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Immunological detection methods and kits for Mycoplasma pneumoniae

A technology for Mycoplasma pneumoniae and immunoassay, which is applied in immunoassay, immunoglobulin, chemical instruments and methods, etc., can solve the problems of unclear specificity and reactivity, and has not yet been determined, and achieves the effect of high sensitivity

Active Publication Date: 2021-02-12
TAUNS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, it has not been determined whether this antiserum can be used in the immunoassay of the present invention, nor is it clear whether it has high specificity and reactivity

Method used

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  • Immunological detection methods and kits for Mycoplasma pneumoniae
  • Immunological detection methods and kits for Mycoplasma pneumoniae
  • Immunological detection methods and kits 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)) , to 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, 6 clones of monoclonal antibody-producing cells against the P30 protein were obtained.

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Abstract

[Problem] An object of the present invention is to provide an antibody specific for Mycoplasma pneumoniae, which is the causative bacterium of mycoplasma pneumonia, which can be easily, rapidly and highly sensitively detected, and an immunological detection method and a kit including the antibody. [Solution] By producing an antibody that recognizes a specific epitope of the P30 protein of Mycoplasma pneumoniae and performing immunological detection using the antibody, the infection of Mycoplasma pneumoniae can be diagnosed more quickly and specifically than conventional methods. According to the present invention, it is possible to detect Mycoplasma pneumoniae and diagnose infection easily and rapidly in a hospital or the like without requiring special equipment and 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 occupy 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 the airway mucous membrane of the pathogen can be seen 2 to 8 days before the onset of symptoms, and reaches a peak level when the 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, str...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00
CPCC07K16/00C12N15/02C12N15/09G01N33/56933G01N2469/10C07K16/1253C12Q1/04G01N33/553G01N33/577G01N33/54387C07K14/30C12Q1/6888G01N2333/30G01N2469/00
Inventor 斋藤宪司
Owner TAUNS CO LTD
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