Patent Description:
An automatic analyzer automates a part of a procedure of an inspection to contribute to a rapid and efficient clinical inspection task. Each of typical automatic analyzers includes a dispensing mechanism for dispensing a predetermined amount of a solution of a sample, a reagent, or the like into a reaction container, and a stirring mechanism that stirs the sample, the reagent, or the like in the reaction container. Among them, an immune system holds a substance to be measured with a carrier obtained by binding a functional group to a surface of a magnetic bead and uses electrochemiluminescence immunoassay (ECLIA), which is among immunoassay methods, to allow a high-accuracy, high-sensitivity, and wide-range inspection.

A high-performance liquid chromatograph mass spectrometer (HPLC/MS) is a device including a combination of a liquid chromatograph and a mass spectrometer.

By combining separation of substances to be measured based on chemical structures and physical properties thereof using the high-performance liquid chromatograph (HPLC) with separation of the substances to be measured based on masses thereof using a mass spectrometer (MS), it is possible to determine the quality/quantity of each of components in a sample. Due to this feature, even when, e.g., a large number of similar substances are mixed with each other as a result of metabolism inside a body such as that of a medical drug in a biological sample, it is possible to determine the quality/quantity of each of substances to be measured, and an application of the HPLC/MS to a clinical inspection field is expected.

In an inspection center, a university hospital, or the like, the HPLC/MS is used to inspect an immunosuppressing agent, an anticancer agent, or a newborn metabolic disorder and perform an inspection such as TDM (Therapeutic Drug Monitoring).

A pretreatment is performed using a test kit or a manual method to supply a sample to the HPLC/MS. Verification (variation) of each of inspection methods is performed under the responsibility of each of inspection institutes to ensure a result of an inspection.

Since a pretreatment step is complicated, depending on a degree of proficiency of a laboratory technician, a result of an inspection varies. In addition, in a pretreatment or HPLC/MS measurement, a human error may cause a defect in a result of an inspection.

Accordingly, it has been requested to expand an automatic analyzer capable of full-automatically performing a batch treatment step including a pretreatment and the HPLC/MS into the clinical inspection field.

As one of such automatic analyzers, an automatic analyzer capable of full-automatically performing the batch step including the pretreatment and the HPLC/MS is disclosed in Patent Literature <NUM>.

A pretreatment step of a typical immune system uses a method which sends, into a solution sending flow path, a solution in a state where a substance to be measured is bonded to a functional group of each of magnetic beads, magnetically collects the magnetic beads in the flow path, and performs measurement using electrochemiluminescence. The ECLIA, which is among methods used in the immune system, is a method in which, after a bead having an antibody bonded thereto is used to react the antibody with an antigen, an antibody labeled with a ruthenium pyridine complex is secondarily reacted with the antigen, and an intensity of emission from the ruthenium pyridine complex is measured using an electrochemical reaction and which uses one type of magnetic bead to which an antibody for one substance to be measured is bonded.

Meanwhile, methods using a plurality of beads include a fluorescent imunnostaining method, DNA microarray analysis, in-situ immuno-hybridization, and the like. The fluorescent immunostaining method is a method in which magnetic beads each having a primary antibody conjugated with a fluorochrome and bonded to a surface thereof are bonded to a substance to be measured, and a labeled secondary antibody is bound to the primary antibody to enhance a fluorescent signal.

The DNA microarray analysis is a method which binds minute beads to a cDNA or rDNA as a plurality of fluorescently labeled nucleic acid templates, reacts the cDNA or rDNA with a target DNA, and observes development of colors.

The in-situ immuno-hybridization is a method which binds a nucleic acid template to a DNA or RNA without extracting the DNA or RNA, and observes development of colors.

As a color development method, organic dyeing such as Cy3 or Cy5 or a solution containing a dye such as used for an inorganic label such as a quantum dot is used. Even in a method using one type of magnetic bead, when a polyclonal antibody, not a highly specific monoclonal antibody, is conjugated with a surface of the magnetic bead, it is possible to allow the one type of magnetic bead to bind to a plurality of substances to be measured.

Patent Literature <NUM> discloses the invention in which the method described above is inclusively implemented in a microreactor.

As a typical pretreatment for the HPLC/MS, it has been practiced to bind a substance to be measured to a filler by using solid phase extraction, and then perform the extraction in multiple stages by using a plurality of types of eluates. For example, in the case of the solid phase extraction in which a C18 filler is sealed, there is a method which separates a substance to be measured from foreign substances by using a plurality of eluates having different organic solvent concentrations.

Related art is disclosed in <CIT> from which the pre-characterising part of claim <NUM> starts out, and in <CIT>.

In a pretreatment method of an automatic analyzer using magnetic beads, it is desired to improve a throughput, reduce a quantity of samples, and improve inspection accuracy.

As a pretreatment method of the automatic analyzer disclosed in Patent Literature <NUM>, a pretreatment method using liquid/liquid extraction and the solid phase extraction is used. However, there is no description of a pretreatment method using magnetic beads, and it is difficult to achieve improved inspection accuracy or the like for a pretreatment using magnetic beads.

In a pretreatment method using magnetic beads in a pretreatment step of an automatic analyzer using the HPLC/MS as a detector, such as used in a magnetic bead immune system, a solution in which only a substance to be measured has been eluted by binding the substance to be measured to a functional group of each of the magnetic beads, performing cleaning, and then adding an eluting solution thereto in a state where the magnetic beads are magnetically collected is supplied as a sample to the HPLC/MS.

In addition, to improve quantity determination accuracy in the MS, it is also required to add a stable isotope substance of the substance to be measured.

Since a pretreatment method using magnetic beads includes the number of steps larger than that of steps included in a typical immune system, a treatment time period is longer than in the typical immune system, and it is required to improve a throughput.

Various methods using beads conjugated with antibodies and a fluorescent label, which are disclosed in Patent Literature <NUM>, measure development of colors from a fluorescently labeled body. Therefore, there is no need to eventually separate the beads from the substance to be measured.

Meanwhile, the present automatic analyzer targeted by the present invention and capable of full-automatically performing the batch step including the pretreatment and the HPLC/MS requires an elution step of separating the beads from the substance to be measured in the pretreatment step to perform separation/detection with the HPLC/MS.

Moreover, in Patent Document <NUM>, there is no disclosure of a method and timing for the elution step required to improve a throughput without degrading inspection accuracy when the plurality of magnetic beads are used.

Note that, in the case of the solid phase extraction, it is unnecessary to use magnetic beads for the filler, and there is no step of performing stirring and magnetically collecting magnetic beads, unlike in the pretreatment step targeted by the present invention.

An object of the present invention is to perform a pretreatment method of an automatic analyzer that binds a substance to be measured to a magnetic bead to perform treatment and full-automatically performs a batch step including pretreatment and a liquid chromatograph mass spectrometer, in which a plurality of the magnetic beads to which a plurality of the substances to be measured can be bound are used to allow the plurality of substances to be measured to be pretreated by a sequential treatment.

To attain the object described above, the present invention is configured as follows.

The invention provides a pretreatment method as set forth in the appended claims.

According to the present invention, it is possible to perform a pretreatment method of an automatic analyzer that binds a substance to be measured to a magnetic bead to perform treatment and full-automatically performs a batch step including pretreatment and a liquid chromatograph mass spectrometer, in which a plurality of the magnetic beads to which a plurality of the substances to be measured can be bound are used to allow the plurality of substances to be measured to be pretreated by a sequential treatment.

Referring to the drawings, a description will be given of embodiments of the present invention. Note that the embodiments described below mainly target an automatic analyzer, but the present invention is applicable to analyzers in general. The present invention is also applicable to, e.g., a gene analyzer or a bacteria tester.

<FIG> is a schematic view of an automatic analyzer that performs a pretreatment method according to First Embodiment of the present invention.

In <FIG>, an automatic analyzer <NUM> includes an analysis section <NUM> for performing an analyzing operation, a control section <NUM> for controlling an operation of the entire analyzer, an input section <NUM> for a user to input information to the analyzer, and a display section <NUM> for displaying information to the user. Note that the input section <NUM> and the display section <NUM> may also be the same section and, as an example thereof, a touch-panel monitor can be used.

The analysis section <NUM> of the automatic analyzer <NUM> includes a pretreatment section <NUM>, a HPLC section <NUM>, and a detector <NUM>.

The analysis section <NUM> includes a sample container transport mechanism <NUM> for transporting sample containers <NUM> containing samples to a sample splitting position, a sample dispensing mechanism <NUM> for ejecting each of the samples, a reaction container mounting rack <NUM> on which reaction containers <NUM> are mounted, a reaction container transport mechanism <NUM> for transporting the reaction containers <NUM>, and a reaction container disk <NUM> capable of holding the plurality of reaction containers <NUM>.

The analysis section <NUM> also includes a reagent disk <NUM> for holding measuring reagent containers <NUM> containing measuring reagents, a reagent dispensing mechanism <NUM> that ejects the measuring reagents into the reaction containers <NUM>, a stirring mechanism <NUM> that stirs a liquid contained in each of the reaction containers <NUM> in non-contact relation, a first magnetism collecting mechanism <NUM> that magnetically collects magnetic beads, a first transport mechanism <NUM> that transports the reaction containers <NUM> between the reaction container disk <NUM>, the stirring mechanism <NUM>, and the first magnetism collecting mechanism <NUM>, and an effluent dispensing mechanism <NUM> that dispenses an effluent from the first magnetism collecting mechanism <NUM>.

The analysis section <NUM> further includes a second magnetism collecting mechanism <NUM> that magnetically collects the magnetic beads, a second transport mechanism <NUM> that transports the reaction containers <NUM> between the reaction container disk <NUM> and the second magnetism collecting mechanism <NUM>, and an eluate dispensing mechanism <NUM> that introduces an eluate into the HPLC section <NUM>.

<FIG> is a schematic configuration view of the HPLC section <NUM>.

In <FIG>, the HPLC section <NUM> includes a pump <NUM> that sends a solution of the sample (pretreated eluate), a pressure sensor <NUM> that measures a system pressure, and a <NUM>-direction <NUM>-position injection valve <NUM> including a sample loop that measures the sample (pretreated eluate).

Meanwhile, the HPLC section <NUM> includes a column <NUM>, a column oven <NUM> that adjusts a temperature of the column <NUM>, a sample vial <NUM> that holds the sample (pretreated eluate), and a syringe <NUM> that introduces the sample from the sample vial <NUM> into the sample loop.

In the case of changing a composition of the sample to allow a substance to be measured to bind to the column <NUM>, the HPLC section <NUM> also includes a mechanism that adds a diluent to the sample vial <NUM>, though not illustrated.

On the detector <NUM> illustrated in <FIG>, a mass spectrometer is mounted. The mass spectrometer includes an ionization section that applies a high temperature and a high voltage to a solution containing the substance to be measured that has been separated by the HPLC section <NUM> and a triple-quadrupole mass spectrometer that separates the substance to be measured from foreign substances depending on a mass number, though illustration thereof is omitted. The triple-quadrupole mass spectrometer is used to measure a component to be measured in a SRM (Selected Reaction Monitoring) mode.

Referring to <FIG>, a description will be given below of an outline of an analysis step by the automatic analyzer.

Prior to analysis, the automatic analyzer <NUM> uses the reaction container transport mechanism <NUM> to transport the reaction containers <NUM> from the reaction container mounting rack <NUM> and place the reaction containers <NUM> on the reaction container disk <NUM>.

The sample dispensing mechanism <NUM> suctions the sample from each of the sample container <NUM> transported by the sample container transport mechanism <NUM> and ejects the sample into each of the reaction containers <NUM> on the reaction container disk <NUM>. As the sample dispensing mechanism <NUM>, a dispensing mechanism involving no replacement of a chip is used in First Embodiment, but it may also be possible to use a disposable dispensing mechanism which uses a dispensing chip attachment/detachment section (not shown) to attach a dispensing chip to a tip portion prior to the suction of the sample. In the case of the disposable dispensing mechanism, when dispensing of the sample from one of the sample containers <NUM> is ended, the sample dispensing mechanism <NUM> discards the dispensing chip into the dispensing chip attachment/detachment section. Each of the reaction containers <NUM> on the reaction container disk <NUM>, into which the sample has been dispensed, is transported by the first transport mechanism <NUM> to the stirring mechanism <NUM>. After stirring of the sample in the reaction container <NUM>, the reaction container <NUM> is returned by the first transport mechanism <NUM> to the reaction container disk <NUM>.

The reagent dispensing mechanism <NUM> suctions the measuring reagent from each of the measuring reagent containers <NUM> on the reagent disk <NUM>, and ejects the measuring reagent into the reaction container <NUM>. An operating portion of the reagent dispensing mechanism <NUM> can access each of the reaction container disk <NUM> and the stirring mechanism <NUM> and, after the stirring mechanism <NUM> begins to stir a liquid contained in the reaction container <NUM>, the reagent dispensing mechanism <NUM> begins to eject the reagent in a state where the reaction container <NUM> is held by the stirring mechanism <NUM>.

For example, the stirring mechanism <NUM> stirs the liquid in the reaction container <NUM> before the reagent dispensing mechanism <NUM> finishes ejecting a predetermined amount of the reagent into the reaction container <NUM>. This reduces a possibility of generation of an insoluble material compared to that in a case where the stirring of the solution is started after the reagent dispensing mechanism <NUM> finishes ejecting a large amount of the reagent.

Note that the predetermined amount of the reagent means the reagent in an amount corresponding to that of a portion of the reagent suctioned by the reagent dispensing mechanism <NUM> from the measuring agent container <NUM>. The reagent dispensing mechanism <NUM> may also operate simultaneously with the stirring mechanism <NUM> or operate while the stirring mechanism <NUM> is stopped.

A solution mixture of the sample and the reagent contained in the reaction container <NUM> is stirred by the stirring mechanism <NUM> to form a flow. The ejection and stirring of the reagent may also be such that, after the reagent dispensing mechanism <NUM> ejects the reagent into the reaction container <NUM> on the reaction container disk <NUM>, the reaction container <NUM> is transported by the first transport mechanism <NUM> to the stirring mechanism <NUM>, and the solution mixture is stirred.

The reaction container <NUM> after the ejection of the reagent by the reagent dispensing mechanism <NUM> and the stirring by the stirring mechanism <NUM> ended is placed again on the reaction container disk <NUM> by the first transport mechanism <NUM>. The reaction container disk <NUM> functions as, e.g., an incubator to incubate the held reaction container <NUM> during a given period of time.

The magnetic beads are stored (contained) in the measuring reagent container <NUM> and ejected using the reagent dispensing mechanism <NUM> into the reaction container <NUM>. In the reaction container <NUM>, the substance to be measured in the sample binds to the magnetic beads. Then, the reaction container <NUM> is transported by the first transport mechanism <NUM> to the first magnetism collecting mechanism <NUM>, and the magnetic beads are adsorbed to a side surface of the reaction chamber <NUM>.

The effluent dispensing mechanism <NUM> has two shippers mounted thereon and has a mechanism capable of suctioning/ejecting an effluent and ejecting a cleaning liquid. The effluent dispensing mechanism <NUM> moves to the first magnetism collecting mechanism <NUM> to suction the solution, and the magnetic beads are extracted from the sample. Then, the effluent dispensing mechanism <NUM> moves to an effluent ejection position to eject the effluent. The effluent dispensing mechanism <NUM> moves to the first magnetism collecting mechanism <NUM> to eject the cleaning liquid into each of the reaction containers <NUM>. The reaction container <NUM> into which the cleaning liquid was ejected is transported by the first transport mechanism <NUM> to the stirring mechanism <NUM>, and stirring is performed.

Then, the reaction container <NUM> is transported by the first transport mechanism <NUM> to the first magnetism collecting mechanism <NUM>, and the suction/ejection of the cleaning liquid is performed by the effluent dispensing mechanism <NUM>. When the cleaning is to be performed a plurality of times, the suction/ejection of the effluent and the ejection of the cleaning liquid is performed a plurality of times. The stirring mechanism <NUM> and the first magnetism collecting mechanism <NUM> are provided at each of two places in each of the mechanisms to allow cleaning treatment to be simultaneously performed in parallel on the plurality of reaction containers <NUM>.

After being cleaned, each of the reaction containers <NUM> is returned by the first transport mechanism <NUM> to the reaction container disk <NUM>. In the reaction container <NUM>, the cleaned magnetic beads are held. An eluate stored in the measuring reagent container <NUM> is ejected by the reagent dispensing mechanism <NUM> into the reaction container <NUM>, and the substance to be measured is separated from the magnetic beads. Then, the reaction container <NUM> is transferred by the first transport mechanism <NUM> to the stirring mechanism <NUM> and returned, after stirring was performed, into the reaction container disk <NUM> to be incubated. The eluate is compliant with a type of the magnetic beads.

By the second transport mechanism <NUM>, the incubated reaction container <NUM> is transported to the second magnetism collecting mechanism <NUM>, and the magnetic beads are adsorbed to the side surface of the reaction container <NUM>. The detector dispensing mechanism <NUM> suctions the eluate in the reaction container <NUM> and transports the eluate to the HPLC section <NUM>.

Using <FIG>, <FIG>, <FIG>, <FIG>, and <FIG> each of which is an explanatory view of an operation of the injection valve <NUM>, a description will be given of a method of introducing the sample into the HPLC section <NUM>.

As illustrated in <FIG>, in a state where the injection valve <NUM> is at a first position (position providing connection between the sample vial <NUM> and the sample loop syringe <NUM>), the injection valve <NUM> drives the singe <NUM> in a direction of the suction to draw the sample in the sample vial <NUM> into the sample loop.

Then, as illustrated in <FIG>, the injection valve <NUM> is shifted to a second position (position at which the sample loop is separated from the sample vial <NUM> and from the syringe <NUM>) to provide a method in which the sample is drawn into the HPLC section <NUM>.

In addition, a method can be provided in which the sample is introduced into the HPLC section <NUM> according to a method illustrated in <FIG>, <FIG>, and <FIG>.

In other words, as illustrated in <FIG>, it is possible to provide a method in which, at the second position of the injection valve <NUM> (position providing connection between the sample vial <NUM> and the syringe <NUM>), the sample is drawn to a syringe side. Then, as illustrated in <FIG>, the injection valve <NUM> is switched to the first position (position providing connection between the sample vial <NUM> and the sample loop syringe <NUM>) to squeeze the sample into the sample loop with the syringe pump <NUM>. Then, as illustrated in <FIG>, it is possible to provide a method in which the injection valve <NUM> is switched to the second position to introduce the sample into the HPLC section <NUM>.

A solution containing the substance to be measured that has been separated by the HPLC section <NUM> is introduced into the detector <NUM>. On the detector <NUM>, a mass spectrometer is mounted, the ionization section ionizes the solution and introduces the ionized solution to the triple-quadrupole mass spectrometer, and measurement is performed. In data analysis, a ratio among area values is acquired and, using a calibration curve produced from a sample having a known concentration, a concentration of the sample is calculated.

As the mass spectrometer, a mass spectrometer of another type, such as a quadrupole mass spectrometer or an ion trap mass spectrometer, can also be used. The detector <NUM> need not necessarily be a mass spectrometer, and may also be a DAD (Diode Array Detector), a UV detector, gas chromatography, or an NMR.

In First Embodiment, a description will be given of an assay protocol in the case of two types of magnetic beads (a magnetic bead (first magnetic bead) having a surface modified with an antibody as a functional group and a magnetic bead (second magnetic bead) having a surface modified with a negative-phase-mode functional group (an example of which is an ODS (octadecylsilyl group))). The first magnetic beads first reacts with the substance to be measured before the second magnetic bead.

The ODS provides a magnetic bead obtained by reacting a silane coupling agent such as a dimethyloctadecylsilane with porous silica gel and having a surface modified with an octadecyl group. The substance to be measured in the sample is bonded to the magnetic bead by hydrophobic interaction to be eluted using an organic solvent or the like.

First, a description will be given of the case of one type of magnetic beads (Functional Groups: ODS Beads).

<FIG> is an explanatory view of an assay protocol when one type of magnetic beads (Functional Groups: ODS Beads) are used. A description will be given of a case where testosterone as a type of steroid hormone is used as the substance to be measured.

In <FIG>, the assay protocol requires a total of <NUM> cycles, and requires <NUM> minutes. Accordingly, a time period of each one of the cycles is set to <NUM> seconds.

In First Embodiment, each one of the cycles is set to <NUM> seconds, but each one of the cycles may also be longer than <NUM> seconds. The following will describe each of the cycles. Treatment steps <NUM> to <NUM> are pretreatment steps when the one type of magnetic beads are used. However, it is also possible to define the treatment steps <NUM> to <NUM> as pretreatment steps when the one type of magnetic beads are used.

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of a sample and an internal standard substance is performed.

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of a first reagent is performed.

In Cycles <NUM>-<NUM> (the treatment step <NUM>), incubation is performed (<NUM> minutes).

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of a second reagent is performed.

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of the magnetic beads is performed.

In Cycles <NUM>-<NUM> (the treatment step <NUM>), addition and stirring of a cleaning liquid is performed.

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of an eluate is performed.

In Cycle <NUM> (the treatment step <NUM>), transfer of the eluate to the HPLC section <NUM> is performed.

Note that the sample added in Cycle <NUM> is blood serum, and <NUM>µL of the blood serum was added. The sample need not necessarily be the blood serum, and may also be blood plasma, whole blood, urine, a cell tissue, a blood cell, or the like. As the internal standard substance, <NUM>µL of <NUM> pg/mL testosterone-<NUM>,<NUM>,<NUM>-13C3 was added. However, the internal standard substance may also be testosterone-D3.

As the first reagent added in Cycle <NUM>, <NUM>µL of a <NUM>% aqueous formic acid solution serving as a pH adjustment reagent was added. The incubation was performed at <NUM>. The second reagent added in Cycle <NUM> is not used in First Embodiment, but typically serves as a second pH adjustment reagent, a protein denaturation reagent, or the like.

The magnetic beads added in Cycle <NUM> were stirred before being added (a stirring mechanism for the magnetic beads is not shown), and then <NUM>µL of the magnetic beads were added. As the cleaning liquid added in Cycles <NUM>-<NUM>, <NUM>µL of pure water was added. As the eluate added in Cycle <NUM>, <NUM>µL of a <NUM>% methanol solution was added.

<FIG> is an explanatory view of an assay protocol in the case of First Embodiment in which two types of magnetic beads (Functional Groups: ODS Beads and Antibody Beads) are used. A description will be given of a case where two types of subjects to be measured, which are testosterone as a type of steroid hormone and gentamicin as a type of aminoglycoside antimicrobial, are to be measured.

The assay protocol requires a total of <NUM> cycles, and requires <NUM> minutes. Accordingly, a time period of each one of the cycles is set to <NUM> seconds. In First Embodiment, each one of the cycles is set to <NUM> seconds, but each one of the cycles may also be longer or shorter than <NUM> seconds. The following will describe each of the cycles. Treatment steps <NUM>-<NUM> are pretreatment steps when the two types of magnetic beads are used. However, it is also possible to define the treatment steps <NUM>-<NUM> and <NUM> as pretreatment steps when the two types of magnetic beads are used.

In Cycles <NUM>-<NUM> (the treatment Step <NUM>), addition and stirring of a sample, a first internal standard substance, and a second internal standard substance is performed.

In Cycle <NUM> (the treatment Step <NUM>), addition and stirring of the first reagent (R1) is performed.

In Cycles <NUM>-<NUM> (the treatment Step <NUM>), incubation is performed (<NUM> minutes).

In Cycle <NUM> (the treatment Step <NUM>), addition and stirring of the second reagent (R2) is performed.

In Cycle <NUM> (the treatment Step <NUM>), addition and stirring of first magnetic beads is performed.

In Cycle <NUM> (the treatment Step <NUM>), addition and stirring of second magnetic beads is performed.

In Cycles <NUM>-<NUM> (the treatment Step <NUM>), addition and stirring of a cleaning liquid is performed.

In Cycle <NUM> (the treatment Step <NUM>), addition and stirring of a first eluate is performed.

In Cycle <NUM> (the treatment Step <NUM>), transfer of the eluate to the HPLC section <NUM> is performed.

In Cycle <NUM> (the treatment Step <NUM>), addition and stirring of a second eluate is performed.

The sample added in Cycles <NUM>-<NUM> (the treatment Step <NUM>) was <NUM>µL of blood serum. As the internal standard substance for gentamicin, <NUM>µL of 1µg/mL tobramycin was added and, as the internal standard substance for testosterone, <NUM>µL of <NUM> pg/mL testosterone-<NUM>,<NUM>,<NUM>-13C3 was added.

As the first reagent added in Cycle <NUM> (the treatment Step <NUM>), <NUM>µL of the <NUM>% aqueous formic acid solution serving as the pH adjustment reagent was used. The incubation was performed at <NUM>. The second reagent added in Cycle <NUM> (the treatment Step <NUM>) is not used in First Embodiment, but typically serves as a second pH adjustment reagent, a protein denaturation agent, or the like.

The magnetic beads <NUM> added in Cycle <NUM> (the treatment Step <NUM>) are magnetic beads each having, in a functional group, an antibody that specifically binds to a structure of an aminoglycoside antimicrobial, and <NUM>µL of the magnetic beads were added. The second magnetic beads added in Cycle <NUM> (the treatment Step <NUM>) are magnetic beads each having the ODS in a functional group, and <NUM>µL of the magnetic beads were added.

The cleaning liquid added in Cycles <NUM>-<NUM> (the treatment Step <NUM>) was pure water, and <NUM>µL of the pure water was added. The first eluate added in Cycle <NUM> (the treatment Step <NUM>) was a <NUM>% glycine sodium solution (pH <NUM>) as a high pH solution, and <NUM>µL of the <NUM>% glycine sodium solution was added. An eluate usable as the eluate for the first magnetic beads each having the antibody in the functional group is limited depending on the type of the second magnetic beads. In First Embodiment, the magnetic beads each having the ODS in the functional group are used as the second magnetic beads, and accordingly a solvent which is a high pH organic solvent cannot be used. Besides <NUM>% glycine sodium solution (pH <NUM>), a <NUM> glycine hydrochloric acid solution (pH <NUM>) as a low pH solution, <NUM> mol/L urea serving as a denaturation agent, or <NUM> mol/L glycine hydrochloride may also be used.

The second eluate added in Cycle <NUM> (the treatment Step <NUM>) was a <NUM>% methanol solution, and <NUM>µL of the <NUM>% methanol solution was added.

In First Embodiment, the two types of magnetic beads each having a surface modified with the functional groups including the ODS and the antibody were used, but another mode may also be used. For example, HILIC, a positive phase, ion exchange, GPC (molecular weight cut-off), or SFC (supercritical fluid chromatography) may also be used. This is appropriately selected depending on physical properties two types of substances to be measured, and a magnetic bead having a high adsorptive specificity is preferably selected for each of the substances to be measured. For example, for a highly hydrophilic substance to be measured, a magnetic bead having a surface modified with a positive-phase-mode functional group is used. For a highly hydrophobic substance to be measured, a magnetic bead having a surface modified with a negative-phase-mode functional group is used.

The first magnetic beads added in Cycle <NUM> (the treatment Step <NUM>) and the second magnetic beads added in Cycle <NUM> (the treatment Step <NUM>) are added such that the magnetic beads modified with more highly specific functional groups are added as the first magnetic beads in cycles having smaller numbers. More highly specific magnetic beads are magnetic beads each having a surface modified with, e.g., an antibody. By adding the more highly specific magnetic beads earlier and treating the substances to be measured in the sample, accuracy of reproducibility is improved.

The respective quantities of the magnetic beads added in Cycle <NUM> (the treatment Step <NUM>) and Cycle <NUM> (the treatment Step <NUM>) are such that <NUM>µL of the first magnetic beads were added and <NUM>µL of the second magnetic beads were added in First Embodiment, but the quantities of the first and second beads can appropriately be changed depending on a quantity ratio between the substance to be measured which are contained in the sample. For example, when the first substance to be measured is on an order of several picograms per milliliters and the second substance to be measured is on an order of several micrograms per milliliters, by adjusting the quantities of the magnetic beads to be added and the concentrations of the magnetic beads to be stored in the measurement reagent containers <NUM>, it is possible to equalize the concentrations of the individual substances to be measured to be contained in the solution after the pretreatment.

When a concentration ratio between the substances to be measured to be contained in the solution after the pretreatment is high (e.g., about <NUM>:<NUM>), peaks after the separation in the HPLC section <NUM> overlap each other and, when the substances to be measured are simultaneously introduced at the same time into the detector <NUM>, the substance to be measured at a higher concentration is preferentially ionized by the ionization section.

Consequently, the efficiency of ionization of the substance to be measured at a lower concentration deteriorates to degrade reproducibility. Accordingly, by equalizing the concentration ratio between the plurality of substances to be measured to be contained in the eluate after the pretreatment, measurement accuracy is improved.

Thus, according to First Embodiment of the present invention, by using the plurality of types of magnetic beads that can bind to the plurality of types of substances to be measured in one assay protocol, it is possible to pretreat the plurality of substances to be measured by a sequential pretreatment. In this case, the number of the cycles in each one assay protocol increases but, since it is possible to treat the plurality of types of substances to be measured, the pretreatment is performed cycle by cycle in parallel to increase the number of tests per time period and allow a throughput improvement.

In First Embodiment, each one of the cycles is set to <NUM> seconds (<NUM> minutes). Accordingly, if one type of magnetic beads are used, since one assay protocol includes <NUM> cycles, <NUM> minutes is required.

By contrast, when two types of magnetic beads are used, since one assay protocol includes <NUM> cycles, <NUM> minutes is required. When the two types of magnetic beads are used, two types of substances to be measured can be treated in each one assay protocol and, accordingly, after the initial sample went through one assay protocol, a throughput is improved to about double a throughput when the one type of beads are used.

In addition, since the quantity of the samples used in one assay protocol is equal, the quantity of the samples for one substance to be measured that can be treated is approximately halved, which can save the quantity of the samples.

In other words, according to First Embodiment, it is possible to perform the pretreatment method of the automatic analyzer that can pretreat the plurality of substances to be measured by a sequential treatment by using the plurality of magnetic beads to which the plurality of substances to be measured can be bonded.

Next, a description will be given of Second Embodiment of the present invention. Note that a pretreatment method according to Second Embodiment is performed by the automatic analyzer illustrated in <FIG>.

<FIG> is an explanatory view of Second Embodiment, which is an explanatory view of an assay protocol when two types of magnetic beads (Functional Groups: Antibody Bead Types (each of the first magnetic beads and the second magnetic beads has a surface modified with an antibody as a functional group)) are used.

A description will be given below of a case where PTH as a type of parathyroid hormone and <NUM>-OH vitamin D3 as a type of fat-soluble vitamin are used as substances to be measured.

The PTH is a factor that adjusts metabolism of calcium and a phosphoric acid in blood, which is a substance contributing to a thyroid disease or cancer. The vitamin D is a factor that adjusts metabolism of an amount of calcium in blood, which is a substance contributing not only to osteoporosis, but also to cancer, diabetes, and the like.

In Second Embodiment, antibody beads capable of selectively capturing the PTH and antibody beads capable of selectively capturing the vitamin D3 are used. The assay protocol requires a total of <NUM> cycles, and requires <NUM> minutes. A time period of each one of the cycles is set to <NUM> seconds. In Second Embodiment, each one of the cycles is set to <NUM> seconds, but each one of the cycles may also be longer or shorter than <NUM> seconds. The following will describe each of the cycles. Treatment steps <NUM>-<NUM> are pretreatment steps when the two types of magnetic beads are used in Second Embodiment. However, it is also possible to define the treatment steps <NUM>-<NUM> as pretreatment steps when the two types of magnetic beads are used in Second Embodiment.

In Cycles <NUM>-<NUM> (the treatment step <NUM>), addition and stirring of the sample, the first internal standard substance, and the second internal substance is performed.

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of the first reagent is performed.

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of the second reagent is performed.

In Cycles <NUM>-<NUM> (the treatment step <NUM>), addition and stirring of the first magnetic beads and the second magnetic beads is performed.

In Cycles <NUM>-<NUM> (the treatment step <NUM>), addition and stirring of the cleaning liquid is performed.

In Cycle <NUM> (the treatment step <NUM>), addition and stirring of the first eluate is performed.

In Cycles <NUM>-<NUM> (the treatment step <NUM>), incubation is performed (<NUM> seconds).

The sample added in Cycles <NUM>-<NUM> (the treatment step <NUM>) is blood serum, and <NUM>µL of the blood serum was added. As the internal standard substance for the PTH, <NUM>µg/mL 15N labeled PTH was used, and <NUM>µL of <NUM>µg/mL 15N labeled PTH was added while, as the internal standard substance for the <NUM>-OH vitamin D3, <NUM> pg/mL <NUM>-OH vitamin D3-d6 was used, and <NUM>µL of <NUM> pg/mL <NUM>-OH vitamin D3-d6 was added.

As the first reagent added in Cycle <NUM> (the treatment step <NUM>), <NUM>µL of the <NUM>% aqueous formic acid solution as the pH adjustment reagent was added. The incubation in the steps <NUM>, <NUM>, <NUM>, and <NUM> was performed at <NUM>.

The second reagent added in Cycle <NUM> (the treatment step <NUM>) is not used in Second Embodiment, but typically serves as the second pH adjustment reagent, a protein denaturation reagent, or the like.

The first magnetic beads added in Cycle <NUM> (the treatment step <NUM>) are magnetic beads each having, in a functional group, an antibody that specifically binds to the PTH, and <NUM>µL of the magnetic beads were added.

The magnetic beads <NUM> added in Cycle <NUM> are magnetic beads each having, in a functional group, an antibody that specifically binds to the <NUM>-OH vitamin D3, and <NUM>µL of the magnetic beads were added.

The cleaning liquid added in Cycles <NUM>-<NUM> (the treatment step <NUM>) is pure water, and <NUM>µL of the pure water was added. The eluate <NUM> added in Cycle <NUM> (the treatment step <NUM>) is a <NUM>% glycine sodium solution (pH <NUM>) as a high pH solution, and <NUM>µL of the <NUM>% glycine sodium solution was added.

Since the substances to be measured in First Embodiment and the substances to be measured in Second Embodiment are different from each other, the two types of magnetic beads are used in each of First and Second Embodiments, but the types of the magnetic beads in Second Embodiment are different from the types of the magnetic beads in First Embodiment.

In First Embodiment, after the first magnetic beads are added in the step <NUM> and stirred, the incubation is performed in the treatment step <NUM>. Subsequently, the second magnetic beads are added in the treatment step <NUM> and stirred, and then the incubation is performed in the treatment step <NUM>.

By contrast, in Second Embodiment, the first magnetic beads and the second magnetic beads are added in the treatment step <NUM> and stirred, and then the incubation is performed in the treatment step <NUM>.

In First Embodiment, since the two types of eluates are used, the addition and stirring of the first eluate is performed in the treatment step <NUM>, the incubation is performed in the treatment step <NUM>, and the transfer of the eluate to the HPLC section <NUM> is performed in the treatment step <NUM>. Then, the addition and stirring of the second eluate is performed in the treatment step <NUM>, the incubation is performed in the treatment step <NUM>, and the transfer of the eluate to the HPLC section <NUM> is performed in the treatment step <NUM>.

By contrast, in Second Embodiment, since the one type of eluate is used, the addition and stirring of the first eluate is performed in the treatment step <NUM>, the incubation is performed in the treatment step <NUM>, and the transfer of the eluate to the HPLC section <NUM> is performed in the treatment step <NUM>.

Accordingly, in First Embodiment, the assay protocol requires a total of <NUM> cycles, and requires <NUM> minutes.

Meanwhile, in Second Embodiment, the assay protocol requires a total of <NUM> cycles, and requires <NUM> minutes. The treatment time period in Second Embodiment is shorter than the treatment time period in First Embodiment.

In other words, according to the present invention, when the substances to be measured differ, it is possible to further reduce the treatment time period.

Next, a description will be given of an example not part of the present invention. Note that a pretreatment method according to this example is also performed by the automatic analyzer illustrated in <FIG>.

Third Embodiment relates to an assay protocol when one type of magnetic beads are used. The magnetic beads in Third Embodiment are of one type, and the magnetic beads each having a surface modified with a plurality of functional groups are used.

<FIG>, <FIG>, and <FIG> are diagrams each illustrating a concept of the magnetic beads having the plurality of functional groups in Third Embodiment.

As illustrated in <FIG>, as the magnetic beads used in Third Embodiment, magnetic beads <NUM> are modified with an antibody A <NUM> (first antibody) and an antibody B <NUM> (second antibody). To the antibody A <NUM>, a substance to be measured A <NUM> (first-type substance to be measured) specifically binds while, to the antibody B <NUM>, a substance to be measured B <NUM> (second-type substance to be measured) specifically binds.

In Third Embodiment, a description will be given of a case where, as the substance to be measured A <NUM>, PTH as a type of thyroid hormone is used and, as the substance to be measured B <NUM>, <NUM>-OH vitamin D3 as a type of fat-soluble vitamin is used.

The surface of each of the magnetic beads <NUM> is modified with an antibody that specifically binds, as the antibody A <NUM>, to the PTH and with an antibody that specifically binds, as the antibody B <NUM>, to the <NUM>-OH vitamin D3.

An able-bodied male has a blood concentration of the PTH which is <NUM>-<NUM> pg/mL and a blood concentration of the <NUM>-OH which is <NUM>-<NUM> ng/ml, and there is an about <NUM>-fold concentration difference therebetween.

Accordingly, as illustrated in <FIG>, a quantity ratio of the antibody A <NUM> with which the surface of each of the magnetic beads <NUM> is modified to the antibody B <NUM> is increased to, e.g., about <NUM>.

Adjustment of the quantity ratio of the antibody A <NUM> to the antibody B <NUM> is not limited to that in the example illustrated in <FIG>. Depending on the blood concentration ratio between the substance to be measured A <NUM> and the substance to be measured B <NUM>, it may also be possible to reduce the quantity ratio of the antibody A <NUM> with which the surface of each of the magnetic beads <NUM> is modified to the antibody B <NUM>, as illustrated in <FIG>.

Alternatively, as illustrated in <FIG>, the quantity ratio of the antibody A <NUM> with which the surface of each of the magnetic beads <NUM> is modified to the antibody B <NUM> may also be adjusted to <NUM>.

<FIG> is a conceptual view illustrating quantity ratios between substances to be measured before and after a pretreatment in which the magnetic bead <NUM> having the plurality of functional groups is used.

In <FIG>, (A) illustrates a graph <NUM> representing a relationship between the substances to be measured and the concentrations thereof in blood (a sample), while (B) illustrates a graph <NUM> representing a relationship between the substances to be measured and concentrations thereof in an eluate after the pretreatment.

At the blood concentrations in blood (in the sample) before the pretreatment illustrated in (A) in <FIG>, a concentration <NUM> of a substance to be measured A is lower than a concentration <NUM> of a substance to be measured B in blood (in the sample).

By pretreating the substances to be measured A and B by using the magnetic beads <NUM> in this example, as illustrated in (B) in <FIG>, it is possible equalize a concentration <NUM> of the substance to be measured A in the eluate and a concentration <NUM> of the substance to be measured B in the eluate.

In other words, by adjusting the quantity ratio between the antibody A and the antibody B of each of the magnetic beads <NUM>, it is possible to equalize a concentration ratio between the plurality of substances to be measured that are contained in the eluate after the pretreatment and improve measurement accuracy.

As illustrated in <FIG>, the assay protocol in this example performs the treatment steps <NUM> to <NUM> when the one type of magnetic beads are used. However, in the case of this example, the one type of magnetic beads <NUM> in use are each modified with the antibodies that bind to the plurality of types of substances to be measured. Therefore, it is possible to treat the plurality of types of substances to be measured, improve a throughput, and equalize the concentration ratio between the plurality of substances to be measured, which allows an improvement in measurement accuracy.

The assay protocol in this example may also perform, depending on the types of the substances to be measured, the treatment steps <NUM> to <NUM> illustrated in <FIG>, and perform treatment the steps <NUM> to <NUM> illustrated in <FIG>.

According to this example, it is possible to not only obtain the same effects as obtained from First and Second Embodiments, but also improve the measurement accuracy, as described above.

Note that, in this example, the description has been given of the magnetic beads <NUM> of one type each having the surface modified with the two types of functional groups. However, the functional groups are preferably of at least two or more types, and may be of, e.g., three types or four types.

According to the present invention described heretofore, it is possible to perform a pretreatment method of an automatic analyzer that binds a substance to be measured to each of magnetic beads to perform treatment and full-automatically performs a batch step including pretreatment and a liquid chromatograph mass spectrometer, in which a plurality of magnetic beads to which a plurality of the substances to be measured can be bound are used to allow a plurality of substances to be measured to be pretreated by a sequential treatment.

The following is a detailed description further given of the effects of the present invention.

The first effect is improving a throughput. In the automatic analyzer capable of full-automatically performing the batch step including the pretreatment and the HPLC/MS, due to introduction into the detector after column separation using the HPLC and mass separation using the MS, even when the plurality of substances to be measured are present in a mixed state in the solution after the pretreatment, the plurality of substances measured can be detected.

Therefore, by using a plurality of types of magnetic beads and a plurality of eluates, the plurality of substances to be measured can be pretreated in a sequential pretreatment (assay protocol).

By using the plurality of types of magnetic beads, the number of cycles in one assay protocol is increased but, since the plurality of substances to be measured can be treated, by performing the pretreatment in parallel, the number of tests per time period increases to improve the throughput. Note that one assay protocol refers to a procedure from addition of a sample, followed by the end of the pretreatment, to introduction of a solution after the pretreatment into a separation/detection step.

One assay protocol includes a plurality of cycles. Each of the cycles takes the same time period and, by continuously performing respective operations in the individual cycles, one assay protocol is performed.

The second effect is allowing a reduction in the quantity of a sample for each of the testable substances to be measured. When a pretreatment is performed using a plurality of magnetic beads in one assay protocol, the quantity of the sample used in one assay protocol is the same, and accordingly a small quantity of the sample is sufficient to allow each of the testable substances to be measured to be inspected.

Finally, it is possible to adjust the concentrations of the substances to be measured in an eluate after the pretreatment and improve inspection accuracy. By using the contents of the plurality of substances to be measured in the sample to adjust the quantity of magnetic beads to be added to each of the substances to be measured, it is possible to adjust the contents of the substances to be measured which are contained in a solution after the pretreatment.

Specifically, the quantity ratio of the magnetic beads to be added to the lower-concentration substance to be measured is increased, while the quantity ratio of the magnetic beads to be added to the higher-concentration substance to be measured is reduced. Alternatively, the concentration of the magnetic beads to be stored in the measuring reagent container relative to the lower-concentration substance to be measured is increased, while the concentration of the magnetic beads to be stored in the measuring reagent container relative to the higher-concentration substance to be measured is reduced. Still alternatively, the concentrations of the individual magnetic beads and the quantities thereof to be added are adjusted.

Since the quantities of the substances to be measured which are contained after the pretreatment can be equalized, it is possible to reduce the influence of ionization efficiency in a MS ion source as a major cause of fluctuations, and consequently inspection accuracy is improved.

Note that, as the magnetic beads, two or more types can be used. Meanwhile, two or more types of substances to be measured may also be used.

Claim 1:
A pretreatment method of an automatic analyzer comprising the steps of: adding (<NUM>, <NUM>; <NUM>) a magnetic bead to a sample containing a substance to be measured; binding (<NUM>, <NUM>; <NUM>) the substance to be measured to the magnetic bead; and extracting (<NUM>; <NUM>) the magnetic bead from the sample,
wherein, in one assay protocol, a plurality of magnetic beads that bind to a plurality of types of substances to be measured are added to the sample, and
wherein the plurality of magnetic beads are a plurality of types of magnetic beads that bind to any one type of a substance to be measured of the plurality of types of substances to be measured,
characterized in that
the plurality of types of magnetic beads have a first magnetic bead and a second magnetic bead, the first magnetic bead being a magnetic bead whose surface is modified with an antibody as a functional group, and the second magnetic bead being a magnetic bead whose surface is modified with a reverse phase mode functional group, and
the plurality of types of substances to be measured are separated (<NUM>, <NUM>; <NUM>) from the magnetic beads by an eluate.