JPH0528344B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field of the Invention] The present invention relates to a dry analytical element for the analysis of albumin in aqueous liquids, and more particularly to a dry analytical element for the quantitative analysis of albumin in biological body fluids, such as blood, cerebrospinal fluid, saliva, lymph fluid, urine, etc. The present invention relates to a manipulable integrated multilayer analytical element. [Prior Art] Albumin in biological fluids, such as blood, spinal fluid, saliva, lymph, urine, etc., can be measured or quantified using a buffered bromcresol green (hereinafter referred to as BCG) solution or USP 3 533 749.
and USP 3 485 587 (USP stands for United States patent).
BCG is a type of acid-base indicator dye belonging to the 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. Buffered BCG ensures that color changes corresponding to PH changes to which it would otherwise be sensitive are avoided. Acid-base indicators such as BCG are not specific for albumin. Globulins present in human body fluids,
Transferrin and other proteins compete for binding with acid-base indicator dyes and interfere with albumin analysis. This competitive interference is sometimes pronounced with albumin at low concentration levels. The total protein concentration in human serum ranges from about 6.0 to about 8.0 g/dl, of which about 2.9 g/dl is globulin and the remainder is mainly albumin. At this albumin concentration competitive interference appears. A dry integrated multilayer analysis element with reduced competitive interference has been proposed in Japanese Patent Application Laid-Open No. 57-50660 (USP 4 333 733). This element consists of a polyethylene terephthalate transparent support and a reagent layer in which BCG and a buffer are dispersed in a non-protein binder polymer, and a porous development layer (reaction layer), which are adhesively laminated in this order. When human serum is spotted on the developing layer, moisture is supplied from the developing layer to the reagent layer, and both layers come into liquid contact, and BCG diffuses from the reagent layer to the developing layer.
Since it preferentially binds to albumin and changes to a color unique to the BCG-albumin bond, it is an element of a dry operation that measures the color density after color change and determines the albumin content using the principle of colorimetry. However, even when this element is used, it has been found that as the measurement time becomes longer, globulin interference appears to a non-negligible extent. [Object of the Invention] An object of the present invention is to provide a dry analytical element for albumin analysis in which errors caused by interference with globulin and other proteins are reduced. 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, and reduces errors due to interference by globulin and other proteins. 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. [Structure of the Invention] The present invention provides a light-transmissive water-impermeable support with:
This is a dry analytical element for albumin analysis, in which a hydrophilic polymer binder layer containing at least one PH buffer and a porous development layer containing an indicator that changes color when bound to albumin are integrally laminated in this order. Examples of porous developing layers used in porous developing layers containing indicators that change color when combined with albumin (hereinafter referred to as reagent developing layers) include JP-A-55-164356;
Fabric development layer (e.g., plain weave of broadcloth, poplin, etc.) described in Japanese Patent Application Laid-Open No. 57-66359, etc., Patent Application No. 59-79158
Knitted fabric development layers (e.g., tricotto, double tricotto, Milanese, etc.) described in, etc., JP-A-57-
148250, Japanese Patent Publication No. 53-21677, USP 3
Non-fibrous isotropic porous materials such as membrane filters (brushed polymer layers), polymer microbeads, glass microbeads, and porous layers containing continuous micropores in which diatomaceous earth is held in a hydrophilic polymer binder as described in 992 158, etc. Deployment layer, JP-A-1987-
A non-fibrous isotropic porous layer consisting of a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure layer) in which the polymer microbeads described in 90859 are bonded in point contact with a polymer adhesive that does not swell with water. A spreading layer or the like 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. Acid-Base Indicators by IM Kolthoff (MacMillan, Inc.) are indicators that change color when combined with albumin.
It is preferable to use acid-base indicator dyes that exhibit protein error, such as those described on pages 350-353 (Published in 1937). Examples of acid-base indicator dyes that indicate protein errors include bromcresol green, bromcresol purple, bromthymol blue, bromphenol blue, chlorphenol red, phenol red, cresol red,
Thymol blue; sulfonphthalein indicator dyes such as cresol phthalein; 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 bromcresol green (BCG) and bromcresol purple (BCP) being most preferred. The amount of acid-base indicator dye to be contained in the reagent developing layer is about 0.2 to about 3.0 g, preferably about 0.5 g per ^m2 .
g to about 1.5 g. Hydrophilic polymer binder layer containing PH buffering agent
A water-absorbing layer whose main components are a PH buffer and a hydrophilic polymer binder that can swell and absorb water upon contact with water (hereinafter referred to as PH-adjusting water-absorbing layer or PH-adjusting layer)
It is. The PH adjustment water absorption layer adjusts the PH of low molecular weight when water in the aqueous liquid sample deposited on the reagent development layer during analysis reaches this layer and the two layers come into liquid contact.
It has a function of supplying a buffer agent to the reagent development layer by diffusion to maintain the region of the reagent development layer where the liquid sample exists at a predetermined constant PH value or a value in the vicinity thereof. The PH buffer contained in the PH adjustment water absorption layer has a low molecular weight PH buffer composition that can maintain the PH value of the reagent development layer in the liquid contact area in the range of about 2.0 to about 4.0, preferably about 3.2 to about 3.4 during analysis operations. It is a substance or a low molecular weight acid. Examples of PH buffers that can be used include low molecular weight carboxylic acids and their salts, such as malic acid, lactic acid, succinic acid, malonic acid, laconic acid, and tartaric acid;
There are other low molecular weight PH buffer compositions, such as those described in USP 3 438 737, "Chemical Handbook Basic Edition" edited by the Chemical Society of Japan (Tokyo, Maruzen, published in 1966), pages 1312-1320. Among these, malic acid is preferred. The content of PH buffer or acid in the PH adjustment water absorption layer is 1
It ranges from about 30 milliequivalents to about 500 milliequivalents, preferably from about 50 milliequivalents to about 300 milliequivalents per m ² . The polymer binder used in the pH-adjusting water-absorbing layer is a natural or synthetic non-protein hydrophilic polymer that has the property of swelling and absorbing water upon contact with water and the ability to form a film. Examples of non-protein hydrophilic polymers include polyacrylamide, JP-A-57-50660;
Acrylamide-N described in JP-A-58-77664 etc.
- Acrylamide-based copolymers such as vinylpyrrolidone copolymers. The content of polymer binder used in the PH adjustment water absorption layer is approximately 7g per ^1m2 .
to about 70 g, preferably from about 10 g to about 50 g. Two or more types of polymer binders may be used in combination, if necessary. The pH-adjusting water absorption layer can contain various components that do not adversely affect the ability of the indicator to bind to albumin, which is the analyte. Examples include nonionic surfactants. Specific examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, p-nonylphenoxy Examples include polyglycidol and octyl glycoside. By including a nonionic surfactant in the PH adjustment layer, the water in the aqueous liquid sample can be absorbed substantially uniformly into the PH adjustment layer during analysis operations, and the liquid can quickly come into contact with the reagent development layer. and become substantially uniform. Two or more pH-adjusting water absorption layers can be provided as necessary. When providing two or more PH-adjusting water absorption layers, a layer close to the reagent development layer contains a low molecular weight PH buffer composition or a low molecular weight acid, and a layer far from the reagent development layer contains a high molecular weight PH buffer composition. It is preferable to contain an agent or a high molecular weight acid. Examples of high molecular weight acids that can be used include known carboxyl group-containing polymers and sulfonic acid group-containing polymers. A water absorbing layer made of a hydrophilic polymer binder can also be provided between the PH adjusting layer and the support. Examples of hydrophilic polymers used in the water absorption layer include non-protein polymers such as polyvinylpyrrolidone, polyacrylamide, and agarose. As the light-transmitting and water-impermeable support, known supports used in conventional integrated multilayer analytical elements can be used. Specific examples thereof include polymers such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), and the thickness is about 50 μm to about 1 mm. , preferably in the range of about 80 μm to about 300 μm,
That is, a smooth, planar support that transmits electromagnetic radiation in the wavelength range of about 200 nm to about 900 nm can be used. A known undercoat layer or adhesive layer can be provided on the surface of the support to strengthen the adhesion with the PH adjusting water absorption tank, the PH adjusting layer, or the water absorption 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. 59-171864, Japanese Patent Application No. 59-79158, Japanese Patent Application No. 1987-79158 as a method for containing an acid-base indicator in a reagent development layer.
59-79159, etc., it is preferable to provide a porous developing layer on the pH-adjusting water absorption layer and then apply an acid-base indicator dye-containing solution, preferably an organic solvent-containing solution, over the developing layer. . The dry analysis element of the present invention has a side of about 15 mm to about 30 mm.
Cut into small pieces such as squares or circles of approximately the same size, and cut into small pieces such as squares or circles of approximately the same size.
501144 and the like and used as a chemical analysis slide for albumin analysis, it is preferable from all viewpoints such as manufacturing, packaging, transportation, storage, and measurement operations. Depending on the purpose of use, it may be used in the form of a long tape and stored in a cassette or magazine, or small pieces may 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 droplet of an aqueous liquid sample such as plasma or serum in the range of 6μ to 15μ is spotted on the reagent development layer, and heated for 1 to 10 minutes, preferably at 37°C.
The optical density of the reagent development layer was prepared in advance by incubation at a nearby temperature and reflection photometry of the optical density of the reagent development layer using light at or near the absorption maximum wavelength of the albumin-acid-base indicator dye bond from the light-transmissive support side. The albumin content in a liquid sample can be determined by the principle of colorimetric measurement using a calibration curve. Quantitative analysis of albumin can be performed with high precision by keeping the amount of aqueous liquid sample spotted, incubation time, and temperature constant. This measurement operation is performed in JP-A-56-77746, JP-A-58-21566, JP-A-58-
161867 etc., it is possible to perform highly accurate measurements with extremely easy operation. Example 1 Elements 1 and 2 Acrylamide-N-vinylpyrrolidone copolymer (monomer weight ratio 1:1; hydrophilic polymer binder; abbreviated as PAVP) on a 180 μm thick colorless transparent polyethylene terephthalate (PFT) film (support) ) and p-nonylphenoxy polyglycidol (containing an average of 10 glycidol units; nonionic surfactant; abbreviated as NPhPG) was applied at the coating amount listed in Table 1 (per 1 m ² ; the same applies hereinafter) and dried. Then, a water absorption layer was provided. On top of that, malic acid (PH buffer), PAVP,
An aqueous solution containing NPhPG was applied in the coating amount shown in Table 1 and dried to provide a pH-adjusting water absorption layer. Next, water was uniformly supplied to the surface of the pH-adjusting water-absorbing layer to wet it, and a woven fabric spreading layer was provided by laminating a cotton broadcloth fabric having a thickness of approximately 140 μm made of 100-count cotton spun yarn thereon. Next, a BCG methanol-water mixed solvent solution with a volume ratio of 1:1 was applied over the textile spreading layer at the coating amount shown in Table 1-1, and dried to form a BCG-containing reagent spreading layer for albumin determination. An integrated multilayer analytical element was prepared. Elements 3 and 4 Poly-2-acrylamide-2-methylpropanesulfonic acid (an acidic polymer that also serves as a hydrophilic polymer binder; abbreviated as PAMPS) was used instead of PAVP in the water absorption layer at the coating amount shown in Table 1-2. The first pH-adjusting water absorption layer is prepared by using
An integrated multilayer analytical element for quantifying albumin was prepared in the same manner as Elements 1 and 2, except that polyacrylamide (hydrophilic polymer binder; abbreviated as PAA) was used instead of PAVP to provide a second PH adjustment water absorption layer. Comparative Example 1 An integrated multilayer analytical element for quantifying albumin shown in Tables 1-3 was prepared according to element 41 described in Example 1 of JP-A-57-50660.

【table】

【table】

[Table] Performance evaluation of each element was performed as follows. Physiological saline without albumin, physiological saline containing 4% human albumin, and physiological saline containing 4% human albumin and 5% human globulin were prepared as aqueous liquid samples. Each of these samples
Place 10μl on the reagent development layer or development layer of each element, incubate for 7 minutes at 37℃, and immediately measure the color density of the reagent development layer or development layer from the PET film side using visible light with a center wavelength of 640nm. The results shown in Table 2 were obtained. Here, D is a color density value, D ₀ : Value of albumin-free physiological saline D _A : Value of albumin-containing physiological saline D _A+G : Value of albumin and globulin-containing physiological saline. The error shows a value based on the formula [(D _{A + G} − D _A /D _A ]×100 (%)).

[Table] From the results in the table above, it is clear that the errors due to globulin coexisting in the liquid sample are reduced to about 1/2 for elements 1 to 4 of the present invention compared to the elements of the comparative example. . Example 2 1 on a colorless transparent PET film with a thickness of 180 μm
The coating amount per m ² is 3.7 g of malic acid, 27 g of PAVP,
An aqueous solution containing these three components was applied in an amount of 1.5 g of NPhPG and dried to form a single pH-adjusting water absorption layer. Next, the PH adjustment water absorption layer was moistened with water, and a cotton broad development layer was provided in the same manner as in Example 1. BCG was applied and impregnated in the same manner as in Example 1, and a reagent development layer was provided. A type multilayer analytical element was prepared. The performance of the obtained elements and the elements of Comparative Example 1 was evaluated in the same manner as in Example 1, and the results shown in Table 3 were obtained.

[Table] From the results in the above table, it can be seen that the element having one PH-adjusting water absorption layer of the present invention reduces the error due to globulin coexisting in the liquid sample to about 1/2 compared to the element of the comparative example. is clear.

Claims (1)

[Scope of Claims] 1. On a light-transparent water-impermeable support, a hydrophilic polymer binder layer containing at least one PH buffer, and a porous spreading layer containing an indicator that changes color when bound as albumin. A dry analytical element for albumin analysis, characterized in that: are integrally laminated in this order. 2. The analytical element according to claim 1, wherein the indicator is an acid-base indicator dye that indicates protein error. 3. The analytical element according to claim 2, wherein the acid-base indicator dye is bromcresol green or bromcresol purple.