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Application of hyperbranched polyglycidyl ether modified nano-magnetic microspheres in chemiluminescence immunoassay

A chemiluminescence immunoassay, glycidyl ether technology, applied in analytical materials, scientific instruments, measuring devices, etc., can solve the problems of low coupling efficiency, inability to break through the maximum theoretical density of single-layer surface, and limiting the amount of antibody coupling, etc. Achieve the effect of reducing non-specific adsorption, optimizing the minimum detection limit, and broadening the detection range

Active Publication Date: 2016-04-06
GETEIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method of magnetic microspheres has the following two significant defects: first, polystyrene is a very hydrophobic material, and direct application will inevitably produce the consequences of non-specific adsorption, which will lead to the adsorption of impurities in the sample On the surface of the microspheres, the interference of impurities will not only increase the background value of the reaction, but also lead to fluctuations in the reaction baseline, and even cause false positive or false negative results in severe cases, directly affecting the reliability of the test, resulting in misdiagnosis or missed diagnosis; Second, the density of the functional groups introduced in any of the above methods cannot break through the maximum theoretical density of the monolayer surface, thus limiting the amount of coupling of antibodies on it
However, the linear PEG polymer has only one or two active sites in the entire polymer structure for coupling due to its structure only having functional groups at the end of the polymer. The binding ability is weak and the coupling efficiency is not high. , which limits its wider application

Method used

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  • Application of hyperbranched polyglycidyl ether modified nano-magnetic microspheres in chemiluminescence immunoassay
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  • Application of hyperbranched polyglycidyl ether modified nano-magnetic microspheres in chemiluminescence immunoassay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation method of diagnostic reagent based on hyperbranched polyglycidyl ether modified magnetic microspheres

[0053] Preparation of Hyperbranched Polyglycidyl Ether

[0054] In a three-neck flask equipped with mechanical stirring and a micro constant flow pump, add 0.24g trimethylolpropane (TMP) as the initiator of the reaction, using 3.7MCH 3 OK (dissolved in methanol) partially deprotonated the hydroxyl group of TMP, the deprotonated part accounted for 10% of the total part, and the excess methanol was removed by heating and evaporating. Then 50mL of glycidol was slowly added dropwise into the system to initiate polymerization, pay attention to the drop rate of glycidol, as dropping too fast may cause implosion. The reaction temperature was 95°C, and the reaction was carried out for 12 hours.

[0055] After the reaction, the product was dissolved in methanol and passed through a strong acid cation exchange resin (AMBERJET TM 1500HResin) neutralization...

Embodiment 2

[0062] In Example 2, the commercial carboxyl magnetic microspheres (manufacturer JSR diagnostic reagent, article number MS160 / Carboxyl) used as a control reagent were coupled to the hGH antibody in the same way as above, only commercial carboxyl magnetic microspheres were used instead of hyperbranched polyglycidyl ether Modified magnetic microspheres.

[0063] Labeling of acridinium esters

[0064] Take hGH‐5801 antibody (manufacturer MedixmAB product number 100041) and add it to 50mMPBS buffer (pH=8.0), add 0.5mmol / L acridinium ester, mix, and react at room temperature for 20min in the dark. Use 10mMPBS buffer (pH=6.5) to remove unbound acridinium esters by dialysis, and finally add 50% redistilled glycerol and store at -20°C.

[0065] Chemiluminescence excitation solution system

[0066] Prepare excitation solution 1 and excitation solution 2: excitation solution 1 is 0.2M nitric acid solution containing 0.5% hydrogen peroxide solution; excitation solution 2 is 0.5M sodium...

Embodiment 3

[0099] Validation of diagnostic reagents developed by hyperbranched polyglycidyl ether modified magnetic microspheres in TPO-Ab project using indirect method in chemiluminescence immunoassay

[0100] The hyperbranched polyglycidyl ether-modified nano-magnetic microsphere-TPO antigen (manufacturer Hytest product number 8RTPO) complex was prepared according to the method of Example 1, and was used for the following TPO-Ab chemiluminescence immunoassay. In this example, the coupling method of commercialized carboxyl magnetic microspheres (manufacturer JSR diagnostic reagent, article number MS160 / Carboxyl) and TPO antigen as a control reagent is the same as that in Example 1, only commercial carboxyl magnetic microspheres are used instead of hyperbranched polymeric microspheres. Glycidyl ether modified magnetic microspheres.

[0101] Add hyperbranched polyglycidyl ether-modified nano-magnetic microspheres-TPO antigen complex to 20 μL of the sample to be analyzed, mix well, and inc...

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Abstract

The invention discloses application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay. By utilizing the hyperbranched polyglycerol modified magnetic nanoparticle microspheres, a nanometer microsphere material of which the surface is hydrophilic and which is enriched in functional groups is prepared. When a chemiluminescence diagnostic agent based on the hyperbranched polyglycerol modified magnetic nanoparticle microspheres is actually used, the reagent can well inhibit nonspecific adsorption of proteins, and the antibody coupling amount is 2-3 times higher than that of commercial magnetic microspheres. In the project-based performance analysis, the diagnostic agent based on the magnetic microspheres can greatly enhance the reaction between antigens and antibodies. When the reagent is compared with a commercial magnetic microsphere reagent, the sensitivity is improved by 4-5 times, and the upper detection limit is improved by 1.3 times, so that the detection range is improved by nearly 6.5 times.

Description

technical field [0001] The invention belongs to the field of biological detection reagents and relates to the application of hyperbranched polyglycidyl ether modified nanometer magnetic microspheres in chemiluminescence immunoassay. Background technique [0002] Chemiluminescence immunoassay is a combination of highly specific immune reaction analysis technology, chemiluminescence reaction system and highly sensitive luminescence assay technology, which is used for the detection and analysis of various antigens, haptens, antibodies, enzymes, hormones and drugs, etc. technology. It is a newest immunoassay technique developed after radioimmunoassay technique, enzyme-linked immunoassay technique, fluorescence immunoassay technique and time-resolved fluorescence immunoassay technique. [0003] The chemiluminescence immunoassay consists of two parts, the immune response system and the chemiluminescence system. The immune response system uses the principle of high specificity of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/553
CPCG01N33/553
Inventor 金晶朱宗哲陈赢苏恩本
Owner GETEIN BIOTECH
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