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Immunological detection method and kit for Mycoplasma pneumoniae

A Mycoplasma pneumoniae, immunoassay technology, applied in immunoassay, biological testing, immunoglobulin, etc., can solve the problems of secondary infection, unable to detect mutant strains of Mycoplasma pneumoniae, etc., and achieve high sensitivity effect

Active Publication Date: 2020-12-29
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 (National Institute of Genetics Database). From the amino acid sequence of the aforementioned P30 protein, the amino acid sequence shown in SEQ ID NO: 2 (AA74-274), which is the extracellular region except the membrane perforating domain, was specified, and the corresponding gene sequence was synthesized. The His-tag expression vector, pET302 / NT-His, was cleaved with restriction enzyme EcoRI and treated with alkaline phosphatase as a dephosphorylation treatment, mixed with the aforementioned gene sequence, and used DAN Ligation Kit Ver .2 (Takara Bio) for ligation reaction. The recombinant P30 plasmid incorporating the target gene was introduced into E.coli BL(DE3)pLysS (product of Novagen), a host for recombinant protein expression. The introduced bacteria were cultured in LB agar plate medium, and the obta...

Embodiment 2

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

[0083]Using the recombinant P30 protein obtained in Example 1 as an antigen for immunization, a monoclonal antibody against the recombinant P30 protein (hereinafter referred to as anti-P30 antibody) was prepared. The preparation of monoclonal antibodies is carried out according to conventional methods. 100 μg of recombinant P30 protein was mixed with an equivalent amount of Aduvant Complete Freund (product of Difco), and mice (BALB / c, 5 weeks old, Japan SLC) were immunized three times, and the spleen cells were used for cell fusion. For cell fusion, Sp2 / 0-Ag14 cells (Shulman et al., 1978), which are bone tumor cells of mice, were used. The following culture medium was used for cell culture: Dulbecco's Modified Eagle Medium (product of Gibco) added L-glutamine 0.3 mg / ml, penicillin G potassium 100 units / ml, streptomycin sulfate 100 μg / ml, Gander Gentacin (Gentacin) 40 μg / ml (hereinafter referred to...

Embodiment 3

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

[0085]The vegetatively propagated cells were intraperitoneally inoculated into mice (BALB / c, retire, Japanese SLC) pre-vaccinated with pristane (2,6,10,14-Tetramethylpentadecane (product of Sigma)), Collect ascites. The ascites fluid is supplied to the protein G column, and the monoclonal antibody is purified. The isotype of the prepared monoclonal antibody was identified using Mouse Monoloconal Antibody Isotyping Reagents (product of Sigma).

[0086]Finally, 4 cloned monoclonal antibody-producing cells against 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 the P30 protein of Mycoplasma pneumoniae and an immunological detection method and kit for Mycoplasma pneumoniae using the antibody.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 of urban pneumonia.[0003]Mycoplasma pneumonia is more common in infants, young children, and adolescents. The incubation period is 2 to 3 weeks. The discharge of pathogens to the airway mucosa is visible 2 to 8 days before the first symptoms are discovered, 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 fatigue, headache, and other cold-like symptoms. It is characterized by a high fever over 38°C and a strong dry c...

Claims

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Application Information

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Patent Type & Authority Patents(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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