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Kit for rapidly coupling proteins, antibodies, enzyme and nucleic acid and preparation method of kit

A kit and antibody technology, applied in the field of biological kit materials, can solve problems such as restricting operation proficiency, improve coupling efficiency and coupling amount, realize rational utilization of resources, and have a wide range of applications.

Inactive Publication Date: 2016-11-09
JIANGSU FLON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, when using carboxyl magnetic beads to couple antibodies in practical applications, the problems often encountered include: which activator to use? What is the amount of activator added, activation temperature and activation time? What are the conditions for antibody conjugation? For non-professionals or beginners, these considerations seriously restrict the proficiency of operation

Method used

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Examples

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Effect test

preparation example Construction

[0039] The preparation method of above-mentioned test kit comprises following main steps:

[0040] 1. The preparation process of activated magnetic microspheres is as follows

[0041] (1) Take 500mL each of N,N-dimethylacetamide (DMAC) and isopropanol and pass through 4A molecular sieve;

[0042] (2) Take the carboxyl magnetic beads and wash them with DMAC two to three times;

[0043] (3) Add DMAC and N-hydroxysuccinimide to the washed carboxyl magnetic beads, and react at 40°C for 0.5h;

[0044] (4) Collect the reaction product, wash it twice with isopropanol, and finally add isopropanol to prepare a 10 mg / mL activated magnetic bead suspension, and store it at 4°C.

[0045] 2. The preparation process of the washing solution is as follows: take 1L of purified water, add concentrated hydrochloric acid to adjust the pH to 2.5-5.0, filter with a 0.22μm filter membrane, and store at 4°C.

[0046] 3. The preparation process of the coupling buffer is as follows: Take 20.926g of 3-(...

Embodiment 1

[0056] Protein A conjugation

[0057] 1. Preparation of Protein A solution: Weigh 10 mg of Protein A with an analytical balance and dissolve in 10 mL of the coupling buffer that comes with the kit to prepare a 1 mg / mL Protein A solution;

[0058] 2. Washing of activated magnetic beads: take 200mg of activated magnetic microspheres, and wash them once with 10mL of the washing solution that comes with the kit, and remove the washing solution after the magnetic beads are attached to the wall by magnetic suction;

[0059] 3. Ligand coupling: Add the Protein A solution prepared in step 1 to the magnetic beads washed in step 2, mix the magnetic beads and ligand solution evenly by shaking, place on a mixer, and rotate slowly. After reacting for 1 hour at room temperature, protein A can be covalently coupled to the magnetic beads;

[0060] 4. Activation of magnetic beads for sealing: Place the magnetic bead suspension coupled with Protein A in step 3 on a magnetic separation rack, an...

Embodiment 2

[0064] Carcinoembryonic Antigen (CEA) Antibody Conjugation

[0065] 1. Preparation of CEA solution: Prepare the CEA antibody at 0.2 mg / mL in the coupling buffer that comes with the kit;

[0066] 2. Activated magnetic beads washing: Take 10mg of activated magnetic microspheres, wash them quickly once with 1mL of the washing solution that comes with the kit, and remove the washing solution after the magnetic beads are attached to the wall by magnetic suction;

[0067] 3. Ligand coupling: Add 0.5mL of the CEA solution prepared in step 1 to the magnetic beads washed in step 2, mix the magnetic beads and ligand solution evenly by shaking, place on a mixer, and rotate slowly , reaction at room temperature for 1 hour, the antibody can be covalently coupled to the magnetic beads;

[0068] 4. Activation of magnetic beads for sealing: Place the magnetic bead suspension coupled with CEA in step 3 on a magnetic separation rack, and after magnetic attraction makes the magnetic beads adher...

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Abstract

The invention relates to a kit for rapidly coupling proteins, antibodies, enzyme and nucleic acid. The kit comprises test tubes which are respectively filled with magnetic microspheres, washing liquid, a coupling buffer solution, a closing buffer solution and a preserving buffer solution, wherein the magnetic microspheres are prepared by the following steps: (1) respectively sieving 500mL of N,N-dimethylacetamide (DMAC) and 500mL of isopropanol by using a 4A molecular sieve; (2) washing carboxyl magnetic beads for 2-3 times with DMAC; (3) adding DMAC and N-hydroxysuccinimide into the washed carboxyl magnetic beads, and reacting at 40 DEG C for 0.5h; and (4) collecting reaction products, washing twice with isopropanol, finally adding isopropanol to prepare 10mg / mL activated magnetic bead suspension liquid, and preserving at 4 DEG C. According to the kit, the problems of tedious and complex operations of a user in processes of magnetic bead activation, ligand coupling, magnetic bead closing and the like are solved, the operation steps are simplified, and reasonable utilization of resources is realized.

Description

technical field [0001] The invention belongs to the field of biological kit materials, and in particular relates to a kit for coupling proteins, antibodies, enzymes and nucleic acids and a preparation method thereof, which can rapidly, simply and efficiently combine proteins, antibodies, enzymes and amino-modified nucleic acids And other molecules with amino groups are covalently coupled to activated magnetic microspheres, and the magnetic beads after coupling ligands can be used for detection, separation and purification, etc. Background technique [0002] Immunomagnetic beads are a new immunological technology developed in recent years. It combines the unique advantages of solidified reagents with the high specificity of immunological reactions. Based on immunology, it penetrates into pathological, physiological, pharmacological, microbial It has been widely used in immunoassay, cell separation, purification of biological macromolecules and molecular biology. [0003] Usi...

Claims

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Application Information

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IPC IPC(8): G01N33/543
CPCG01N33/54326
Inventor 谭建雄奚伟红
Owner JIANGSU FLON BIOTECH
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