Apparatus for auto-pretreating sugar chain

摘要

To provide an autoanalyzer for analyzing a sugar chain contained in a biological sample, in particular, serum. Namely, it is intended to provide a method of analyzing a sugar chain in a sample, which comprises the following steps: A) the sugar chain-releasing step of releasing the sugar chain in the sample; B) the detection sample-preparing step of preparing the released sugar chain for detection; and, in the case of conducting mass spectrometry using a plate, C) the step of forming a plate for the mass spectrometry having the captured sugar chain dotted thereon which comprises the step of providing the tagged sugar chain sample solution obtained in the step B) on a collection plate; and, if required, the step of conducting an operation in a solid phase support-enclosed plate to form the plate for mass spectrometry; and D) the step of analyzing the sugar chain to be assayed.

主权项

1. A method for analyzing a sugar chain in a sample, the method comprising the following steps:
A) a sugar chain releasing step of releasing a sugar chain in a sample, the step comprising the following steps:
A-1) a step of providing the sample on a plate for reaction;A-2) a step of adding a solubilizing agent to the sample to thereby place the sample under a reaction condition;A-3) a step of adding a reducing agent to the sample to thereby place the sample under a reaction condition;A-4) a step of adding an —SH protecting agent to the sample to thereby place the sample under a reaction condition;A-5) a step of adding a proteolytic enzyme to the sample to thereby place the sample under a reaction condition;A-6) a step of deactivating the proteolytic enzyme; andA-7) a step of adding a sugar chain releasing enzyme to the sample to thereby release the sugar chain; B) a detection sample preparing step of preparing the released sugar chain for use in detection, the step comprising the following steps:
B-1) a step of contacting the sample prepared in the step (A) with a sugar chain-capturing bead to thereby place the sample under the conditions allowing the released sugar chain in the sample to bind to the bead, and thus producing a captured sugar chain sample;B-2) a step of adding a protein denaturing agent to the captured sugar chain sample to thereby place the captured sugar chain sample under a reaction condition;B-3) a step of washing the captured sugar chain sample, and then discarding the residual washing liquid by suction;B-4) a step of adding a salt releasing agent for the sugar chain capturing agent on beads to the captured sugar chain sample, and then discarding the salt releasing agent by suction;B-5) a step of adding a protective agent to the captured sugar chain sample to thereby place the captured sugar chain under a reaction condition;B-6) a step of adding an acid to the captured sugar chain sample, and discarding the acid by suction;B-7) a step of adding an organic reaction solvent to the captured sugar chain sample;B-8) a step of removing the solvent and the moisture in the bead;B-9) a step of adding a methyl esterifying agent to the captured sugar chain sample to thereby place the captured sugar chain sample under a reaction condition, and alkylating the carboxylic acid of sialic acid;B-10) a step of adding the organic reaction solvent to the captured sugar chain sample, and discarding the organic reaction solvent by suction;B-11) a step of washing the captured sugar chain sample, and subsequently discarding the residual washing liquid by suction;B-12) a step of releasing a sugar chain sample from the captured sugar chain sample, wherein when an analysis requiring tagging is conducted, the sugar chain in the captured sugar chain sample is tagged with a labeling reagent and is released from the bead; andB-13) a step of dissolving the released sugar chain sample to produce a sugar chain sample solution; C) when performing mass spectrometry using a plate, a step of producing a plate for mass spectrometry having the captured sugar chain dotted thereon, the step comprising:
C-1) a step of disposing the tagged sugar chain sample solution obtained in the step (B) on a plate for recovery;C-2) a step of disposing the tagged sugar chain sample solution from the plate for recovery by adding an organic solvent to the plate and mixing so as to obtain a concentration at which the sugar chain adsorbs to a solid phase, wherein the concentration at which the sugar chain adsorbs to the solid phase is 80 to 90% in the organic solvent; and wherein the step optionally comprises the steps C-3) to C-6):C-3) a step of providing a solid phase carrier-enclosed plate;C-4) a step of activating the solid phase carrier-enclosed plate according to the phase of the solid phase carrier-enclosed plate, and washing the solid phase carrier-enclosed plate;C-5) a step of adding the tagged sugar chain sample solution to the solid phase carrier-enclosed plate, and conditioning the tagged sugar chain sample solution to a solvent having a polarity appropriate for the phase of the solid phase carrier-enclosed plate;C-6) a step of recovering the tagged sugar chain sample solution by suction from the solid phase carrier-enclosed plate to a second plate for recovery; and when subjecting the tagged sugar chain sample solution to MALDI-TOF MS, comprising the following step (C-7):
C-7) a step of mixing the tagged sugar chain sample solution with a matrix for mass spectrometry, and dotting the mixture on a plate for MALDI-TOF MS determination; and D) a step of conducting an analysis of the sugar chain to be determined.