Method for producing an isolated monoclonal antibody specifically binding to a glycolipid antigen of mycoplasma pneumoniae
Inventors
Matsuda, Kazuhiro • Shingu, Yuko
Assignees
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Publication Number
US-10870669-B2
Publication Date
2020-12-22
Expiration Date
Abstract
The present invention provides a novel glyceroglycolipid produced by Mycoplasma pneumoniae. The glyceroglycolipid can be used as a diagnostic marker for a disease caused by Mycoplasma pneumoniae.
Core Innovation
The document describes isolation and structural determination of a novel Mycoplasma pneumoniae-derived glyceroglycolipid antigen. The antigen includes Galβ-6Galβ-3DAG and Glcβ-6Galβ-3DAG, wherein the diacylglycerol portion is characterized by specified sugar linkages and R3 acyl groups.
A specific marker for diagnosing Mycoplasma pneumoniae-related diseases is described using the disclosed glyceroglycolipid antigen. The document links the antigen to diagnostic contexts including mycoplasma pneumonia, asthma, and nervous diseases, and frames the detection around antigen/antibody-based analysis.
Diagnostic embodiments described in the document include compositions and diagnostic agents/kits configured to detect autoantibodies by immunological assays. The document describes detecting and measuring anti-glyceroglycolipid antibodies using formats including enzyme-linked immunosorbent assay, immunostaining, and immunochromatography, with experimental support including patient anti-glycolipid antibody detection in Guillain–Barré syndrome.
Claims Coverage
The document provides one independent claim covering a method for producing an isolated monoclonal antibody specifically binding to a Mycoplasma pneumoniae glycolipid antigen. The independent claim is supported by dependent claims that narrow the antibody-producing cell source, constrain R3 substituent choices, and require chemical synthesis of the glycolipid antigen; overall, the claim coverage centers on antigen-defined monoclonal antibody generation and verification of specific binding by ELISA.
Isolated monoclonal antibody production for Mycoplasma pneumoniae glycolipid antigen
A method for producing an isolated monoclonal antibody specifically binding to a Mycoplasma pneumoniae glycolipid antigen by immunizing a mammal with a glycolipid antigen, obtaining an antibody-producing cell, fusing a myeloma cell with the antibody-producing cell to form a hybridoma cell line, screening the hybridoma cell line for monoclonal antibody specific binding that is verifiable by an enzyme-linked immunosorbent assay using the glycolipid antigen, culturing the screened hybridoma cell line, and isolating the monoclonal antibody.
Defined isolated glycolipid antigen scaffold with R1/R2/R3 and n constraints
The glycolipid antigen used in immunization is an isolated compound represented by one of two general formulas, with R1 and R2 and R3 constrained as specified and n restricted to 12, 14, 16 or 18, including variants where R1 is OH and R2 is H and where R1 is H and R2 is OH, together defining the antigen structure for monoclonal binding.
Spleen cell as the antibody-producing cell
The method further specifies that the antibody-producing cell obtained from the mammal is a spleen cell.
Specific R3 acyl substituent pattern
At least one R3 is —(CH2)14CH3 and the other R3 is either —(CH2)14CH3 or —(CH2)16CH3.
Chemical synthesis of the glycolipid antigen
The method is characterized by chemically synthesizing the glycolipid antigen.
Claim coverage focuses on generating an isolated monoclonal antibody that specifically binds to a structurally defined Mycoplasma pneumoniae glycolipid antigen, with specific ELISA-based verification of binding. Dependent features further narrow the antibody-producing cell to spleen cells, constrain R3 substituent choices, and require chemical synthesis of the glycolipid antigen.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Diagnosing Mycoplasma pneumoniae-related diseases including mycoplasma pneumonia, asthma, and nervous diseases using a specific marker based on the disclosed glyceroglycolipid antigen and detection of anti-glyceroglycolipid antibodies.
Detecting patient anti-glycolipid antibodies in Guillain–Barré syndrome using immunostaining, including TLC-immunostaining, and immunoassay formats including ELISA.
Use in diagnostic agents/kits and diagnostic methods for antigen/antibody-based detection, including ELISA, immunostaining, and immunochromatography test strip formats for measuring anti-glyceroglycolipid antibodies.
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