JPS6227664A - Dry analytical element for analyzing albumin - Google Patents

Abstract

PURPOSE:To make it possible to simply analyze albumin with high accuracy by reducing the obstruction of protein such as globulin, by using glutaric acid, adipic acid, pimelic acid or a derivative thereof as a pH buffer. CONSTITUTION:Glutaric acid, adipic acid, pimelic acid or a derivative obtained by substituting the methylene group of each of said acids with 1-2 1-3C alkyl groups is used as a pH buffer to hold pH of an aqueous liquid specimen such as serum or urine to 2.5-4.2. When the pH buffer is contained in a hydrophilic polymer binder layer, said buffer is generally used in an amount of 1.5-20g/m<2>. 6-15mul of an aqueous liquid specimen such as plasma or serum is dripped on a porous developing layer to be incubated for 1-10mn at about 37 deg.C and the optical density of the reagent developing layer is subjected to reflection photometry from the side of a light permeable support by using the absorption max. wavelength of albumin indicator color bonding and the albumin content in a liquid specimen is measured by a colorimetric method using a preliminarily calculated calibration curve.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a dry analytical element for albumin analysis in aqueous liquids, and more particularly to biological body fluids, such as aqueous liquids such as blood, cerebrospinal fluid, saliva, lymph, and urine. The present invention relates to a dry-operable integrated multilayer analytical element for quantitative analysis of albumin.

[Conventional technology]

The measurement or quantification of albumin in biological body fluids, such as aqueous fluids such as blood, cerebrospinal fluid, saliva, lymph, and urine, can be performed using buffered bromcresol green (hereinafter referred to as BCG) fluid or USP 3,533,749 and USP 3. ，
485,587 (USP stands for United States patent), etc. BCG is a type of acid-base indicator dye belonging to sulfonophthalein dyes that changes color when combined with proteins. BCG changes color depending on the amount of protein, such as albumin, present in the aqueous liquid. A buffered BCG ensures that color changes corresponding to voice changes that would otherwise be sensitive are avoided.

Acid-base indicators such as BCG are not specific for albumin. Globuli/transferrin and other proteins present in human body fluids bind to acid-base indicator dyes and interfere with albumin analysis. This competitive interference is particularly pronounced with albumin at low concentration levels. The total protein concentration in human serum is approximately 6. - In the range of about 8.0 g/dt, about 2.91 g/dt is globulin, and the remainder is mainly albumin. At this aldimine concentration competitive interference appears.

A dry integrated multilayer analysis element that reduced competitive interference was published in Japanese Patent Publication No. 5
Publication No. 7-50660 (USP 4,333,733)
has been proposed. This element consists of a polyethylene terephthalate transparent support and a reagent layer in which BCG and a buffer agent are dispersed in a non-protein binder polymer and a porous development layer (reaction layer), which are adhesively laminated in this order.

Compounds with large dissociation constants among organic acids such as malic acid, lactic acid, cono-phosphoric acid, malonic acid, citric acid and tartaric acid are used as the above-mentioned buffer.

When human serum is spotted on the developing layer, water is supplied from the developing layer 1d to the reagent layer and both layers come into liquid contact, and BCG diffuses from the reagent layer to the developing layer and binds preferentially to albumin. Since it changes to a color specific to the BCG-albumin bond, it is an element of a dry operation in which the albumin content is determined by the principle of colorimetry by measuring the color density after the color change.

Previously, the present inventors have developed a dry method for albumin analysis that solves the interference caused by globulin (especially the interference caused by globulin that appears when the measurement time is prolonged) in the dry integrated multilayer analytical element for albumin analysis described in JP-A-57-50660. The analysis element was proposed in Japanese Patent Application No. 85906/1983. Although competitive interference by globulins and other proteins is definitely reduced in the previously proposed dry analytical element for albumin analysis, the accuracy and reproducibility still sometimes deteriorate to perform high-precision quantitative analysis of albumin. This was experienced. Surprisingly, it was discovered that this problem could be solved by dicarboxylic acid, which has not been used in the past, and the present invention was achieved.

[Problem that the invention seeks to solve]

An object of the present invention is to provide a dry analytical element for albumin analysis that allows albumin to be analyzed easily, reduces errors due to interference by globulin and other proteins, and improves reproducibility and reproducibility.

Another object of the present invention is to provide a dry analytical element for albumin analysis that has a simple layer structure, is easy to manufacture, reduces errors due to interference with globulin and other proteins, and has improved accuracy and reproducibility.

Another object of the present invention is to provide a dry analytical element for albumin analysis that allows selective measurement of albumin to be carried out continuously.

[Means for solving problems]

The present invention provides a dry analytical element for albumin analysis that achieves the above object, and includes an indicator and a buffer that change color when combined with alzomine.

It is characterized in that Gourmet/l/acid, adipic acid, pimelic acid or any derivative thereof is used as the buffer.

Indicators that change color when combined with albumin include "Acid-Base Indicators" by Koltthoff (1, M.).
(MacM111an company.

It is preferable to use an acid-base indicator dye that exhibits protein error as described in P. 350-353 (Published in 1937). Examples of acid-base indicator dyes that indicate protein errors are bromcresol green, bromcresol purple, and bromthymol blue.

Bromophenol blue, chlorophenol red, phenol red, cresol red, thymol blue;
There are sulfonophthalein indicator dyes such as cresolphthalein, indigoid dyes such as indigo carmine, and azo dye indicators such as methyl red and methyl orange. Among these indicators, sulfonephthalein indicator dyes are preferred, with fromcresol green (BCG) and dobromcresol group (BCP) being most preferred.

The length of the acid-base indicator dye contained in the reagent development layer is 1 m.
About 0.2 from this, about 3.0 from F. F, preferably about 0
．． It ranges from 5I to about 1.5I.

One buffering agent used in the present invention is glutaric acid, adipic acid, pimelic acid or their methylene group having 1 carbon atom.
It is a derivative of ~3 in which one to two alkyl groups are substituted, and the - of aqueous liquid samples such as serum or urine is 2.5 to 46.
2, preferably maintained at pi (3.2 to 3.8, with water solubility of 150 and 1.0, P/
water Zoo- or more, and the first dissociation constant pK1 (25℃
) is preferably 3.5 or more.

Examples of derivatives include α-methylglutaric acid, β-methylglutaric acid, α, α′-dimethylglutaric acid, β, β
-tumethylglutaric acid, α-ethylglutaric acid, β-ethylglutaric acid, α, α′-diethylglutaric acid, β,
β-diethylglutaric acid, α-ethyl-α′-methylglutaric acid, β,β-ethylmethylglutaric acid, α-propylglutaric acid, β-propylglutaric acid, β,β-methylglopylgluta/L'[ , β, β-ethylglobilglutaric acid, 2-methyladipic acid, 3-methyladipic acid, 2-ethyladipic acid, and the like. - When included in the hydrophilic polymer binder layer, the amount of buffering agent varies depending on the μ value to be buffered, but is usually 1.5 to 20 1F/m'' and 2.5 to 121
1/m2 is preferred.

The dry analytical element for albumin analysis of the present invention is usually provided on a light-transparent water-transmissive support with at least one layer of a hydrophilic polymer layer containing the above-mentioned voice buffering agent to uniformly diffuse the sample. It consists of a porous spreading layer that holds the surface. An indicator that changes color when combined with albumin may be contained in the hydrophilic polymer binder layer or may be contained in the porous development layer. A typical example of such a multilayer analytical element for measuring aqueous liquids is U.S. Pat.
No. 158 and No. 4,042,335. In some cases, it may be a single zone element as in US Pat. No. 3,485,587.

Examples of the porous spreading layer include woven fabric spreading layers (e.g., plain weave such as broad and boblin) described in JP-A-55-164356 and JP-A-57-66359, etc.;
A knitted fabric development layer (e.g., tricot knit, double tricot knit, Milanese knit, etc.) described in JP-A No. 79158, etc., and a spread layer made of paper containing organic polymer fiber pulp described in JP-A-57-148250. , Special Public Service 1973-21
677, USP 3.992, 158, etc., a membrane filter (plush polymer single layer), a continuous microvoid-containing porous layer in which polymer micropeas, glass microbeads, and diatomaceous earth are held in a hydrophilic polymer inder. Non-fibrous isotropic porous spread layer such as
A porous layer containing continuous micropores (three-dimensional lattice-like granular structure layer) in which polymer micropeas described in Japanese Patent No. 90859 are bonded in point contact with a polymer adhesive that does not swell with water.
Non-fibrous isotropic porous spread layer consisting of, and other JP-A-56
-24576 publication, Japanese Patent Application Laid-open No. 57-94658,
Those described in JP-A-57-125847 can be used.

Among these porous spreading layers, fibrous spreading layers, such as woven fabric spreading layers and knitted fabric spreading layers, are preferred because they can easily contain and retain the indicator. By providing such a multi-light spreading layer, an aqueous liquid sample such as serum or urine can be uniformly diffused, thereby improving analysis precision and reproducibility.

- The buffering agent is coated on the support together with a natural or synthetic hydrophilic polymer binder to form a layer structure and its release amount is controlled.The hydrophilic polymer binder layer containing the PH buffer is in contact with the buffering agent and water. This is a water-absorbing layer (hereinafter referred to as a PH-adjusting water-absorbing layer) whose main component is a hydrophilic polymer binder that can swell and absorb water. The PH adjustment water absorption layer supplies a buffering agent to the reagent development layer by diffusion when the water in the aqueous liquid sample spotted on the porous development layer reaches this layer during analysis operations and both layers come into liquid contact. It has a function of maintaining the region of the porous development layer where the liquid sample exists at a predetermined constant value or a value in the vicinity thereof. Preferred hydrophilic polymers are water soluble since fluids such as serum or urine are aqueous. Acrylamide, vinyl alcohol, N-vinyl-2-pyrrolidone, hydroxyefl'7 acrylate, N-vinylcaprolactam, methyl vinyl ether, ethyl oxide, acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid, etc. Mention may be made of homopolymers and copolymers of hydrophilic vinyl monomers, starch and cellulose, and derivatives thereof. Among these, water-soluble vinyl polymers are preferred. Particular preference is given to polymers consisting of repeating units of acrylamide and/or vinylpyrrolidone, especially poly(vinylpyrrolidone-coacrylamide) containing 20 to 80% by weight of vinylpyrrolidone and correspondingly 80 to 20% by weight of acrylamide. ,
Alternatively, it is a copolymer of acrylamide and vinylpyrrolidone made from a monomer blend of equal weights of acrylamide and vinylpyrrolidone. A method for producing an acrylamide-N-vinylpyrrolidone copolymer is described in JP-A-58-77664. Hydrophilic polymer binder content ranges from about 79 to about 7 per m”
0g, preferably in the range of about 10I to about 50.9I. Two or more types of hydrophilic polymer binders can be used in combination, if necessary.

A nonionic surfactant can be used in the albumin analysis element of the present invention.

Such ingredients are nonionic surfactants.

Representative surfactants include, for example, alkylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, p-nonylphenoxypolyglycidol, octyl glucoside, and the like. By including a nonionic surfactant in the monotoning water absorption layer, the water in the aqueous liquid sample can be absorbed substantially uniformly into the monotoning water absorption layer during analytical operations, and the liquid can come into contact with the porous spreading layer. becomes quickly and substantially uniform.

Two or more adjustment water absorption layers can be provided as necessary. When providing two or more monotonous water absorption layers, a layer close to the reagent spreading layer contains a low molecular weight PT' buffer composition or a low molecular weight acid, and a layer far from the reagent spreading layer contains a high molecular weight PT'. It is preferred to include a buffer or a high molecular weight acid. Examples of high molecular weight acids that can be used include known cargexyl group-containing polymers and sulfonic acid group-containing polymers.

A water absorption layer consisting of a hydrophilic polymer binder can also be provided between the regulating water absorption layer and the support.

Examples of hydrophilic polymers used in the water absorption layer include non-protein polymers such as polyvinylpyrrolidone and polyacrylamide P17.

As the light-transmitting and water-permeable support, any known support used in conventional integrated multilayer analysis elements can be used. Specific examples include polyethylene terephthalate, polycarzenate of bisphenol A, polystyrene, cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.)
a smooth planar support having a thickness of from about 50 μm to about 100 μm, preferably from about 80 μm to about 300 μm, transparent, i.e. transparent to electromagnetic radiation in the wavelength range from about 200 nm to about 900 nm; A known subbing layer or adhesive layer can be provided on the surface of the support to strengthen the adhesion with the regulating water absorbing layer or the water absorbing layer.

The dry analytical element of the present invention can be prepared by the known methods described in the aforementioned patent specifications.

Japanese Patent Application Laid-open No. 1983 (1983) as a method for containing an indicator in a reagent spreading layer
- As described in Japanese Patent Application No. 171864, Japanese Patent Application No. 59-79158, Japanese Patent Application No. 59-79159, etc., after providing a porous spreading layer on the regulating water absorbing layer, It is preferred to apply an indicator dye-containing solution, preferably an organic solvent-containing solution, from above.

The dry analytical element of the present invention ranges from about 15 x m to about 30 m5 on each side.
Cut the square pieces into circular pieces of approximately the same size,
JP-A-54-156079, JP-A-56-142
454, Japanese Unexamined Patent Publication No. 57-63452, Japanese Utility Model Application No. 58-32350, Japanese Patent Publication No. 58-501144, etc., and used as a chemical analysis slide for albumin analysis. It is preferable from all viewpoints such as tearing, packaging, transportation, storage, and measurement operations. Depending on the purpose of use, it can be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.

The dry analytical element of the present invention can analyze albumin in a liquid sample by the operations described in the above-mentioned patent specifications. That is, a sample tube of aqueous liquid such as plasma or serum in the range of 6 μl to 15 μl is placed on the porous development layer, and
The optical density of the reagent development layer is reflected using light at or near the maximum absorption wavelength of the albumin-indicator dye bond from the light-transmissive support side. The albumin content in a liquid sample can be determined by photometry and the principle of colorimetric measurement using a calibration curve prepared in advance.

By keeping the amount of aqueous liquid sample spotted, incubation time, and temperature constant, quantitative analysis of albumin can be performed with high precision. This measurement operation was performed in Japanese Patent Application Laid-Open No. 56-7774.
6, JP-A-58-21566, JP-A-58
-161867 etc. (The chemical analyzer described in this publication can perform highly accurate measurements with extremely easy operation.

[For production]

In conventional dry analytical elements for albumin analysis, one buffer,
Among organic acids such as malic acid, lactic acid, succinic acid, malonic acid, citric acid, and tartaric acid, compounds with large dissociation constants are used to suppress the reaction between the indicator and albumin.
As a result, it was the cause of interference from Globulin.

In the present invention, instead of conventionally used organic calgenic acids such as malic acid, lactic acid, malonic acid, and citric acid, which have a relatively large dissociation constant, we use organic acids that maintain a high Using carbic acid avoided globulin interference.

〔Example〕

Example 1 Colorless transparent polyethylene terephthalate with a thickness of 180 μm)
(PET) support with one of the above-mentioned laxatives.
．． 0,! i'/m''-containing acrylamide/N-vinylpyrrolidone copolymer (50150vrtL%)
5Ii/m", and on top of that a buffer layer containing 1
A broad woven fabric made of 50 μm PET spun yarn (equivalent to number 100) was laminated. BC, an albumin indicator
G's methanol-water volume ratio 1:1 mixed general medium solution was applied to the fabric with a BCG coating amount of 0.7! It was coated and dried at a ratio of J/m2. JP-A-57-5066 as a comparative sample
A sample using phosphoric acid as a buffering agent, which was commonly used in Example No. 0, was prepared in the same manner.

10 ttl of physiological saline, 4% human albumin, and 4% human albumin containing 1.25 times more human globulin than albumin were spotted on each of the prepared samples, and incubated for 37 minutes for 6 minutes.
After incubation at ℃, the reflected light was measured at 640 nm. As a result, it is clear that when the buffer of the present invention is used, the color density increases and the deviation from the aqueous liquid sample containing globulin is halved.

Example 2 Albumin analysis indicator BCG and pl (same 1
It has a one-layer structure similar to the embodiment of JP-A No. 57-50660, which is included in the research, and the - of the buffer layer is 3.4 with 1 num of sodium hydroxide.
A sample was created accordingly. As a comparison sample, a sample prepared according to the example of JP-A No. 57-50660 was used.

10 μE of physiological saline (equivalent to an aqueous liquid without albumin), 4% human albumin containing 1.25 times more human globulin than albumin were spotted on each of the prepared samples, and incubated at 37°C for 6 minutes. After that, the reflected light at 640 nm was measured. The results shown in the following table show that when the buffering agent of the present invention was used, the degree of color development increased and the deviation from the aqueous liquid containing globulin was significantly reduced.

Example 3 The following single-layer albumin test paper was prepared using Toyoji paper A-52. As a comparative example (referred to as Comparative Example-3), BC described in Example-12 of U.S. Patent No. 3,485,587
A G-tartaric acid system was used. The same test as in Examples 1 and 2 was conducted and the following results were obtained. It was found that even with a single zone test paper, when the buffer of the present invention was used, the deviation with respect to globulin was halved.

〔Effect of the invention〕

The dry analytical element for albumin analysis of the present invention greatly reduces interference from globulin and other proteins, and allows albumin to be analyzed easily and with high precision. Furthermore, since the structure is simple, it is easy to manufacture, and if a machine is used, it is possible to continuously measure a large number of specimens.

Claims (4)

[Claims] (1) Indicator and pH that changes color when combined with albumin
In a dry analytical element for albumin analysis containing a buffer,
A dry analytical element for albumin analysis, characterized in that glutaric acid, adipic acid, pimelic acid, or any derivative thereof is used as the pH buffering agent. (2) The analytical element according to claim 1, wherein the indicator that changes color when combined with albumin is a sulfonephthalein indicator. (3) The analytical element according to claim 2, wherein the sulfonephthalein indicator is bromcresol green or bromcresol purple. (4) The dry analysis element has a hydrophilic polymer binder layer containing at least one pH buffering agent and a porous development layer containing an indicator that changes color when combined with the albumin, on a light-transparent water-transmissive support. The analytical element according to claim 1, which is integrally laminated in this order.