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Tripolycyanamide single-reagent polarization fluoroimmunoassay

An immunoassay method and fluorescence polarization technology, applied in measuring devices, instruments, scientific instruments, etc., can solve problems such as time-consuming and complicated operation

Inactive Publication Date: 2012-10-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to establish a single-reagent fluorescence polarization immunoassay method for melamine in view of technical deficiencies such as complex operation and time-consuming in the existing melamine immunoassay technology. The whole detection process only needs one step and is easy to operate.

Method used

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  • Tripolycyanamide single-reagent polarization fluoroimmunoassay
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  • Tripolycyanamide single-reagent polarization fluoroimmunoassay

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: the preparation of fluorescent tracer

[0037] Step 1: Synthesis of fluorescein isothiocyanate ethylenediamine derivatives.

[0038] Prepare a 1% (volume percentage concentration) triethylamine solution with methanol to obtain methanol triethylamine solution;

[0039] Add 20 mg (0.3 μmol) of ethylenediamine into 5 mL of methanolic triethylamine solution to obtain ethylenediamine solution;

[0040] Then 11.7 mg (0.03 μmol) of fluorescein isothiocyanate was dissolved in 1 mL of methanolic triethylamine solution, added dropwise to the ethylenediamine solution, stirred at room temperature in the dark for 1 h, concentrated, and subjected to silica gel column chromatography. The eluent is ethyl acetate + methanol (3:1, v / v), and the yellow substance that flows out is collected to obtain the fluorescein isothiocyanate ethylenediamine derivative, identified by mass spectrometry, MS (EI) m / z: 448.1 , corresponding to fluorescein isothiocyanate ethylenediamine d...

Embodiment 2

[0051] Example 2: Preparation of Single Reagent Immune Complex

[0052] Use boric acid buffer (50mmol / L, pH 8.0. Mix 70mL of 0.2mol / L boric acid solution with 30mL of 0.05mol / L borax solution, dilute to 400ml with distilled water to obtain boric acid buffer with pH 8.0) As a diluent for melamine polyclonal antibody. Mix 10 mL of melamine fluorescent tracer with a concentration of 1 nmol / L (see Example 1 for the preparation method) and an equal volume of 100-fold diluted melamine polyclonal antibody (for the preparation method, refer to Yin et al., J Agric Food Chem 2010, 58, ( 14), 8152-8157.) The single-reagent immune complex can be obtained by mixing and reacting for 1 to 15 minutes. The state of the reaction is monitored by continuously measuring the fluorescence polarization value (mP), and the binding kinetics curve is obtained, see appendix figure 1 shown. The results showed that after 12 minutes of mixed reaction, the fluorescence polarization value basically remai...

Embodiment 3

[0054] Embodiment 3: the drawing of standard curve

[0055] Take several 180 μL portions of spare single-reagent immune complexes prepared in Example 2, add 20 μL respectively, and the concentrations are 0 ng / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL , 500 ng / mL, 1000 ng / mL, 5000 ng / mL, 10000 ng / mL melamine standard (products purchased from Sigma, USA), and boric acid buffer solution (50 mmol / L, pH 8.0, same as Example 2) As a blank control (0ng / mL), continuously measure the fluorescence polarization value to monitor the status of the displacement reaction and obtain the kinetic curve of the competition reaction, see attached figure 2 shown. The results showed that the fluorescence polarization value of the melamine standard substance at the same concentration remained basically at the same level after the displacement reaction for 15 minutes, that is, the displacement reaction reached an equilibrium state at this time, which could ensure the stability and accuracy of...

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Abstract

The invention discloses a tripolycyanamide single-reagent polarization fluoroimmunoassay. A stable and sensitive single-reagent immunocomplex is prepared by the method, and can be stably preserved for at least more than one month at the temperature below 4 DEG C. For the tripolycyanamide polarization fluoroimmunoassay established on this basis, washing and separation are not required in the reaction process, and the detection result can be obtained just through a short-time one-step reaction between a specimen to be detected and the single-reagent immunocomplex, wherein the detection limit is 3.2 ng / mL, and the detection range is 12.2-1012.4 ng / mL. Thus, the tripolycyanamide single-reagent polarization fluoroimmunoassay is a fast and convenient high-flux tripolycyanamide detection method, is very suitable for the practical detection application of tripolycyanamide, and lays a foundation for developing an on-site detection kit in future.

Description

technical field [0001] The invention belongs to the technical field of food safety, in particular to a melamine single-reagent polarization immunoassay (Single-reagent Polarization Fluoroimmunoassay, SRPFIA). Background technique [0002] Melamine (Chemical formula: C 3 h 6 N 6 ), commonly known as melamine, protein essence, named "1,3,5-triazine-2,4,6-triamino" by the International Union of Theoretical and Applied Chemistry (IUPAC), is a triazine nitrogen-containing Cyclic organic compounds are usually used as chemical raw materials. In recent years, melamine has been illegally added to food and feed, especially for protein fraud in dairy products. Ingestion of melamine may lead to reproductive damage, kidney stones and even bladder cancer. In order to ensure food safety and the health of consumers, five departments including the Ministry of Agriculture and the Ministry of Health jointly issued the "Announcement on the Limit Value of Melamine in Food" in 2011, which cl...

Claims

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Application Information

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IPC IPC(8): G01N33/533
Inventor 孙远明雷红涛王强王弘沈玉栋杨金易徐振林吕书为唐秋实
Owner SOUTH CHINA AGRI UNIV
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