Connection method of antibody and microsphere

A connection method and antibody technology, applied in chemical instruments and methods, carrier binding/immobilization of peptides, peptides, etc., can solve problems such as limited sensitivity and reduced detection effect, and achieve the effect of improving flexibility

Inactive Publication Date: 2013-06-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of this method is limited, and the non-specific adsorption of antibodies on microbeads will greatly reduce its detection effect

Method used

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  • Connection method of antibody and microsphere
  • Connection method of antibody and microsphere
  • Connection method of antibody and microsphere

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the connection of rabbit immunoglobulin (IgG) and magnetic beads:

[0052] 1) Cross-linking rabbit immunoglobulin (IgG) with nucleotides

[0053] A. Modification and desalting of rabbit immunoglobulin (IgG)

[0054] Dilute 50 μg of rabbit immunoglobulin (Rabbit Immunoglobulin, referred to as Rabbit IgG) with modification buffer (100 mM phosphate, 150 mM sodium chloride, pH7.2-7.4) to a concentration of 6 mg / mL; then use SANH solution (concentration of 6.89×10 -2 mol / L), the molar ratio of SANH to Rabbit IgG is 60:1. After uniformly mixing the SANH solution and the Rabbit IgG solution, place it in a shaker at 25°C and incubate for modification at a speed of 100 rpm for 4 hours. After incubation, transfer the product into Millipore's YM-100 filter tube, wash with 0.1mM EDTA solution of pH 8, centrifuge (5000 rpm) and filter 3 times to remove excess SANH modifier, and then centrifuge the filter tube upside down (5000 rpm) to collect the antibody-modified ...

Embodiment 2

[0081] Example 2 Connection of Transgenic Cry1Ac Protein Monoclonal Antibody to the Surface of Magnetic Beads

[0082] 1) Transgenic Cry1Ac protein monoclonal antibody cross-linked with nucleotides

[0083] A. Antibody modification and desalting

[0084]Take 50 μg of transgenic Cry1Ac protein monoclonal antibody (prepared by Hangzhou Yikang Company), and adjust the concentration to 4 mg / mL. SANH powder (Succinimidyl4-hydrazinonicotinate acetone hydrazone, that is, succinimidyl 4-hydrazinonicotinate acetone hydrazone. Purchased from the US SoluLink company S-HyNic (SANH) Kit product number (S-9002-2)) with DMF (N,N-Dimethylformamide, N-formamide dimethylamide) solvent dissolved into a solution (concentration of 6.89×10 -2 mol / L) modification. Take the SANH solution with 60 times the molar amount of the antibody and mix it with the antibody solution, place it in a shaker at 25°C, and incubate for modification at a speed of 100 rpm for 4 hours. After incubation, transfer the ...

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Abstract

The invention discloses a connection method of antibody and microsphere, which comprises crosslinking of the antibody and nucleotide, combination of nucleotide and microsphere, and hybridization of a nucleotide complementary chain. The crosslinking of the antibody and nucleotide comprises modification and desalination of the antibody, modification and desalination of nucleotide, crosslinking and purifying of the modified antibody and nucleotide, and the like. The connection method takes a double functional group of nucleotide and the antibody as modification, and the nucleotide and the antibody are combined through an aldehyde group and an amino group to form hydrazone bond combination. According to the invention, the combination of the antibody and microsphere is completed by microsphere coated with avidin and biotin which is modified on a nucleotide chain through specifically adsorption; the hybridization of the nucleotide complementary chain is completed under certain temperature condition through a specific hybridization liquid; the incubation and identification for whether the reaction on the microsphere successes or not can be carried out under the specific temperature condition through Cy3 labeled secondary antibody and the like. The connection method takes oligonucleotide as a support arm for separating the antibody and the magnetic bead for a certain distance, and is in favor of avoiding the destroy of a structure of the antibody, and thereby the antibody activity can be protected.

Description

technical field [0001] The invention relates to a method for linking antibodies and microbeads. Background technique [0002] Since polystyrene microbeads were successfully magnetized and linked to antibodies in 1979, it has become an excellent separation method, triggering a revolution in bioseparation technology. The method has the following advantages: (1) fast separation speed, high efficiency, and good repeatability; (2) simple operation and no need for expensive equipment; (3) large specific surface area, which can improve detection sensitivity; (4) no Affect the biological properties and functions of isolated cells or other biological materials. [0003] With the development of science and technology and the promotion of nanotechnology, nanomaterials have played an important role in the field of life science research. As a new type of nanotechnology, magnetic nano-beads have been successfully applied to systems such as nucleic acid extraction, wastewater treatment, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/00
Inventor 郑晓冬殷赟沙莎
Owner ZHEJIANG UNIV
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