JPH04316497A - Analytical element and its production - Google Patents

Abstract

PURPOSE:To obtain an analytical element, having high simultaneous reproducibility and excellent in preservation quality. CONSTITUTION:The subject analytical element is an analytical element, having a reagent layer containing a coupler and an enzyme on a support and useful for analyzing specific ingredients in a biological fluid sample. A protein in an amount of about >=10wt.% based on the solid substance of the aforementioned reagent layer is contained therein.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical element containing an enzyme such as sarcosine oxidase.

0002

BACKGROUND OF THE INVENTION For example, creatininase, creatinase, and
Analytical elements that measure creatinine using enzymes such as sarcosine oxidase and peroxidase can be prepared by dissolving these enzymes in water and then adding them to the gelatin solution, or by directly adding the enzymes to the gelatin solution. It is manufactured by coating the enzyme-containing reagent layer solution obtained by the following steps on a support.

[0003] However, the analytical elements obtained in this manner have the problem of large variations in performance. In particular, in the reagent layer solution containing the enzyme, for example, 7-chloro-3-(2-hexyldecylsulfonylethyl)-
When a coupler such as 6-methyl-pyrazolo-[3,2-c]-s-triazole is dispersed and contained using the oil protection method, there is a problem that the sensitivity decreases significantly and the simultaneous reproducibility is poor. There is a point.

[0004] Furthermore, when coating such a coupler-containing reagent solution, there are other problems such as coating failures such as repelling and the like, and large variations in the thickness of the coating film.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an analytical element with high simultaneous reproducibility and good storage stability. The object of the present invention is an analytical element for analyzing a specific component in a biological fluid sample, which has a reagent layer containing a coupler and an enzyme dispersed, for example, by an oil protection method, on a support. This is achieved by an analytical element characterized in that the reagent layer contains protein in an amount of about 10% by weight or more based on the solid content of the reagent layer.

[0006] Also, a method for producing an analytical element for analyzing a specific component in a biological fluid sample, which has a reagent layer containing a coupler and an enzyme dispersed by, for example, an oil protection method on a support. , the coupler, enzyme and protein-containing solution dispersed by the oil protection method prepared by adding the enzyme to a protein-containing solution blended so that the solid content of the reagent layer is about 10% by weight or more; This is achieved by a method for manufacturing an analytical element, which is characterized in that the analytical element is coated on a support.

[0007] When the above analytical element is an analytical element for analyzing creatine and/or creatinine in a biological fluid sample, creatinase is contained in at least one reagent layer and the developing layer. It is preferable that sarcosine oxidase is contained in the reagent layer, and in the case of a creatinine analysis element, it is preferable that creatininase is further contained in at least one reagent layer. It is preferable that the content of creatinase is higher in the developing layer than in the reagent layer, and in particular, the ratio of the content of creatinase in the reagent layer to the content of creatinase in the developing layer is 1:10 to 100. In addition, the content of creatinase is 25,000 to 20,000
0I. U. /m2, and the creatininase content is 1/m2 relative to the creatinase content.
It is preferable that it is 2000 to 1/15.

A case where the present invention is applied to an analytical element for measuring creatine and/or creatinine will be explained as follows. Analysis of creatine and/or creatinine is performed by the following series of reactions (1) to (4).

[0009]

[Chemical formula 1]

These reactions are catalyzed by creatininase, creatinase, sarcosine oxidase and peroxidizing substances, respectively. Creatinase and creatinase are commercially available from a number of sources. Several types of each enzyme isolated from various microbial sources are known in the literature. Creatininase with a pH optimum of 6.5 at 37°C and creatinase with a pH optimum of 7.7 at 37°C, both obtained from strains of Flavobacterium, are preferred.

Sarcosine oxidase is also obtained from a number of sources. For example, Japanese Patent Application Laid-Open No. 55-34001,
JP-A-56-92790, JP-A-61-1621
It is described in Publication No. 74. Peroxidizing substances include peroxidases. Peroxidases useful in the present invention are of plant or microbial origin.

The coupler as a dye precursor used in the present invention is disclosed in Japanese Patent Application Laid-open No. 57-94653 and Japanese Patent Application Laid-open No. 57-94653.
-94654 publication, JP-A-57-94655 publication,
JP-A-60-47696, JP-A-63-1271
Those disclosed in Japanese Patent Application Laid-open No. 58, Japanese Patent Application Laid-Open No. 63-205564, and Japanese Patent Application Laid-open No. 1-35369 can be used. Incidentally, it is preferable to use a diffusion-resistant coupler described in JP-A-60-47696. The content of coupler varies depending on the type of coupler, but is preferably about 1 to 5 g/m2.

When the coupler is contained in the reagent layer, it can be added to the coating solution after being dissolved in an organic solvent. When a coupler, particularly a diffusion-resistant coupler, is also included in the reagent layer, it is preferable to add it by an oil protection method used in photographic technology. Specifically, a diffusion-resistant coupler is dissolved in an organic solvent together with a high-boiling point solvent, mixed with an aqueous solution containing a hydrophilic colloid such as gelatin containing an anionic surfactant and/or a nonionic surfactant, and a high-speed Emulsification and dispersion may be performed using a rotary mixer, a colloid mill, an ultrasonic dispersion device, or the like. At this time, dibutyl phthalate or the like is preferably used as the high boiling point solvent, and the amount thereof is preferably 2 to 8% by weight based on the solid content of the reagent layer.

In the present invention, enzymes such as sarcosine oxidase and 7-chloro-3-(2-hexyldecylsulfonylethyl)-6-methyl-pyrazolo-[3,2
A coupler such as -c]-s-triazole is contained in the reagent layer.
Contains 5% by weight or more. In order to contain proteins such as bovine serum albumin in the reagent layer, the solid content of the reagent layer should be approximately 10
An enzyme such as sarcosine oxidase is dissolved in a solution containing protein such as bovine serum albumin in a concentration of % by weight or more, this solution is added to and mixed with the coupler-containing hydrophilic colloid aqueous solution, and this is applied onto a support. It should be applied to.

[0015] The reagent layer coated in this manner is less likely to cause uneven coating such as repelling during coating, has less variation in the thickness of the coated film, and is furthermore free from a decrease in the enzyme activity of sarcosine oxidase. It is difficult for this to occur, and an analytical element with high sensitivity and little variation can be obtained. Moreover, since there is little variation,
The production lot can be made larger, and therefore the production cost can be lowered and variations can also be reduced.

The analytical element of the present invention may contain one or more other additives used in general analytical elements for manufacturing or operational advantages. Such additives include surfactants, ion chelators, buffers, solvents, hardeners, antioxidants, and the like. Surfactants can be used regardless of whether they are nonionic or ionic. Nonionic surfactants are useful coating aids because they dissolve well in both organic solvents and water, and they also have biological properties. This is particularly preferred since it is effective in improving the development speed when a fluid sample is dropped onto the porous development layer. Specific examples useful as surfactants include Triton X-1
00 (octylphenoxypolyethoxyethanol, manufactured by Rohm and Haas), surfactant 10G (p
-nonylphenoxy polyglycidol, manufactured by Orin)
, Alkanol
-248 (perfluorooctyl sulfonic acid tetraethylammonium salt, manufactured by Bayer AG), Florado FC-
Mention may be made of nonionic and ionic surfactants such as 170C (perfluoroalkylpolyoxyethylene ethanol, manufactured by 3M). In particular, the surfactant contained in the porous spreading layer is preferably a nonionic surfactant, and the following R-O-(CH2-CH2-O-)n H (in the formula R
represents a phenyl group substituted with an alkyl group having 1 to 9 carbon atoms or an alkyl group having 1 to 20 carbon atoms, and n is an integer of 1 to 70). 2
Preferably, it contains 0g/m2.

[0017] As a buffering agent, please refer to "Chemical Handbook Basic Edition" edited by the Chemical Society of Japan (Tokyo, Maruzen Co., Ltd., published in 1966).
pp. 1312-1320, R. M. C. “Data for Biochemical” edited by Dawson et al.
Research” 2nd edition (Oxford at the Clendon Press, 1969) pp. 476-508, “Biochemistry”
Volume 5, page 467 (1966), “Analyti
104, pages 300-310 (1980).

Specific examples of pH buffers in the range of pH 5.5 to 9.0 include buffers containing tris(hydroxymethyl)aminomethane (Tris), buffers containing phosphate, and borate. Buffers containing citric acid or citrate, buffers containing glycine, N,N-bis(2-hydroxyethyl)glycine [Bicine (Bic
ine)], N-2-hydroxyethylpiperazine-N
'-2-Hydroxypropane-3-sulfonic acid (HEP
PS) Na salt or K salt, etc., N-2-hydroxyethylpiperazine-N'-3-sulfonic acid (EPPS) Na salt or K salt, etc., N-[tris(hydroxymethyl)methyl]
-3-aminopropanesulfonic acid (TAPS) Na salt or K salt, etc., N-2-hydroxyethylpiperazine-N'
-2-ethanesulfonic acid (HEPES) Na salt or K salt, etc., N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) Na salt or K salt, etc., and can be combined with any of these if necessary There are acids, alkalis or salts. Specific examples of preferred buffers include:
Potassium dihydrogen phosphate-disodium hydrogen phosphate, tris-sodium borate, triscitric acid, citric acid-sodium dihydrogen phosphate, bicine, HEPPS, HEPP
S sodium salt, HEPES potassium salt, EPPS, E
Examples include PPS sodium salt, TAPS, TAPS sodium salt, TES, TES sodium salt, TES potassium salt, and the like.

Since the pH buffer is preferably present in the enzyme-containing layer, the pH buffer is preferably contained in the enzyme-containing porous development layer or reagent layer. Since low molecular weight pH buffers can migrate between layers as water permeates, which is the medium of the applied aqueous liquid sample, pH buffers need to be included in all layers containing the enzyme and coloring reagent compositions. For example, p is only present in the hydrophilic or water absorbing layer.
H buffering agent may be included.

Dibutyl phthalate or the like is preferably used as the high boiling point solvent added when dispersing the coupler in the oil protection method in the reagent layer, and the amount thereof is 1 to 4 g.
/m2 is preferred. Incorporation of creatinase or sarcosine oxidase into the porous spreading layer in the practice of the present invention can be accomplished in many ways. For example, when coating is used as a method for forming a porous development layer, it is common to dissolve it in a solvent of a coating liquid (for example, when the solvent is water). If the solubility of the reagent in the solvent is not appropriate, it may be dispersed in the solvent without being dissolved. For various methods regarding dispersion, see New Experimental Chemistry Course, Volume 18,
The method described in "Interfaces and Colloids" (Maruzen Co., Ltd., edited by the Chemical Society of Japan, published in 1976), pages 340 to 355, can be used. In addition, regardless of whether a solvent is used or not, creatinase or sarcosine oxidase can be physically or chemically adsorbed onto the porous developing layer constituent material in advance, or simply as part of the porous developing layer constituent material. You can also mix them. Preferably, a nonionic surfactant, a porous developing layer constituent material, and creatinase or sarcosine oxidase are added to an organic solvent that is liquid at room temperature, such as benzene, toluene, xylene, ether, hexane, and its derivatives, and the mixture is stirred using a sand stirrer. , a homogenizer, an ultrasonic homogenizer, etc., and then applying and drying the dispersion liquid to form a porous spread layer.

Although the porous spreading layer of the dry analytical element of the present invention is self-supporting (ie, composed of a material sufficiently rigid to maintain its original shape), it is preferably formed of another non-porous material. placed on a sexual support. Such supports have adequate dimensional stability, are preferably non-porous, and
It is a transparent (ie, radiation-transparent) material that is capable of transmitting electromagnetic radiation of wavelengths between 200 and 900 nm. The choice of support for a particular analytical element should be adapted to the mode of detection (transmission or reflection spectrophotometry) to be detected. Useful supports include paper, metal foil, polystyrene, polyester (eg polyethylene terephthalate), polycarbonate or cellulose ester (eg cellulose acetate), and the like.

The porous spreading layer in the present invention is described in US Pat. No. 4,292,272 and US Pat. No. 3,992,158.
specification, U.S. Patent No. 4,258,001, U.S. Patent No. 4,430,436, and JP-A-57-1017
Fibers or non-fibrous substances as described in Japanese Patent Publication No. 61-61347, or a mixture of both.
It is prepared from a fabric such as that described in the publication.

The analytical element of the present invention can have various functional layers and layer configurations as desired. For example, reagent layers, reflective layers, subbing layers as described in US Pat. No. 3,992,158, radiation blocking layers as described in US Pat. No. 4,042,335, barrier layers as described in US Pat. No. 4,066,403, US Pat. No. 4,144,306. Registration layer as described in US Pat. No. 4,166
The migration prevention layer described in No. 093, the scintillation layer described in U.S. Pat. No. 4,127,499, the scavenger layer described in JP-A-55-90859, the breakable pod-shaped member described in U.S. Pat. No. 4,110,079, etc. It is possible to construct a multilayer analytical element that meets the purpose of the present invention by combining them arbitrarily.

The various layers mentioned above can be formed into layers of arbitrary thickness by using slide hopper coating, extrusion coating, dip coating, etc., which are known in the photographic industry. In addition, as for the layer structure, for example, JP-A-51-4
0191 (Japanese Patent Publication No. 58-18628), U.S. Patent No. 4110079, JP-A-58-13156
It is possible to select the layer structure described in Publication No. 5 and the like.

The analytical element manufactured in this way can be made into various shapes depending on the analytical method. For example, it can be of various shapes including tape, sheet or slides mounted on plastic mounts of the desired width. The present invention will be specifically explained below, but the present invention is not limited to this example.

[0026]

EXAMPLE A reagent layer shown in Table 1 below was provided on a transparent undercoated polyethylene terephthalate support having a thickness of about 180 μm. Table 1 (Reagent layer) R-1
R-2 R-3
R-4A 20
Same left
Same left Same left B
0.9 Same left
Same left Same left C 1.8 Same left Same left
Same left D 3.0
Same left Same left
Same left E
0.09 Same left Same left Same left F
30000 (0.14g) Same as left
Same left Same
Left G 14000 (0.50g) Same left Same left
Same left H 6500 (0.23g)
Same left Same left
Same left I 1500 (0.
14g) Same as left Same as left
Left Same left J
6.2 Same as left
Same left Same left K
1.5 Same left Same left Same left L 6.2
3.9 1.0
- In Table 1, A is gelatin (g/m2)
, B is sodium triisopropylnaphthalene sulfonate (g/m2), C is dibutyl phthalate (g/m2)
), D is 7-chloro-3-(2-hexyldecylsulfonylethyl)-6-methyl-pyrazolo-[3,2-c
]-s-triazole (g/m2), E is 1,2-bis(vinylsulfonyl)ethane (g/m2), F is peroxidase (I.U./m2, specific activity 209.3)
U/mg), G is sarcosine oxidase (I.U./mg), G is sarcosine oxidase (I.U./mg),
m2, specific activity 28.1 U/mg), H is creatininase (I.U./m2, specific activity 278 U/mg), I
is creatinase (I.U./m2, specific activity 10.8
U/mg), J is N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (g/m2),
K is potassium hydroxide (g/m2), and L is bovine serum albumin (g/m2). Further, in Table 1, the content of bovine serum albumin in reagent layer R-1 is approximately 20% by weight, the content of bovine serum albumin in reagent layer R-2 is approximately 10% by weight, and the content of bovine serum albumin in reagent layer R-3 is approximately 20% by weight. The albumin content is approximately 3% by weight. This reagent layer is created using the oil protection method used in the field of photography.
First, 7-chloro-3-(2-hexyldecylsulfonylethyl)-6-methyl-pyrazolo-[3,2-c]-
Dissolve s-triazole and dibutyl phthalate in an appropriate amount of ethyl acetate, mix this solution with an aqueous gelatin solution containing sodium triisopropylnaphthalene sulfonate (surfactant), and emulsify using a high-speed rotating mixer and an ultrasonic dispersion device. 1,2-bis(vinylsulfonyl)ethane and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer were added and mixed to this emulsified dispersion solution, and on the other hand, peroxidase, sarcosine oxidase, Creatininase and creatinase were added to an aqueous solution of bovine serum albumin at approximately 10°C (adjusted to pH 8.0 with KOH).
After dissolving this in the coupler-containing solution, this was added to the coupler-containing solution, and this reagent layer solution was coated on a polyethylene terephthalate support. When constructing this reagent layer, the one applied 15 minutes after the addition of the enzyme (R-
1a, R-2a, R-3a, R-4a) and those applied 45 minutes after enzyme addition (R-1b, R-2b, R-3
b, R-4b) were created. Note that when bovine serum albumin was not used, distilled water was used instead of the bovine serum albumin aqueous solution.

Next, 1.8 g/m of vinylpyrrolidone-vinyl acetate copolymer (polymerization ratio 2:8) was applied onto this reagent layer.
2 to form an adhesive layer, and a porous spreading layer was further coated on this adhesive layer in the following manner. That is, Triton X-
100 and 11.0 g/m2 of styrene.
After dissolving glycidyl methacrylate copolymer (polymerization ratio 9:1) in 280 g of xylene, fiber for filter paper raw material was added to give a weight of 106 g/m2, and 4-N,N-diethylamino-hydrochloric acid was added to give a weight of 0.2 g/m2. 2-(2'-methanesulfonamidoethyl) aniline, 1.7g/m
2 of 5,5-dimethyl-1,3-cyclohexanedione was added and stirred, and then 12,000 I. U
．． /m2 ascorbic acid oxidase,
112500I. U. /m2 of creatinase and bovine serum albumin of 2.0g/m2 are added, dispersed with a homogenizer, the resulting dispersion is applied over the adhesive layer, and dried to form a porous spreadable layer. was constructed.

The structure of the analytical element thus constructed is shown in Table 2. For the analytical elements in each of the above examples, add 10 μl of control serum N "Roche" (manufactured by F. Hoffmann-La Roche).
The solution was added dropwise and incubated at 37° C., and the difference in reflection density at 546 nm between 3 minutes and 30 seconds and 7 minutes was determined. The results are shown in Table 3.

According to this, even if it takes a long time from the addition of sarcosine oxidase to the application, the product of the present invention has almost no adverse effect, whereas the product of the comparative example has a long time from the addition of sarcosine oxidase to the application. As the time required for this process increases, sensitivity decreases.

In addition, the analytical elements-1 and -3 of the present invention and the analytical elements-1 and -3 of the comparative example were stored at 40°C for 3 days for forced deterioration, and then control serum N "Roche" was added at 10°C.
μl was added dropwise, incubated at 37° C., and the difference in reflection density at 546 nm between 3 minutes and 30 seconds and 7 minutes was determined. The results are shown in Table 4. Table 4
Reflection density difference
Before forced degradation After forced degradation Analytical element of the present invention-1 0
．． 0309 0.0297
Analytical element of the present invention-3 0.0291 0
．． 0272 Analytical element-1 of comparative example
0.0256 0.0204
Analytical element-3 of comparative example 0.0224
0.0148 According to this, it can be seen that the analytical element of the present invention exhibits stable characteristics and has excellent storage stability.

[0031] In addition, control serum N'
10 μl of ``Roche'' was added dropwise, incubated at 37°C, the difference in reflection density at 546 nm for 3 minutes and 30 seconds and 7 minutes was determined, and the concentration was determined using a conversion formula prepared in advance using human serum containing various creatinine. converted to a value. This was performed for each element with N=18 to examine simultaneous reproducibility, and the results are shown in Table 5.

From Table 5, it can be seen that the analytical element of the present invention has excellent simultaneous reproducibility.

[0033]

[Effects] The analytical element of the present invention has excellent sensitivity and simultaneous reproducibility, and has good storage stability.

Claims (2)

[Claims] 1. An analytical element for analyzing a specific component in a biological fluid sample, comprising a reagent layer containing a coupler and an enzyme on a support, the reagent layer having a solid content of about An analytical element characterized by containing 10% by weight or more of protein. 2. A method for producing an analytical element for analyzing a specific component in a biological fluid sample having a reagent layer containing a coupler and an enzyme on a support, the reagent layer having a solid content of about 10 A method for manufacturing an analytical element, characterized in that the coupler prepared by adding the enzyme to a protein-containing solution blended at a weight % or more, and the enzyme- and protein-containing solution are coated on the support. .