Chemistry: analytical and immunological testing – Tracers or tags
Patent
1997-03-13
1998-07-07
Alexander, Lyle A.
Chemistry: analytical and immunological testing
Tracers or tags
422 61, 436 97, 436170, G01N 3372
Patent
active
057767793
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an integral multi-layer element for analyzing bile acid sulfate contained in aqueous liquid samples. In particular, the present invention relates to an integral multi-layer element for quantitatively analyzing bile acid sulfate in aqueous liquid samples, for example, body fluids such as blood (whole blood, blood plasma and serum), cerebrospinal fluid, lymph, saliva, urine, and the like, which element can operate in a dry state and is advantageous in clinical use.
2. Description of the Related Art
JP-A-2-145183 discloses as an analytical method for bile acid sulfate, a solution method comprising converting bile acid sulfate to 3.beta.-hydroxybile acid with bile acid sulfate sulfatase (hereinafter referred to as "BSS"), reacting 3.beta.-hydroxybile acid with 3.beta.-hydroxysteroid dehydrogenase (hereinafter referred to as "3.beta.-HSD") and nicotinamide adenine nucleotides (hereinafter referred to as "NADs") or nicotinamide adenine nucleotide phosphates (hereinafter referred to as "NADPs") as coenzymes, and measuring the amount of NADHs (reduced NADs) or NADPHs (reduced NADPs) which is formed in proportional to the amount of bile acid sulfate.
However, this method has a drawback that an amount of developed color is insufficient for being used in dry analysis since it uses tetrazolium salts in a color-developing system.
A highly sensitive method for analyzing bile acid uses enzymatic cycling (see JP-A-3-224498). This method enables the highly sensitive quantitative analysis of a-type bile acid using .alpha.-HSD in the liquid system.
It has been found that it is possible to detect color development of thio-NADH with a relatively simple agent composition when the above two methods are combined and 3.beta.-hydroxybile acid, which has been desulfated with BSS, as the substrate is quantitatively measured with 3.beta.-HSD.
However, the analysis in a liquid system is troublesome, and therefore, it has been desired to develop a dry type integral multi-layer analytical element which is simple and has high sensitivity.
When dry type integral multi-layer analytical elements which use the enzymatic cycling method are produced simply by the conventional methods for producing the dry type test elements, the following problems arise: develops in the production process, which may be due to color development of reduced thio-NAD (thio-NADH). solution and cannot be dispersed homogeneously in the solution. Therefore, no homogeneous solution is obtained, and measurement errors may arise. in a wide range between 4 and 9. However, it is difficult to find a buffer which can buffer the samples in this wide pH range and can be used in a dry system.
Therefore, it has been difficult to apply the agent system for the above solution method in the dry analytical element with maintaining the highly sensitive quantitative analyzing properties of the enzymatic cycling.
The present invention intend to enable the production of an integral multi-layer element for quantitatively analyzing bile acid sulfate with a high sensitivity.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides an integral multi-layer analytical element for analyzing bile acid sulfate comprising a support member, a reagent layer on said support member and a porous developing layer on said reagent layer, at least one of said reagent layer and said developing layer containing bile acid sulfate sulfatase, 3.beta.-hydroxysteroid dehydrogenase and a combination of thio-NAD.sup.+ and reduced NADs or reduced NADPs, wherein said reagent layer contains a water-soluble polymer, a buffer and at least one component selected from the group consisting of sugar alcohols and Mn.sup.2+.
BRIEF DESCRIPTION OF DRAWING
FIG. 1 is a graph showing the results in Example 1.
DETAILED DESCRIPTION OF THE INVENTION
When the test samples are urine, they have large variety of pH values depending on individuals as explained above. Thus, it is necessary to use a buffer which has the buffering abi
REFERENCES:
Caplus 1991:78186 includes a characterization of the abstract from JP 89-17564.
Chosa Satoshi
Tamura Hiroshi
Yonehara Satoshi
Alexander Lyle A.
Kyoto Dai-ichi Kagaku Co. Ltd.
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