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Method for determining antibody amino acid sequence in blood

An amino acid and antibody technology, applied in the field of biochemistry, can solve the problems of inability to prepare antibodies, impossible to achieve, difficult amino acid sequences, etc., and achieve the effect of a wide range of sample sources.

Inactive Publication Date: 2017-12-29
张效龙 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, hybridoma technology has major limitations: first, the technology of producing antibodies by hybridoma cells can only be applied to a few kinds of animals, mainly mice and rats, and those skilled in the art believe that hybridoma technology cannot produce Antibodies of human origin, which are widely used in the diagnosis and treatment of diseases
Second, monoclonal antibodies obtained by hybridoma technology often have low affinity, and cells that can produce high-affinity antibodies may be eliminated during the screening process of hybridoma cells
It is very difficult to isolate specific antibodies against a certain antigen from blood and determine the amino acid sequences of these specific antibodies, and it is generally recognized by those skilled in the art that it is impossible to achieve

Method used

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  • Method for determining antibody amino acid sequence in blood
  • Method for determining antibody amino acid sequence in blood
  • Method for determining antibody amino acid sequence in blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Preparation of human protein PD-L1 protein

[0121] Programmed death protein 1 (PD1, Programmed death 1) is a type I transmembrane glycoprotein that regulates the body's immune response, and programmed death protein ligand 1 (PD-L1, Programmed death 1 ligand) is a ligand for PD1 protein. We cloned the gene of the extracellular part of human PD-L1, constructed an expression vector, and expressed the extracellular part of human PD-L1 in Escherichia coli. The expressed human PD-L1 protein (the amino acid sequence of the human PD-L1 protein is shown in SEQ ID NO: 5) is mainly in the inclusion body of Escherichia coli. The expressed Escherichia coli was collected by centrifugation at 5000 rpm at 4°C. The Escherichia coli was lysed with an ultrasonic instrument and centrifuged at high speed to obtain inclusion bodies. The collected inclusion bodies were dissolved with 8M urea, and the protein was refolded by step-by-step dialysis. The renatured protein is purified with an ...

Embodiment 2

[0124] Preparation of Human Protein PD-L1 Antigen Affinity Chromatography Column

[0125] We linked the expressed and purified PD-L1 protein in Example 1 to the surface of the activated agarose colloid through a chemical reaction, thereby obtaining a PD-L1 antigen affinity chromatography column. The preparation process is as follows: in the first step, the expressed and purified PD-L1 protein is linked to a biotin group using the SULFO-NHS-BIOTIN kit of Thermo Fisher (THERMO); in the second step, the biotin group is linked to The aggregated PD-L1 protein was attached to a STREPTAVIDIN column (HITRAPSTREPTAVIDIN HP column produced by General Electric Company).

Embodiment 3

[0127] Immunization of rabbits to obtain anti-human protein PD-L1 antibody

[0128] The highly active and high-purity PD-L1 protein purified in Example 1 was used to immunize white rabbits. After multiple immunizations for about three months, the enzyme-linked immunosorbent assay (Elisa) test confirmed that the rabbit blood already contained a high amount of anti-PD-L1 antibody. Rabbit blood was collected and centrifuged at low temperature to remove blood cells to obtain plasma containing PD-L1 antibody. The amount of blood collected was approximately 40 mL per animal, and was used for the next experiment after freeze-drying.

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Abstract

The invention relates to a method for determining an antibody amino acid sequence in blood. The invention aims to directly determine the antibody amino acid sequence in human or animal blood through a biological chemistry method. The method comprises the following steps: A) separating a specific antibody combined with a specific antigen from blood or a blood product; B) generating an antibody fragment by digesting the separated specific antibody with protease; C) separating the acquired antibody fragment in the manners of SDS-PAGE electrophoresis and ion exchange chromatography, positive chromatography, reverse chromatography, two-dimensional electrophoresis or isoelectric focusing electrophoresis, thereby acquiring the component of the antibody fragment with at most five different amino acid sequences; D) cutting the separated antibody fragment into 5-60 amino acid peptide fragments; E) determining the amino acid sequences of the peptide fragments; F) splicing the determined amino acid sequences of the peptide fragments into an antibody sequence through the overlapped parts.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for determining the amino acid sequence of antibodies in blood. Background technique [0002] Antibodies are a type of special protein produced by the differentiation of B cells under the action of antigenic substances, which can specifically bind to the corresponding antigen. Antibodies exist in body fluids such as blood and on the cell membrane surface of B cells, and are weapons used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. [0003] The properties of an antibody are determined by its amino acid sequence, so determining the amino acid sequence of an antibody is an important part of studying its chemical and biological properties. [0004] Over the past few decades, in order to determine the amino acid sequence of specific antibodies, attention has been turned to antibody-producing B cells. Because each specific antib...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N27/62
CPCG01N33/6824G01N33/6848G01N33/6854
Inventor 张效龙金茜
Owner 张效龙
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