparation of lead ion artificial immunization antigen

[0042] Preparation of artificial complete antigens, including immune antigen Pb, by the isothiocyanate method 2+ -ITCBE-BSA and coating antigen Pb 2+ -ITCBE-OVA. coated with antigen Pb 2+ -ITCBE-OVA as an example, its preparation method is as follows.

[0043] (1) Weigh 20 mg of ovalbumin OVA and dissolve in 1 mL of HEPES buffer solution with a concentration of 0.01 mol / L and a pH value of 9.0 to form a carrier protein solution;

[0044] (2) Weigh 10mg of isothiocyanate benzylethylenediaminetetraacetic acid chelating agent (ITCBE) and dissolve it in 1mL dimethyl sulfoxide (DMSO) to form a metal chelating agent solution;

[0045] (3) Add 0.5mL metal chelator solution dropwise to the carrier protein solution, mix well, add 100uL, 1.5moL / L tri-n-butylamine, react at room temperature on a shaker for 24h, and make OVA-ITCBE solution;

[0046] (4) Dissolve 11.25mg of lead nitrate in 100uL of ...

Embodiment 2

[0048] Embodiment two, lead ion artificial immunization antigen identification

[0049] Determination of Pb by the method of bisquinolinecarboxylic acid 2+ -Concentration of carrier protein BSA in ITCBE-BSA, with BSA as the standard protein, made into a concentration gradient of 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, with bisquinoline formic acid The standard curve of concentration detection was constructed by the method, and the linear equation of the standard curve of BSA concentration is: y=0.0022x+0.008, 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.

[0050] Determination of Pb by ICP-AES 2+ -Pb in ITCBE-BSA 2+ concentration. 100 μg / mL of Pb 2+ -Pb in ITCBE-BSA 2+ The standard stock solution was diluted with 2% nitric acid to a concentration gradient of 0 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, and 0.5 μg / mL, and the instrument software automatically d...

Embodiment 3

[0051] Embodiment three, the preparation of lead ion monoclonal antibody

[0052] (1) Immunization of animals. with Pb 2+ -ITCBE-BSA was used to immunize 5 Balb / c mice at a dose of 50 μg·0.2 mL / mouse, and injected subcutaneously at multiple points on the back. For the first shot, dilute Pb with sterile phosphate buffered saline (PBS) 2+ -ITCBE-BSA, emulsified with an equal volume of complete Freund's adjuvant (CFA); for booster immunization, dilute Pb with sterilized PBS 2+ -ITCBE-BSA, emulsified with an equal amount of Freund's incomplete adjuvant (IFA), the second immunization was performed 3 weeks after the first immunization, and then immunized once every 3 weeks, a total of 5 immunizations, 10 days after the third immunization Blood was collected by docking the tail, placed in a water bath at 37°C for 30 minutes, placed overnight at 4°C, centrifuged at 800 r / min at 4°C for 5 minutes, and the supernatant was taken and stored at -20°C for later use.

[0053] (2) Alter...

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Abstract

The invention relates to an indirect competitive ELISA kit for detecting lead ions and a preparation method thereof. The box body is provided with an enzyme-labeled plate coated with an antigen coated with lead ions, a lead ion monoclonal antibody, an enzyme-labeled secondary antibody, Substrate chromogenic solution, stop solution, lead ion standard solution, washing solution concentrate and sample treatment solution. The lead ion indirect competitive ELISA kit of the present invention can detect lead ions in trace amounts, can be used for the detection of lead ion pollution residues in the environment, soil, water, food and utensils, and has the advantages of fast, simple, sensitive, specific and economical and other characteristics, less detection steps, saving detection time, reducing operational errors; time-sensitive, can be used for on-site detection; it can be used not only for the screening of large batches of samples, but also for the rapid detection of small batches of samples. , Environmental pollution monitoring and evaluation, etc. provide effective means.

Description

technical field [0001] The invention relates to ELISA and ion detection, in particular to an indirect competitive ELISA kit for detecting lead ions and a preparation method thereof. Background technique [0002] Lead (Pb) is a blue-gray heavy metal, one of the well-known environmental toxicants, widely distributed in nature. With the development of industry and agriculture, lead is used more and more widely, and it can be found in air, soil, food, tableware, and toys, but lead pollution cannot be ignored. Lead is a systemic poison with high accumulation and multi-affinity, and can be absorbed by the body through digestion and breathing. Acute lead poisoning is often manifested as stomach pain, headache, tremor, nervous irritability, and in severe cases, coma and even death. Chronic lead poisoning is mostly manifested as neurotoxicity, causing certain damage to the blood system, immune system, kidneys, etc. Lead is one of the most threatening environmental pollutants to ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/531
Inventor 王自良王爱萍葛亚明范国英张海棠张慧辉
Owner HENAN INST OF SCI & TECH
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