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Method for detecting loading capacity of streptavidin magnetic microspheres combined with free biotin

A technology of streptavidin and magnetic microspheres, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of complex detection methods and low accuracy of detection data, and achieves low cost, simple operation, and high sensitivity. high sex effect

Pending Publication Date: 2020-09-04
南京瑞贝西生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is to provide a kind of detection method that streptavidin magnetic microsphere binds free biotin load, to solve the detection method complex of streptavidin magnetic nanometer microsphere bind biotin load in the prior art , Defects with low detection data accuracy

Method used

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  • Method for detecting loading capacity of streptavidin magnetic microspheres combined with free biotin
  • Method for detecting loading capacity of streptavidin magnetic microspheres combined with free biotin
  • Method for detecting loading capacity of streptavidin magnetic microspheres combined with free biotin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Step 1: After pretreatment of the 50nm magnetic nanosphere solution, add it to the 0-60ng / 100uL multi-group gradient biotin solution configured above for complexation;

[0133] (1.1) Take a 1.5mL EP tube, add 75μL (0.75mg) of SA magnetic nanospheres vortexed and mixed, perform magnetic separation on a magnetic stand, and discard the supernatant;

[0134] (1.2) Add 1 mL of 1×PBST to the EP tube, resuspend the magnetic nanospheres, and magnetically separate to remove the supernatant. Repeat this step once, then add 1.5mL 1×PBST to resuspend the magnetic nanospheres, and the concentration of the microspheres is 0.5μg / μL;

[0135] (1.3) Take a 96-well ELISA plate, and add 50 μL of the above-mentioned SA magnetic nano-microspheres in order to the wells. Place the 96-well plate on a 96-well microplate magnetic separator, magnetically separate, and remove the supernatant;

[0136] Add gradient concentrations of D-Biotin working solution to the microtiter plate sequentially, ...

Embodiment 2

[0153] Step 1: After pretreatment of the 300nm magnetic nanosphere solution, add it to the 0-60ng / 100uL multi-group gradient biotin solution configured above for complexation;

[0154] (1.1) Take a 1.5mL EP tube, add 75μL (0.75mg) of SA magnetic nanospheres vortexed and mixed, perform magnetic separation on a magnetic stand, and discard the supernatant;

[0155] (1.2) Add 1 mL of 1×PBST to the EP tube, resuspend the magnetic nanospheres, and magnetically separate to remove the supernatant. Repeat this step once, then add 1.5mL 1×PBST to resuspend the magnetic nanospheres, and the concentration of the microspheres is 0.5μg / μL;

[0156] (1.3) Take a 96-well ELISA plate, and add 50 μL of the above-mentioned SA magnetic nano-microspheres in order to the wells. Place the 96-well plate on a 96-well microplate magnetic separator, magnetically separate, and remove the supernatant;

[0157] Add gradient concentrations of D-Biotin working solution to the microtiter plate sequentially,...

Embodiment 3

[0174] Step 1: After pretreatment of the 1000nm magnetic nanosphere solution, add it to the 0-60ng / 100uL multi-group gradient biotin solution configured above for complexation;

[0175] (1.1) Take a 1.5mL EP tube, add 75μL (0.75mg) of SA magnetic nanospheres vortexed and mixed, perform magnetic separation on a magnetic stand, and discard the supernatant;

[0176] (1.2) Add 1 mL of 1×PBST to the EP tube, resuspend the magnetic nanospheres, and magnetically separate to remove the supernatant. Repeat this step once, then add 1.5mL 1×PBST to resuspend the magnetic nanospheres, and the concentration of the microspheres is 0.5μg / μL;

[0177] (1.3) Take a 96-well ELISA plate, and add 50 μL of the above-mentioned SA magnetic nano-microspheres in order to the wells. Place the 96-well plate on a 96-well microplate magnetic separator, magnetically separate, and remove the supernatant;

[0178] Add gradient concentrations of D-Biotin working solution to the microtiter plate sequentially...

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Abstract

The invention discloses a method for detecting the loading capacity of streptavidin magnetic microspheres combined with free biotin. The method comprises the steps of pretreating streptavidin magneticnano-microsphere solutions, respectively adding the pretreated streptavidin magnetic nano-microsphere solutions into a plurality of groups of biotin solutions for affinity adsorption to form a relatively stable complex, and cleaning the plurality of groups of complex products with a cleaning solution; adding horse radish peroxides (Biotin-HRP) labeled by biotin for adsorption; adding a color developing solution into the marked product to carry out a color developing reaction; measuring the OD (optical density) value of the solution after the chromogenic reaction; and calculating according tothe obtained multiple groups of OD values to obtain the loading capacity of streptavidin magnetic nano-microspheres combined free biotin. According to the detecting method, the loading capacity of streptavidin combined with free biotin can be detected more accurately and quickly, a basis is provided for the use amount of downstream streptavidin magnetic nano-microspheres, and the method is easy and convenient to operate, low in cost and high in sensitivity.

Description

technical field [0001] The invention relates to the technical field of nanometer microsphere detection and molecular biology application, in particular to a detection method for streptavidin magnetic microspheres combined with free biotin loading. Background technique: [0002] Biotin, also known as vitamin H, is a water-soluble vitamin and belongs to the B vitamins. [0003] Streptavidin magnetic nanospheres are covalently bonded with superparamagnetic microspheres and high-purity streptavidin. The streptavidin-biotin (SA-Biotin) system has a very high binding affinity (Kd=10^-15), and has a wide range of applications in the biological field. Streptavidin magnetic nanospheres can be easily, quickly and effectively combined with many biotinylated complexes, such as: small molecules, peptides, proteins, antibodies, carbohydrates, lectins, oligonucleotides, DNA / RNA Wait. [0004] The application of streptavidin magnetic nanospheres is more and more widely, and there are usu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/58G01N21/78
CPCG01N33/54326G01N33/581G01N21/78
Inventor 余浩程思瑶
Owner 南京瑞贝西生物科技有限公司
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