Indirect competition enzyme linked immunoreagent kit for detecting lead ions and manufacturing method thereof

An enzyme-linked immunosorbent reagent and lead ion technology, applied in the field of enzyme-linked immunosorbent assay and ion detection, can solve problems such as complex operation process

Inactive Publication Date: 2013-12-25
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 competition enzyme linked immunoreagent kit for detecting lead ions and manufacturing method thereof
  • Indirect competition enzyme linked immunoreagent kit for detecting lead ions and manufacturing method thereof
  • Indirect competition enzyme linked immunoreagent kit for detecting lead ions and manufacturing method thereof

Examples

Experimental program
Comparison scheme
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 ovalbumin OVA and dissolve it in 1 mL 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 dis...

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 tail docking, 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 collected and stored at -20°C for later use.

[0053] (2) Alter...

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Abstract

The invention relates to an indirect competition enzyme linked immunoreagent kit for detecting lead ions and a manufacturing method of the kit. An elisa plate enveloped by lead ion envelope antigens, lead-ion monoclonal antibodies, elisa second antibodies, a substrate color developing solution, a stop solution, a lead ion standard solution, a washing liquor concentrated solution and a sample treating liquid are arranged in the kit. The indirect competition enzyme linked immunoreagent kit can detect the lead irons in a trace mode, is used for detecting the lead iron contamination residual in the environment, the soil, water, foods and containers, and has the advantages of being rapid, easy and convenient to use, sensitive, peculiar, economical and the like. The kit is few in detection step and high in timeliness, saves detection time, reduces operation errors and can perform field detection. The kit not only can be used for screening samples in large batch, but also can perform rapid detection on samples in small batch, and provides effective means for food import and export inspection, food inspection, monitoring and evaluation of environmental pollution and the like.

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 Applications(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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