Anti-human C-reactive protein antibody 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: 2017-05-24
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-human C-reactive protein antibody and application thereof
  • Anti-human C-reactive protein antibody and application thereof
  • Anti-human C-reactive protein antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1. Preparation of anti-human C-reactive protein hybridoma cell line

[0042] 1. Animal immunization

[0043] BALB / c female mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) were immunized with C-reactive protein (purchased from HyTest Company) extracted from human plasma 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.

[0044] 2. Cell Fusion

[0045] (1). Preparation of spleen cells

[0046] The immunized mice were plucked from the eyeballs to take blood, put to death by breaking the cervical spine, soaked in 75% (v / v) alcohol for 10 minutes, took out the spleen in a sterile operating table, placed it in a cell mesh, a...

Embodiment 2

[0056] Example 2. Determination of the variable region sequence of the hybridoma cell line antibody

[0057] The variable region sequences of the above-mentioned hybridoma cell lines M20 and M02 antibodies were determined.

[0058] a. Extraction of RNA: Extract the total RNA of the above-mentioned hybridoma cell lines M20 and M02 with reference to the instructions of the Total Cell RNA Extraction Kit (purchased from Roche Company) and perform reverse transcription immediately;

[0059] b. Reverse transcription of RNA into DNA: Refer to Thermo Scientific Reverted First strand cDNASynthesis 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;

[0060] 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 general prime...

Embodiment 3

[0064] Example 3. Recombinant expression and purification of single-chain antibody

[0065] According to the sequencing results in Example 2, a connecting peptide (GGGGS) was added between the heavy chain and light chain variable regions of the M20 and M02 antibodies, respectively. 3 , and its whole gene was synthesized, and the expression vector construction and recombinant expression of the single-chain antibody were carried out. The expressed antibodies were named as antibody P20 and antibody P02 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:

[0066] a) Construction of single-chain antibody P20 and P02 expression plasmids

[0067] The nucleotide sequence of the single-chain antibody P20 is shown in SEQ ID NO: 19, the amino acid sequence is shown in SEQ ID NO: 17, the nucleotide sequence of the single-chain antibody P02 is sh...

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Abstract

The invention relates to anti-human C-reactive protein (CRP) antibody, a preparation method thereof and application of the antibody in anti-human C-reactive protein detection. An inventor prepares a variety of antibodies, and antibody combinations (P20 and P02) capable of meeting the sensitivity and specificity demands are obtained through pairing and screening. In addition, large-scale production of the antibodies is promoted, and the future large-scale clinic application demand can be met. A colloidal gold immunochromatography quantitative determination card simple and convenient to operate and having sensitivity, specificity and relevant detection properties capable of meeting human clinic sample detection, of a detected human C-reactive protein can be obtained through the detection system debugging and optimizing work to the antibody combinations.

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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IPC IPC(8): C07K16/18C12N15/13C12N15/63C12P21/00G01N33/68G01N33/558
CPCC07K16/18C07K2317/565C07K2317/622G01N33/558G01N33/68G01N2333/4737
Inventor 马永赵利利王安良
Owner ZONHON BIOPHARMA INST
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