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sH2a monoclonal antibody hybridoma and monoclonal antibody and application thereof

A monoclonal antibody and hybridoma cell technology, which is applied in the field of protein detection, can solve the problems of insufficient sensitivity, specificity, insufficient antibody efficiency, and low titer, and achieve shortened reaction time, sufficient and rapid response, and short reaction time Effect

Active Publication Date: 2017-07-21
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

H2a and H2b only differ in the structure of the extra pentapeptide in the extracytoplasmic domain immediately adjacent to the transmembrane fragment. Studies have shown that sH2a is cleaved into a 35KD fragment immediately adjacent to the pentapeptide, including the complete extracellular domain, which is secreted. The soluble form of the receptor (sH2a), the patent application number 200480013005.2 discloses that sH2a is used as a marker for liver disease diagnosis, the patent application number 201310062296.5 discloses a quantitative detection kit for sH2a, and the patent application number 201410042011.6 discloses a serum Time-resolved immunofluorescence analysis method and detection kits for sH2a content, but these existing technologies have more or less problems, such as the antibodies used are not efficient enough, the titer is not high, the detection method and the matching kit sensitivity, There are still deficiencies in specificity and other aspects

Method used

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  • sH2a monoclonal antibody hybridoma and monoclonal antibody and application thereof
  • sH2a monoclonal antibody hybridoma and monoclonal antibody and application thereof
  • sH2a monoclonal antibody hybridoma and monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, expression, purification and identification of sH2a recombinant protein

[0069] 1. Prokaryotic protein expression

[0070] Using liver cancer tissue cDNA as a template, the upstream primer 5'-GGATCCGCACAGCTGCAAGCCGAGCTGCG-3' (SEQ ID No.1) and the downstream primer 5'-GAATTCTCAGGCCACCTCGCCGGTGGCA-3' (SEQ ID No.2) were used as primers to amplify the sH2a gene fragment by PCR ( figure 1 ), after gel cutting, recovery and purification, it was connected to the cloning vector pCR2.1 and sequenced for identification, and then the sH2a gene fragment with the correct sequence identification was cloned into the prokaryotic expression vector pET30a(+), and the restriction sites were BamH I and EcoR I , to obtain the recombinant plasmid pET30a-sH2a. The recombinant plasmid pET30a-sH2a was transformed into Escherichia coli BL21(DE3), and the successfully transformed clones were picked, cultured at 37°C until the OD600nm value was about 0.6, and 1mM IPTG was added to...

Embodiment 2

[0076] Embodiment 2, preparation of anti-sH2a monoclonal antibody

[0077] 1. Antigen-immunized mice

[0078] Two 6-week-old BALB / c female mice were taken and immunized according to the following steps:

[0079] First immunization: mix the sH2a prokaryotic recombinant protein solution with Freund’s complete adjuvant at a volume ratio of 1:1, fully emulsify, and inject 0.1ml (30 μg / mouse of sH2a recombinant protein) subcutaneously at two points on the back of the mouse;

[0080] The second immunization: after an interval of 14 days, mix the sH2a recombinant protein solution with Freund's incomplete adjuvant at a volume ratio of 1:1, fully emulsify, and inject 0.1ml (sH2a recombinant Protein 50μg / piece);

[0081] The third immunization: after an interval of 14 days, mix the sH2a recombinant protein solution with Freund’s incomplete adjuvant at a volume ratio of 1:1, fully emulsify, and inject 0.1ml subcutaneously at the back of the shoulder and abdomen on both sides of the mou...

Embodiment 3

[0093] Example 3, paired screening of anti-sH2a monoclonal antibodies

[0094] 1. Antibody Pair Screening

[0095] The double-antibody sandwich method was used for pairwise paired experiments. The above-mentioned five anti-sH2a monoclonal antibodies with higher titers were respectively coated in solid-phase microwell plates, the antibody concentration was 5ug / ml, 100ul / well, blocked with 2% BSA phosphate buffer for 2h, and the sH2a prokaryotic The recombinant protein was diluted to 500ng / ml, and continued to be diluted to 6 concentrations. The diluted solution was used as a blank control, and reacted at 37°C for 1 hour, and the other 4 anti-sH2a strains labeled with horseradish peroxidase (HRP) were added except itself. Monoclonal antibody (1:10000), detect the absorbance value (wavelength 450nm). The results show that the pairing of anti-sH2a monoclonal antibody 1F2D2 and 5A9B8 can detect a significant concentration gradient of the protein, which can be used to establish a ...

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Abstract

The invention discloses an sH2a monoclonal antibody hybridoma and a monoclonal antibody and application thereof. A hybridoma cell line include 5A9B8 and 1FD2D which are preserved at the Chinese typical culture collection center of Wuhan university (on March 29, 2017), the preservation numbers are CCTCC NO.C201751 and CCTCC NO.C201750 respectively. The hybridoma can secrete sH2a monoclonal antibodies which can be specifically bound with sH2a recombinant proteins, is high in titer and good in affinity, can be used for testing sH2a and auxiliary diagnosis of liver diseases such as liver damage, hepatitis and liver fibrosis.

Description

technical field [0001] The invention belongs to the technical field of protein detection, in particular to sH2a monoclonal antibody hybridoma cells, and also relates to the monoclonal antibody produced by the cell and the application of the monoclonal antibody. Background technique [0002] The human sialoglycoprotein receptor (ASGPR) is expressed only in hepatocytes and is responsible for the clearance of asialoglycoprotein from plasma. ASGPR is composed of two related amino acid sequence subunits H1 (46KD) and H2 (50KD). H2a and H2b are two alternative splice forms of the H2 subunit of ASGPR. H2a and H2b only differ in the structure of the extra pentapeptide in the extracytoplasmic domain immediately adjacent to the transmembrane fragment. Studies have shown that sH2a is cleaved into a 35KD fragment immediately adjacent to the pentapeptide, including the complete extracellular domain, which is secreted. The soluble form of the receptor (sH2a), the patent application numbe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/28G01N33/577G01N33/576G01N33/535G01N33/532G01N33/543
CPCC07K16/2851G01N33/532G01N33/535G01N33/54326G01N33/576G01N33/577G01N2800/085
Inventor 王弢周小进曹丽娟杜帅
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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