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Anti-C-reactive-protein antibodies and application thereof

A technology of reactive proteins and antibodies, applied in the biological field, can solve problems such as insufficient risk prediction, and achieve the effect of easy operation and convenient mass production

Active Publication Date: 2016-09-21
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CRP is usually measured by antibody turbidimetry or immunoturbidimetry, and their detection ability is above 3-5 mg / L. This level is only suitable for the prediction of infection, and the risk prediction for coronary artery and cerebrovascular is far from enough

Method used

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  • Anti-C-reactive-protein antibodies and application thereof
  • Anti-C-reactive-protein antibodies and application thereof
  • Anti-C-reactive-protein antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1. Preparation of anti-human C-reactive protein hybridoma cell line

[0043] 1. Animal immunization

[0044] BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with C-reactive protein extracted from human plasma (purchased from HyTest Company) according to the general immunization procedure. For specific immunization conditions, please refer to the "Experimental Guidelines for Antibody Preparation and Use". The serum titer of immunized mice was tracked by indirect ELISA method, and the immunized mouse with the highest serum titer was selected for fusion experiment of mouse splenocytes and mouse myeloma cells.

[0045] 2. Cell Fusion

[0046] (1). Preparation of spleen cells

[0047] Take the immunized mice, remove their eyeballs, take blood, put them to death by breaking the cervical spine, soak them in 75% (v / v) alcohol for 10 minutes, take out their spleens in a sterile operating table, place them in a cell ...

Embodiment 2

[0057] Example 2. Determination of the variable region sequence of the hybridoma cell line antibody

[0058] The sequences of the antibody variable regions of the above-mentioned hybridoma cell lines M24 and M03 were determined.

[0059] a. Extraction of RNA: Extract the total RNA of the above-mentioned hybridoma cell lines M24 and M03 with reference to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company) and perform reverse transcription immediately;

[0060] b. Reverse transcription of RNA into DNA: Refer to Thermo Scientific Reverted First strand cDNA Synthesis Kit (purchased from Thermo Company) to reverse transcribe the total RNA extracted in the previous step to obtain cDNA, and freeze it at -20°C for later use;

[0061] c. PCR amplification and recovery of the variable region sequence: the cDNA obtained in the above step is used as a template, and the variable region sequence of the heavy chain and light chain is sequenced with the genera...

Embodiment 3

[0065] Example 3. Recombinant expression and purification of single-chain antibody

[0066] According to the sequencing results in Example 2, a linking peptide (GGGGS) was added between the heavy chain and light chain variable regions of the hybridoma cell lines M24 and M03 antibody respectively 3 , introduce six histidine tags SEQ ID NO: 17, and perform recombinant expression of single-chain antibodies by codon-optimizing the whole gene fusion histidine tags according to the preference of the Pichia pastoris expression system. The expressed antibodies were named as antibody P24 and antibody P03 respectively, and their structures and compositions are shown in the attached figure 2 shown. The recombinant expression of the above-mentioned single-chain antibody is specifically as follows:

[0067] a) Expression plasmid construction of fusion protein gene

[0068] The nucleotide sequence of the codon-optimized antibody P24 is shown in SEQ ID NO:18, and the amino acid sequence ...

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Abstract

The invention relates to anti-C-reactive-protein antibodies and an application thereof. Various antibodies are prepared, and pairing and screening are carried out to obtain antibody combinations (P24 and P03), the sensitivity and specificity of which can meet requirements. Massive production of the antibodies becomes convenient, and the antibodies can meet requirements of clinical applications on a large scale. The antibody combinations are subjected to debugging and optimization of a detection system, and a time-resolved fluoroimmuno-chromatography quantitative detection card of C-reactive protein is obtained, wherein the card is easy to operate and has sensitivity, specificity and related detection performance which can meet detection of a human clinical sample.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to two anti-human C-reactive protein (CRP) antibodies, a preparation method thereof and the application of the antibodies in the detection of human C-reactive protein. Background technique [0002] C-reactive protein (C reactive protein, CRP) is a member of the protein family, and it is an acute phase reaction protein. The plasma concentration rises sharply when tissue damage and bacterial infection occur. It is an important defense molecule of the body and is mainly produced by the liver. produced and secreted. CRP is composed of five identical spherical monomers combined by non-covalent bonds to form a stable disc structure, which belongs to the regular pentamer family. CRP is a symmetrical pentahedron in structure, its monomer is composed of 206 amino acids, its molecular weight is about 23KDa, and the total molecular weight of CRP is about 118KDa. CRP participates in v...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13G01N33/68
CPCC07K16/18C07K2317/565
Inventor 马永赵利利杨芸王安良范宇
Owner ZONHON BIOPHARMA INST
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