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

A technology of Mycoplasma pneumoniae and monoclonal antibody, applied in the field of immunology, can solve the problems of difficulty in rapid identification, long time, and incompatibility.

Active Publication Date: 2019-12-06
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 Mycoplasma pneumoniae 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; , is a fast, sensitive, and specific technique, but at present, this technique 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Preparation of recombinant human mycoplasma pneumoniae surface protein P1

[0036] 1) Cloning and expression of human Mycoplasma pneumoniae P1 gene

[0037]Bioinformatic analysis of human Mycoplasma pneumoniae surface protein P1 (accession number in NCBI protein database is AAK92040.1) gene, combined with GC content, codon bias, mRNA secondary structure, RNA instability motif, mRNA freedom Considering performance stability, 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 EcoRI at the 3' end (the whole sequence synthesis was handed over to Kim Completed by Sirui Biotechnology Co., Ltd., the artificially synthesized gene fragment was connected to the vector pUC57 at the time of delivery), denoted as P1'. The full sequence of the gene and the encoded amino acid sequence are shown i...

Embodiment 2

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

[0042] 1) Immunization of New Zealand purebred rabbits

[0043] The recombinant P1 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 P1 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] The recombinant P1 protein was used as ...

Embodiment 3

[0053] The preparation of embodiment 3 human mycoplasma pneumoniae surface protein monoclonal antibody Mp-7#

[0054] 1) Immunization of animals

[0055] Using the recombinant human mycoplasma pneumoniae surface protein P1 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 after the...

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PUM

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Abstract

The invention discloses a human mycoplasma pneumoniae surface protein monoclonal antibody and an antigen capture ELISA kit. The human mycoplasma pneumoniae surface protein monoclonal antibody is secreted by a hybridoma cell line with a preservation number being CCTCC NO: C2017217. The human mycoplasma pneumoniae surface protein monoclonal antibody can be used for detecting human mycoplasma pneumoniae. The invention further discloses the human mycoplasma 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 mycoplasma pneumoniae surface protein monoclonal antibody, a hybridoma cell line and an antigen capture ELISA kit. Background technique [0002] Mycoplasma pneumoniae (M.Pneumonia, Mp) is the pathogen of human mycoplasma pneumonia. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchial pneumonia, which is called primary atypical pneumonia. It is mainly transmitted by droplets, with an incubation period of 2 to 3 weeks, and the highest incidence rate is among adolescents. It can occur all year round, but mostly in autumn and winter. The clinical manifestations and chest X-ray examination of mycoplasma pneumonia are not characteristic, and the diagnosis cannot be made based on clinical manifestations and chest X-ray examination alone. Definitive diagnosis requires detection of the pathogen. [0003] At present, the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12C07K14/30C12N15/31C12N15/70G01N33/569G01N33/543G01N33/577
CPCC07K14/30C07K16/1253C07K2317/35C12N15/70G01N33/54306G01N33/56933G01N33/577G01N2469/10
Inventor 胡征
Owner 湖北云璐生物工程有限公司
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