Human Streptococcus pneumoniae surface protein monoclonal antibody and antigen capture ELISA kit

A Streptococcus pneumoniae and monoclonal antibody technology, applied in the field of immunology, can solve problems such as long time, difficult to meet rapid identification, unsuitable

Active Publication Date: 2021-04-09
湖北云璐生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the method for detecting the pathogenic microorganism in the respiratory tract is mainly based on the traditional method, that is, the separation and identification method. This method takes a long time, usually 2-3 days, and it is difficult to meet the needs of rapid identification; the developed in recent years PCR technology is a fast, sensitive, and specific technology, but at present, this technology still relies on the pre-enrichment step of the traditional method, and there are often PCR inhibitors in the enrichment solution, which affects the effect of amplification
At the same time, this technology also requires professional testing equipment, which is not suitable for bedside testing

Method used

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  • Human Streptococcus pneumoniae surface protein monoclonal antibody and antigen capture ELISA kit
  • Human Streptococcus pneumoniae surface protein monoclonal antibody and antigen capture ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Preparation of recombinant human Streptococcus pneumoniae surface protein PspA

[0036] 1) Cloning and expression of human Streptococcus pneumoniae PspA gene

[0037]Bioinformatic analysis of human Streptococcus pneumoniae surface protein PspA (accession number in NCBI protein database is AAF27703.1) gene, combining GC content, codon preference, mRNA secondary structure, RNA instability motif, mRNA Considering the stability of free energy, etc., the DNA coding sequence was optimized, and at the same time, the whole gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end, the termination signal TAA and the restriction site XhoI at the 3' end (the whole sequence synthesis was handed over to Completed by GenScript Biotechnology Co., Ltd., the artificially synthesized gene fragment was connected to the vector pUC57 at the time of delivery), denoted as PspA'. The full sequence of the gene and the encoded amino acid seq...

Embodiment 2

[0041] The preparation of embodiment 2 human Streptococcus pneumoniae surface protein polyclonal antibody

[0042] 1) Immunization of New Zealand purebred rabbits

[0043] The recombinant PspA protein prepared in Example 1 was mixed with complete Freund's adjuvant, emulsified and used as an immunogen to immunize 2 male New Zealand rabbits, each rabbit was subcutaneously injected with a total amount of 2ml, and the total amount of antigen was 2mg / rat. Afterwards, the emulsion formed by recombinant PspA protein and Freund's incomplete adjuvant was used to immunize once every two weeks, for a total of 5 times, and the amount of antigen was the same as that of the first immunization. 3-5 days after the fifth immunization, a large amount of blood was taken from the heart, placed at 37°C for 1 hour, then placed in the refrigerator at 4°C overnight, and serum was collected the next day.

[0044] 2) Determination of polyclonal antibody titer

[0045] Recombinant PspA protein was use...

Embodiment 3

[0053] The preparation of embodiment 3 human Streptococcus pneumoniae surface protein monoclonal antibody Sp-10#

[0054] 1) Immunization of animals

[0055] Using the recombinant human Streptococcus pneumoniae surface protein PspA prepared in Example 1 as an antigen, 5 8-week-old BALB / c mice were immunized, and 2 were not used as immunized mice as negative controls. After the initial immunization antigen was fully emulsified with an equal amount of Freund's complete adjuvant, the mice were subcutaneously injected into the back of the immunized mice at a rate of 100 μg / mouse. After that, the second immunization was performed by intraperitoneal injection with the same dose of antigen fully emulsified with Freund's incomplete adjuvant at an interval of three weeks, and the third time was injected intraperitoneally with the same dose of antigen fully emulsified with Freund's incomplete adjuvant at an interval of 2 weeks. immunity. Blood was collected from the tail vein 15 days ...

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Abstract

The invention discloses a human Streptococcus pneumoniae surface protein monoclonal antibody and an antigen capture ELISA kit. The human Streptococcus pneumoniae surface protein monoclonal antibody is secreted and produced by a hybridoma cell line with the preservation number CCTCC No: C2017211. The human Streptococcus pneumoniae surface protein monoclonal antibody can be used to detect the human Streptococcus pneumoniae. The invention also discloses a human Streptococcus pneumoniae surface protein capture ELISA kit based on the monoclonal antibody.

Description

technical field [0001] The invention belongs to the field of immunology, and relates to a human Streptococcus pneumoniae surface protein monoclonal antibody, a hybridoma cell line and an antigen capture ELISA kit. Background technique [0002] Streptococcus pneumoniae was first isolated from patient sputum in France and the United States by Louis Pasteur and G.M.Sternberg in 1881, respectively. It is a Gram-positive bacterium with spearhead-like diplococci arranged in pairs or short chains. There are polysaccharide capsules outside the virulent strains. 5% to 10% of normal people carry this bacterium in the upper respiratory tract. Virus strains are important pathogenic bacteria that cause human diseases. In pyogenes, the pathogenicity of Streptococcus pneumoniae is second only to Staphylococcus aureus. Streptococcus pneumoniae mainly causes lobar pneumonia, bronchitis, otitis media, meningitis, pleurisy, endocarditis, sepsis and other diseases. Acute lung infection caus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/12C07K14/315C12N15/31C12N15/70G01N33/569G01N33/543
CPCC07K14/3156C07K16/1275C07K2317/35C12N15/70G01N33/54306G01N33/56944G01N2469/10
Inventor 胡征
Owner 湖北云璐生物工程有限公司
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