JP3192401B2 - Method for separating liquid sample components and apparatus used for the method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating a plurality of components in a liquid sample by utilizing a difference in adsorption power to an adsorbent, and a separation apparatus used in the method.

[0002]

2. Description of the Related Art As a method for separating a plurality of components in a liquid sample by using a difference in adsorption power to an adsorbent, a method using column chromatography is generally known.
In this method, a general method for introducing a sample into a column is to directly introduce a sample onto a column carrier or using a sample introduction device (injector). This is performed upstream with respect to the elution direction of the sample.

[0003]

In the above method, since the sample is introduced from the upstream of the column, when the same column is repeatedly used for separation / analysis of a sample containing insoluble matter such as serum which cannot pass through the column, Since a very small amount of insolubles in the sample clogs the column, the pressure in the column increases and the separation becomes poor. As a result, the life of the column is shortened. Therefore, generally, before introducing the sample,
Pretreatment such as filtration was performed to remove insolubles and separation was performed. However, the operation was complicated, and there was a problem that work efficiency was not improved.

In addition, when a sample introduction device is used, there is a problem that a very small amount of sample remains in the sample introduction device and causes contamination (carry over) of a sample to be analyzed next. . Therefore, after the sample is introduced, it is necessary to repeatedly wash the sample introduction path with a large amount of the washing liquid.

According to the present invention, a carry-over can be avoided even if the sample introduction section is not washed with a washing liquid, and even if a large number of liquid samples are separated many times using the same separation device, the separation device can be used. It is an object of the present invention to provide a method for separating a liquid sample component and a separation apparatus used in the method, which can prevent a phenomenon in which the internal pressure is increased or separation is deteriorated.

[0006]

According to the present invention, there is provided a separation method for separating a plurality of components in a liquid sample by utilizing a difference in adsorption power to an adsorbent. Inject into the separation device from the direction opposite to the elution direction of the separation device holding the reagent, and adsorb the other to the adsorbent under conditions where either the target substance or impurities affecting the measurement are almost completely eluted Then, the adsorbed substance is separated and eluted from the elution tip together with the eluent.

Further, the separation device of the present invention is provided with a separation device holding an adsorbent and one end of the separation device located on the elution side.
A liquid sample introduction section containing an insoluble matter that does not pass through the column, and a means for reducing or depressurizing and pressurizing the inside of the separation device connected to the other end of the separation device, wherein the adsorbent
Are the measurement target A and the impurity B
Adsorbent with a large difference in adsorption power
There is a feature.

In short, according to the present invention, a small amount of insoluble matter adheres to the adsorbent supporting portion at the lower end (elution tip) of the separation device by introducing the liquid sample into the separation device from the direction opposite to the elution direction. Since the substance is removed at the beginning of elution and does not remain in the separation device, it is possible to avoid the phenomenon that the pressure inside the separation device increases or separation is deteriorated even if the separation operation is performed many times. The gist of the present invention is that carry-over can be avoided since the sample introduced into the sample introduction path, even if it remains, can be removed by an elution operation.

[0009]

Next, an embodiment of the present invention will be described. In the method of the present invention, since the liquid sample is introduced into the separation device in a direction opposite to the elution direction, most of the insoluble matter in the liquid sample is first eluted by flowing the eluent by the principle of backwashing. It is eluted and removed together with the liquid. The components adsorbed on the adsorbent are separated by a difference in adsorption power.

As the adsorbent used in the present invention, it is preferable to use an adsorbent having a large difference in adsorption power between the object A to be measured and the impurity B (which may be plural) which adversely affects the measurement. For example, when separating the measurement target A and the impurity B, an adsorbent that adsorbs and elutes a measurable amount or more of A under a certain condition, but adsorbs and elutes only a B amount or less that does not affect the measurement. Or, A is the same as above, but for B, even if adsorbed more than the amount that does not affect the measurement, the adsorbent that elutes less than the amount that does not affect the measurement, and A hardly adsorbs , B may be selected from adsorbents that adsorb most of them and elute less than the amount that does not affect the measurement. The impurity B 'which does not adversely affect the measurement may or may not be separated from the measurement target A.

The most effective effect of the present invention is to select an adsorbent which is almost completely adsorbed on one of the object to be measured and the impurity under the condition that the other is eluted. This includes, for example, those having the following properties. The almost completely adsorbed component may be eluted according to the type of adsorption as described below. In this case, either the object to be measured or the impurity may be adsorbed. However, since the impurity generally contains various components, the object to be measured is preferably adsorbed.

(Ion exchange) It is used for separation between substances having ion differences. In this case, elution of the adsorbed substance changes the ionicity of the eluent (increases salt concentration) or
It may be performed by a method such as changing the pH. (Hydrophobic adsorption) Separation between substances having hydrophobic differences. The adsorbed substance is eluted by lowering the hydrophobicity of the eluent (reducing the salt concentration, for example, adding an organic solvent such as methanol, ethanol, acetonitrile, etc.).

(Specific affinity) Affinity-chromatography of antigen-antibody reactions, substrates and enzymes, receptors and receptor-reacting substances (eg, hormones), lectins and sugar chains, avidin (streptavidin) and biotin, etc. −
It is used for separation using the substance having specific adsorption used in the above. The elution of the adsorbed substance in this case is
What is necessary is just to carry out by the well-known method which dissociates the bond of specific adsorption.

The adsorbent used in the present invention is preferably
Examples of the adsorbent that can be adsorbed by the above-mentioned ion exchange, hydrophobic adsorption or specific affinity, such as an ion exchange resin, a hydrophobic carrier, hydroxyapatite, and an affinity carrier on which protein A is immobilized, are exemplified. In addition, the thickness of the adsorbent layer does not need to be particularly large, and a membrane or a filter paper having an ion exchange ability or the like can be used.

When serum, urine, or the like is used as a liquid sample, for example, it is preferable to dilute the sample with a reagent such as a buffer serving as a solvent and then introduce the diluted sample into a separation instrument in order to improve the separation. As the buffer used for such a purpose, a buffer usually used in this field may be used. In this case, when an antibody against the measurement object is added, the “measurement object” forms an immune complex of “measurement object + antibody”, so that the hydrophobicity changes and separation is facilitated by hydrophobic adsorption. Further, if an ionic group is added to this antibody, an immune complex of “measurement target + antibody + ionic group” is formed, so that the antibody is further easily separated by ion exchange.

Further, when a label is bound to an antibody other than the ionic group-bound antibody, and the labeled antibody and the ionic group-bound antibody are bound to the object to be measured, the ionic group and the label are introduced into the object to be measured. It will be. If a label that is not present in the biological component is used, the specificity of the detection of the measurement target can be improved. Further, according to the method of the present invention, the measurement target in the biological component can be effectively separated from free labeled antibodies and substances that may be contained in the measurement target and that hinder detection, thereby improving the accuracy of measurement. Can be improved.

FIG. 1 is a schematic view showing an embodiment of a separation apparatus used in the separation method of the present invention. A sample introduction tube is connected to an elution tip of a column which is a separation instrument, and the other end of the column is provided. Is connected to a pump for reducing and increasing the pressure in the column via a pipe.

The separation device (separation tube) used in the present invention comprises:
A sample introduction port (elution port) and an opening capable of depressurizing (suctioning) the inside of the separation instrument for sample introduction may be used as long as it can hold an adsorbent inside. There is no particular limitation. That is, for example, a so-called cylindrical column filled with an adsorbent, which is generally used in column chromatography, is generally used.For example, a sheet-shaped adsorbent such as filter paper or a membrane-shaped adsorbent is separated. The sample introduction part is located at one end of the separation device held in the outward bulge (for example, a disk-shaped bulge) formed in the tube, and the other end is connected to a decompression means or an opening in contact with the atmosphere. May be formed. In addition, a centrifugal filtration tube (made by Nippon Millipore Limited) or the like in which a film-shaped adsorbent is held can be used.

The separation device is usually used vertically so that the elution tip is at the lower end. However, when one of the measurement object and the impurity is almost completely adsorbed, the elution tip is horizontal even if the elution tip is at the upper end. No problem. In order to introduce a liquid sample from the elution tip of the separation instrument, as shown in FIG. 1, the pressure inside the separation instrument (column) is reduced by a pump, and the liquid sample is introduced into the separation instrument from the sample introduction tube into the elution direction. Sucks in the opposite direction. Even if the eluent is allowed to flow as it is, the elution tip and the sample introduction tube (sample introduction path) are washed with the eluent, so that carry-over can be avoided.

However, after aspirating the liquid sample, air,
It is preferable to aspirate a liquid or gas such as pure water or an eluent, preferably more than the capacity of the sample introduction path, and to flow the liquid sample attached to the suction flow path into the separation tube, because the measurement sensitivity is improved. preferable. That is, after the sample is introduced, almost all of the sample can be introduced into the adsorbent in the separation instrument by introducing a gas or liquid of a pipe volume or more from the sample introduction port to the adsorbent in the separation instrument. Sensitivity can be improved.

The liquid or gas used is not particularly limited as long as it does not adversely affect separation and measurement. When inhaling gas more than the bed amount of the separation device, it is preferable to select an adsorbent that does not lower the separation ability due to inhalation of the gas. When introducing a liquid, it is advisable to optimize the suction speed or the like in order to reduce the diffusion of the sample.

After the sample is adsorbed on the adsorbent as described above, the eluent is flowed from the upstream of the separation instrument toward the elution tip, that is, in the elution direction. As the eluent, those usually used in column chromatography may be used according to the kind of the adsorbent. The eluent may be introduced from the upstream of the separation device, that is, in the case of the apparatus shown in FIG. 1, into a pipe between the pump and the column (separation device) or from a sample introduction tube. When the sample is introduced from the sample introduction tube, it is possible to simultaneously introduce the sample adhered to the suction channel, and there is an advantage that the apparatus is simplified.

After the eluent is introduced into the column, the inside of the column is pressurized by a pump, and the eluent is eluted together with the sample components directly from the elution tip or through a sample introduction tube. The pump used in the present invention may be any pump that can suck air into the separation device and discharge the air.
There is no particular limitation.

When a pump is used, it is preferable to select an adsorbent such that the load on the pump is 30 kgf / cm ² or less under the selected conditions. By selecting such an adsorbent, the sample can be smoothly introduced from the sample introduction tube, and the separation and elution performed by pressurizing the inside of the separation instrument can be smoothly performed. .

The means for reducing and increasing the pressure may not be a pump. For example, like a syringe, a rod-shaped body may be closely fitted to an outer cylinder so as to be slidable, and a liquid sample may be sucked and discharged. In this case, the outer cylinder is
The separating device itself or another device different from the separating device may be used. Further, like a pipette, the cap may be expanded and sucked, and the cap may be crushed and discharged. In the present invention, the pressurizing means is not always necessary, and the above-described depressurizing and pressurizing means may be removed in a sucked state to discharge under the atmospheric pressure.

When separating impurities B and insolubles which adversely affect the measurement from the measurement target A in the liquid sample, for example,
This can be done as follows. The above A as an adsorbent
(Or B) using an adsorbent that adsorbs all or most of the liquid sample, inhaling a liquid sample from the elution tip, and then inhaling a predetermined amount of eluent from the elution tip that does not elute the adsorbed A (or B); Pressurize and flow the eluent in the elution direction,
Most of the insolubles and B (or A) are removed. Next, a predetermined amount of eluent for eluting the adsorbed A (or B) is sucked from the elution tip, and the eluent is flowed in the elution direction under pressure to elute A (or B) adsorbed on the adsorbent. You just have to take it out.

To elute A and insolubles together,
The eluate may be divided into a plurality of fractions to separate A, or the eluate may be filtered to separate A. Since the components adsorbed on the adsorbent as described above are all washed away, the above operation can be repeated for another liquid sample using the above-mentioned separation device.

In the above embodiment, the eluent is introduced from the elution tip, but may be introduced from the upstream of the separation device. In the above embodiment, the eluent is eluted by applying pressure. However, the eluent may be eluted by suction at normal pressure such as natural dropping, or by suction from the elution tip.

In the above embodiment, the sample introduction tube is connected to the elution tip. However, if the sample can be directly sucked due to the shape of the elution tip, the sample introduction tube may be omitted. In the above embodiment, the eluent is eluted by suctioning the liquid sample under reduced pressure and then applying pressure. The sample can be introduced with the elution tip of the separation instrument upward, and then the eluent can be injected from the upper end with the elution tip at the lower end and dropped, and can be carried out at normal pressure. The introduction of the liquid sample into the elution tip of the separation instrument may be performed by injecting the liquid sample into the elution tip of the separation tube using an instrument such as a syringe. Since the separation can be performed quickly, it is preferable to use the first embodiment.

The sample applied to the method and apparatus of the present invention may be a liquid sample generally used for column chromatography, and is particularly useful for separating biological components such as serum containing a trace amount of insoluble matter. It is.

[0031]

Next, the present invention will be further described with reference to examples and comparative examples, but the present invention is not limited to these examples. Example 1 (1) Preparation of reagents used Antibody solution Peroxidase-labeled anti-α-fetoprotein antibody Fab ′
Fragment (Fab'-POD, Wako Pure Chemical Industries, Ltd.)
36.2 μg / 10 ml and Fab′-PO
Anti-α-fetoprotein antibody confirmed to recognize an epitope different from D-sulfated tyrosine peptide [Ala
_{- (Thy (SO 3))} 8] bound Fab 'fragments (Fab'-YS8, manufactured by Wako Pure Chemical Industries (Ltd.)) 3.7μ
g / 10 ml containing 50 mM N- (2-
Acetamide) -2-aminoethanesulfonic acid buffer (p
H6.5) was prepared and used as an antibody solution.

Sample α-fetoprotein (AFP, Wako Pure Chemical Industries, Ltd.)
Was diluted with 50 mM N- (2-acetamide) -2-aminoethanesulfonic acid buffer (pH 6.5) to give 1
It was prepared to be 00 ng / milliliter and used as a sample. Substrate Solution A 10 mM citrate buffer (pH 6.5) containing 32 mM 4-acetamidophenol and 38 mM hydrogen peroxide was prepared and used as a substrate solution.

(2) Analysis Method 100 microliters of the above antibody solution and 10 microliters of the sample were mixed and reacted at 8 ° C. for 5 minutes. 80 microliters of this mixture was introduced into a POROS-DEAE column (0.46 × 1 cm, manufactured by Perceptive) by the following methods. Then, a 50 mM tris (hydroxymethyl) aminomethane buffer (pH 8.
After washing the column with 10 ml of 0,0.3 M sodium chloride, 50 mM Tris (hydroxymethyl) was added.
In 3 ml of aminomethane buffer (pH 8.0, containing 3M sodium chloride), Fab'-POD, AFP and Fa
The immune complex of b'-YS8 was eluted. To 2 ml of the obtained eluate, 200 μl of the above substrate solution was added, reacted at 37 ° C. for 10 minutes, and the fluorescence intensity was measured at an excitation wavelength of 328 nm and a fluorescence wavelength of 432 nm. FIG. 3 shows the obtained results. Eluent (buffer)
Were introduced from the pump side of the column.

(2) Sample Injection Method Comparative Example As shown in FIG. 2, a pump (syringe pump SIL10) was used.
A, manufactured by Shimadzu Corporation) with Injector (Model 7)
125 type, manufactured by RHEODYNE), POROS-DE
Using an apparatus connected in the order of an AE column (separation device), 80 microliters of a sample was introduced into the column by an injector. Invention Method-1) As shown in FIG. 1, POROS-
Directly connect DEAE column, volume 1 at column elution outlet
An apparatus with a sample introduction tube of 00 microliter was used. After aspirating 80 microliters of the sample from the sample introduction tube with a pump, 100 microliters of air was aspirated.

Inventive method-2) Using the same apparatus as described above, a sample of 80 microliters was sucked from the sample introduction tube by a pump, and then 200 microliters of air was sucked. Inventive Method-3) Using the same apparatus as above, 80 microliters of a sample was sucked from a sample introduction tube by a pump, and then 200 microliters of purified water was sucked.

(3) Results As is apparent from FIG. 3, the method of the present invention-2) and -3)
Is the same as that of the comparative example, but the fluorescence intensity obtained by the method 1) of the present invention is about 10% lower than that of the comparative example. From this, after sample introduction,
It can be seen that by sufficiently washing the introduction path, measurement sensitivity equivalent to that of the conventional method can be obtained.

Although the data are not shown, the method of the present invention was applied to α-phytoprotein solutions of various concentrations.
The fluorescence intensity was determined according to 1), -2) and -3), and α-
When a calibration curve representing the relationship between the phytoprotein concentration and the fluorescence intensity was prepared, a calibration curve having good linearity was obtained by any of the methods. From this, it can be seen that the method of the present invention-1) can accurately analyze the target component.

Example 2 100 microliters of a human plasma specimen sample in which turbidity was observed was introduced into the same POROS-DEAE column as in Example 1 by the following methods (introduction methods 1) and 2)), and a pump was used for each introduction. A 50 mM tris (hydroxymethyl) aminomethane buffer solution (pH 8.0, containing 3M sodium chloride) was introduced from the pump side, flowed through the column at a flow rate of 3 ml / min, and the pressure applied to the column was measured. FIG. 4 shows the results.

[Introduction Method 1]] (Comparative Example) As shown in FIG.
Using the apparatus used for comparison in Example 1 coupled in the order of the OS-DEAE column (separation device), 100 microliter of the sample was introduced by an injector, and 50 mM
Tris (hydroxymethyl) aminomethane buffer (pH
8.0 mL of 3M sodium chloride) was discharged.

[Introduction Method 2] (Method of the Present Invention) As shown in FIG. 1, a POROS-DEAE column was directly connected to a pump, and a sample introduction tube having a capacity of 100 microliters was connected to the column elution outlet. The same equipment was used. A sample of 100 microliters is sucked from a sample introduction tube by a pump, and 200 microliters of purified water is further sucked. Then, the sample is pressurized by a pump and a 50 mM tris (hydroxymethyl) aminomethane buffer solution (pH 8. 0, 3M sodium chloride) was discharged in an amount of 1 ml.

As is clear from FIG. 4, the pressure increases as the number of introductions increases in the introduction method 1) (conventional method) of the comparative example, but in the introduction method 2) of the method of the present invention, the pressure increases even if the number of introductions increases. Is hardly noticeable. From these results, it can be seen that according to the method of the present invention, even a sample containing insolubles can be directly introduced into a column (separation device), and the life of the column is significantly improved.

Example 3 (1) Antibody solution, sample and substrate solution Same as in Example 1. (2) Washing solution and eluent Washing solution: 50 mM tris (hydroxymethyl) aminomethane buffer, pH 8.0, containing 0.3 M sodium chloride. Eluent: 50 mM Tris (hydroxymethyl) aminomethane buffer, pH 8.0, containing 3M sodium chloride.

(3) Analysis method and results 100 μl of the antibody solution and 10 μl of the sample were mixed and reacted at 8 ° C. for 5 minutes. In addition, a 5 ml syringe is filled with a Sartobind ^™ Membrane Adsorber D5F.
D5F, abbreviated name MAD5F, manufactured by Sartorius Co., Ltd.), and an apparatus was prepared in which an eluate of the MAD5F was further connected to an introduction tube having a capacity of 100 microliters.

By pulling the syringe of the syringe,
80 microliters of the mixture is introduced into the introduction tube,
Next, 5 mL of the washing liquid was sucked into the MAD5F from the introduction tube by further pulling the syringe, and then the syringe was pushed to discharge the washing liquid, thereby washing the MAD5F. This washing operation was repeated three times.

Next, 3 ml of the eluent was similarly sucked and discharged, and the immune complex of Fab'-POD, AFP and Fab'-YS8 was eluted. To 2 ml of the obtained eluate, 200 μl of the substrate solution was added and reacted at 37 ° C. for 10 minutes, and the fluorescence intensity was measured at an excitation wavelength of 328 n.
m, at a fluorescence wavelength of 432 nm, a fluorescence intensity equivalent to that of the inventive method-3) of Example 1 was obtained. From the above, it can be seen that a similar result can be obtained even when an injection cylinder is used instead of the pump.

Example 4 Measuring Apparatus Apparatus configuration The apparatus configuration will be described with reference to FIGS. 5 to 7 and the piping system diagram of FIG. The reaction disk 1 is a turntable having a triple line configuration. The inner periphery is a sample container 2, the middle is an antigen-antibody reaction container 3 and an eluent A storage container 4,
The outer periphery is provided with an enzyme reaction cell 5. The sample container 2, the antigen-antibody reaction container 3, and the eluent A storage container 4 are kept cool at 8 ° C. by the cooler unit 27, and the temperature of the enzyme reaction cell 5 is controlled at 40 ° C. by the heater unit 28.

The dispensing unit having the first probe 6 includes the sample contained in the sample container 2 and the first reagent (R1).
8. The second reagent (R2) 9 and the third reagent (R3) 10 are sucked and dispensed into the reaction container. The first probe 6 includes a quantitative syringe pump 12 and a washing syringe pump 1.
The suction and discharge of the probe and cleaning are performed by the syringe pump. Note that the inside of this pipe is filled with pure water from the tank 23. Cleaning pot 7
Is used for cleaning the first probe 6. Further, the first probe 6 can also suck the additional sample 11 in the additional sample container installation section.

The dispensing unit having the second probe 14 is provided for aspirating the antigen-antibody reaction solution in the antigen-antibody reaction container 3 and introducing the solution into the column 18, washing with the eluent A20 and eluent A
Dispensing to the storage container 4, elution of the immune complex with the eluent B21 and / or eluent C22, and dispensing of the eluate to the enzyme reaction cell 5 are performed. This second probe 14
The syringe pump 17 for the column is connected via a pipe, and the syringe pump 17 performs each suction and discharge. This pipe is also filled with pure water from the tank 23. The multiple valve 19 is a flow path switching valve that changes the eluent A / B / C. The detergent 16 is used for cleaning the inside of the column and the second probe 14 piping. The washing pot 15 is used for washing the second probe 14 and discharging the eluate not used for analysis.

The detector 25 is a fluorescence meter for measuring the fluorescence intensity of the enzyme reaction cell 4. The detector 25 irradiates the reaction liquid with excitation light and measures the amount of fluorescence emitted from the reaction liquid by a photomultiplier (photomultiplier). Photomaru can adjust the gain in accordance with the amount of fluorescence.

The discharge nozzle 26 is a waste liquid processing section for draining the reaction liquid after the analysis by a pump (not shown). The waste liquid is stored in the drain tank 24. Pure water tank 23
Contains pure water used for cleaning each probe and piping.

The drain tank 24 stores each probe and a waste liquid of the reaction solution. The display unit 29, the keyboard 30, and the printer 31 are device control units for requesting analysis, displaying results, and starting operations.

2. Analysis Sequence The analysis sequence shows the flow of the operation of each unit of the apparatus when analyzing one sample. In other words, it indicates at what timing the sample / reagent is dispensed / reacted / metered / discharged in a series of analysis operations.

(1) In the case of one reagent and two eluents: As a pretreatment, an antigen-antibody reaction was performed with only the first reagent (R1), and as an analysis, an operation of eluting the immune complex with only the eluent B was performed. The analysis sequence in this case is shown in FIG.

1) Preparation A reagent, an eluent A / B, pure water, a detergent, and a column to be used for analysis are set. The sample container 2 is set on the inner periphery of the reaction disk 1 and kept cool. The antigen-antibody reaction container 3 and the eluent A storage container 4 are set on the middle circumference of the reaction disk 1 and kept cool.
The enzyme reaction cell 5 is set on the outer periphery of the reaction disk 1 and 40
Adjust the temperature to ° C. 1st probe 6, 2nd probe 14
Is cleaned with pure water.

2) Start after performing an analysis request operation in the device control unit. (A) The reaction disk 1 operates, and the eluent A storage container 4 moves to the access position of the second probe 14 [AD].
Next, the second probe 14 dispenses the eluent A into the eluent A storage container 4 [A]. (B) The reaction disk 1 operates and the sample container 2 moves to the access position of the first probe 6 [SD]. Next, the first probe 6 sucks the sample [S].

(C) The reaction disk 1 operates, and the antigen-antibody reaction container 2 moves to the access position of the first probe 6 [SD]. Next, the first probe 6 discharges the sample into the antigen-antibody reaction container 3 [S]. (D) The reaction disk 1 operates and the antigen-antibody reaction vessel 3
Moves to the access position of the first probe 6 [S
D].

(E) The first probe 6 has the first reagent (R
1) is aspirated and dispensed into the antigen-antibody reaction container 3 [R1]. (F) The antigen-antibody reaction starts and the reaction proceeds for about 4.5 minutes. (G) The reaction disk 1 operates and the antigen-antibody reaction vessel 3
Moves to the access position of the second probe 14 [I
D]. Next, the second probe 14 aspirates the antigen-antibody solution [I].

(H) The reaction disk 1 is operated and the eluent A
The storage container 4 moves to the access position of the second probe 14 [AD]. Next, the antigen-antibody solution is introduced into the column 18 while the second probe 14 aspirates the eluent A [I]. (I) The second probe 14 is moved to the access position of the washing pot 15, and the column 18 is washed with the eluent A [A
Liquid washing].

(J) The second probe 14 moves to the access position of the washing pot 15 and performs washing of the column with the eluent B [solution B elution]. (K) The reaction disk 1 operates and the enzyme reaction cell 5
To the access position of the probe 14
D]. Next, the second probe 14 performs elution and fractionation of the immune complex into the enzyme reaction cell 5 [fractionation of B fraction].

(L) The second probe 14 is moved to the access position of the washing pot 15, and the column is washed with the eluent B [solution B elution]. (M) The reaction disk 1 operates, and the enzyme reaction cell 5 moves to the access position of the first probe 6 [R3D]. Next, the first probe 6 sucks the reagent 3 and the enzyme reaction cell 5
[R3].

(N) At this point, the enzymatic reaction of the eluate starts. (O) The reaction disk 1 operates, and the enzyme reaction cell 5 moves to the detector access position [photometry D]. Next, the fluorescence intensity of the enzyme reaction cell 5 is measured [photometry]. (P) The fluorescence intensity is measured for about 10 minutes.

(Q) During the measurement of the fluorescence intensity, the second probe 14 moves to the access position of the washing pot 15 to wash the column 18 and the second probe 14 [column washing]. (R) As for each photometric data obtained from a plurality of gains by an apparatus data processing unit (not shown), data of a gain whose fluorescence intensity change falls within a measurable range is selected and calculated, and measurement in a sample is performed. Calculate the concentration of the object. at the same time,
The result is output to the display unit and the printer.

(S) The reaction disk 1 operates and moves to the discharge nozzle access position [reaction liquid discharge D]. (T) The analysis reaction end liquid is drained by a pump (not shown). Thus, a series of sample analyzing operations is completed.

In this analysis sequence, the operation of the first reagent (R1) can be changed to the operation of the second reagent (R2), and the operation of the eluent B can be changed to the operation of the eluent C. Further, the second reagent (R2) can be used during the operation of the first reagent (R1) and the eluent C can be used during the operation of the eluent B, and vice versa.

(2) Analysis sequence of 2 reagents and 3 eluents In the above analysis sequence, the first reagent (R
1) The operations of the second reagent (R2) and the eluents B and C can all be included. FIG. 11 shows an example of the analysis sequence.

In the present example, after dispensing the first reagent (R1),
8.9 minutes later, the second reagent (R2) was dispensed, and then 1
After 8.6 minutes, the reaction solution is introduced into the column 18. Then, after washing the eluent A solution, elution with the eluent B, eluent C
, The enzymatic reaction of each eluate is measured photometrically. The result data is calculated and calculated for the amount of the immune complex by the eluents B and C, respectively. Further, using the two result data, the total amount of the immune complex and each ratio can be calculated.

3. Operation Sequence A plurality of samples can be continuously analyzed by repeating the analysis sequence. However, by simply repeating the analysis sequence, only one analysis result is obtained for each time necessary for executing a series of analysis sequences. Therefore, in order to increase the processing capacity, it is general practice to operate the apparatus by further incorporating the operation of another analysis sequence into one analysis sequence. A sequence in which the operation of another analysis sequence is incorporated in one analysis sequence is called an operation sequence.

The operation sequence of the present apparatus is made based on the operation of the reaction disk unit and the time occupied by the operation of other units. The operation sequences shown in FIG. 10 and FIG. 12 may incorporate the operation of another analysis sequence to the maximum in one analysis sequence,
The time required to execute one operation sequence is 150 seconds. When a plurality of samples are continuously analyzed by repeating this operation sequence, an analysis result is obtained every 150 seconds. That is, by setting the operation sequence in this way, the processing capacity is increased 7 times in the case of the analysis sequence processing of one reagent and two eluents, and 20 times in the case of the analysis sequence processing of two reagents and three eluents. It will be.

4. Operation of Each Unit in Operation Sequence Details of the operation of each unit will be described below. (1) Operation of the reaction disk. (A) [AD] shows an operation of moving the eluent A storage container 4 to the access position of the second probe 14 in order to dispense the eluent A.

(B) The [ID] indicates that the antigen-antibody reaction container 3 is connected to the second probe 14 to aspirate the antigen-antibody reaction solution.
The operation of moving to the access position of FIG. (C) [Photometry] is for the enzyme reaction cell 5 to perform photometry.
Is moved to the detector 25 access position.
This photometric operation is repeated at the same cycle (every 30 seconds in this example).

(D) [SD] is used to aspirate the sample,
The operation of moving the sample container 2 to the access position of the first probe 6 and then moving the antigen-antibody reaction container 3 to the access position of the first probe 6 for discharging the sample will be described. (E) [R1D] indicates an operation of moving the antigen-antibody reaction container 3 to the access position of the first probe 6 in order to dispense the first reagent (R1).

(F) [B fraction dispensing D] and [C fraction dispensing
D] shows an operation of moving the enzyme reaction cell 5 to the access position of the second probe 14 in order to dispense the eluent B or the eluent C. (G) [R3D] indicates an operation of moving the enzyme reaction cell 5 to the access position of the first probe 6 in order to dispense the third reagent (R3).

(H) [R2D] shows the operation of moving the antigen-antibody reaction container 3 to the access position of the first probe 6 in order to dispense the second reagent (R2). (I) [Stirring D] indicates an operation of moving the antigen-antibody reaction container 3 to the access position of the first probe 6 to stir the liquid. Note that this operation is not selected in an analysis that does not require mixing of liquids. (J) [Reaction liquid drainage D] indicates an operation of moving the antigen-antibody reaction container 3 to the access position of the discharge nozzle 26 in order to drain the reaction liquid after the analysis.

(2) Operation of the first probe 6 (a) [S] moves to the access position of the sample container 2,
The operation of aspirating the sample, then moving to the access position of the antigen-antibody reaction container 3 and dispensing is shown. Also, an operation of moving to the access position of the cleaning pot 7 and performing probe cleaning by [S cleaning] will be described.

(B) [R1] shows an operation of moving to the access position of the first reagent (R1), aspirating the reagent, and then moving to the access position of the antigen-antibody reaction container 3 for dispensing. . [Agitation] indicates an operation of sucking and discharging the reaction liquid and mixing and stirring the liquid. Note that this operation is not selected in an analysis that does not require liquid mixing. Furthermore, [R1 cleaning]
The operation of moving to the access position of the cleaning pot 7 and performing probe cleaning will be described.

(C) [R2] indicates an operation of moving to the access position of the second reagent (R2), aspirating the reagent, and then moving to the access position of the antigen-antibody reaction container 3 for dispensing. . [Agitation] indicates an operation of sucking and discharging the reaction liquid and mixing and stirring the liquid. However, this operation is not selected in an analysis that does not require mixing of liquids. Furthermore, [R2 cleaning]
Indicates an operation of moving to the access position of the cleaning pot 7 and performing probe cleaning.

(D) [R3] indicates an operation of moving to the access position of the third reagent (R3), aspirating the reagent, and then moving to the access position of the enzyme reaction cell 5 for dispensing. [R3 cleaning] indicates an operation of moving to the access position of the cleaning pot 7 and performing probe cleaning.

(3) Operation of the second probe 14 (a) [A] moves to the access position of the washing pot 15 and elutes the inside of the pipe by sucking and discharging the eluent A by the column syringe pump 17. Replace with liquid A, and then move to the access position of eluent A storage container position 4, where eluent A
Shows the operation of dispensing.

(B) [I] moves to the access position of the antigen-antibody reaction container 3, aspirates the antigen-antibody reaction solution, then moves to the access position of the eluent A storage container 4, and then moves to the eluent A The operation of introducing the antigen-antibody reaction solution into the column 18 while aspirating is shown. (C) [Washing of solution A] indicates an operation of moving to the access position of the washing pot 15 and repeating the suction and discharge of the eluent A by the column syringe pump 17, thereby washing the column with the eluent A.

(D) [solution B elution] is performed by moving to the access position of the washing pot 15 and repeating the suction and discharge of the eluent B by the column syringe pump 17, whereby the immunosorbent adsorbed on the column by the eluent B is used. The operation of eluting the complex is shown. [B fraction dispensing] indicates an operation of moving to the access position of the enzyme reaction cell 5 and discharging the eluate. (E) [C solution elution] is to move to the access position of the washing pot 15 and repeat the suction and discharge of the eluent C by the column syringe pump 17 to elute the immune complex bound to the column with the eluent C. The operation to be performed will be described. [C fraction dispensing] indicates an operation of moving to the access position of the enzyme reaction cell 5 and discharging the eluate.

(F) [Column washing] is the washing pot 15
To the access position, and the column syringe pump 17
After the column 18 and the second probe 14 are washed by repeating the suction and discharge of pure water in step 1, the cleaner 16 is moved to the access position of the detergent 16, the detergent 16 is sucked and introduced to the column 18, and then the washing pot 15 Then, the operation is performed in which the column syringe pump 17 repeats the suction and discharge of pure water to wash the detergent.

(4) Operation of Discharge Nozzle 26 (a) In [a reaction liquid drainage], a discharge nozzle is inserted into the antigen-antibody reaction container 3 that has moved to the access position of the discharge nozzle 26, and drainage of the reaction liquid after the analysis is completed. The operation to be performed will be described.

(5) Operation of Detector 25 (a) [photometry] indicates an operation of performing fluorescence photometry of the enzyme reaction cell 5 that has moved to the access position of the detector 25.
This [photometry] is defined as one continuous photometry of the enzyme reaction cell 5 during the enzyme reaction, and is repeated five times (in this example, every 30 seconds) in one cycle at the same interval.

5. Note The operation time, reaction time, number of sample containers and various reaction containers, suction and discharge amount of each solution, set temperature, analysis sequence, operation sequence, etc. of each unit in this description are not limited to this content. Of course, various changes can be made.

Example 5 Measurement of AFP (1) Apparatus used The apparatus of Example 4 was used.

(2) Preparation of Reagents Used Antibody Solution The same antibody solution as in Example 1 was used.
Was set at the position of the reagent (R1). Samples Human serum with AFP concentration of 420 ng / ml in physiological saline 1/10, 2/1
The sample was diluted by a factor of 0, 3/10, 4/10, 5/10, 6/10, 7/10, 8/10, or 9/10 and used as a sample. In addition, a standard product (manufactured by Wako Pure Chemical Industries, Ltd.) having an AFP concentration of 100 ng / ml was used as a standard solution.

Substrate Solution The same substrate solution as in Example 1 was used.
Was set at the position of the reagent (R3). Eluent Eluent A: 50 mM Tris (hydroxymethyl) aminomethane buffer (pH 8.0, containing 0.3 M sodium chloride) Eluent B: 50 mM Tris (hydroxymethyl) aminomethane buffer (pH 8.0, 3.0 M sodium chloride) (Contains) These two were used as eluents and set at the positions of eluent A and eluent B in the apparatus of Example 4, respectively.

(3) Analytical Method Using the analytical sequence 1 (one reagent, two eluents) of Example 4, the analysis was performed under the following analytical conditions, and the change in the fluorescence intensity per minute was measured for each sample. The AFP concentration in each sample was calculated from a comparison between the obtained measured value and the measured value of the AFP standard solution. The measurement was performed five times for each sample.

Reagent 1: 100 μl Sample: 3 μl Antigen-antibody reaction solution Column introduction volume: 20 μl Base mass: 100 μl Fractionation volume: 1 ml Column washing volume: 18 ml Column: POROSE-DEAE column (5.5 × 6.9 mm)

Results The relationship between the dilution ratio and the measured value is shown in FIG. As is clear from FIG. 13, it is understood that a good linear relationship passes through the origin. The coefficient of variation of the measured value of each sample was 0.8% to 6.0%, which was a good value.

Example 6: Separation measurement of AFPs having different sugar chain structures (1) Apparatus used The apparatus of Example 4 was used.

(2) Preparation of Reagents Used First Reagent Lentil bean lectin (LCA, manufactured by Honen Corporation), 10 mg / 10 ml, and a sulfated tyrosine peptide having five sulfate residues [ Ala- (Tyr (SO ₃ ) ₅ )]
Was bound to an anti-α-fetoprotein antibody Fab ′ fragment (Fab′-YS5, manufactured by Wako Pure Chemical Industries, Ltd.) at 1.75 nmol / 1.
50 mM N- (2-acetamide) containing 0 ml
-2-aminoethanesulfonic acid (ACES) buffer (pH 6.
5) was prepared, and this was used as the first reagent, and set at the position of the first reagent (R1) in the apparatus of Example 4.

Second Reagent A peroxidase-labeled anti-α-fetoprotein antibody Fab ′ fragment (Fab′-POD, manufactured by Wako Pure Chemical Industries, Ltd.), 402 pmol / 1 mm, in which the recognition site was different from that of Fab′-YS5 Litter and Fab'-YS5, a sulfated tyrosine peptide having eight sulfate residues in the Fab 'fragment of the anti-α-fetoprotein antibody confirmed to have a different recognition site from Fab'-POD [Ala- (Tyr (SO ₃ ) A 50 mM ACES buffer (pH 6.5) containing 72 pmol / 1 milliliter (Fab'-YS8, manufactured by Wako Pure Chemical Industries, Ltd.) to which ₈ )] was bound, was prepared. The second reagent was set at the position of the second reagent (R2) in the apparatus of Example 4.

The antibody used for producing Fab′-YS8 has a property of binding only to AFP to which LCA is not bound. In contrast, Fab'-YS5 and Fab'-YS5
The antibody used to produce POD has the property of binding to all AFPs regardless of the presence or absence of LCA binding.

Samples Human serum having an AFP concentration of 690 ng / ml and an AFP-L3 fraction ratio (%) of 46% was diluted with physiological saline to 1/10, 2/10, 3/10, 4/10, 5/1.
Samples were diluted 0, 6/10, 7/10, 8/10, 9/10 times. In addition, at a AFP concentration of 200 ng / ml, a standard product having an AFP-L3 fractionation ratio (%) of 0%, and at an AFP concentration of 200 ng / ml, AF
A calibration curve was prepared using a standard product having a P-L3 fraction ratio (%) of 100% (all manufactured by Wako Pure Chemical Industries, Ltd.) as a standard solution. Substrate solution The same substrate solution as in Example 1 was used.
Was set at the position of the reagent (R3).

Eluent Eluent A: 50 mM Tris (hydroxymethyl) aminomethane buffer (pH 8.0, containing 0.3 M sodium chloride) Eluent B: 50 mM Tris (hydroxymethyl) aminomethane buffer (pH 8.0, 0.78) Eluent C: 50 mM Tris (hydroxymethyl) aminomethane buffer (pH 8.0, containing 3.0 M sodium chloride) These three were used as eluents, each of which was eluent A of the apparatus of Example 4. , Eluent B and eluent C.

(3) Analytical method Using the analytical sequence 2 of Example 4 (two reagents, three eluents), analysis was performed under the following analytical conditions. The immune complex 1 (Fab'-POD-AFP-Fab'-YS5) was eluted from the column with the eluent B, and the immune complex 2 (Fab'-POD-AFP-Fa
b'-YS8-Fab'YS5) is eluted from the column. 1 for each of the obtained immunocomplex 1 fraction and immunocomplex 2 fraction
The change in the fluorescence intensity per minute was measured, and the AFP concentration in each sample was calculated from the total of the obtained measured values and the measured value of the AFP standard solution.

Further, the measured value for the immunocomplex 1 fraction and the measured value for the immunocomplex 2 fraction were substituted into the following equation to determine the fractionation ratio (%) of each sample. The AFP-L3 in each sample was fitted to a calibration curve prepared using the fractionation ratio obtained in the same manner using a standard solution having an L3 fractionation ratio of 0% and an AFP standard solution having an L3 fractionation ratio of 100%. Fractionation ratio (%)
I asked.

Fractionation ratio (%) = measured value of one immunocomplex fraction / (measured value of one immunocomplex fraction + measured value of two immunocomplex fractions)

First reagent: 100 μl Second reagent: 10 μl Sample: 10 μl Amount introduced into the column of antigen-antibody reaction solution: 80 μl Base mass: 100 μl 1 fraction of immunocomplex: 1 ml 2 fraction of immunocomplex: 1 ml Column wash volume: 18 ml Column: POROSE-DEAE column (5.5 x 6.9 mm)

Results The relationship between the dilution ratio and the measured value is shown in FIG. As is clear from FIG. 14, it is understood that the AFP concentration has a good linear relationship passing through the origin. Also, it can be seen that the AFP-L3 fraction ratio (%) does not change even when the sample is diluted. This is because the ratio of AFP that reacts with a specific lectin (has a specific sugar chain structure) does not change even when the sample is diluted.

[0102]

According to the present invention, it is possible to avoid a phenomenon in which the pressure in the separation device increases or the separation is deteriorated even if the separation is performed many times, so that the life in the separation device is remarkably improved and the carry is improved. The present invention solves the problem that the conventional technique of preventing over-generation does not occur, and is therefore an extremely innovative invention. In the case of elution under pressure using a pump, according to the present invention, since the sample is introduced from the free end of the elution tip, there is obtained an advantage that a special sample introduction device is not required.

[0103]

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a separation device of the present invention.

FIG. 2 is a schematic diagram showing a conventional separation device.

FIG. 3 is a bar graph showing the measurement results of the fluorescence intensity of Example 1.

FIG. 4 is a line graph showing the measurement results of fluctuations in column pressure in Example 2.

FIG. 5 is a partially cutaway plan view showing an embodiment of the separation device of the present invention.

FIG. 6 is a front view showing an embodiment of the separation device of the present invention.

FIG. 7 is a side view showing an embodiment of the separation device of the present invention.

FIG. 8 is a piping diagram showing an embodiment of the separation device of the present invention.

FIG. 9 shows an analysis sequence of the separation device of the present invention in the case of one reagent and two eluents.

FIG. 10 shows an operation sequence of the separation device of the present invention in the case of one reagent and two eluents.

FIG. 11 shows an analysis sequence of the separation device of the present invention in the case of two reagents and three eluents.

FIG. 12 shows an operation sequence of the separation device of the present invention in the case of two reagents and three eluents.

FIG. 13 is a calibration curve diagram showing a relationship between a dilution factor and an AFP concentration measurement value according to Example 5.

FIG. 14 is a calibration diagram showing the relationship between the dilution factor and the measured AFP concentration, and the dilution factor and AFP-L3 according to Example 6.
FIG. 4 is a calibration diagram showing a relationship with a fractionation ratio (%).

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Reaction disk 2 Sample container 3 Antigen-antibody reaction container 4 Eluent A storage container 5 Enzyme reaction cell 6 First probe 7 Washing pot 8 First reagent R1 (antibody solution) 9 Second reagent R2 (antibody solution) 10 Third reagent R3 (fluorescent substrate solution) 11 Additional sample 12 Quantitative syringe pump 13 Washing syringe pump 14 Second probe 15 Washing pot 16 Detergent 17 Column syringe pump 18 Column 19 Multiple valve 20 Eluent A 21 Eluent B 22 Eluent C 23 Pure water tank 24 Drain tank 25 Detector 26 Discharge nozzle 27 Cooler unit 28 Heater unit 29 Indicator 30 Keyboard 31 Printer

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masaka Hayashi 1 at Nishinokyo Kuwaharacho, Nakagyo-ku, Kyoto-shi, Kyoto Inside Shimadzu Sanjo Works Co., Ltd. (72) Inventor Hideka Arashima 1 Nishinokyo-kuwaharacho, Nakagyo-ku, Kyoto, Kyoto Stock Shimadzu Corporation Sanjo Plant (72) Inventor Tetsushi Okuyama 1 Kyoto Nishi-no-Kyowa-cho, Nakagyo-ku, Kyoto, Japan Shimadzu Sanjo Plant (72) Inventor Nobuhiro Hanabusa 1-Nishi-no-Kyowabara-cho, Nakagyo-ku, Kyoto, Kyoto Shimazu, Ltd. Inside the Sanjo Plant of the Works (72) Koji Tanimizu, Inventor 1 at the Kuwaharacho Nishinokyo-ku, Nakagyo-ku, Kyoto City, Kyoto Prefecture Inside the Sanjo Plant of Shimadzu Corporation (56) References JP-A-63-21551 (JP, A) JP-A-7-209272 (JP, A) JP-A-5-188047 (JP, A) JP-A 8-313510 (JP, A) JP-A JP-A-60-207054 (JP, A) JP-A-57-28253 (JP, A) JP-A-1-120903 (JP, U) JP-A-1-120904 (JP, U) (58) Fields investigated (Int) .Cl. ⁷ , DB name) G01N 30/40 G01N 30/00

Claims (14)

(57) [Claims] 1. A method for separating a plurality of components in a liquid sample containing insolubles that cannot pass through a column by utilizing a difference in adsorption power to an adsorbent, wherein the liquid sample is separated from the liquid sample by holding an adsorbent. Introduced into the separation device from the direction opposite to the elution direction of
Most of the detrimental impurities are eluted by the other
Adsorbed on the adsorbent under the following conditions, then the adsorbed substance
The method of separating a liquid sample components, characterized in that for the separate elution from the elution front end with eluent. 2. The separation method according to claim 1, wherein the liquid sample is sucked and introduced into the separation device. 3. The separation method according to claim 1, wherein the eluent is sucked and introduced into the separation device from a direction opposite to the elution direction of the separation device. 4. The separation method according to claim 2, wherein after sucking the liquid sample, a gas or a liquid is sucked, and the liquid sample attached to the suction channel flows into the separation device. 5. A pressurized in said separation instrument elution direction, the method of separation according to any one of claims 1 to 4 for separation elution of a liquid sample the adsorbed. 6. A and adsorption to the adsorbent of the liquid sample eluted and, using the same said separation instrument, method of separation according to any one of claims 1 to 5, repeated for another fluid sample . Wherein said separation instrument holding the adsorbent, a method of separating according to any one of claims 1 to 6, which is a column filled with an adsorbent. 8. The separation device holding the adsorbent is a separation device holding a sheet-like or film-like adsorbent in a separation tube or an outwardly bulging portion formed in the separation tube. the method of separation according to any one of 1-6. 9. A separation device holding an adsorbent, and an insoluble matter that cannot pass through a column located at an elution side at one end of the separation device .
A liquid sample introducing part including the separation other end connected isolation instrument instrument and means for pressurizing reduced pressure or vacuum and pressure, wherein the adsorbent given an adverse effect on the measurement and the measurement object A
Of the adsorbing power for the impurity B
An apparatus for separating components in a liquid sample by using a difference in adsorption power with respect to an adsorbent, which is characterized in that the adsorbent has a large difference . 10. The adsorbent having a large difference in the adsorbing power,
When separating the measurement target A and the impurity B, under certain conditions,
Adsorbs and elutes more than the measurable amount of A, but affects the measurement
An adsorbent that adsorbs and elutes only B in an amount that does not give
A is the same as above, but B is measured
Even if the adsorption is larger than the amount that does not affect the measurement, it will affect the measurement.
About adsorbent that elutes less than or less than
Hardly adsorbs, but adsorbs most of B and
Adsorbent that elutes less than the amount that does not affect the measurement
The device according to claim 9, which is: Is wherein said separating device in a reduced pressure or vacuum and pressurizing means, air in the separation device - obtained was aspirated and the air - device of claim 9 or 10 is a pump capable of ejecting . 12. A load applied to the pump is 30 kgf.
/ Cm ² or less, wherein the adsorbent is selected to be not more than / cm ^2.
An apparatus according to claim 11 . 13. The separation device which holds the adsorbent, any one of claims 9 to 12, which is a column filled with an adsorbent
The device according to item . 14. The separation device holding the adsorbent is a separation device holding a sheet-like or film-like adsorbent in a separation tube or an outwardly bulging portion formed in the separation tube. apparatus according to any one of 9-13.