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Method for realizing directional coupling by utilizing glycosyl of antibody and solid-phase carrier material

A solid-phase carrier and antibody technology, which is applied in the analysis of materials, material inspection products, and biological material analysis, can solve problems such as reducing the activity of antibody-binding antigens, blocking active sites, and affecting the stability and sensitivity of immunoassay methods. Effects of capture ability and extraction efficiency, increase in binding capacity, and increase in sensitivity

Active Publication Date: 2021-04-09
UNIV OF SCI & TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The carboxyl group or amino group on the antibody is abundant, and the distribution in the tertiary structure is uniform, so the coupling of the antibody to the solid-phase carrier material is non-directional, and the amino group or carboxyl group on the solid-phase carrier material will be randomly coupled to the antibody, Changing the conjugated structure of the antibody is likely to block the active site where the antibody binds to the antigen due to steric hindrance, thereby reducing the antigen-binding activity of the antibody and affecting the stability and sensitivity of the immunoassay method

Method used

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  • Method for realizing directional coupling by utilizing glycosyl of antibody and solid-phase carrier material
  • Method for realizing directional coupling by utilizing glycosyl of antibody and solid-phase carrier material
  • Method for realizing directional coupling by utilizing glycosyl of antibody and solid-phase carrier material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Directional coupling of antibody to aflatoxin B1 and time-resolved fluorescent microspheres (solid phase carrier material).

[0048] S1. Modification of time-resolved fluorescent microspheres:

[0049] 1) Take out 0.5mg of time-resolved fluorescent microspheres, centrifuge at 12000r for 15min at 4°C, and remove the protective solution;

[0050] 2) Add 0.2 mg of bovine serum albumin to the precipitate after centrifugation, and incubate at 25° C. for 2 hours. Centrifuge at 12000r for 15min at 4°C to remove excess bovine serum albumin. Wash twice with phosphate buffer (PB, 20mol / L, pH 7.2), centrifuge at 12000r for 15min at 4°C, and remove the liquid phase;

[0051] 3) Add 250 microliters of preservation solution (0.1% Proclin300) to the precipitate after centrifugation.

[0052] S2. Antibody activation:

[0053] 1) Prepare 2L dialysate 1mmol / L pH4.4 sodium acetate solution, add acetic acid to adjust the pH to 4.4, and pre-cool at 4°C;

[0054] 2) Prepare 0.1mol / L sod...

Embodiment 2

[0074] The antibody of C-peptide is coupled with immunomagnetic beads for the extraction of C-peptide in purified serum.

[0075] S0, magnetic bead pretreatment:

[0076] S0.1 Washing of Magnetic Beads

[0077] 1) Vortex to resuspend the carboxyl magnetic beads, pipette 100 μL magnetic beads into a 2 mL centrifuge tube, magnetically separate and discard the supernatant;

[0078] 2) Add 500 μL of coupling buffer (50 mmol / L MES, pH 6.0, 0.01% Triton X-100) to the tube, vortex for 20 s to wash the magnetic beads, place the centrifuge tube on the magnetic stand for 60 s, magnetically separate and discard clear; repeat the washing step three times;

[0079] Activation of S0.2 Magnetic Beads

[0080] 1) Prepare EDC (50mg / mL) and NHS (50mg / mL) with coupling buffer (ready-to-use), add 60 μL of coupling buffer, 20 μL of newly prepared EDC solution and 20 μL of newly prepared NHS solution, vortexed to mix.

[0081] 2) After incubating at room temperature for 15 minutes, place the c...

Embodiment 3

[0108] Directional coupling of zearalenone antibody to quantum dots for detection of zearalenone in edible oil.

[0109] S1. Quantum dot modification:

[0110] 1) Take out 0.5mg of quantum dots, centrifuge at 12000r for 15min at 4°C, and remove the protective solution;

[0111] 2) To activate, add 250 μL of MES buffer solution (50mmol / L, pH 5) containing 5mg EDC and 1.44mg NHS to the quantum dots, pipette up and down, shake at 25°C for 20min, centrifuge at 12000r for 15min at 4°C, and remove the liquid phase;

[0112] 3) Wash, add 250 μL of MES buffer (50 mmol / L, pH 5) to the precipitate after centrifugation, pipette up and down, sonicate for 3 minutes, centrifuge at 12000 r for 15 minutes at 4°C, and remove the liquid phase;

[0113] 4) Add 0.1 mg of bovine serum albumin to the precipitate after centrifugation, and incubate at 25° C. for 2 hours. Centrifuge at 12000r for 15min at 4°C to remove excess bovine serum albumin. Wash twice with phosphate buffer (PB, 20mmolL, pH 7...

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Abstract

The invention relates to the field of biochemical synthesis, and provides a method for realizing directional coupling by utilizing glycosyl of an antibody and a solid-phase carrier material, a directional conjugate and an application. The method comprises the following steps that a solid-phase carrier material is modified by utilizing a modification reagent, so that the surface of the solid-phase carrier material is modified with a binding arm with a certain length and rich in amino groups, and the binding arm can extend into an Fc region of an antibody and react with the glycosyl of the antibody; the glycosyl of the antibody is oxidized into an aldehyde group; and the aldehyde group reacts with the amino group of the modified solid-phase carrier material to form Schiff base, and the Schiff base is reduced into an amine compound, thereby obtaining a directional conjugate. According to the invention, the problem that a solid-phase carrier material can be hardly coupled to glycosyl in an antibody is solved; the problem of random immobilization of an antibody and a solid phase carrier material is solved, and an active part, combined with an antigen, of an antibody can be fully exposed; and when the directional conjugate constructed by the invention is applied to immunoassay, more antigens can be recognized only by using a small amount of the directional conjugate, and the sensitivity of immunoassay determination is improved.

Description

technical field [0001] The present invention relates to the field of biochemical synthesis, in particular to a method for realizing directional coupling by utilizing sugar groups of antibodies and solid-phase carrier materials, directional conjugates and applications. Background technique [0002] Protein coupling is to link small molecular substances (haptens, etc.) or macromolecular substances (enzymes, etc.) Wait. Antibodies have been widely used in immunoassay and detection technology. Antibody conjugation is the use of covalent or non-covalent binding of antibodies to solid-phase carrier materials (such as enzymes, fluorescent complexes, and biotin). By using these methods to optimize specific antibodies, the specific recognition of antigens and antibodies is used. , making it easier to track in complex mixtures and providing quantifiable and intuitive detection results. [0003] There are many methods for labeling antibodies on solid-phase carrier materials, such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/533G01N33/577G01N33/58G01N33/53G01N33/543G01N33/68G01N33/558
CPCG01N33/531G01N33/533G01N33/577G01N33/582G01N33/585G01N33/5308G01N33/54326G01N33/6893G01N33/588G01N33/558G01N2800/042Y02P20/55
Inventor 时国庆毛心怡于冰儿弓爱君李旭琴
Owner UNIV OF SCI & TECH BEIJING
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