JPS60241898A - Determination of biological components with immobilized enzymes - Google Patents

Abstract

PURPOSE:Biological components in a small amounts of a solution is passed through a column of immobilized dehydrogenase and a column of immobilized oxidoreductase to measure the products whereby rapid and accurate determinations become possible. CONSTITUTION:A sample solution containing biological components is passed through a column filled with immobilized dehydrogenase and the effluent is passed through a column filled with immobilized oxidoreductase. Then, the effluent is subjected to measurement. In the meantime, the sample solution is passed through the control column filled with only the support without dehydrogenase and the effluent is passed through the column of immobilized oxidoreductase and subjected to measurement. The difference between these measurement is calculated to determine biological components accurately. When the biological component is steroid, the dehydrogenase is preferably hydroxysteroid- dehydrogenase and the oxidoreductase is preferably diaphorase.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a method for quantifying biological components using immobilized enzymes.

(Prior Art) When quantifying the biological component 5, such as bile acid present in trace amounts in serum, it may be difficult to directly detect the biological component with a detector such as a fluorophotometer. In such cases, a method is known in which the biological component and the reaction component are subjected to an enzymatic reaction in the presence of an enzyme and the resulting product is measured. One such method is to introduce a sample solution containing biological components and reaction components into a column packed with a carrier on which enzymes are immobilized (immobilized enzyme column), and detect the resulting products with a detector. There is.

For example, US Pat. No. 4,153,513 discloses a method for quantifying biological components contained in serum. According to this, macromolecular components that can interfere with measurement must be removed from a sample solution containing biological components using a dialyzer in advance. Moreover, this sample solution undergoes an enzymatic reaction in the immobilized enzyme column, is measured by a colorimeter, and is discharged out of the system; It can pass through two types of yarn paths: a yarn path for blanks that is discharged to the outside of the system. in this way,
Along with a dialysis machine being essential.

Unless the dialysis and the enzyme reaction within the immobilized enzyme column enter a steady state and the values detected by the detector become stable, biological components cannot be accurately measured. Therefore, it takes time to quantify biological components and requires a large amount of sample solution. Furthermore, no matter which thread path is switched to -h, the sample solution must pass through the immobilized enzyme column, so the immobilized enzyme is quickly deactivated by biological components contained in the sample solution.

Another method for quantifying biological components by measuring enzymatic reaction products is, for example, a method for quantifying hydroxy acids in biological components (Japanese Patent Laid-Open Nos. 56-144096 and 151499-1982). Publication No.).

Hydroxy acid is a steroid hormone containing hydroditosic acid that is contained in serum, etc. To quantify this hydroxy acid, first, the hydroxy acid and nicotinamide adenine dinucleotide (NAD') as a coenzyme are combined with the hydroxy steroid. Dehydrogenase (II SO
). NAD" is reduced to reduced nicotinamide adenine dinucleotide (NADll)
becomes. When this NADH is reacted with a tetrazolium salt in the presence of diaphorase, NADH is oxidized again and N
AD” and at the same time formazan is produced.The resulting formazan is measured colorimetrically at 540 nm.In this way, 5
A quantification of the hydroxy acid is made. Among the above-mentioned publications, JP-A-56-151499 describes that non-steroidal hydroxy dehydrogenase is inactivated by adding an inhibitor such as oxamic acid or pyruvic acid. Non-steroidal dehydrogenases include lactate dehydrogenase (LDI+), which is present in serum. Above H3
Reactions involving D5 typically take about 10 minutes, during which time nonsteroidal hydroxy dehydrogenase reacts with NAD'' to form NADll. The amount of NADll produced is offset by subtracting the blank value. However, since the blank value is high, the measurement error becomes large.Therefore, it is necessary to add an inhibitor to inactivate it.

In such a method, it is time-consuming to add the inhibitor; the expensive 115D is used for only one measurement and is disposable, so the measurement reagents to be supplied are expensive. There are drawbacks such as.

On the other hand, in Japanese Patent Publication No. 56-39+99, bile acids contained in a sample solution are quantified by using resazurin in place of the tetrazolium salt described in 1. and measuring the fluorescence of the resulting resorufin. In this method, measurements are performed by reducing the amount of expensive H2O used and increasing the reaction time to several tens of minutes. Nonsteroidal hydroxy dehydrogenase can also be inactivated by heating the sample solution to about 37°C during the reaction. However, this method also requires heating; takes a long time to measure; requires an expensive fluorometer. It has drawbacks such as:

(Objective of the Invention) An object of the present invention is to provide a method for accurately measuring biological components in a small amount of sample solution in a short time using enzymes. Another object of the present invention is to provide a method for quantifying biological components that uses an immobilized enzyme and can be used for a long period of time without being deactivated. Still another object of the present invention is to provide a method for accurately measuring biological components without using an i3 analyzer and without being affected by interfering components. Still another object of the present invention is to
The object of the present invention is to provide a method for accurately measuring biological components in a Ne81 solution in a single test in a short time without using special inhibitors or processing methods.

(Structure of the Invention) In the present invention, a sample solution containing a biological component and a reaction component that can react with the biological component is passed through a column in which no enzyme is immobilized to measure its blank value, while the same amount of the same sample solution is immobilized. This method was completed based on the inventor's knowledge that biological components can be quantified with high precision from the measured values and blank values by passing the enzyme reaction product through an enzyme column and measuring the enzymatic reaction products generated therein. Therefore, the method for quantifying biological components using the immobilized enzyme of the present invention is (1
(1) A step of introducing a certain amount of sample solution containing biological components into one or more immobilized dehydrogenase columns packed with immobilized dehydrogenase, (2) Dehydrogenase flowing out from the immobilized dehydrogenase column. a step of introducing a sample solution containing a reaction product to an immobilized oxidoreductase column filled with an immobilized oxidoreductase; (3) a sample solution containing a oxidoreductase reaction product flowing out from the oxidoreductase column; (4) A control column filled only with the carrier on which the dehydrogenase is not supported is subjected to the step (1).
A step of introducing a sample solution of the same kind and amount as the sample solution of (
5) a step of guiding the sample solution flowing out from the control column to the immobilized oxidoreductase column; (6) a step of measuring the sample solution flowing out from the oxidoreductase column.

and (7) calculating the difference in measured values between the sample solution that passes through the dehydrogenase column and flows out from the oxidoreductase column and the sample solution that passes through the control column and flows out from the oxidoreductase column. Process.

, thereby achieving the above objective.

Next, the quantitative method of the present invention is embodied by, for example, the quantitative device shown in FIG.

This device is shown in 3 Figures 1 and 2.

A buffer tank 1 containing a buffer solution is connected to a syringe 3 via a constant flow pump 2. The syringe 3 is connected via a six-way valve 4 to a control column 5 and an immobilized dehydrogenase column 6. These columns 5 and 6 are further connected to an immobilized oxidoreductase column 7 via this six-way valve 4. The immobilized oxidoreductase column 7 is connected to a visible absorption photometer 8 as a detector. The visible absorption photometer 8 is connected to a recorder 9, and the measured value is displayed.

The above buffer contains a coenzyme, which is a reaction component that can react with the biological component to be measured through the action of dehydrogenase; Contains reactive components. For example, when a bile acid is placed in place, the coenzyme corresponds to NAD'', and the component capable of reacting with the reduced coenzyme corresponds to a tetrazolium salt.

This buffer is supplied to columns 5 and 6 by constant flow pump 2. A sample containing biological components is prepared into a sample solution using the buffer solution supplied to the syringe 3.

Columns 5 and 6 are also fed. The immobilized dehydrogenase column 6 is filled with a desired dehydrogenase immobilized on a suitable carrier. For example, when quantifying 3α-bile acids, 3α-hydroxysteroid dehydrogenase (3α-11sD) is used as the dehydrogenase. Due to the catalytic action of 3α-11sD, 3α-bile acid reacts with NAD', which is present in large excess, and NAD' is reduced to become NA.The number of moles of NAD11 produced is 3α.
-corresponds to the number of moles of bile acids. The sample Fl solution that has passed through columns 5 and 6 is supplied to immobilized oxidoreductase column 7. When measuring hydroxysteroids such as llj acid, diaphorase is immobilized on column 7.

N A D II contained in the sample solution and a large excess of tetrazolium salt react with the catalytic action of diaphorase,
NADll is oxidized to IIAD' and the tetrazolium salt becomes formazan. The number of moles of formazan produced corresponds to NADll. In other words, it corresponds to the number of moles of 3α-bile acid. The formazan contained in the sample solution is derived from the NA produced by the above reaction and NAD' and NMDll that are present from the beginning in a sample such as serum. This formazan is measured by a visible absorption photometer 8 and displayed on a recorder 9. 3α-bile acids can also be quantified by directly measuring the amount of NADH with 8I fluorescence.

However, in this method, fluorescent substances such as bilirubin and albumin contained in the sample solution are also measured at the same time, resulting in a very high blank value. Therefore, the target biological component cannot be quantified accurately. In the method of the present invention, a visible absorption photometer is used instead of an expensive fluorescence photometer, so biological components can be measured at low cost. The sample solution after measurement is led to the waste liquid tank IO. According to the quantitative method of the present invention, the time required for measurement is as short as about 2 minutes or less. Therefore, the measurement is performed with almost no influence of non-steroidal hydroxy dehydrogenase contained in the sample such as serum, so the blank value is small and accurate quantification can be performed.

In particular, the material of the carrier packed in each column is 5til.
It is preferable to use a substance such as cellulose, which does not cause any interaction with the substance in the sample solution, since the measurement time can be shortened.

When measuring a plurality of biological components contained in a sample solution, by selecting an appropriate carrier that has the opposite interaction, it becomes possible to elute the plurality of components in fractions.

The control column 5 is filled with only a carrier that does not support an enzyme. By switching the six-way valve 4 and guiding the sample solution to this control column 5 instead of the immobilized dehydrogenase column 6, it is possible to know the blank value of the absorbance of the sample solution. The blank peak recorded on the recorder 9 is based on the original light intensity of the sample solution and the absorbance of formazan derived from NAD and NADH that are originally present in the sample solution. The value obtained by subtracting the peak area of the recorder 9 obtained via the control column 5 from the peak area of the recorder 9 obtained corresponds to formazan resulting from the enzyme reaction in the immobilized oxidoreductase column 7. This value can be applied to a pre-prepared 2 calibration curve to obtain the constant I value of the desired biological component, in this case 3α-bile acid.
, there is no particular 1β order when leading to 6.

The immobilized dehydrogenase column 6 may come first or the control column 5 may come first.

When measuring two components present in the same biological sample, two immobilized dehydrogenase columns are provided in parallel, as shown in FIGS. 3 to 5. For example, when quantifying each of 3α-bile acids and 3β-bile acids among bile acids contained in serum, as shown in FIG. An immobilized dehydrogenase column 22 packed with and 3β-H2O is also installed. Column 15 is a control column. Six-way valve 14
The sample solution is prepared by sequentially switching between 1 and 21.

As shown in FIGS. 4 and 5, column 15,
16 and 22. In this way, a plurality of biological components, in this case 3α-bile acids and 3β-bile acids, can be sequentially quantified. If you follow this.

Measuring three or more components in a sample solution3 Both are possible.

The control column in the present invention performs the following functions.

For example, when the sample is serum, oil ingested through meals may be suspended in the serum as oil droplets. When such a sample solution is introduced into the measurement system without passing through the control column, the sample solution passes only through the immobilized oxidoreductase column and reaches the detector. On the other hand, if the sample solution is guided to the immobilized dehydrogenase column and then to the immobilized oxidoreductase column, the sample solution will pass through the two columns and reach the detector. Each column is filled with a packing material having a diameter of several tens of μm. Oil droplets in the sample solution.

As it passes through the filler, which has a diameter of several tens of micrometers, it breaks down and becomes oil droplets with a small diameter. The size of the oil droplets differs between the sample solution that does not pass through the control column and the sample solution that passes through the control column. The size of these oil droplets causes errors when performing colorimetric determination.

Blank 4 if no control column is provided During measurement, the sample solution passes only through the immobilized oxidoreductase column and enters the detector, so the sample solution passes through the detector cell within 1 minute after sample injection.

On the other hand, when measuring through an immobilized dehydrogenase column and an immobilized oxidoreductase column, the sample solution diffuses more between the columns, so the passage time for the same amount of sample solution is about twice the above time or more. be. As a result.

Regarding the respective peaks recorded in the record 81, the former is a relatively sharp peak, and the latter is a gentle peak.

Since the size of the peak is proportional to the amount of biocomponents contained, the areas of both peaks are essentially the same regardless of the shape, except for the portion contributed by the enzyme reaction. However, when the peak area is measured with an integrating needle using a microcomputer, it is 1.
Since the shapes of the two peaks are significantly different, it becomes difficult to calculate the areas, which should originally be equal, to be equal. This is because when the microcomputer recognizes the starting point and ending point of a peak, there is too much difference in the scent between the two peaks, resulting in a reading error. It is difficult to create a program to accurately measure such peak areas. For these reasons, it can be understood that the control column for measuring blank values plays an important role. Because this column exists,
Biological components can be accurately measured even in milky specimens.

There are no differences in peak shape due to differences in detector transit time, and there are no errors in reading by the microcomputer (
Become.

The method of the present invention can be widely used for quantifying various steroids in addition to bile acids.

(Example) The present invention will be explained below using examples.

5 m/ml of cellulose particles having a particle diameter of about 80 μm were used as a carrier, and 5 m/IQ ml of a 2M sodium carbonate aqueous solution of ion-exchanged water was added thereto and stirred.

1 m7 of acetonitrile with 2 g of cyanide bromide dissolved in advance! was added and reacted for 90 seconds with vigorous stirring. The cellulose particles activated in this way were quickly dissolved in <0.1M carbonate buffer (pH 9.5).
, ion-exchanged water, and 0.1M carbonate buffer (pH 9.5) containing 0.5M sodium chloride]6 ram. 3α-hydroxysteroid dehydrogenase (3α-11sD) derived from a bacterium of the genus Pseudomonas testosterone 2 was dissolved in O.1M carbonate buffer containing 0.5M sodium chloride (pit 9.5
) 5 ml was added and stirred at room temperature for 2 hours to complete the reaction, and 3α-11SD was immobilized on the carrier. In order to block the active sites still present on the surface of the carrier on which 3α-SD was immobilized, 0.1M) Lis-HCl buffer (pH 8.0) containing 0.05% 2-mercaptoethanol was added. The reaction was carried out at 4°C for 2 hours.

The dehydrogenase immobilized carrier obtained in this way was
0.1 M acetate buffer (pH 5) containing M sodium chloride
, 0), ion-exchanged water, O, 1M carbonate buffer containing 0.5M sodium chloride (pH 9,5) sequentially.

Washed repeatedly. This was packed into a column with a length of 100 mm and an inner diameter of 4 mm to obtain a column 6 filled with 3α-H5D immobilized enzyme (immobilized dehydrogenase column).

On the other hand, homogeneous cellulose particles were mixed with 0.1M acetate buffer (PIT 5.0) containing 0.5M sodium chloride, ion-exchanged water, and 0.1M containing 0.5M sodium chloride.
After repeated washing with carbonate buffer (pi-19,5).

A control column 5 was obtained by filling a column with a length of 100 mm and an inner diameter of 4 mm.

An immobilized oxidoreductase column 7 was obtained by using 44 diaphorase instead of 3α-H2O.

Obtained as described above. control column.

An immobilized dehydrogenase column and an immobilized oxidoreductase column were connected as shown in FIG. ■＾D゛199■ and nitro bluete in ll!・Razurium (3.3
° -(3,3゛-dimethoxy-4,4゛-diphenylene)bis[2-(p-nitrophenyl)-5-phenyl-tetrazolium chloride])
Put M phosphate buffer (pit 8.0) into buffer tank 1,
The valve 4 was placed in the state shown in FIG. With constant flow pump 2,
1mA! The liquid is pumped at a flow rate of /min, and when the liquid feeding is stabilized,
0.01 ml of serum collected from a healthy person was injected using syringe 3.

mixed with buffer. The sample solution obtained in this way flows through the control column 5 and the immobilized oxidoreductase column 7 in the flow path shown by the arrow in Figure 1, and within 2 minutes after injection, the absorbance of all the samples has increased. Passed 8 in total. Immediately, valve 4 was switched to one INIa as shown in FIG. 0.01 ml of the same serum was injected again using syringe 3. This sample underwent an enzymatic reaction on the immobilized dehydrogenase column 6, and all the samples passed through the spectrophotometer 8 2 minutes after injection. The absorption photometer 8 uses a visible absorption photometer manufactured by Shimadzu Corporation, and has an absorption wavelength of 540 nin.
Measurements were made with.

An absorbance intensity one-hour curve shown in FIG. 6 was obtained. Based on the difference between the area of peak b and the area of peak a in Figure 6.

From the separately prepared calibration curve, determine the concentration of 3α-sex hormone, the main component of which is testosterone, in the test serum.

10μmol/7! It was calculated that

Example-η A 3α-11SD immobilized enzyme column 16 was obtained in the same manner as in Example 1. Furthermore, instead of 3α-11SD, 7α-
A 7α-H5D immobilized enzyme column 22 was prepared using 11sD. Control column 15 and immobilized oxidoreductase column 17 were prepared in the same manner as in Example 1.

The above four types of columns were connected as shown in FIG.

The same buffer solution as in Example 1 was put into the buffer solution tank 1.1, the valves 14 and 21 were set to the state shown in FIG. When the fluid delivery became stable, 0.01r+1 serum collected from cirrhotic patients in the early morning on an empty stomach was injected using the syringe 13 and mixed with the buffer solution. The sample solution thus obtained flows through the control column 15 and the immobilized oxidoreductase column 17 in the flow path shown by the arrow in FIG. It has passed. Immediately the valve 21 was switched as shown in FIG. Same serum 0.0I117! was injected again using the syringe 13. This sample was subjected to an enzyme reaction in a 3α-H5[)-immobilized enzyme column 16, and after reinjection, all of the samples passed through the spectrophotometer 18 in 2 minutes. Immediately valves 4 and 21 were switched as shown in FIG. Same serum 0.
01 val was injected using the syringe 13. This sample was subjected to an enzymatic reaction on a 7α-H3D immobilized enzyme column 22, and all the samples passed through the spectrophotometer 18 in 2 minutes after injection. Detection using the spectrophotometer 18 was carried out under the same conditions as in Example 1, and the absorption intensity one-hour curve shown in FIG. 7 was obtained. Peak C in FIG. 7 corresponds to the blank value passed through column 15. The concentrations of 3α-bile acid and 7α-bile acid in the serum sample were calculated using a separately prepared calibration curve based on the differences in area between peak d and peak C, and between peak e and peak C.

20.2 μmol/! each! , was calculated to be 18.5 μmol/l.

(Effects of the Invention) According to the present invention, biological components can be accurately quantified in a short time using an immobilized enzyme and a small amount of sample. Since only a small amount of sample solution is required, the enzyme will not be deactivated early. Even if the sample is not purified in advance by means such as dialysis, it will not be affected by interfering components during measurement. Furthermore, since a column with no enzyme immobilized was provided in the flow path during blank measurement, biological components can be accurately measured even when using an emulsified sample. An immobilized oxidoreductase column is provided to colorimetrically quantify the enzymatic reaction products, thereby eliminating the need for expensive fluorometers. Since a fluorophotometer is not used, unintended substances with fluorescence are not measured.
Therefore, the blank value becomes smaller, and biological components can be quantified more accurately. The peaks appearing on the recorder can be read more accurately by the microcomputer, allowing efficient measurements.

[Brief explanation of drawings]

Fig. 1 is a flow diagram showing an example of a quantitative device for quantifying one type of biological component to embody the method of the present invention; Fig. 2 is a flow diagram of main parts showing other connection states of the device in Fig. 1. Diagram: Figures 3 to 5 are flow diagrams showing an example of a quantitative device for quantifying two types of biological components to embody the method of the present invention, and other connection states of the device in Figure 3, respectively. Main part flow diagram; Figure 6 is the absorption intensity one-hour curve obtained using the apparatus shown in Figures 1 and 2 above; Figure 7 is the absorption intensity obtained using the apparatus shown in Figures 3 to 5. One hour curve: 2 or more Applicant Sekisui Chemical Co., Ltd. 3 Figure 2 Figure 4 1 day 2 Figure 5 Figure 6 Figure 7 Q12341. . Ding-HE Neichi J-E Yoi: (Voluntary) February 20, 1985'') 1. Indication of the incident 1988 Patent Application No. 9830.1 2. Name of the invention Biological component using immobilized enzyme Relation to the quantitative method 3, 7+!i-correct case Patent applicant postal code 530 Address 2-4-4 Nishitenma, Kita-ku, Osaka Patent Department TF
, l, Osaka (06) 365-2181 Patent Department Tokyo (03) 434-95524, Detailed explanation of the invention of the specification subject to amendment 5, Contents of amendment (1) Description @ page 19 In lines 12 and 13, the statement ``The concentration of 3α-sex hormones whose main component is testosterone in the test serum is'' should be corrected to ``The concentration of 3α-bile acids in the test serum is.''that's all

Claims (1)

[Claims] A step of introducing a fixed amount of a sample solution containing 1 μl of a biological component to one or more immobilized dehydrogenase columns packed with immobilized dehydrogenase. (2) A step of introducing a sample solution containing a dehydrogenase reaction product flowing out from the immobilized dehydrogenase column to an immobilized oxidoreductase column packed with immobilized oxidoreductase. (3) A step of measuring the sample solution containing the oxidoreductase reaction product flowing out from the oxidoreductase column. (4) A step of introducing a sample solution of the same type and in the same amount as the sample solution in step fl+ into a control column filled with only the carrier on which no dehydrogenase is supported. (5) A step of introducing the sample solution flowing out from the control column to the immobilized oxidoreductase column. (6) measuring the sample solution flowing out from the oxidoreductase column; and (7) measuring the sample solution flowing out from the oxidoreductase column after passing through the dehydrogenase column and the sample solution flowing out from the oxidoreductase column after passing through the dehydrogenase column; A process of calculating the difference between the measured value and the sample solution flowing out from the oxidoreductase column. A method for quantifying biological components using immobilized enzymes including 2. The biological component is a steroid compound. 2. The method according to claim 1, wherein the dehydrogenase is hydroxysteroid dehydrogenase and the oxidoreductase is diaphorase. 3. Claim 2, wherein the steroid compound subjects the reduced nicotinamide adenine dinucleotide and tetrazolium salt produced by the dehydrogenase reaction to an oxidoreductase reaction using diaphorase, and the resulting formazan is measured with an absorptiometer. The method described in.