JPS61243364A - Dry type analysis element for albumin analysis - Google Patents

Abstract

PURPOSE:To reduce errors due to disturbance by protein, by using an analysis element in which a hydrophilic polymer binder layer containing a pH buffer agent and a porous development layer containing an indicator adapted to change in the color being bonded to albumin are laminated sequentially on a light transmitting non-water permeable support. CONSTITUTION:This invention relates to a dry type analysis element for albumin analysis in which at least one layer of hydrophilic polymer binder layers containing a pH buffer agent and a porous development layer containing an indicator adapted to change in the color being bonded to albumin are laminated integral on a light transmitting non-water permeable support sequentially. The porous development layer is preferably a fibrous development layer represented by a weaving development layer and a knitting development layer in that the indicator can be contained and held easily. The indicator preferably employs acid base indicator pigment indicating errors due to protein. The pH buffer agent shall be a low molecular weight of pH buffer agent component or acid that is allowed to maintain the pH value of a reagent development layer in the range of about 2.0-4.0.

Description

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

[Prior Art] Albumin in biological body fluids, such as blood, aqueous fluids, 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 usp3
485, 587 (USP stands for United States Patent), etc., using the test piece described. 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. Buffered BCG is.

In the case of no buffering, color changes corresponding to sensitive pl+ changes can be reliably avoided.

Acid-base indicators such as BCG are not specific for albumin. Globulin 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 particularly pronounced with albumin at low concentration levels. The total protein concentration in human serum is approximately 6.0-8.
Within the range of 0 g/d+, about 2.9 g/d+ is globulin, and the remainder is mainly albumin. At this albumin concentration competitive interference appears.

A dry integrated multilayer analysis element that reduced competitive interference was published in Japanese Patent Publication No. 5
No. 7-50660 (IJsP 4333733'). 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. When human serum is spotted on the developing layer, water 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 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. 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.

[Components of the invention The present invention is applied on a light-transparent water-impermeable support.

This is a dry analytical element for albumin analysis, in which a hydrophilic polymer binder layer containing at least one p)l 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 and JP-A-57.
-66359 etc., the textile spreading layer (e.g., broad).

(plain weave such as boblin, etc.), knitted fabric development layers (e.g., tricot knit, double tricot knit, Milanese knit, etc.) described in Japanese Patent Application No. 59-79158, organic polymer fiber bulb-containing paper described in JP-A-57-148250. Developing layer made of paper, Special Publication No. 53-21677, tJsP 399215
8 etc. Membrane filter (plush polymer single layer).

Non-fibrous isotropic porous spread layer such as continuous microvoid-containing porous layer comprising polymer microbeads, glass microbeads, diatomaceous earth held in a hydrophilic polymer binder, JP-A No. 5
A non-fibrous isotropic layer comprising a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure layer) in which the polymer microbeads described in 5-90859 are bonded in point contact with a polymer adhesive that does not swell with water. A porous spreading layer or the like can be used. Among these porous spreading layers, the fibrous spreading layer represented by the woven fabric spreading layer 9 knitted fabric spreading layer is preferred since it is easy to contain and retain the indicator.

As an indicator that changes color when combined with albumin, Acid-Base Indicators J.
MacMillan Co., published in 1937) 350-3
Protein error described on page 53 etc.
It is preferable to use an acid-base indicator dye that exhibits or). 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 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 bromcresol green (BCG) and bromcresol purple (BCP) being most preferred.

I of the acid-base indicator dye contained in the reagent developing layer is ll1
About 2g to about 3.0g per 12, preferably about 0.5g
g to about 1.5 g.

The hydrophilic polymer binder layer containing pH buffering agent is f) H
A water-absorbing layer consisting mainly of a buffer and a hydrophilic polymer binder that swells and absorbs water when it comes in contact with water;
(referred to as a regulating water absorption layer or a pH regulating layer). The pH adjustment water absorption layer diffuses a low molecular weight pH buffer into the reagent development layer when the water in the aqueous liquid sample spotted on the reagent development layer during analysis reaches this layer and the two layers come into liquid contact. It has a function of supplying the liquid sample to a predetermined constant OpH value or a value in the vicinity thereof by supplying the liquid sample in the reagent spreading layer.

The I)H buffer contained in the pH adjusting water absorption layer adjusts the p)l value of the reagent development layer in the liquid contact area from about 2.0 to about 4.0. Preferred are low molecular weight I)H buffer compositions or low molecular weight acids that can be maintained in the range of about 3.2 to about 3.4. Examples of pH buffering agents that may be used include low molecular weight carboxylic acids and their salts such as malic acid, lactic acid, succinic acid, malonic acid, tartaric acid, etc., USP 343
8737. Complete Japanese Chemistry “Chemical Handbook Basic Edition” (Tokyo,
There are other low molecular weight p)l buffer compositions, such as those described in Marukubi, published in 1966, pp. 1312-1320. Among these, malic acid is preferred. The content of pH buffers or acids in the p)l-adjusted water absorption layer is approximately 3 per ff12
It ranges from 0 milliequivalents to about 500 milliequivalents, preferably from about 50 milliequivalents to about 300 milliequivalents.

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. An example of a non-proteinaceous hydrophilic polymer is polyacrylamide, as described in JP-A-57-50660. There are acrylamide copolymers such as acrylamide-N-vinylpyrrolidone copolymer described in JP-A-58-77664 and the like. The content of the polymer binder used in the pH-adjusting water absorption layer is 1n.
range from about 7 g to about 70 g, preferably from about log 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 the analyte albumin. Examples include nonionic surfactants. Specific examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, and p-nonylphenoxypolyglycidol.

Examples include octyl glucoside. By including a nonionic surfactant in the pH adjustment layer, water in an aqueous liquid sample can be absorbed substantially uniformly into the pH adjustment layer during analytical operations, and liquid contact with the reagent spreading layer can be prevented. becomes quickly and substantially uniform.

Two or more pH-adjusting water absorption layers can be provided as necessary. When two or more pH-adjusting water absorption layers are provided, 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.

It is also possible to provide a water-absorbing layer consisting of a hydrophilic polymer binder between the pl+ regulating 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 include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, and cellulose esters (eg, cellulose diacetate, cellulose triacetate).

(cellulose acetate propionate, etc.) with a thickness of about 50 μm to about 11 μm. Preferably about 80
It is transparent in the range of μm to about 300 μm, ie transparent to electromagnetic radiation in the wavelength range of about 200 nm to about 900 nm. A smooth planar support can be used. A known undercoat layer or adhesive layer is provided on the surface of the support.
Adjustment water absorption layer + The adhesion with the pH adjustment layer or water absorption layer can be strengthened.

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

A method for incorporating an acid-base indicator into a reagent spreading layer is disclosed in Japanese Patent Application Laid-Open No. 171864/1982, Japanese Patent Application No. 79158/1983,
As described in Japanese Patent Application No. 59-79159, etc., after a porous developing layer is provided on the pH-adjusting water absorption layer, a solution containing an acid-base indicator dye, preferably a solution containing an organic solvent, is applied over the developing layer. It is preferable.

The dry analysis element of the present invention has a side length of about 15 mm to about 30 mm.
It is cut into small pieces such as + squares or circles of approximately the same size, and is cut into small pieces such as ``+'' squares or circles of approximately the same size. Japanese Patent Publication No. 56-14245
4. Japanese Patent Publication No. 57-63452. Utsukai Showa 58-32350
．． It is preferable to use it as a chemical analysis slide for albumin analysis by storing it in a slide frame as described in Japanese Patent Publication No. 58-501144 etc. from all viewpoints such as manufacturing, 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 aforementioned patent specifications. That is, a droplet of an aqueous liquid sample such as plasma or serum in the range of 6 μm to 15 μm is spotted on the reagent development layer, incubated for 1 to 10 minutes, preferably at a temperature around 37° C., and then placed on the light-transparent support side. The optical density of the reagent layer is measured by reflection photometry using light at or near the absorption maximum wavelength of the albumin-acid-base indicator dye bond, and the liquid is measured using the principle of colorimetry using a calibration curve prepared in advance. The albumin content in the sample can be determined. 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 is described in Japanese Patent Application Laid-Open No. 56-77746. Japanese Patent Application Publication No. 5
The chemical analysis apparatus described in JP-A No. 8-21566, JP-A No. 58-161867, etc. allows highly accurate measurement with extremely easy operation.

Example 1 Elements 1 and 2 Acrylamide-N-vinylpyrrolidone copolymer (monomer weight ratio 1:l; l'JI aqueous polymer binder; P.A.V.P.
An aqueous solution containing p-nonylphenoxy polyglycidol (containing an average of 10 glycidyl units; a nonionic surfactant; abbreviated as NPhPG) was coated at the coating amount listed in Table 1 (per 1 m2; the same applies hereinafter). A water absorption layer was formed by coating and drying.

Additionally, Nimalic acid (pH 1lt buffer), PAVP, N
An aqueous solution containing PhPG 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 PTL adjustment water absorbing layer to wet it, and a cotton broadcloth fabric having a thickness of about 140 μm made of 100 count cotton spun yarn was laminated thereon by pressure bonding to provide a fabric spreading layer. Next, a first layer is applied from above the textile spreading layer using a mixed solvent solution of BCG in a methanol-water volume ratio of 1:1.
An integrated multilayer analytical element for quantifying albumin was prepared by applying the coating amount as shown in Table 1 and drying to form a BCG-containing reagent development layer.

Elements 3 and 4 Poly-2-acrylamide-2-methylpropanesulfonic acid (wI polymer that also serves as a hydrophilic polymer binder; abbreviated as PAMPS) instead of PAVP in the water absorption layer with the coating amount shown in Table 1-2. A first pH-adjusting water absorption layer is provided using + polyacrylamide (hydrophilic polymer binder;
An integrated multilayer analytical element for albumin determination was prepared in the same manner as Elements 1 and 2, except that a second pH-adjusting water absorption layer was provided using PAA (abbreviated as PAA).

Comparative Example 1 An integrated multilayer analytical element for quantifying albumin shown in Table 1-3 was prepared according to Element 41 described in Example 1 of JP-A-57-50660.

(Margin below) Table 1-1 Table 1-2 Table 1-3 Performance evaluation of each element was conducted as follows.

As an aqueous liquid sample, the main one without albumin was prepared. 10 μm of each of these samples was spotted on the reagent development layer or development layer of each element, and after incubation for 7 minutes at 37°C, the color of the reagent development layer or development layer was immediately detected from the PET film side using visible light with a center wavelength of 640 nm. The density was measured by reflection photometry and the results shown in Table 2 were obtained. Here, D is the color density value.

Do=value due to albumin-incompatible physiological saline D^: value due to albumin-containing physiological saline DA+G: value due to albumin and globulin-containing physiological saline.

The error is calculated using the formula [(D A4G - D A)/D AI
The value is shown in 100 ($).

From the results shown in Table 2 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 m2 on a colorless transparent PET film with a thickness of 180 μm
The amount of coating per unit is 3.7 g of malic acid, P A V P
An aqueous solution containing these three components was coated in an amount of 5 g of 278° NPhPGl and dried to form one pi-adjusting water absorption layer. Next, the pH-adjusting water absorption layer was moistened with water, a cotton broad development layer was provided in the same manner as in Example 1, BCG was coated and impregnated in the same manner as in Example 1, a reagent development layer was provided, and a layer for albumin determination was prepared. A body type multilayer analysis element was manufactured by W@.

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.

(Leaving space below) From the results shown in Table 3 above, the element having one pH-adjusting water absorption layer of the present invention has a reduction in error due to globulin coexisting in the liquid sample to about 1/2 compared to the element of the comparative example. It is clear that

Claims (3)

[Claims] (1) 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 integrated in this order on a light-transparent water-impermeable support. A dry analytical element for albumin analysis characterized by being laminated with. (2) The analytical element according to claim 1, wherein the indicator is an acid-base indicator dye that shows protein error. (3) The analytical element according to claim 2, wherein the acid-base indicator dye is bromcresol green or bromcresol purple.