Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The method according to the present invention for preparing peptide fragments comprises bringing an antibody, which includes a Fc domain immobilized in pores of a porous body, into contact with a protease immobilized on surface of microparticles. Thus, the Fab domain of the antibody is site-specifically cleaved by the protease and a sample containing Fab domain-derived peptide fragments at a high concentration can be obtained. The protease to be used in the present invention is an animal-derived trypsin contaminated with chymotrypsin wherein the chymotrypsin is inactivated and the trypsin is chemically modified at amino group in lysine residues.

Abstract: 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.

Abstract: The present invention provides a method for detecting 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; and (c) a step of detecting, by a liquid chromatography mass spectrometry (LC-MS), peptide fragments obtained by the selective protease digestion, wherein the selective protease digestion of step (b) is conducted at pH 8 to 9 in the presence of a chaotropic reagent and a reducing agent.

Abstract: 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.

Abstract: Certain embodiments are directed to methods for preparing or processing FFPE samples for subsequent analysis of antibodies or other proteins contained within the FFPE sample. In certain aspects, preparing or processing a FFPE sample can include, but is not limited to (i) deparaffinizing a portion of the FFPE specimen, (ii) decrosslinking the deparaffinized sample portion, (iii) homogenizing the decrosslinked portion, and (iv) fractionating the homogenized portion, wherein isolated antibodies or proteins having a higher order structure that can be bound by capture agents, e.g., immunoglobulin capture agents.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The present invention provides a technique that can simultaneously analyze a plurality of antibody drugs and can be validated. The present invention provides a method of bringing a porous body in which monoclonal antibodies to be measured are immobilized in pores into contact in a liquid with nanoparticles on which proteases are immobilized and performing selective proteolysis of monoclonal antibodies to detect peptide fragments each comprising unique amino acid sequence derived from the Fab region of the monoclonal antibodies via liquid chromatography-mass spectrometry (LC-MS), wherein peptide fragments of two or more types of monoclonal antibodies in the same biological sample are simultaneously quantified.

Abstract: 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.

Abstract: There are numerous proteins having different in vivo effects depending on variants. In order to gain a more accurate understanding of an in vivo effect of a protein, when the protein in a sample is quantified, it is necessary to measure an amount of each variant that can be present. Provided is a method for performing parallel quantification of variants of a protein having 2 or more variants using mass spectrometry. The method includes: protease-digesting a protein in a sample; detecting, using mass spectrometry, among peptides obtained by the protease digestion, peptides having amino acid sequences specific to the variants without separating the peptides; and determining amounts of the variants in the sample based on results of the detection.

Abstract: The present invention provides a method for quantifying a monoclonal antibody, the method including: a step of performing selective protease digestion of a monoclonal antibody as a measurement target by bringing a porous body in which the monoclonal antibody is immobilized in pores into contact with fine particles onto which a specific protease is immobilized; and a step of detecting a peptide fragment that is obtained from the digestion and contains an amino acid derived from a CDR2 region of a heavy chain or a light chain of the monoclonal antibody.

Abstract: 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.

Abstract: The present invention provides a highly active protease that can be used in sample preparation for mass spectrometry of a protein and has excellent stability against a change in an external environment. The present invention provides an immobilized protease characterized in that a crudely purified protease or a protease that has not been subjected to a self-digestion resistance treatment is immobilized on surfaces of nanoparticles, and provides a method for producing the immobilized protease. The immobilized protease of the present invention can maintain high activity without being subjected to a change in an external environment, and thus is effective, for example, in preparing a sample to be supplied for mass spectrometry of a protein in a clinical specimen.