JP3464234B2 - Enzyme immobilization carrier and immobilized enzyme - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme immobilization carrier and an immobilized enzyme used in a microanalysis method for body fluid components applicable to clinical tests used for diagnosis of diseases.

[0002]

2. Description of the Related Art For quantifying a specific substance in a body fluid such as serum, plasma or urine, it has been carried out with high sensitivity without utilizing the specificity of an enzyme to separate the substance. . Above all, the importance of a method of producing hydrogen peroxide by an oxidase and quantifying the hydrogen peroxide under the action of peroxidase using an oxidizable color dye or a fluorescent dye is increasing. However, enzymes are expensive and have limited uses. At present, it has been put into practical use in various fields to immobilize an enzyme and repeatedly use it as a bioreactor.

There are adsorption method, entrapment method, cross-linking method and covalent method for immobilizing the enzyme. The adsorption method or the entrapment method is easy to operate, but it is difficult to prevent the separation of the enzyme during the reaction, and the cross-linking method requires a large amount of enzyme and is low in efficiency.
While the activity of the enzyme is likely to decrease during immobilization, the enzyme immobilized by the covalent bond method exhibits high activity and has the advantage that the release of the enzyme is extremely small. Is optimal for use as a bioreactor. As a carrier for immobilizing an enzyme by a covalent bond method, various carriers having different materials, ionic properties, particle sizes, pore sizes, etc. are known. On the other hand, when a bioreactor is used as a detector of high performance liquid chromatography (HPLC) or flow injection analysis method (FIA), and a body fluid component is quantified, a quantification method using peroxidase and a chromogenic substrate is used.

[0004]

However, depending on the method, it is often difficult to quantify a trace amount of components in a body fluid.

[0005]

Means for Solving the Problems The inventors of the present invention have made earnest studies on a method capable of detecting a trace component in a body fluid with high sensitivity. As a result, a specific dye and an enzyme immobilization carrier used for colorimetric determination have been obtained. The present invention has been completed by finding that it is possible to quantify a trace component in a body fluid with high sensitivity by using a combination of.

That is, the present invention is (1) basic or neutral, which is used for detecting a trace component in a body fluid using an immobilized enzyme and a dye whose basicity (cationicity) is increased by an oxidation reaction. (2) The carrier according to (1) above, wherein the enzyme is peroxidase, (3) the carrier according to (1) or (2) above, wherein the dye is a leuco dye.
(4) The present invention relates to the immobilized enzyme according to the above (1), (2) or (3), wherein the enzyme is immobilized.

A feature of the present invention is that when a trace amount component in a body fluid is quantified using a dye whose basicity (cationicity) is increased by an enzyme reaction with an immobilized enzyme, the enzyme is used with a basic or neutral carrier. Is to be fixed.

According to the present invention, a peak obtained from a detector is obtained by colorimetric determination by HPLC or FIA using a bioreactor using a dye whose basicity increases by an oxidation reaction such as leuco dye. It becomes sharp and microquantification becomes possible.

The immobilized enzyme of the present invention is packed in a column by a known method to form a bioreactor, which is connected to an HPLC or FIA device according to a known method before use. These devices include at least a liquid feed pump, a sample injection device, a detector, an instruction recording device, and pipes that form a flow path.

The buffer solution and the column size of the bioreactor used when performing quantification using these devices are as follows.
It is appropriately selected depending on the enzyme and dye used. For example, as the buffer solution, phosphate, citrate, borate, tris hydrochloride, Good's buffer and the like can be used. Further, a surfactant and the like are added as necessary.

The column size of a bioreactor is usually 1
It is used in an amount of 0 μl to 10 ml, preferably a column volume of about 50 to 500 μl. The reaction temperature in the bioreactor is usually in the range of 0 to 80 ° C, preferably 4 to 40 ° C.

In the present invention, examples of the basic or neutral carrier include a carrier having an amino group, an epoxy group, a hydroxyl group or the like as a functional group, or a compound having two hydroxyl groups at the αβ position such as a polysaccharide. Particularly preferred is a carrier having an amino group or an epoxy group. In addition, a carrier having an active group (for example, a tresyl group) such that an acidic functional group is generated as a side reaction during the enzyme immobilization reaction, if the acidic functional group is completely blocked and can be controlled to be neutral or basic. It can be used. Further, a carrier having a functional group (eg, formyl group, tresyl group, etc.) capable of generating an acidic group during storage of the carrier can be used as long as it is just after production, or such a carrier can be used at the time of use. A functional group (formyl group, tresyl group, etc.) may be introduced and used.

Specific preferred examples of the basic or neutral carrier include aminotoyopearl (manufactured by Tosoh Corporation) and aminocellulofine (Seikagaku Corporation).
Made), Aminopropyl-CPG (sold by Funakoshi Co., Ltd.), AH Sepharose 4B (Pharmacia Co., Ltd.)
), Epoxy Toyopearl (manufactured by Tosoh Corporation), Epoxy-activated Sepharose 6B (manufactured by Pharmacia Co., Ltd.),
DEAE-Cellulofine (manufactured by Seikagaku Corporation), D
EAE-Toyopearl (manufactured by Tosoh Corporation), Shodex Gel DEA (manufactured by Showa Denko KK), CNBr-activated Sepharose 4B (manufactured by Pharmacia Co., Ltd.), Tresyl Toyopearl (manufactured by Tosoh Corporation), etc. However, the present invention is not limited to this. 1 to 300μ as particle size
The functional group concentration of 1 to 500 μmol / ml-gel is generally used.

Various known methods can be applied to the method of immobilizing the enzyme on the carrier, but the covalent method is preferred. Examples of the covalent bond method include a method of oxidizing the enzyme with periodate and then binding to a carrier having an amino group, a method of reacting glutaraldehyde with a carrier having an amino group to bind the enzyme, a dimethylaminoethyl group, and diethylamino. A method of reacting epichlorohydrin with a carrier having a tertiary amino group such as an ethyl group to bond an enzyme, a method of bonding an enzyme to a carrier having a functional group such as an epoxy group or a tresyl group, and a method such as a polysaccharide in which α2 position is 2 A method of reacting a compound having one hydroxyl group with bromocyan and binding it to an enzyme can be applied.

The enzyme used in the present invention is peroxidase, but its origin and origin are not limited, and it may be plant,
Peroxidase derived from animals or microorganisms can be used.

An oxidase which produces hydrogen peroxide can be used in combination with peroxidase. For example, oxidase and peroxidase can be separately packed in a column and used by connecting them. When the carrier of the present invention is used, the immobilized oxidase and the immobilized peroxidase can be packed in the same column and used as one bioreactor, or each enzyme can be immobilized together and used.

Pyranose oxidase (PROD) (EC 1.1.3.10), as an oxidase for producing hydrogen peroxide, which can be used in combination with peroxidase,
L-sorbose oxidase (EC 1.1.3.1
1), glucose oxidase (GOD) (EC 1.
1.3.4), uricase (EC 1.7.3.3),
Cholesterol oxidase (EC 1.1.3.
6), putrescine oxidase (EC 1.4.3.1)
0) and the like, but not limited thereto, and any oxidase that generates hydrogen peroxide can be used.

In the present invention, examples of the dye whose basicity increases by the oxidation reaction include leuco dyes, and specifically, N- (Carboxymethylaminocarbonyl) -4,
4'-bis (dimethylamino) -diphenylamine sodium salt
(DA-64), 10- (Carboxymethylaminocarbonyl) -3,
7-bis (dimethylamino) -phenothiazine sodium salt (D
A-67), 4,4'-bis (dimethylamino) diphenylamin
e, 10-N-methylcarbamyl-3,7-bis (dimethylamino) -10H-
phenothiazine, Bis (3-bis (4-chlorophenyl) methyl-4-d
Examples include imethyl-aminophenyl) amine and the like. Especially preferred is N- (Car, which has a high molar extinction coefficient and high water solubility.
boxymethylaminocarbonyl) -4,4'-bis (dimethylamino)-
diphenylamine sodium salt (DA-64), 10- (Carb
oxymethylaminocarbonyl) -3,7-bis (dimethylamino) -phe
Examples thereof include nothiazine sodium salt (DA-67), but are not particularly limited. It is suitable to use these dyes at a concentration of 0.1 to 1000 μM when quantifying a trace amount of components, and particularly preferably 1 to 250 μM.

Examples of the body fluid include serum, plasma, urine, spinal fluid, saliva and the like, and when quantifying a trace amount of the component, the body fluid can be measured as it is or after pretreatment as necessary.

Examples of trace components to be measured (detected) include 1,5-anhydro-D-glucitol, glucose, uric acid, cholesterol and polyamine.

According to the present invention, when an enzyme immobilized using a specific carrier is used for colorimetric determination using a dye whose basicity increases due to color development in the quantitative determination using an enzymatic reaction, It becomes possible to measure trace components in body fluids with high sensitivity.

[0022]

EXAMPLES The present invention will be specifically described below with reference to comparative examples and examples, but the present invention is not limited to these examples.

Example 1 Horseradish Peroxidase (HRP) 250
00 unit 4m of 0.3M sodium hydrogencarbonate aqueous solution
Dissolve in 1 l, 60 mM sodium metaperiodate 4 ml
Was added and reacted at room temperature for 30 minutes, 400 μl of 1.6 M glycerin was added, and the mixture was stirred at room temperature for 30 minutes. Then, 2 L of 0.01 M carbonate buffer (pH 9.5) was dialyzed at 4 ° C. 6 ml of aminotoyopearl (manufactured by Tosoh Corporation) equilibrated with the above carbonate buffer was added to the solution, and the mixture was shaken at 4 ° C. for 4 hours and allowed to stand at that temperature for 24 hours. Then, the carrier particles were thoroughly washed with distilled water and 1M NaCl in this order. Furthermore, in order to block the active groups, the carrier particles were transferred to 0.1 M Tris-HCl buffer (pH 8), reacted at 25 ° C. for 2 hours, washed well with distilled water and 1 M NaCl in this order, and immobilized HRP was removed. Prepared.

The immobilized HRP has an inner diameter of 4.6 mm and a length of 35.
mm column with 0.1M phosphate buffer (pH 7.5)
It was filled with. The obtained immobilized HRP reactor was a Tosoh CCPM pump, a Shimadzu SPD-10AV detector, a Tosoh SC-8010 data processor, and Rheodyne 7161.
It was installed in front of the detector of the HPLC system consisting of the sample injection device. 0.1 ml of a phosphate buffer (pH 7.5) containing 0.2 mM DA-64 as an eluent at 1 ml / m
The solution was sent at a flow rate of in and 10 μl of 0.01 to 5 mg / L sodium perborate (stable hydrogen peroxide) was injected. The absorbance at 727 nm due to the dye produced in the column was measured as a chromatogram. The measurement was performed at 25 ° C. At this time, the minimum sodium perborate concentration recognized as a peak was 0.01 mg / L. Further, when the area-to-height ratio (A / H) of the chromatogram was obtained as an index of the peak shape, the A / H of sodium perborate 5 mg / L was 11.6, which was a sharp waveform. The results are shown in Table 1.

Example 2 HRP was immobilized on a carrier in the same manner as in Example 1, but aminocellulofine (manufactured by Seikagaku Corporation) was used as the carrier. This immobilized HRP was packed in a column as in Example 1, and sodium perborate was measured by the same method as in Example 1.

As in Example 1, the minimum sodium perborate concentration recognized as a peak and A / H were determined. The results are shown in Table 1. As in Example 1, the peak was sharp and the minimum detection limit of sodium perborate was 0.01 mg / L.
And was good.

Example 3 Immobilization of HRP on a carrier was carried out in the same manner as in Example 1, except that aminopropyl-CPG pore size 1 was used as a carrier.
400 (sold by Funakoshi Co., Ltd.) was used. This fixed H
The column was packed with RP in the same manner as in Example 1, and sodium perborate was measured in the same manner as in Example 1.

As in Example 1, the minimum sodium perborate concentration and A / H recognized as peaks were determined. The results are shown in Table 1. As in Example 1, the peak was sharp and the minimum detection limit of sodium perborate was 0.01 mg / L.
And was good.

Example 4 HRP25000 unit was dissolved in 4 ml of 0.3 M sodium hydrogen carbonate solution and reacted with 4 ml of 60 mM sodium metaperiodate at room temperature for 30 minutes, 400 μl of 1.6 M glycerin was added, and the mixture was stirred at room temperature for 30 minutes. did.
Then 0.01 M carbonate buffer (pH 9.5) 2 L, 4
It was dialyzed at ° C. 100 mg of hexamethylenediamine was added to the liquid and the reaction was carried out while stirring at 4 ° C. for 24 hours.
Furthermore, 0.5 M phosphate buffer (pH 7.2) 2 L, 4 ° C
It dialyzed with. To this, 0.3 g of Epoxy Toyopearl (manufactured by Tosoh Corporation) was added and shaken at 37 ° C. for 4 hours. Then, the carrier particles were distilled water, 1M NaCl, 1 / 15M
Immobilize H by washing well in the order of phosphate buffer (pH 7.2)
RP was prepared.

The column was packed in the same manner as in Example 1, and sodium perborate was measured in the same manner as in Example 1.

As in Example 1, the minimum sodium perborate concentration recognized as a peak and the A / H were determined. The results are shown in Table 1. As in Example 1, the peak was sharp and the minimum detection limit of sodium perborate was 0.01 mg / L.
And was good.

Example 5 Peroxidase (AR from Arthromyces ramosus
Immobilized ARP was prepared in the same manner as in Example 1 using 25,000 units (P, sold by Funakoshi Co., Ltd.).

This was packed in a column in the same manner as in Example 1,
Sodium perborate was measured in the same manner as in Example 1.

As in Example 1, the minimum sodium perborate concentration and A / H recognized as peaks were determined. The results are shown in Table 1. As in Example 1, the peak was sharp and the minimum detection limit of sodium perborate was 0.01 mg / L.
And the detection of low value was also good.

Example 6 Using the immobilized enzyme prepared in Example 1, the same test as in Example 1 was carried out using 0.2 mM DA-67 instead of DA-64. The measurement wavelength is 666 nm. Example 1
Similarly to the above, the minimum sodium perborate concentration and A / H recognized as peaks were determined. The results are shown in Table 1.

As in Example 1, the peak was sharp and the minimum detection limit of sodium perborate was as low as 0.02 mg / L.

Example 7 40 ml of HRP25000 unit was added to 0.1 M phosphate buffer (containing 0.5 M NaCl) (pH 7.0).
Dissolved in Tresyl Toyopearl (manufactured by Tosoh Corporation) 1
g was added and shaken at 4 ° C. overnight. Then, the carrier particles were thoroughly washed with distilled water and 1M NaCl to immobilize HRP. Furthermore, in order to block active groups, 0.1 M Tris-HCl buffer (containing 0.5 M NaCl) (pH 8.
At 0), the mixture was reacted at 25 ° C. for 1 hour and washed in the same manner to prepare immobilized HRP.

The column was packed in the same manner as in Example 1,
Sodium perborate was measured in the same manner as in Example 1.

As in Example 1, the minimum sodium perborate concentration recognized as a peak and the A / H were determined. The results are shown in Table 1.

Although the peak was slightly broader than that of Example 1, the minimum detection limit of sodium perborate was 0.05 mg / L, which was a good detection value.

Comparative Example 1 1.0 g of an acidic carrier, carboxy-cellulofine (manufactured by Seikagaku Corporation), was swollen with 20 ml of distilled water, and 1-ethyl-3- (3-dimethylaminopropyl hydrochloride was added. ) Carbodiimide (EDC) 5 mg / ml aqueous solution 1
0 ml was added and the pH was adjusted to 4.0 to 5.0 with dilute hydrochloric acid. When there is no change in pH, suction filtration is performed, and the product is thoroughly washed with distilled water. Immediately thereafter, separately prepared HRP2
0.1 M acetate buffer solution (pH: 5000 units)
4.5) 10 ml was added and the mixture was reacted with stirring at 4 ° C. overnight.
To block the active groups, 1.5 ml of 10% ethanolamine was added, and the mixture was reacted at room temperature for 2 hours. afterwards,
Immobilize HRP by washing carrier particles thoroughly with distilled water, 1M NaCl, 1 / 15M phosphate buffer (pH 7.2) in this order.
Was prepared.

The immobilized HRP was packed in a column as in Example 1, and sodium perborate was measured by the same method as in Example 1.

As in Example 1, the minimum sodium perborate concentration recognized as a peak and the A / H were determined. The results are shown in Table 1. The peak was considerably broader than that of the example, and the minimum detection limit of sodium perborate was 1.0.
It was difficult to detect as low as mg / L.

Comparative Example 2 Using the immobilized HRP prepared in Comparative Example 1 and using 0.2 mM DA-67 instead of DA-64, the same test as in Example 1 was conducted. The measurement wavelength is 666 nm.

As in Example 1, the minimum sodium perborate concentration recognized as a peak and A / H were determined. The results are shown in Table 1.

The peak was considerably broader than that of the example, and the minimum detection limit of sodium perborate was 1.5 mg / L.
And low value was difficult to detect.

Next, the case where it is combined with an oxidase which produces hydrogen peroxide is shown.

Example 8 PROD as an enzyme for producing hydrogen peroxide and 1,5-anhydro-D-glucitol (1,5-A as a substrate)
G) was used.

PROD 3400 units (protein concentration 3
7 mg / ml) in the same manner as in the HRP immobilization method of Example 7, 0.1 M phosphate buffer (containing 0.5 M NaCl) (p
H7.0) was dissolved in 20 ml, Tresyl Toyopearl (manufactured by Tosoh Corporation) 0.5 g was added, and the mixture was shaken at 4 ° C. overnight. Thereafter, the same operation as in Example 7 was carried out to carry out the immobilized PROD.
Was prepared.

The immobilized PROD has an inner diameter of 4.6 mm and a length of 3
0.1 M phosphate buffer (pH 7.
Filled in 5). Example 1 for PROD packed reactor
It was installed in front of the HRP-filled reactor (immobilized HRP reactor) of the above apparatus.

A 0.1 M phosphate buffer solution (pH 7.5) containing 0.2 mM DA-64 was fed at a flow rate of 1 ml / min to obtain 10 μl of 0.01-5 mg / L 1,5-AG. The absorbance at 727 nm of the injected dye was measured as a chromatogram. The minimum 1,5-AG concentration recognized as a peak at this time was 0.01 mg / L. In addition, the area-to-height ratio (A / H) of the chromatogram was calculated as an index of the peak shape, and 1,5-AG 5 mg / L
The peak A / H of the concentration was 12.3, a sharp waveform was obtained, and the minimum detection limit was 0.01 mg / L, and the detection of the low value was good. The results are shown in Table 2.

Example 9 PROD 7000 units (protein concentration 37 mg / m
Immobilized PROD was prepared by using 1) and operating with 1.0 g of carboxy-cellulofine as in the HRP immobilization method of Comparative Example 1.

The immobilized PROD was packed in a column in the same manner as in Example 8, and 1,5-AG was measured by the same method as in Example 8.

As in Example 8, the minimum 1,5-AG concentration recognized as a peak and A / H were determined. The results are shown in Table 2.
It was shown to. Sharp peaks were obtained as in Example 8,
The minimum detection limit was as low as 0.01 mg / L, and detection was low.

Example 10 PROD7000 units (protein concentration 37 mg / m
10 ml of 0.5M phosphate buffer (pH 7.2)
, 0.5 g of Epoxy Toyopearl (manufactured by Tosoh Corporation) is added, and the mixture is shaken and stirred at 37 ° C. for 4 hours. After the reaction, the carrier particles were thoroughly washed with distilled water and 1M NaCl. Further, in order to block the active groups, 0.5 M Tris-HCl buffer (pH 8.0) was reacted at 25 ° C. for 1 hour and washed in the same manner to prepare immobilized PROD.

The immobilized PROD and the immobilized HRP obtained in Example 4 were mixed at a ratio of 1: 1 and the mixture was mixed with an inner diameter of 4.6.
The column was packed in a column having a length of 70 mm and a length of 70 mm, and was installed in place of the immobilized HRP reactor of the apparatus of Example 1, and 1,5-AG was measured by the same method as in Example 8.

As in Example 8, the minimum 1,5-AG concentration recognized as a peak and A / H were determined. The results are shown in Table 2.
It was shown to. Sharp peaks were obtained as in Example 8,
The minimum detection limit was 0.02 mg / L, and detection of low values was good.

Example 11 To measure 1,5-AG in serum, a pretreatment column of Rana AG (anhydroglucitol) kit (manufactured by Nippon Kayaku Co., Ltd.) was used according to the operating method. Pretreated.
That is, the filling solution of the kit pretreatment column was drained, washed with 1 ml of distilled water and conditioned. Standard 1,5-AG (0 μg / ml, 5 μg / ml and 5
0 μg / ml) and human serum No. 50 μl of 1-10
Was added and eluted with 0.5 ml of distilled water twice. Using the apparatus of Example 8, 0.01-5 mg / L of 1,5-AG 10 μl
Inject 20 μl of the eluate from the above column instead of
Others were measured in the same manner as in Example 8. Standard 1,
A calibration curve was prepared from the peak area value of 5-AG, and the 1,5-AG concentration was calculated from the area value of each serum. The results and the measured values of Lana AG (anhydroglucitol) are shown in Table 3.

Example 12 In Example 11, instead of the apparatus of Example 8, Example 9 was used.
The measurement was performed in the same manner as in Example 11 except for using the above apparatus. The results are shown in Table 3.

Example 13 In Example 11, Example 1 was used instead of the apparatus of Example 8.
The measurement was performed in the same manner as in Example 11 except that the apparatus of No. 0 was used. The results are shown in Table 3.

[0061]

[Table 1]

[0062]

[Table 2]

[0063]

[Table 3]

[0064]

EFFECTS OF THE INVENTION By using the immobilized enzyme according to the present invention, it becomes possible to measure trace components in body fluid with high sensitivity.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yumiko Tsukagoshi 901 Kaminami-enokicho, Takasaki-shi, Gunma Prefecture (72) Inventor Yoshihiko Umeka Yoshihiko, Shonandai, Fujisawa, Kanagawa Prefecture 26-205 (72) Inventor Yoshihisa Furuyashi Kanagawa 4-26-5-304 Shonandai, Fujisawa City, Japan (72) Inventor Takashi Kitamura 1100-179 (56) Nishikatsumahara, Kumage-machi, Kumage-gun, Yamaguchi Reference JP-A-57-2700 (JP, A) Anal. Chem. Act a. , September 1992, Vol. 266, No. 2, p. 309-315 Mikrochim Acta, 1985, Vol. 2, No. 3, p. 211-221 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-1/66 C12N 9/08 C12N 11/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A bioreactor filled with immobilized peroxidase and a basic (cationic) substance by an oxidation reaction.
A carrier for immobilizing a basic or neutral peroxidase having an amino group or an epoxy group as a functional group, which is used when detecting a trace component in a body fluid using a leuco dye of increasing number. 2. An immobilized peroxidase for a bioreactor, which is immobilized on the carrier according to claim 1.