An enzyme-linked immunosorbent assay method based on biotinylated antigen or antibody-inorganic salt hybrid nanoflowers

A technology of biotinylated antigen and enzyme-linked immunosorbent adsorption, which is applied in the field of immunological detection, can solve problems such as overnight reaction, and achieve the effect of high sensitivity, high applicability, and wide detection range

Active Publication Date: 2018-10-26
WUHAN UNIV
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method utilizes the rapid and efficient specificity of streptavidin and biotin to quickly couple biotinylated antigens or antibody-inorganic salt hybrid nanoflowers to streptavidin-modified microtiter plates, solving the problem of The problem that the traditional ELISA antibody coating needs to react overnight; secondly, due to the high stability of biotinylated antigen or antibody-inorganic salt hybrid nanoflowers, it still has good activity under normal temperature conditions; finally , the synthesized biotinylated antigen or antibody-inorganic salt hybrid nanoflower has a three-dimensional layered structure, which is more efficient for subsequent antibody or antigen capture, which is conducive to improving detection sensitivity ( figure 1 )

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • An enzyme-linked immunosorbent assay method based on biotinylated antigen or antibody-inorganic salt hybrid nanoflowers
  • An enzyme-linked immunosorbent assay method based on biotinylated antigen or antibody-inorganic salt hybrid nanoflowers
  • An enzyme-linked immunosorbent assay method based on biotinylated antigen or antibody-inorganic salt hybrid nanoflowers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] [Example 1] The biotinylated antibody-inorganic salt hybrid nanoflower of the present invention is used for rapid and efficient detection of the cancer marker alpha-fetoprotein (AFP) test (3D ELISA)

[0041] 1) Add 4 μL copper sulfate solution to 600 μL phosphate buffer solution containing biotinylated alpha-fetoprotein (AFP) monoclonal antibody, and place at room temperature for 18 hours to obtain biotinylated antibody-inorganic salt hybrid nanoflowers;

[0042] The concentration of the phosphate buffer solution is 0.1M, pH 7.4; the concentration of the copper sulfate solution is 120mM; the concentration of the antibody is a biotinylated alpha-fetoprotein monoclonal antibody is 0.016mg / mL.

[0043] 2) The above mixture was centrifuged to obtain a precipitate, washed three times with deionized water to obtain the washed biotinylated antibody-inorganic salt hybrid nanoflower complex; the centrifugation speed was 10000r / min, and the centrifugation time was 10min.

[0044]...

Embodiment 2

[0053] [Example 2] Traditional 2D ELISA is used to detect the cancer marker alpha-fetoprotein (AFP) test

[0054] 1) Add 50 μL alpha-fetoprotein (AFP) monoclonal antibody (20 μg / mL) to the well of a Thermo Fisher Scientific Inc. (Cat. No.: 446469) microtiter plate and incubate overnight at 4°C.

[0055] 2) Pour off the solution in the coated microtiter plate, add 300 μL of BSA solution (2%), and block at 37° C. for 2 hours.

[0056] 3) Add different concentrations of AFP standard solutions to the antibody-coated ELISA plate, shake gently to mix, cover or cover the ELISA plate, and react at 37°C for 45 minutes.

[0057] 4) Discard the liquid, wash five times, and pat dry. Add 50 μL of HRP-labeled anti-AFP antibody at a concentration of 20 μg / μL to each well, and react at 37° C. for 45 minutes.

[0058] 5) After incubation, discard the liquid in the wells, wash the plate 5 times, and pat dry. Add 50 μL of TMB chromogenic working solution to each well, and develop color at 37°...

Embodiment 3

[0063] [Example 3] Coating time optimization of the biotinylated antibody-inorganic salt hybrid nanoflower synthesized in Example 1

[0064] 1) The biotinylated antibody-inorganic salt hybrid nanoflowers obtained in Example 1 were uniformly dispersed in PBST buffer solution, placed in a streptavidin-modified well plate, and incubated at 37°C for 5, 10, After 30, 60, 90, and 120 min, wash five times with washing solution, then add 300 μL of 2% BSA solution, and block at 37°C for 45 min to obtain an ELISA plate coated with biotinylated antibody-inorganic salt hybrid nanoflowers ( "3D Substrate" for short), and stored at 4°C for future use. The washing solution is PBST (10 mM, 150 mM NaCl, 0.05% Tween 20, pH 7.2).

[0065] 2) Add 50 microliters of 25ng / mL AFP standard solution to the microtiter plate coated with biotinylated antibody-inorganic salt hybrid nanoflowers, shake gently to mix, cover or cover the microtiter plate, and keep at 37°C React for 45 minutes.

[0066] 3) D...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
adsorption capacityaaaaaaaaaa
Login to view more

Abstract

The invention discloses an enzyme-linked immunosorbent assay method based on biotinylated antigen or antibody-inorganic salt hybridized nanoflowers, and belongs to the field of immunological assays. According to the method, through fast and efficient specific effects of streptavidin and biotin, the biotinylated antigen or antibody-inorganic salt hybridized nanoflowers are rapidly coupled to an enzyme-labeled plate modified by the streptavidin, so that the problem that conventional ELISA antibody coating needs overnight reaction is solved; secondly, as the biotinylated antigen or antibody-inorganic salt hybridized nanoflowers has high stability, the biotinylated antigen or antibody-inorganic salt hybridized nanoflowers still has good activity when placed in the normal temperature condition; finally, the synthetic biotinylated antigen or antibody-inorganic salt hybridized nanoflowers are of a three-dimensional stratified structure which is efficient for subsequent capture of antibodies or antigens and beneficial to improvement of the assay sensitivity. According to the method, it is only necessary to provide diffenrent biotinylated antigens or antibodies for achievement of assays of the different antigens or antibodies, and the applicability is high.

Description

technical field [0001] The invention relates to an immunological detection method, in particular to an enzyme-linked immunosorbent assay method based on biotinylated antigen or antibody-inorganic salt hybrid nanoflowers. Background technique [0002] Enzyme-linked immunosorbent assay (ELISA) refers to the qualitative and quantitative detection method of immune response by adsorbing soluble antigen or antibody to a solid-phase carrier and using antigen-antibody binding specificity. Since the invention of Engvall and Perlmann in 1971, the method has been developed rapidly and has been widely used in the detection of various antigens and antibodies. [0003] Sandwich ELISA method is the most commonly used antigen and antibody detection method at present. This method has the advantages of simple operation, rapidity and sensitivity. The basic principle is that when detecting antibodies, the corresponding antigen molecule is coated on a polystyrene solid phase carrier, the sample...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 何治柯刘宇澄吉邢虎
Owner WUHAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap