Immunological detection method and kit for mycoplasma pneumoniae

A technology of Mycoplasma pneumoniae and detection method, applied in immunoassay, biological test, immunoglobulin and other directions, can solve the problems of secondary infection, unable to detect Mycoplasma pneumoniae variant strains, etc., and achieve the effect of high sensitivity

Active Publication Date: 2017-11-28
TAUNS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be considered that the P30 protein is a protein specific to Mycoplasma pneumoniae, but using an antibody that recognizes the C-terminal region of the P30 protein, it is impossible to detect mutant strains of Mycoplasma pneumoniae, and there is a concern that Mycoplasma pneumoniae infection may be overlooked and appropriate treatment cannot be implemented, which in turn may cause Worries about secondary infection

Method used

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  • Immunological detection method and kit for mycoplasma pneumoniae
  • Immunological detection method and kit for mycoplasma pneumoniae
  • Immunological detection method and kit for mycoplasma pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] (Example 1: Expression and purification of recombinant P30 protein)

[0081] The amino acid sequence of P30 protein of Mycoplasma pneumoniae M129 strain was obtained from DDBJ (Database of National Institute of Genetics). From the amino acid sequence of the aforementioned P30 protein, the amino acid sequence (AA74-274) shown in SEQ ID NO: 2, which is the extracellular region except the membrane-penetrating domain, was specified, and the corresponding gene sequence was synthesized. The His-tag expression carrier, pET302 / NT-His, was cleaved with the restriction endonuclease EcoRI, treated with alkaline phosphatase as a dephosphorylation treatment, and mixed with the aforementioned gene sequence, using DAN Ligation Kit Ver .2 (Takara Bio) for the ligation reaction. The recombinant P30 plasmid incorporating the gene of interest was introduced into E. coli BL(DE3)pLysS (manufactured by Novagen), a host for recombinant protein expression. The introduced bacteria were cultur...

Embodiment 2

[0082] (Example 2: Preparation of monoclonal antibody against recombinant P30 protein)

[0083] The recombinant P30 protein obtained in Example 1 was used as an antigen for immunization to prepare a monoclonal antibody against the recombinant P30 protein (hereinafter referred to as anti-P30 antibody). Production of monoclonal antibodies is carried out according to conventional methods. Mice (BALB / c, 5-week-old, Japanese SLC) were immunized three times by mixing 100 μg of recombinant P30 protein with an equal amount of Aduvant Complete Freund (manufactured by Difco), and the spleen cells were used for cell fusion. Sp2 / 0-Ag14 cells (Shulman et al., 1978), which are mouse myeloma cells, were used for cell fusion. The following culture solution was used for the culture of the cells: 0.3 mg / ml of L-glutamine, 100 units / ml of potassium penicillin G, 100 μg / ml of streptomycin sulfate, gander Gentacin (Gentacin) 40 µg / ml (hereinafter referred to as DMEM) was added with fetal bovine ...

Embodiment 3

[0084] (Example 3: Preparation of monoclonal antibody)

[0085] The clonally propagated cells were inoculated intraperitoneally into mice (BALB / c, retired, Japan SLC) previously inoculated with pristane (2,6,10,14-Tetramethylpentadecane (manufactured by Sigma)), Collect ascites. This ascitic fluid was supplied to a protein G column, and the monoclonal antibody was purified. The isotype of the prepared monoclonal antibody was identified using Mouse Monoloconal Antibody Isotyping Reagents (manufactured by Sigma).

[0086] Finally, 4 clones of monoclonal antibody-producing cells against the P30 protein were obtained.

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Abstract

The present invention aims at providing a specific antibody that can simply and rapidly detect Mycoplasma pneumoniae which is a causative bacterium of mycoplasma pneumonia, with high sensitivity, and also an immunological detection method and a kit containing the same antibody. The present invention makes it possible to diagnose infection with Mycoplasma pneumoniae more rapidly and specifically than the conventional method, by producing an antibody recognizing a specific epitope of P30 protein of Mycoplasma pneumoniae and performing an immunological detection using the antibody. Also, the present invention enables easy and rapid detection of Mycoplasma pneumoniae and diagnosis of infection with the same at a hospital or the like without need of specialized instruments or skilled techniques.

Description

technical field [0001] The present invention relates to an antibody against P30 protein of Mycoplasma pneumoniae and an immunological detection method and kit for Mycoplasma pneumoniae using the same. Background technique [0002] Mycoplasma pneumonia is atypical pneumonia caused by Mycoplasma pneumoniae. Mycoplasma pneumonia and chlamydia pneumonia together account for 30-40% of atypical pneumonia, and account for a high proportion in urban pneumonia. [0003] Mycoplasma pneumonia is more common in infants, young children, and adolescence. The incubation period is 2 to 3 weeks. The discharge of pathogens to the airway mucous membrane can be seen 2 to 8 days before the onset of symptoms, and reaches a peak level when clinical symptoms are discovered. After about 1 week, the discharge continues for more than 4 to 6 weeks. . The main clinical symptoms are fever, general malaise, headache, and other cold-like symptoms. It is characterized by high fever exceeding 38°C, stron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569C07K14/30G01N33/53G01N33/543G01N33/553G01N33/577C12N15/09C12Q1/04
CPCC07K14/30C12N15/09G01N33/56933G01N2469/10C07K16/1253C12Q1/04G01N33/553G01N33/577G01N33/54387C12Q1/6888G01N2333/30G01N2469/00
Inventor 斋藤宪司
Owner TAUNS CO LTD
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