Shimadzu Corporation

Keio University

Kyoto University

National Cancer Center

Takashi Shimada

Noriko Iwamoto

Taka-Aki Sato

Chikage Aoki

Atsuhiko Toyama

Megumi Takanashi

Atsushi Suzuki

Koichi Tanaka

Daisuke Aoki

Hiroyuki Nomura

Yutaka Aoki

Yukari Umino

Ikuko Nishimura

Tomoo Shimada

Noriko Fukao

Akinobu Hamada

Tetsuyoshi Sugita

Nobuyuki Susumu

A sample preparation kit related to the present invention provides a significantly versatile analytical technique that is not affected by the diversity of antibodies, difference in species, matrix and the like. For preparing a sample to be used for detection of a monoclonal antibody through high-performance liquid chromatography-mass spectrometry (LC-MS), the kit includes a porous body for immobilizing a monoclonal antibody to be detected; nanoparticles with an immobilized protease; a reaction vessel for selectively digesting the monoclonal antibody by bringing the porous body and nanoparticles into contact; a buffer to be introduced into the reaction vessel along with the nanoparticles and porous body so that a protease reaction is carried out; and a filtration membrane to remove the porous body and nanoparticles after the proteolysis so as to extract the reaction product and the buffer.

Kit for preparing sample for detecting monoclonal antibody

The present invention provides a detection method for a monoclonal antibody in a sample, comprising: (a) a step of capturing the monoclonal antibody in the sample and immobilizing the monoclonal antibody in pores of a porous body; (b) a step of bringing the porous body in which the monoclonal antibody is immobilized with nanoparticles on which protease is immobilized to conduct selective protease digestion of the monoclonal antibody; (c) a step of detecting, by a liquid chromatography mass spectrometry (LC-MS), peptide fragments obtained by the selective protease digestion; and (a') a step of conducting a reduction reaction under an acidic condition after the step (a). By the present invention, further applicability of the detection method for the monoclonal antibodies using mass spectrometry is expected.

Method for improving result monoclonal antibody detection

A method of proteolyzing a protein, including immobilizing a protein in at least one pore of a porous body, and contacting the protein immobilized in the pore and a protease immobilized on a solid surface such that the protease selectively accesses a site of the protein and proteolyzes the protein at the site.

Method for preparing peptide fragments, kit for preparing peptide fragments to be used therein, and analysis method

The present invention provides a method in which a porous body having a monoclonal antibody to be measured immobilized in pores thereof is brought into contact with nanoparticles having a protease immobilized thereonto in a liquid to perform selective protease digestion of the monoclonal antibody and a peptide fragment obtained by the digestion is detected by liquid chromatography mass spectrometry (LC-MS), wherein the monoclonal antibody is digested with the protease in the presence of an antibody specifically binding to the monoclonal antibody or a target molecule of the monoclonal antibody.

Method for quantifying monoclonal antibody having antigen or anti-antibody bonded thereto

A container set including: a filter unit; and a closing unit that is attachable to and detachable from the filter unit. The filter unit includes: a housing having a sample introduction opening provided at one end, and a liquid discharging opening provided at the second end; and a filter fixed to the housing. The housing has a first joining part, and the closing unit has a second joining part configured to be joinable with the first joining part. The first joining part and the second joining part are configured such that the liquid discharging opening is spatially closed in a state in which the two joining parts are joined together. A space between the closing unit and the filter is made into a positive pressure relative to a pressure in the sample holding space by pushing the filter unit and the closing unit such that the distance therebetween is shortened.

Container set and sample preparation method using same

The present invention provides a method for detecting a monoclonal antibody in a sample, the method comprising: (a) a step of capturing and immobilizing, in pores of a porous body, the monoclonal antibody in the sample; (b) a step of performing selective protease digestion of the monoclonal antibody for 30 min or longer by contacting the porous body having the monoclonal antibody immobilized thereon with nanoparticles having a protease immobilized thereon; and (c) a step of detecting a peptide fragment obtained by the selective protease digestion, using liquid chromatography mass spectrometry (LC-MS), wherein step (b) is carried out under stirring condition for 10 sec to 5 min in the initial reaction stage, and then under static condition. According to the present invention, the detection method of a monoclonal antibody using mass spectrometry is simplified and can be applicable to multisample analysis.

Simplified monoclonal antibody quantification method

A method is provided for more easily detecting and quantifying a protein by regioselectively digesting a variable region of an Fab domain of a monoclonal antibody while suppressing proteolysis of an Fc domain. In the method, a porous body in which a monoclonal antibody is immobilized in pores and nanoparticles on which a protease is immobilized are brought into contact with each other in a liquid to perform selective proteolysis of the monoclonal antibody, and a resulting peptide fragment is detected using liquid chromatography-mass spectrometry (LC-MS), and a peptide having an amino acid sequence that includes an amino acid derived from a CDR2 region of a heavy chain or a light chain of the monoclonal antibody is detected.

Method for detecting monoclonal antibody using mass spectrometry

A thiol probe for a protein, which promotes ionization in proteomic analysis using mass spectrometry and can be used even for a protein that has a high degree of hydrophobicity and quickly turns over, and is represented by the following formula (I): In formula (I), Rrepresents a linker group, and Rrepresents a substituted ammonium group or a substituted amino group. A mass spectrometry method for a protein, includes the steps of obtaining a modified protein by reacting the thiol probe with a protein to be subjected to mass spectrometry and subjecting the modified protein to mass spectrometry.

Hydrophilic thiol probe

It is provided a method for identification of the morbidity of epithelial ovarian cancer based on a tissue-type in view of molecular typing which is different from a conventional histopathology, and a marker for identification of a tissue-type of epithelial ovarian cancer. A method for identification of the morbidity of epithelial ovarian cancer based on a tissue-type, comprising: subjecting a sample originated from an individual of interest to a treatment for detecting at least one selected from the group consisting of biological molecules specifically showing an upregulation in expression in a specific tissue-type of epithelial ovarian cancer, and/or at least one selected from the group consisting of biological molecules specifically showing a downregulation in expression in a specific tissue-type of epithelial ovarian cancer, and identifying whether or not the significant detection of the protein is achieved, thereby identifying the tissue-type.

Marker for identification of tissue type of epithelial ovarian cancer, and method for determination of the occurrence of epithelial ovarian cancer based on tissue type by using the marker

It is intended to provide a method of preparing sample for matrix-assisted laser desorption/ionization mass spectrometry using a matrix capable of generating preferred crystals that cause effective ionization of a molecule to be measured. A method of preparing a sample for matrix-assisted laser desorption/ionization mass spectrometry comprising the steps of: preparing a solution of 2,5-dihydroxybenzoic acid of 40 mg/mL to saturated concentration as a matrix solution; and dispensing the matrix solution to a sample to be analyzed by using an inkjet mechanism, to crystallize the 2,5-dihydroxybenzoic acid.

Method of preparing sample for matrix-assisted laser desorption ionization mass spectrometry and matrix-assisted laser desorption ionization mass spectrometry

Growing community of inventors

Kyoto, Japan

Takashi Shimada