JPH0379997B2 - - Google Patents

Description

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

Conventionally, when quantifying biological components such as bile acids that exist in trace amounts in serum, it is difficult to directly detect the components to be measured such as bile acids with a detector such as a fluorometer. utilizes the catalytic action of an enzyme to transfer a sample solution to which a reactant that reacts with the component to be measured in the presence of the enzyme into a column filled with an enzyme immobilized on a carrier. (referred to as an enzyme column), where the component to be measured reacts with the reactant to produce a reduced coenzyme, which is detected by a fluorescence detector and the amount of the component to be measured contained in the sample solution is calculated. is being carried out.

For example, when quantifying bile acids, nicotinamide adenine dinucleotide (NAD+
The sample solution to which the enzyme 3α-hydroxysteroid dehydrogenase (hereinafter referred to as 3α-HSD) has been added is
) is passed through an immobilized enzyme column packed with a carrier on which bile acids are immobilized, and the bile acids and NAD+ are reacted there. As a result, the fluorescent substance NADH is generated in an equimolar amount with the bile acids, and the NADH is It can be detected with a fluorometer, or the NADH generated above is oxidized to NAD by the action of the enzyme diaphorase in the presence of resazurin, and at the same time resazurin is reduced to produce resorufin, a fluorescent substance. The fluorescence of resorufin has been measured.

However, the amount of the component to be measured cannot be accurately quantified unless the blank value, which is the measured value shown when the biological sample solution does not pass through the immobilized enzyme column, is measured. A measurement device without an immobilized enzyme column was prepared separately. However, in this case, in order to measure the blank value and obtain the detected value of the biological sample liquid that has passed through the immobilized enzyme column, it is necessary to collect the biological sample twice, and the detectors are separate. This method has disadvantages in that it tends to cause weighing errors and also takes a long time to measure.

The purpose of the present invention is to eliminate such drawbacks, and its gist is to immobilize a biological sample solution containing a component to be measured between a branch flow path to which an immobilized enzyme column is connected. Diverting the flow into a branch channel to which the enzyme column is not connected, and bringing the biological sample liquid flowing through the branch channel to which the immobilized enzyme column is connected into contact with the immobilized enzyme within the column to cause a reaction with the component to be measured; The biological sample liquid flowing through each of the branch channels described above is led to a confluence channel and sequentially reaches two detectors connected in series to the confluence channel, and one detector detects the reaction product due to contact with the immobilized enzyme. The invention consists in a method for quantifying biological components using an immobilized enzyme, which is characterized in that the other detector detects a substance that is not changed by the immobilized enzyme.

Next, a method for quantifying biological components using the immobilized enzyme of the present invention will be described in more detail with reference to the drawings.

Reference numeral 1 denotes a buffer tank, and the buffer in the tank contains a component capable of reacting with the component to be measured by the action of an enzyme, such as NAD+ in the case of bile acid analysis.
Reference numeral 2 denotes a constant flow pump, which is provided to feed the buffer solution at a constant flow rate.

Reference numeral 3 denotes an injector for a biological sample liquid containing a component to be measured, and the biological sample liquid and the buffer solution are combined in the injector 3.

Both the biological sample liquid and the buffer are mixed uniformly. After the biological sample liquid mixed with the buffer solution exits the syringe 3, it is divided into branch channels 4 and 5. An immobilized enzyme column 7 is connected to the branch flow path 4 via a coiled flow line 6, and a coiled flow line 8 having the same diameter as the flow line 6 is connected to the branch flow path 5. It is formed. The branch channels 4 and 5 are both provided in parallel and have approximately the same length.

The biological sample liquid diverted to the branch flow path 4 is introduced into the immobilized enzyme column 7 through the coiled flow path 6, and when it comes into contact with the immobilized enzyme within the column 7, it is subjected to the catalytic action of the immobilized enzyme. The measured component reacts with the reactive component in the liquid to generate a reaction product. The biological sample liquid branched into the branch flow path 5 flows through a coiled flow conduit 8 . The coiled flow pipes 6 and 8 are, for example, wound stainless steel tubes with an inner diameter of 0.2 mm and a length of 10 m, and the pressure loss caused by this is significantly larger than that with the immobilized enzyme column 7, so branch flow pipes are used. The ratio of the flow rates of 4 and 5, that is, the division ratio, is close to 1:1.

However, the split flow ratio of the biological sample liquid is not always exactly 1:1 due to the viscosity of the biological sample liquid, the pulsating flow of the constant flow pump 2, etc. Even if you try to determine the amount of the component to be measured from the blank value and the detected value of the reaction product upon contact with the immobilized enzyme, if the split flow ratio is not exactly 1:1, the measurement will fail. This will result in erroneous determination of the value.

However, according to the present invention, the branch channels 4, 5
Even if the diversion ratio of the biological sample liquid flowing through the sample is not exactly 1:1, the amount of the component to be measured can be determined accurately by obtaining a correction value based on the diversion ratio. For this purpose, in the present invention, the detector 9 and the detector 10 are connected in series to the confluence channel 11, so that the biological sample liquid flowing through the branch channel 5 first reaches the detector 9 and the detector 10. Then, the biological sample liquid that has flowed through the branch channel 4 reaches the detectors 9 and 10 with a slight time difference.

Here, the detector 9 detects only substances that are not changed by the immobilized enzyme present in the biological sample solution and do not detect reaction products due to contact with the immobilized enzyme. The ratio of the amount of the biological sample liquid that has passed through the branch channel 5 to the amount of the biological sample liquid that has passed through the branch channel 4, that is, the diversion ratio is determined. The detection result by detector 9 is recorded by recorder 1.
Recorded by 2.

The detector 10 detects a reaction product caused by contact with the immobilized enzyme in the immobilized enzyme column 7 in the branch channel 4, and the biological sample liquid that has passed through the branch channel 5 first reaches the reaction product. A blank value of the biological sample liquid is measured, and then the biological sample liquid that has passed through the branch flow path 4 arrives after a slight time lag, thereby obtaining a detected value of the biological sample liquid containing the reaction product. The detection result by the detector 10 is recorded by the recorder 13.

Note that the detector 9 and the detector 10 do not need to be connected in series in this order, and the detector 9 and the detector 10 may be connected in the reverse order.

In this way, the detector 9 detects substances present in the biological sample liquid that are not affected by the immobilized enzyme, and the detector 10 detects reaction products resulting from contact with the immobilized enzyme.

From the detection result by the detector 9, the quotient of the amount of biological sample liquid that has passed through the branch channel 5 and the amount of the biological sample liquid that has passed through the branch channel 4 is determined. Next, the detection value of the reaction product obtained by the detector 10 is multiplied by the quotient to obtain a detection value with the branching ratio corrected. In addition, the detected value obtained by this and the detector 10
Calculate the difference between the blank values obtained by Then, the amount of the component to be measured can be determined from a calibration curve that is measured separately.

According to the method of the present invention, even when the amount of biological sample liquid flowing through the branch flow path 4 and the branch flow path 5 is not exactly 1:1, it is possible to obtain the detected value of the reaction product with the split flow ratio corrected. Furthermore, the analyte component can be quantified by applying the value obtained from the difference from the blank value to a separately measured calibration curve.

Furthermore, according to the method of the present invention, the biological sample only needs to be collected once, making it possible to shorten the measurement time.

Example 1 Cellulose fine particles with a particle size of about 80 microns were used as a carrier, and 5 ml of ion-exchanged water was added to 5 ml of the fine particles.
After adding 10 ml of a 2M aqueous sodium carbonate solution and stirring, 1 ml of acetonitrile in which 2 g of bromide cyanide had been dissolved in advance was added, and the mixture was allowed to react for 90 seconds while stirring vigorously.

After quickly washing the activated cellulose microparticles with 0.1M carbonate buffer (PH9.5), ion exchange water, and 0.1M carbonate buffer (PH9.5) containing 0.5M sodium chloride, 44mg of 3α-HSD was added. 5 ml of 0.1M carbonate buffer (PH9.5) containing dissolved 0.5M sodium chloride was added, and the mixture was stirred at room temperature for 2 hours to react. Next, by the above processing, 3α−HSD
In order to block active sites still present on the surface of the immobilized microparticles, 0.1 M Tris-HCl buffer (PH) containing 0.05% 2-mercaptoethanol was added.
8.0) for 2 hours at 4°C.

The enzyme-immobilized microparticles thus obtained were mixed with a 0.1M acetate buffer (PH5.0) containing 0.5M sodium chloride,
Contains ion-exchanged water and 0.5M sodium chloride
After repeated washing with 0.1M carbonate buffer (PH9.5), it was packed into a column with a length of 100 mm and an inner diameter of 4 mm to prepare an immobilized enzyme-packed column.

The column obtained as above was used as column 7 in Figure 1, and 1 contained 199 mg of NAD+.
Pour 0.1M pyrophosphate buffer (PH9.5) into buffer tank 1, then pump the liquid at a flow rate of 1.2 ml/min with constant flow pump 2. When the liquid feeding stabilizes, add serum from a healthy person.
0.01 ml of 200 μM formazan in pyrophosphate buffer was mixed with 0.01 ml, and a total of 0.02 ml was injected from syringe 3 and mixed with the buffer solution. The sample solution thus obtained is divided into two parts and distributed to branch channels 4 and 5, respectively, and the sample solution branched to branch channel 5 is distributed through a coiled distribution pipe 8 with an inner diameter of 2 mm and a length of 10 m. , and the sample liquid diverted to branch channel 4 has an inner diameter of 2 mm and a length of 10 mm.
The enzyme flowed from the coiled flow pipe 6 of the pipe 6 through the immobilized enzyme column 7 and reached the confluence channel 11.

Next, a detector 9 connected in series to the merging path 11
and the substances that have reached the detector 10 and are not changed by the immobilized enzyme flowing through the branch channels 4 and 5 are sequentially detected by the detector 9, and the blank value of the reaction product and the immobilized enzyme are detected by the detector 10. Detection values of reaction products due to contact with were sequentially detected. A spectrophotometer was used as the detector 9, and the absorption wavelength was 540 nm, and the absorption intensity-time curve shown in FIG. 2 was obtained. From this curve, the quotient of the area of peak (a) and the area of peak (b) (b/a) of 1.05 was obtained. Also, while injecting a large number of samples, I learned that this quotient fluctuates in the range of 1.00 to 1.11 due to the viscosity of the sample and the influence of the pulsating flow of the constant flow pump. Detection by the detector 10 uses a fluorometer, with an excitation wavelength of 360 nm and a fluorescence wavelength of 360 nm.
The fluorescence intensity-time curve shown in FIG. 3 was obtained by using 460 nm. Multiply the area of peak (c) of this curve by the quotient (b/a) obtained earlier to obtain the area after correcting the diversion ratio, and subtract the area of peak (d) from this to calculate the amount of bile acids in serum. The corresponding area value was obtained, and from the calibration curve measured separately, the blood bile acid value was 8 μmol/
obtained the value of

When the same serum above was repeatedly measured 20 times,
An average measurement value of 8.1 μmol/with a coefficient of variation of 1.8% was obtained.

Comparative Example 1 In FIG. 1 of Example 1 above, the detector 9 was removed, and the value obtained by subtracting the area of peak (d) from the area of peak (c) in FIG. By calculating the blood bile acid level from the line,
When the serum of Comparative Example 1 was repeatedly measured 20 times,
The average value of the measurements remained unchanged at 8.0 μmol/, but the coefficient of variation was 5.3%.

Example 2 In order to cover the detectors 9 and 10 of Example 1 with one device, a detector as shown in FIG. 4 was devised. As shown in FIG. 4, this is a structure in which two lights, one for a spectrophotometer and one for a fluorometer, are extracted from the same light source. In FIG. 4, 14 is a xenon lamp, which can provide sufficient light intensity in the wavelength range from 250 nm to 600 nm or more. Light from the xenon lamp 14 passes through an optical path 15 and reaches a half mirror 16. Here, about 1% of the arriving light is reflected, passes through the optical path 17, and reaches the 540 nm metal interference filter 18, and only the 540 nm wavelength component reaches the flow cell 19. A buffer solution and a sample solution are flowed into the flow cell 19 through a pipe 31, and after passing through the flow cell 19, they pass again through a flow cell 22 for a fluorescence detector, and then are discharged to the outside of the system through the pipe.

Now, the 540 nm light that passes through 18 is absorbed by the sample in the flow cell, and the change in the amount of light is converted into an electrical signal by the photoprinter 20, amplified by the amplifier 27, and recorded on the recorder 29.

On the other hand, 99% of the light that has passed through the half mirror 16 travels straight through the optical path 21 and reaches the 360 nm metal interference filter 21, and only the 360 nm wavelength component reaches the flow cell 22. in the sample
Of the fluorescence generated in all directions by NADH, a component 23 perpendicular to the optical path 21 is guided to a 460 nm metal interference filter 24. Wavelength 460nm passed through 24
The change in the light intensity of the fluorescence originating from the sample is converted into an electrical signal by the photomultiplier 26, and then sent to the amplifier 27.
The signal is amplified and recorded on the recorder 30.

When the above detectors were replaced with the detectors 9 and 10 in FIG. 1 of Example 1 and the same procedure as in Example 1 was carried out, the same results as in Example 1 were obtained.

Example 3 The absorption wavelength of the detector 9 in FIG. 1 of Example 1 is
Only the same serum as in Example 1 was injected except that the wavelength was changed to 280 nm, and the rest of the procedure was the same as in Example 1.

In this case, the detector 9 detected absorption at 280 nm of serum protein, which is a component in serum, instead of the dye added to the sample in advance in Example 1. As a result, the same results as in Example 1 were obtained.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing an embodiment of the method of the present invention, FIG. 2 is an absorption intensity-time curve obtained in Example 1, and FIG. 3 is a fluorescence intensity-time curve obtained in Example 1. The curve in FIG. 4 shows an explanatory diagram of the detector used in Example 2. Explanation of symbols, 1... Buffer tank, 2... Constant flow pump, 3... Syringe, 4, 5... Branch flow path, 6,
8... Coiled flow pipe, 7... Immobilized enzyme column, 9, 10... Detector, 11... Combiner, 1
2, 13...Recorder, 14...Xenon lamp,
15, 17, 25... optical path, 16... half mirror, 18, 21, 24... metal interference filter,
19,23...flow cell, 20,26...photomar, 27,28...amplifier, 29,30...
Recorder.

Claims (1)

[Claims] 1. Separate the biological sample solution containing the component to be measured into the branch channel to which the immobilized enzyme column is connected and the branch channel to which the immobilized enzyme column is not connected, and separate the branch channel to which the immobilized enzyme column is connected. The flowing biological sample liquid is brought into contact with the immobilized enzyme in the column to cause a reaction with the component to be measured, and the biological sample liquid that has flowed through each of the branch channels is guided to a confluence channel, and the column is connected in series to the confluence channel. One detector detects a reaction product resulting from contact with the immobilized enzyme, and the other detector detects a substance that is not changed by the immobilized enzyme. A method for quantifying biological components using immobilized enzymes.