Abstract:
A column is described, as representative one of which includes: a disposable column used in a measuring sample preparator, comprising: a support immobilized a substance capable of conjugating a protein, a support holding part for holding the support, a fluid connecting part for connecting to the sample preparator, and a liquid storing part for receiving a liquid through the support held by the support holding part and storing the liquid to be collectively from the top.

Description:
[0001]     This application claims priority under 35 U.S.C. § 119 to Japanese Patent Application No. 2005-172501 filed Jun. 13, 2005, the entire content of which is hereby incorporated by reference.  
       FIELD OF THE INVENTION  
       [0002]     The present invention relates to a column, a sample preparation apparatus using the column, and auto analyzer.  
       BACKGROUND  
       [0003]     Sample mini columns are known which can bind an analysis object material in a sample to a substructure within the column by having a funnel for storing sample at the top end of a column, introducing sample into the funnel, and injecting sample into the column by sealing the top part of the funnel and increasing the pressure (for example, refer to WO98/03264). Intemational Laid-Open Patent Publication No. WO98/03264 discloses an auto analyzer that injects a solvent into a column through an inlet nozzle to dissociate an analyte from a substructure by connecting the inlet nozzle to the top end of the column, and introducing the solvent containing the analyte into a detection device through an outlet nozzle connected to the bottom end of the column so as to automatically detect the analyte in order to analyze an analyze analyte material held in a column.  
         [0004]     This auto analyzer is provided with an inlet nozzle and outlet nozzle for each of a plurality of columns. The inlet nozzle is connected to a pump via a rotating valve, and liquid is fed into an object column from the pump via the rotating valve and inlet nozzle by switching the rotating valve. The outlet nozzle is connected to a detecting device through a rotating valve, and liquid is fed to the detecting device from the object column via the outlet nozzle and rotating valve.  
         [0005]     In the above auto analyzer with columns, however, a plurality of columns are provided, and the flow paths from the pump to the detection device via the columns are complicated in order to analyze an analyte material held in a different column switching the flow path from a column using rotating valves, such that in order to analyze an analyte material in a different column, the residue of the previous analyte remaining in the complex of flow paths must be washed therefrom.  
       SUMMARY  
       [0006]     The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.  
         [0007]     In view of this information, the present invention provides a column and sample preparation apparatus and an auto analyzer having simple constructions appropriate for auto analyzers.  
         [0008]     A first aspect of the present invention is a disposable column used in a measuring sample preparator, and includes: a support immobilized a substance capable of conjugating a protein, a support holding part for holding the support, a fluid connecting part for connecting to the sample preparator, and a liquid storing part for receiving a liquid through the support held by the support holding part and storing the liquid to be collectively from the top.  
         [0009]     A second aspect of the present invention is a liquid sample preparation apparatus, and includes: a mounting part for mounting a column comprising a support holding part for holding a support immobilized a substance capable of conjugating a protein, a liquid storing part communicating with the support holding part and for storing a liquid to be collectively from the top, and a connecting part for connecting the mounting part, and a fluid driving part for driving a liquid in the column mounted on the mounting part.  
         [0010]     A third aspect of the present invention is an auto analyzer, and includes: a mounting part for mounting a column comprising a support for isolating a target substance in a first liquid sample, a support holding part for holding the support, and a liquid storing part for collecting a second liquid sample prepared when a specific fluid passes through the support and storing the second liquid sample to be collectively from the top, a collecting part for collecting the second liquid sample stored in the liquid storing part of the column mounted on the mounting part, and an analyzing part for analyzing the collected second liquid sample and obtaining information related to the target substance. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0011]      FIG. 1  is a top view of the column of the present invention;  
         [0012]      FIG. 2  is a side view of the column of the present invention;  
         [0013]      FIG. 3  is a cross sectional view on the A-A view of  FIG. 1 ;  
         [0014]      FIG. 4  is a perspective view of the sample preparing unit of the present invention;  
         [0015]      FIG. 5  is a top view of a fluid manifold of the present invention;  
         [0016]      FIG. 6  is a cross sectional view on the B-B view of  FIG. 5 ;  
         [0017]      FIG. 7  is a fluid circuit diagram of the sample preparing unit shown in  FIG. 4 ;  
         [0018]      FIG. 8  is a perspective view of the auto measuring mechanism of the present invention;  
         [0019]      FIG. 9  is a block diagram showing the control system for controlling the auto measuring mechanism of the auto measuring apparatus;  
         [0020]      FIG. 10  is a flow chart showing the operation of the control system of  FIG. 9 ;  
         [0021]      FIG. 11  is a block diagram showing the hardware structure of the controlling part of the auto measuring apparatus;  
         [0022]      FIG. 12  is a cross sectional diagram showing a column with caps installed on the top and bottom openings;  
         [0023]      FIG. 13  is a cross sectional view showing the bottom cap removed from the column of  FIG. 12 ; and  
         [0024]      FIG. 14  is a flow chart showing the operation of another embodiment of the control system shown in  FIG. 9 .  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0025]     Although the present invention is described in details hereinafter based on the embodiments shown in the drawings, the present invention is not limited to these embodiments.  
         [0026]     Column  
         [0027]      FIG. 1  is a top view of a column used in the present embodiment,  FIG. 2  is a side view of the column shown in  FIG. 1 , and  FIG. 3  is a cross sectional A-A view of the column shown in  FIG. 1 .  
         [0028]     As shown in the drawings, the column  1  is cylindrical in shape and made of vinyl chloride resin, and has in its interior a support holding part  2  for holding a support  6  used for isolating a target substance in a liquid sample, a liquid introducing part  3  for introducing a liquid sample to the support holding part  2 , and a liquid storing part  4  for receiving and storing a liquid sample from the support holding part  2 . The column  1  is a precision cut, solid molded part of vinyl chloride resin. When mass produced, solid molding may also be accomplished by a molding process. Polyprene resin and polyacetal resin with scant protein absorption may be used instead of vinyl chloride resin. The column  1  is disposable as the column  1  is composed of above material.  
         [0029]     The liquid storing part  4  of the column  1  has a 300 μL capacity, and a top of the liquid storing part  4  has an opening  5  for collecting liquid sample by an external source. The column has a diameter of 10 mm, and a length of 35 mm. A column diameter of 1˜15 mm is desirable, as is a column length of 10˜50 mm. The support is a cylindrical monolithic silica gel measuring 2.7 mm in diameter by 3 mm in height. The monolithic silica gel has different particle supports, and has a structure of a three-dimensional network framework with integrated empty spaces. The support  6  is inserted into the support holding part  2  from the bottom opening, and supported by an elastic pressure provided by an anchor pipe  8  through an O-ring  7 .  
         [0030]     The anchor pipe  8  is press-fitted from the bottom opening of the column  1 , and the pipe  8  and the hole of the O-ring  7  form a liquid introducing part  3 .  
         [0031]     The bottom end of the column  1  is provided with a charging flange  9  for charging and anchoring the column  1  in a sample preparing unit described later. The flange  9  is a planed flange with horizontal notch, such that the bilateral width of the disk-like flange of diameter D is (W&lt;D).  
         [0032]     Sample Preparing Unit  
         [0033]      FIG. 4  is a perspective view of the sample preparing unit of the present embodiment.  
         [0034]     As shown in the drawing, a sample preparing unit  11  is provided with an L-shaped support plate  12 , and fixedly attached to the support plate  12  are a fluid manifold  13 , syringe  14 , and speed reducing stepping motor  15 .  
         [0035]     A screw shaft  16  is connected to the output shaft of the stepping motor  15 . A drive arm  17 , which screws onto the screw shaft  16 , is connected to the tip of a piston  18  of the syringe  14 . When the screw shaft  16  is rotated by the stepping motor  15 , the piston  18  is moved vertically. The syringe  14  and fluid manifold  13  are connected via delivery tube  18  through connectors  19  and  20 . The syringe  14  is also connected to a liquid holding chamber  34  described later by a delivery tube  20   b  via a connector  20   a . The syringe  14 , stepping motor  15 , screw shaft  16 , drive arm  17 , and piston  18  configure a syringe pump.  
         [0036]     Liquid Manifold  
         [0037]      FIG. 5  is a top view of the fluid manifold, and  FIG. 6  is a cross sectional B-B view of  FIG. 5 . As shown in these drawings, the fluid manifold  13  is formed of vinyl chloride resin, and is provided with a column connecting part  21  for connecting to the liquid introducing part  3  of the column  1 , and a liquid sample receiving part  22  for receiving a liquid sample.  
         [0038]     The fluid manifold  13  is provided with an internal flow path  23 , and at the bottom surface is provided with an electromagnetic valve  24  that operates between the liquid sample introducing part  22  and the flow path  23 , and an electromagnetic valve  25  that operates between the flow path  23  and the column connecting part  21 . The fluid manifold  13  has a threaded connector hole  26  ( FIG. 5 ) for connecting the connector  20  to the side surface, and the threaded hole  26  connects to the flow path  23 .  
         [0039]      FIG. 7  is a flow circuit diagram of the sample preparing unit  11 , and shows the syringe  14  connected to the fluid manifold  13  via the connector  20 . The liquid holding chamber  34  is connected to the syringe  14  via an electromagnetic valve  33  provided on the fluid operating part  71  (refer to  FIG. 8 ) described later, and a positive pressure is applied from a positive pressure source  35  to the liquid holding chamber  34 .  
         [0040]     The method by which the column  1  is loaded in the fluid manifold  13  is described below.  
         [0041]     As shown in  FIGS. 5 and 6 , a column receiving concavity  27  is formed on the top surface of the fluid manifold  13  in order to accommodate the bottom end of the column  1 , and an O-ring  28  is fitted on the circumference of the bottom surface while allowing passage through the center of the bottom of the concavity  27  to the column connecting part  21 . Attached to the top surface of the fluid manifold  13  are two pressing plates  29  and  30  having L-shaped cross sections are attached in parallel with a gap wider than W and narrower than D therebetween as shown in  FIG. 1 , and centered on the column receiving concavity  27 .  
         [0042]     The column  1  is loaded in the column receiving concavity  27  so that the flange  9  passes between the pressing plates  29  and  30 , and rotated 90 degrees in either a clockwise or counter clockwise direction. Thus, the D diameter part of the flange  9  engages the pressing plates  29  and  30 , and the flange  9  is attached by the pressing plates  29  and  30  via the elasticity of the O-ring  28 . To remove the column  1 , the column  1  is pressed and rotated 90 degrees in either left or right directions.  
         [0043]     When the column  1  is loaded in the fluid manifold  13  of the sample preparing unit  11 , the concavity  27  of the manifold  13  is filled with liquid to prevent the introduction of air bubbles, and fluid may overflow depending on the capacity when the tip of the column  1  is inserted in the concavity  27 , as described later. In order to prevent this liquid from flowing to the margins, an overflow storage concavity  31  (refer to  FIGS. 5 and 6 ) is formed at the perimeter of the column loading concavity  27 , and a concavity  32  which is deeper than the concavity  31  is provided at part of the concavity  31 .  
         [0044]     When the sample preparing unit  11  is installed in an auto measuring mechanism as described later, the overflow liquid is suctioned and drained from the concavity  32  by a dispensing pipette  62 .  
         [0045]     Auto Measuring Apparatus  
         [0046]      FIG. 8  is a perspective view of an auto measuring apparatus of the present embodiment. The auto measuring apparatus is configured by an auto measuring mechanism  51 , and a personal computer  80  connected to the auto measuring mechanism so as to enable communication.  
         [0047]     The auto measuring mechanism  51  is provided with a frame  52  for moving a pipette in the X direction, a frame  53  for moving a pipette in the Y direction, and a block  54  for moving a pipette in the Z direction.  
         [0048]     The frame  52  is provided with screw shaft  55  for moving the block  54  in the arrow X direction, a slide shaft  56  for supporting and oscillating the block  54 , and a stepping motor  57  for rotating the screw shaft  55 .  
         [0049]     The frame  53  is provided with a screw shaft  58  for moving the frame  52  in the arrow Y direction, parallel slide shafts  59  and  60  for supporting and oscillating the frame  52 , and a stepping motor  61  for rotating the screw shaft  58 .  
         [0050]     The block  54  is provided with a screw shaft  67  for moving the arm  68  that supports the dispensing pipette  62  in the arrow Z direction, slide shaft  69  for supporting and oscillating the arm  68 , and a stepping motor  70  for rotating the screw shaft  67 .  
         [0051]     Within the frame  52  are provided six sample preparing units  11 , a specimen/reagent placement part  63  for storing specimen containers and reagent containers at appropriate temperatures, washing part  64  for washing the dispensing pipette  62 , waste part  65  for accommodating waste fluid, and a detecting part  66  for detecting a placed detection container. At the back part of the frame  52  is provided a fluid operating part  71  for operating the fluids, and which is connected to the washing part  64 , and each sample preparing unit  11  and the like. The fluid operating part  71  is provided with electromagnetic valves  24  and  25  of each of the sample preparing units  11 , electromagnetic valve  33  for controlling the fluid when fluid is loaded in the syringe from the liquid storing chamber, electromagnetic valves for controlling fluid when liquid is suctioned and discharged by the dispensing pipette  62 , electromagnetic valves for controlling fluid when waste fluid is suctioned from the dispensing pipette in the waste part  65 , and electromagnetic valves for controlling fluid when washing the dispensing pipette in the washing part  64 .  
         [0052]     The auto measuring mechanism  51  is provided with a liquid holding chamber  34 , positive pressure source  35 , and at the back is provided with a receiving unit accommodating a washing liquid tank  73  described later, waste tank  74 , pure water tank  75 .  
         [0053]     Furthermore, at the side of the frame  53  is provided a drive circuit  76  for supplying drive signals to each of the sample preparing units  11 , specimen/reagent placement part  63 , stepping motors  57 ,  61 ,  70 , and fluid operating part  71 .  
         [0054]     The personal computer  80  is provided with a control part  77  connected to the drive circuit  76 , input part  78  for inputting data and the like to the control part  77 , and a display part  79  for displaying analysis results and the like.  
         [0055]     The structure of the control part  77  of the personal computer  80  is described below. The control part  77  is provided with a CPU  91   a , ROM  91   b , RAM  91   c , I/O interface  91   d , and image output interface  91   e . The ROM  91   b  stores an operating system, control programs for controlling the operations of the apparatus, and data needed for the execution of the control programs. The CPU  91   a  loads the control program into the RAM  91   c , or directly executes the control program from the ROM  91   b . Thus, the result data processed by the CPU  91   a  are transmitted to the drive circuit  76  of the auto measuring mechanism  51  through the I/O interface  91   d , and the data requiring processing by the CPU  91   a  is received from the drive circuit  76  of the auto measuring mechanism  51  through the I/O interface  20   d . The CPU  91  a controls the drive circuit  76  of the auto measuring mechanism  51  by executing the control program. The CPU  91   a  calculates analysis data reflecting the activity based on the fluorescent light intensity obtained by the detecting part  66  in a detecting step described later, and displays the analysis data on the display part  79 .  
         [0056]     Control System  
         [0057]      FIG. 9  is a block diagram of the control system for controlling the auto measuring mechanism  51  of the auto measuring apparatus shown in  FIG. 8 . This control system is provided with a drive circuit  76  that has driver circuits for drive each part of the auto measuring mechanism  51 , as shown in the drawing. The control part  77  for controlling the drive circuit  76  and analyzing detection results from the detecting part  66 , the input  78  for inputting data and the like to the control part  77 , and the display part  79  for displaying analysis results and the like analyzed by the control part  77 .  
         [0058]     The control part  77  outputs drive signals for driving the stepping motor  15  of each of the sample preparing units  11 , drive signals for temperature adjustment of the specimen/reagent placement part  63 , drive signals for driving the stepping motors  57 ,  61 , and  70 , and drive signals for driving the electromagnetic valves of the fluid operating part  71  from the drive circuit  76  by controlling the drive circuit  76 . The control part  77  also acquires detection signals from the detecting part  66  through the drive circuit  76 .  
         [0059]     The washing solution tank  73  for supplying the washing solution is connected to the washing part  64 , the waste liquid tank  74  for accommodating waste liquid is connected to the waste part  65 , and a pure water tank  75  for supplying pure dilution water for the specimens and reagents is connected to the fluid operating part  71 .  
         [0060]     Measurement Preparation  
         [0061]     When starting a measurement, a stock solution is preloaded in the fluid manifold  13  of the sample preparing unit  11  as described below. In the flow path shown in  FIG. 7 , first, stock solution is introduced to the syringe  14  from the stock solution chamber  34  by opening the electromagnetic valves  24  and  33 , and the stock solution flows to the liquid sample receiving part  22  via the electromagnetic valve  24 . Then, the electromagnetic valves  24  and  33  are closed and the electromagnetic valves  25  and  33  are opened, and stock solution flows from the stock solution chamber  34  and is delivered to the column receiving concavity  27  via the electromagnetic valve  33 , syringe  14 , and electromagnetic valve  25 , such that the stock solution fills the concavity  27 .  
         [0062]     Thereafter, the electromagnetic valves  25  and  33  are closed.  
         [0063]     In this condition, the column  1  is mounted.  
         [0064]     An antibody that specifically bonds to the enzyme that is the activity measurement object is solid phased beforehand in the support (monolithic silica gel) within the column  1 . Then, stock solution  103  is loaded in the column  1  so as to prevent any contact of the antibody with the air, and caps  101  and  102  are respectively attached to the top and bottom of the column  1  which is then sealed ( FIG. 12 ). First, the bottom cap  102  of the column  1  is removed, and brought near the column loading concavity  27  of the sample preparing unit  11 . At this time, a droplet  104  forms at the tip due to the weight of the liquid in the column  1  ( FIG. 13 ). Then, the droplet  104  at the tip of the column  1  and the liquid filling the column loading concavity  27  come into contact as the column  1  is inserted into the concavity  27 , whereupon the column  1  is rotated 90 degrees and fixed in place. Thereafter, the top cap  101  of the column  1  is removed. Thus, measurement preparation is completed.  
         [0065]     Measurement Operation  
         [0066]     After the measurement preparation is completed, measurement is started by pressing the start key on the input part  78  (keyboard) of the personal computer  80 . The measurement operation is described below using the flow chart of  FIG. 10 .  
         [0067]     1. Immunoprecipitation Pre-buffer Solution Delivery  
         [0068]     (1) The dispensing pipette  62  is inserted into the liquid storing part  4  to a fixed depth C (refer to  FIG. 3 ), the stock solution within the liquid storing part  3  is suctioned and discharged to the waste part  65 .  
         [0069]     (2) The exterior and interior of the dispensing pipette  62  is washed by the washing part  64 .  
         [0070]     (3) 100 μL of immunoprecipitation pre-buffer is suctioned from the specimen/reagent placement part  63  within the apparatus, and injected to the sample liquid receiving part  22  of the fluid manifold  13 .  
         [0071]     (4) The electromagnetic valve  24  is opened, and 100 μL immunoprecipitation pre-buffer is suctioned at a rate of 100 μL/min by the syringe pump, the electromagnetic valve  24  is closed and the electromagnetic valve  25  is opened, and the pre-buffer is discharged to the column  1  at a rate of 100 μL/min, then the electromagnetic valve  25  is closed.  
         [0072]     (5) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, the discharged liquid is suctioned through the column  1 , and discharged to the waste part  65 .  
         [0073]     (6) The dispensing pipette  62  is washed by the washing part  64 .  
         [0074]     2. Immunoprecipitation (Step S 2 )  
         [0075]     (1) 150 μL of specimen is suctioned from the specimen/reagent placement part  63 , and injected into the sample liquid receiving part  22  of the fluid manifold  13 .  
         [0076]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0077]     (3) The electromagnetic valve  24  is opened, and 150 μL of specimen is suctioned at a rate of 100 μL/min by the syringe pump, and thereafter suctioning temporarily stops.  
         [0078]     (4) Since the remaining specimen flows into the flow path  23  ( FIG. 6 ), 30 μL of washing solution is dispensed into the sample liquid receiving part  22  by the dispensing pipette  62 .  
         [0079]     (5) 30 μL of washing solution is suctioned by the syringe pump at a rate of 100 μL/min.  
         [0080]     (6) The electromagnetic valve  24  is closed and the electromagnetic valve  25  is opened, and 50 μL of the liquid is delivered to the column  1  at a rate of 50 μL/min by the syringe pump is temporarily stopped.  
         [0081]     (7) Since the 50 μL discharged to the liquid storing part  4  is the immunoprecipitation pre-buffer that remained within the column  1 , the dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, the discharged liquid is suctioned, and discharged to the waste part  5 .  
         [0082]     (8) 130 μL of the liquid is delivered to the column  1  at a rate of 50 μL/min by the syringe pump. The target protein contained in the specimen binds to the support  6  as an immunoreaction begins in the support  6 .  
         [0083]     (9) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, and the liquid is suctioned and discharged to the waste part  65 . Thus, after the reaction, the specimen can be collected and reused as the specimen in another column  1 .  
         [0084]     (10) The dispensing pipette  62  is washed by the washing part  64 .  
         [0085]     3. Enzyme Reaction Pre-buffer  1  Solution Delivery (Step S 3 ) Enzyme Reaction Pre-buffer  1  Solution Delivery (step S 3 )  
         [0086]     (1) 100 μL of enzyme reaction pre-buffer  1  is suctioned from the specimen/reagent placement part  63 , and injected into the sample liquid receiving part  22  of the fluid manifold  13 .  
         [0087]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0088]     (3) The electromagnetic valve  24  is opened and the electromagnetic valve  25  is closed, and 100 μL of enzyme reaction pre-buffer  1  is suctioned at a rate of 100 μL/min by the syringe pump and temporarily stopped.  
         [0089]     (4) The electromagnetic valve  24  is closed and the electromagnetic valve  25  is opened, and 100 μL of liquid is delivered to the column  1  at a rate of 100 μL/min.  
         [0090]     (5) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, and the liquid is suctioned and discharged to the waste part  65 .  
         [0091]     (6) The dispensing pipette  62  is washed by the washing part  64 .  
         [0092]     4. Enzyme Reaction Pre-buffer  2  Solution Delivery (step S 4 )  
         [0093]     (1) 100 (L of enzyme reaction pre-buffer  2  is suctioned from the specimen/reagent placement part  63 , and injected into the sample liquid receiving part  22  of the fluid manifold  13 .  
         [0094]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0095]     (3) The electromagnetic valve  24  is opened and the electromagnetic valve  25  is closed, and 100 (L of enzyme reaction pre-buffer  2  is suctioned at a rate of 100 μL/min by the syringe pump and temporarily stopped.  
         [0096]     (4) The electromagnetic valve  24  is closed and the electromagnetic valve  25  is opened, and 100 μL of liquid is delivered to the column  1  at a rate of 100 μL/min.  
         [0097]     (5) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, and the liquid is suctioned and discharged to the waste part  65 .  
         [0098]     (6) The dispensing pipette  62  is washed by the washing part  64 .  
         [0099]     5. Enzyme Reaction (step S 5 )  
         [0100]     (1) 100 μL of substrate solution is suctioned from the specimen/reagent placement part  63 , and injected into the sample liquid receiving part  22  of the fluid manifold  13 .  
         [0101]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0102]     (3) The electromagnetic valve  24  is opened and the electromagnetic valve  25  is closed, and 100 μL of substrate solution is suctioned at a rate of 100 μL/min by the syringe pump and temporarily stopped.  
         [0103]     (4) The electromagnetic valve  24  is closed and the electromagnetic valve  25  is opened, and 100 μL of liquid is delivered to the column  1  at a rate of 100 (L/min.  
         [0104]     (5) Since the 50 μL discharged to the liquid storing part  4  is enzyme reaction pre-buffer  2  that remained in the column  1 , the dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, and the liquid is suctioned and discharged to the waste part  65 .  
         [0105]     (6) The dispensing pipette  62  is washed by the washing part  64 .  
         [0106]     (7) 50 μL of liquid is delivered to the column  1  at a rate of 10 μL/min by the syringe pump. An enzyme reaction starts between the substrate in the substrate solution and the target enzyme trapped in the support  6 . As a result, a product reflecting the activity of the target enzyme is extracted to the liquid storing part  4 .  
         [0107]     Since the enzyme reaction was sufficiently produced, the substrate solution repeatedly delivered by the support  6  at an increased flow rate,  
         [0108]     6. Fluorescent labeling reaction (step S 6 )  
         [0109]     (1) 20 μL of fluorescent labeling reagent is suctioned from the specimen/reagent placement part  63 , and injected into the liquid storing part  4  of the column  1 .  
         [0110]     (2) The dispensing pipette  62  is inserted into the liquid storing part  4  and repeatedly suctioned and discharged to mix the contents.  
         [0111]     (3) The dispensing pipette  62  is washed by the washing part  64 .  
         [0112]     (4) The contents are allowed to stand for 20 minutes while the product of the enzyme reaction reacts with the fluorescent labeling reagent.  
         [0113]     7. Process for Terminating the Labeling Reaction (step S 7 )  
         [0114]     (1) 200 μL of labeling reaction terminating reagent is suctioned from the reagent placement part  63 , and injected into the liquid storing part  4  of the column  1 .  
         [0115]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0116]     (3) The contents are allowed to stand for 3 minutes while the surplus fluorescent labeling reagent reacts with the labeling reaction terminating reagent.  
         [0117]     8. Dispensing to the Detecting Apparatus Container (step S 8 )  
         [0118]     (1) The dispensing pipette  62  is inserted into the liquid storing part  4 , and suctions 50 μL of the produce of the fluorescent labeling process.  
         [0119]     (2) The dispensing pipette  62  is moved to the container of the detecting part  66  and discharged the liquid.  
         [0120]     (3) The dispensing pipette  62  is washed by the washing part  64 .  
         [0121]     9. Detection (step S 9 ) 9. Detection (step S 9 )  
         [0122]     (1) The fluorescent intensity of the product is measured by the detecting part  66 .  
         [0123]     (2) Numeric data reflecting the activity is calculated by the control part  77  from the measured fluorescent intensity.  
         [0124]     Thus, the enzyme activity of the target enzyme contained in the specimen is measured.  
         [0125]     Another embodiment of the enzyme activity measurement of a target enzyme contained in a specimen using the auto measuring apparatus is described below.  
         [0126]     Measurement Preparation  
         [0127]     When starting a measurement, a stock solution is preloaded in the fluid manifold  13  of the sample preparing unit  11  as described below. In the flow circuit shown in  FIG. 7 , first, stock solution is introduced to the syringe  14  from the stock solution chamber  34  by opening the electromagnetic valves  25  and  33 , and the stock solution flows to the column receiving concavity  27  via the electromagnetic valve  25 , such that the stock solution fills the column receiving concavity  27  and overflows from the concavity  27  and is collected and stored in a collecting cavity  32  through a cavity  31 . Then, stock solution is suctioned and discharged by the dispensing pipette  62  from the cavity  32 . The stock solution collected in the cavity  27  is suctioned and discharged by the dispensing pipette  62 , such that the stock solution fills the space to the open end of the column connecting part  21 . Thereafter, the electromagnetic valves  25  and  33  are closed.  
         [0128]     Next, the electromagnetic valve  25  is opened, and approximately 16 μl of air is suctioned from the column connecting part  21  by the syringe pump. Thereafter, the syringe pump is stopped, and the electromagnetic valve  25  is closed.  
         [0129]     Then, approximately 150 μl of stock solution is injected into the column receiving concavity  27  by the dispensing pipette  62 . An air gap is formed within the column connecting part  21 .  
         [0130]     In this condition, the column  1  is mounted.  
         [0131]     An antibody that specifically bonds to the enzyme that is the activity measurement object is solid phased beforehand in the support (monolithic silica gel) within the column  1 . Then, stock solution  103  is loaded in the column  1  so as to prevent any contact of the antibody with the air, and caps  101  and  102  are respectively attached to the top and bottom of the column  1  which is then sealed ( FIG. 12 ). First, the bottom cap  102  of the column  1  is removed, and brought near the column loading concavity  27  of the sample preparing unit  11 . At this time, a droplet  104  forms at the tip due to the weight of the liquid in the column  1  ( FIG. 13 ). Then, the droplet  104  at the tip of the column  1  and the liquid filling the column loading concavity  27  come into contact as the column  1  is inserted into the concavity  27 , whereupon the column  1  is rotated 90 degrees and fixed in place. Thereafter, the top cap  101  of the column  1  is removed. Thus, measurement preparation is completed.  
         [0132]     Measurement Operation  
         [0133]     After the measurement preparation is completed, measurement is started by pressing the start key on the input part  78  (keyboard) of the personal computer  80 . The measurement operation is described below using the flow chart of  FIG. 14 .  
         [0134]     1. Immunoprecipitation Pre-buffer Solution Delivery (step S 11 )  
         [0135]     (1) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C (refer to  FIG. 3 ), and stock solution is suctioned from the liquid storing part  4  and discharged to the waste part  65 .  
         [0136]     (2) The exterior and interior of the dispensing pipette  62  is washed by the washing part  64 .  
         [0137]     (3) 100 μL of pre immunoprecipitation pre-buffer is suctioned from the specimen/reagent placement part  63  within the apparatus, and injected to the liquid storing part  4  of the column  1 .  
         [0138]     (4) The electromagnetic valve  25  is opened, and 100 μL immunoprecipitation pre-buffer is suctioned at a rate of 100 μL/min by the syringe pump, and the pre-buffer is discharged to the liquid storing part  4  of the column  1  at a rate of 100 μL/min, then the electromagnetic valve  25  is closed.  
         [0139]     (5) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, the discharged liquid is suctioned through the column  1 , and discharged to the waste part  65 .  
         [0140]     (6) The dispensing pipette  62  is washed by the washing part  64 .  
         [0141]     2. Immunoprecipitation (step S 12 )  
         [0142]     (1) 150 μL of specimen is suctioned from the specimen/reagent placement part  63 , and injected into the sample liquid receiving part  22  of the fluid manifold  13 .  
         [0143]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0144]     (3) The electromagnetic valve  25  is opened, and 150 μL of specimen is suctioned at a rate of 10 μL/min by the syringe pump, and 150 μL of the specimen is discharged into the liquid storing part  4  of the column  1 , then the electromagnetic valve  25  is closed. The target protein contained in the specimen binds to the support  6  as an immunoreaction begins in the support  6 .  
         [0145]     (4) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth, and the liquid is suctioned and discharged to the waste part  65 . Thus, after the reaction, the specimen can be collected and reused as the specimen in another column  1 .  
         [0146]     (5) The dispensing pipette  62  is washed by the washing part  64 .  
         [0147]     3. Enzyme Reaction Pre-buffer  1  Solution Delivery (step S 13 )  
         [0148]     (1) 100 μL of enzyme reaction pre-buffer  1  is suctioned from the sample/reagent placement part  63  and injected into the liquid storing part  4  of the column  1 .  
         [0149]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0150]     (3) The electromagnetic valve  25  is opened, and 100 μL enzyme reaction pre-buffer is suctioned at a rate of 100 μL/min by the syringe pump, and 100 μL of the pre-buffer is discharged to the liquid storing part  4  of the column  1  at a rate of 100 μL/min, then the electromagnetic valve  25  is closed.  
         [0151]     (5) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, and the liquid is suctioned and discharged to the waste part  65 .  
         [0152]     (5) The dispensing pipette  62  is washed by the washing part  64 .  
         [0153]     4. Enzyme Reaction Pre-buffer  2  Solution Delivery (step S 14 )  
         [0154]     (1) 100 μL of enzyme reaction pre-buffer  2  is suctioned from the sample/reagent placement part  63  and injected into the liquid storing part  4  of the column  1 .  
         [0155]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0156]     (3) The electromagnetic valve  25  is opened, and 100 μL enzyme reaction pre-buffer is suctioned at a rate of 100 μL/min by the syringe pump, and 100 μL of the pre-buffer is discharged to the liquid storing part  4  of the column  1  at a rate of 100 μL/min, then the electromagnetic valve  25  is closed.  
         [0157]     (4) The dispensing pipette  62  is inserted into the liquid storing part  4  of the column  1  to a fixed depth C, and the liquid is suctioned and discharged to the waste part  65 .  
         [0158]     (5) The dispensing pipette  62  is washed by the washing part  64 .  
         [0159]     5. Enzyme Reaction (step S 15 )  
         [0160]     (1) 100 μL of substrate solution is suctioned from the specimen/reagent placement part  63 , and injected into the liquid storing part  4  of the column  1 .  
         [0161]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0162]     (3) The electromagnetic valve  25  is opened, and 100 μL of substrate solution is suctioned at a rate of 10 μL/min by the syringe pump, and 100 μL of the liquid is discharged into the liquid storing part  4  of the column  1 , then the electromagnetic valve  25  is closed. An enzyme reaction starts between the substrate in the substrate solution and the target enzyme trapped in the support  6 . As a result, a product reflecting the activity of the target enzyme is extracted to the liquid storing part  4 .  
         [0163]     Since the enzyme reaction was sufficiently produced, the substrate solution may repeatedly delivered by the support  6  at an increased flow rate.  
         [0164]     6. Fluorescent Labeling Reaction (step S 16 )  
         [0165]     (1) 20 μL of fluorescent labeling reagent is suctioned from the specimen/reagent placement part  63 , and injected into the liquid storing part  4  of the column  1 .  
         [0166]     (2) The dispensing pipette  62  is inserted into the liquid storing part  4  and repeatedly suctioned and discharged to mix the contents.  
         [0167]     (3) The dispensing pipette  62  is washed by the washing part  64 .  
         [0168]     (4) The contents are allowed to stand for 20 minutes while the product of the enzyme reaction reacts with the fluorescent labeling reagent.  
         [0169]     7. Labeling Reaction Termination Process (step S 17 )  
         [0170]     (1) 200 μL of labeling reaction terminating reagent is suctioned from the sample/reagent placement part  63 , and injected into the liquid storing part  4  of the column  1 .  
         [0171]     (2) The dispensing pipette  62  is washed by the washing part  64 .  
         [0172]     (3) The contents are allowed to stand for 3 minutes while the surplus fluorescent labeling reagent reacts with the labeling reaction terminating reagent.  
         [0173]     8. Dispensing to the Detecting Apparatus Container (step S 18 )  
         [0174]     (1) The dispensing pipette  62  is inserted into the liquid storing part  4 , and suctions 50 μL of the produce of the fluorescent labeling process.  
         [0175]     (2) The dispensing pipette  62  is moved to the container of the detecting part  66  and discharges the liquid.  
         [0176]     (3) The dispensing pipette  62  is washed by the washing part  64 .  
         [0177]     9. Detection (step S 19 )  
         [0178]     (1) The fluorescent intensity of the product is measured by the detecting part  66 .  
         [0179]     (2) Numeric data reflecting the activity is calculated by the control part  77  from the measured fluorescent intensity.  
         [0180]     Thus, the enzyme activity of the target enzyme contained in the specimen is measured.  
         [0181]     The sample is prepared, for example, by a general method that homogenizes and centrifuges biological tissues.  
         [0182]     In the present invention, when a target enzyme is captured and the enzyme activity measured, sepharose beads and the like may be used instead of monolithic silica gel as the support placed in the column.  
         [0183]     Furthermore, CDK-1 (cyclin-dependent protein kinase 1) is offered as an example of a target enzyme. When CDK-1 is the target enzyme, the antibody used will be CDK-1 antibody.  
         [0184]     Examples of the liquids and reagents used in the measurement process are provided below.  
         [0185]     Stock Solution, Washing Solution 
        Tris-HCL, pH 7.4, 25 mM     NaCl, 150 mM        
 
         [0188]     Immunoprecipitation Pre-Buffer 
        Tris-HCL, pH 7.4, 50 mM     Nonidet-P40, 0.1%        
 
         [0191]     Enzyme Reaction Pre-Buffer  1  
        Nonidet-P40, 1%     Tris-HCL, pH 7.4, 50 mM     NaCl, 300 mM        
 
         [0195]     Enzyme Reaction Pre-Buffer  2  
        Tris-HCL, pH 7.4, 50 mM        
 
         [0197]     Substrate Solution 
        Tris-HCL, pH 7.4, 40 mM     Triton X-100, 0.1%     MgC12, 20 mM     ATPγS-4Li, 2 mM     Histone H1, 10 μg        
 
         [0203]     Fluorescent Labeling Reagent 
        5-lodoacetamide, 2 mM     Fluorescein (5-IAF)     DMSO (dimethyl sulfoxide)     Tris-HCL, pH 7.4, 25 mM     EDTA-2Na, 5 mM        
 
         [0209]     Labeling Reaction Terminating Reagent 
        2ME (2-mercaptoethanol), 5%     Tris-HCL, pH 7.4, 25 mM     NaCl, 150 mM        
 
         [0213]     Although a support having an antibody that specifically binds to the enzyme solid-phased beforehand was used in the present embodiments, the antibody may be fixed in the support by using a support capable of fixing an antibody that specifically binds to the enzyme, and flushing a liquid containing this antibody through the support.  
         [0214]     Although a support for trapping enzyme in the support and measuring the activity of the enzyme is used in the above embodiments, an affinity chromatography support, ion exchange chromatography support, hydrophobic chromatography support, gel filtration support and reverse phase chromatography support and like supports used in chromatography may be used as a support for isolating proteins, nucleic acids, hormones, neural transmitting substances, and vitamins and the like.