JPH02232561A - Novel reagent composition for detecting occult blood - Google Patents

Abstract

PURPOSE:To enable measurement with a high sensitivity and accuracy in a case of not only a hemolysis sample but also non-hemolysis sample by adding cytolysin with a high hemolyic activity to a reagent composition for detecting occult blood. CONSTITUTION:By a normal method, an absorbing carrier such as paper and fiber is impregnated with reagents composed of an indicator that is oxidized accompanying a change in color with the presence of a complementary blood molecule group in blood, an oxidation agent effective for oxidization thereof, a buffer agent, an activator, cytolysin with a high hemolytic activity and the like being divided once or several times and dried. The carrier is cut to a proper size to make a disc or that cut to a proper size is further fixed on a support with a double-side adhesive tape to be used as stick. The cytolysin herein used is preferably soluble well in water with a high hemolyic activity while being stable in a range of pH 4-7. The use of a reagent composition thus obtained enables the lowering of a drop in coloration sensitivity remarkably when a non-hemolysis sample is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION "Industrial Application Field" The present invention relates to the qualitative analysis of blood in body fluids and body exudates (including excreta) such as urine, vomit, gastrointestinal contents, cerebrospinal fluid, feces, etc. The present invention relates to an improved reagent composition for detecting occult blood useful for quantitative determination.

"Background of the Invention" Detection of occult blood in body fluids and body excreta (including excreta; the same applies hereinafter) and knowing its amount provide valuable information for diagnosing diseases and observing the progress of prognosis. Currently, it has become one of the important tests indispensable in clinical medicine.

That is, in diseases involving mucosal erosion such as ulcers and cancer, blood is found in the stomach contents and vomit, and latent blood is often found in feces. In addition, blood components or blood complement molecules are excreted in the urine due to typhus fever, scurvy, purpura, cerebral hemorrhage, nephritis, kidney stones, burns over large parts of the body, or the effects of various hemolytic toxins. Ru. From the perspective of clinical medicine, a highly sensitive occult blood detection method is required that can reliably detect blood in body fluids or body excreta, even if the amount is so minute that it cannot be detected by blunt microscopic identification. Ru.

Various methods have been known to date for detecting occult blood in body fluids or biological excreta, but currently the main method is to detect blood components such as hemoglobin, hemoglobin, etc.
It takes advantage of the ability of myoglobin or its decomposition products to catalyze the transfer of oxygen from an oxygen source to a receptor, and if the receptor is a dye precursor, it will be in the oxidation state K. Utilizes the property that it is colorless but develops color when oxidized, it detects the presence of blood components such as hemoglobin or its decomposition products from the developed color tone, and detects the presence or absence of bleeding, and Sarak detects the amount. It is something to know. The rate of color change, intensity or density of the color provides a means of quantifying the amount of blood present.

However, these test methods have drawbacks such as requiring measurement instruments or devices and being complicated to operate.
In place of this, a diagnostic test strip method has been developed that allows even beginners to easily measure κ.

However, many of the conventional test strips for detecting occult blood cannot detect hemolyzed blood (
Although the color development speed and sensitivity with hemoglobin or myoglobin were satisfactory, the color development speed with non-hemolyzed specimens (red blood cells) was slow and the sensitivity was not sufficient. There were five problems in that there was a difference in coloration between the hemolyzed sample and the non-hemolyzed sample.

``Object of the Invention'' The present invention was made in view of the current situation as described above, and provides a novel reagent composition for detecting occult blood that enables highly sensitive measurement of not only hemolyzed specimens but also non-hemolyzed specimens. The purpose is to provide.

``Structure of the Invention'' The present invention provides an indicator that is oxidized with a color change in the presence of a blood complementing molecular group present in blood, an oxidizing agent effective for oxidizing the indicator, and a pH range of 4 to 7. This is a reagent composition for detecting occult blood in body fluids and body excreta, which comprises a buffer to maintain, an activator to increase sensitivity, and cytolysin with high hemolytic activity.

That is, the present inventors have conducted intensive research to develop a reagent composition for detecting occult blood that is easy to prepare and capable of highly sensitive measurement of not only hemolyzed specimens but also non-hemolyzed specimens. An indicator that is oxidized with a color change due to the presence of a complementary group of molecules present in the blood, an oxidizing agent that is effective in oxidizing the indicator, a buffer that maintains the pH in the range of 4 to 7, and an activation agent that increases sensitivity. The present inventors have found that the objective can be achieved by adding cytolysin (cytolysin), which has high hemolytic activity, to a known reagent composition for detecting occult blood which contains a reagent composition K, which is known in the art, for detecting occult blood.

The reagent composition for detecting occult blood of the present invention can be easily prepared by using a known method for preparing a reagent composition for detecting occult blood.

In other words, an indicator that is oxidized with a color change in the presence of a blood complement group present in blood, an effective oxidizing agent, a buffer, an activator, and a substance with high hemolytic activity that oxidizes the indicator.
Each of the reagents constituting the reagent composition for detecting occult blood according to the present invention, such as IJ Thin, is impregnated into an absorbent carrier such as paper or fiber in one to several batches using a conventional method and dried. Cut this into an appropriate size and make a disk, or cut it into an appropriate size and fix it on a suitable support using double-sided adhesive tape to make a stick. is optional.

Cytolysins with high hemolytic activity used in the present invention include, for example, saponin, melittin, and gramici. Preferred examples include saponin, polymyxin B, etc., which have high hemolytic activity, are well soluble in water, and are stable in the pH range of 4 to 7. Of these, saponin is particularly preferred.

Saponin is a glycoside that is widely distributed in the plant kingdom, and is a general term for a group of compounds that have steroids or tritellinoid as the non-sugar moiety. Show action.

Specific examples of steroidal saponins include: nogitonin, solanine, sarsasaponin, osladine, timosaponin, ammonin, cammonin, sabotoxin,
Specific examples of triterbenoid saponins include cyossin, Jl'nin, Suzuko, and chasaponins, tupax saponins, panaxilone, mucrodisaponin, spinach saponin, esculin, etc., but all of these are limited to K. It's not something you can do.

These cytolysins with high hemolytic activity according to the present invention may be used alone or in an appropriate combination of two or more, and the concentration used is such that it can be dissolved in the detection reagent solution. Although it is not particularly limited as long as it is within the range, it is usually 0.01 to 5η to %, preferably 0.05
~2. It is added in an Ow/v% range.

On the other hand, even if a compound is known to have a hemolytic effect, for example, anionic surfactants such as sodium lauryl sulfate have low hemolytic activity. However, when these are used at such concentrations, inverse K coloration inhibition is observed, resulting in disadvantages. That is, even if the compound has hemolytic activity, K cannot be used for the purpose of the present invention if it is a compound with low hemolytic activity.

Other reagents used in the reagent composition for detecting occult blood of the present invention, that is, indicators (dyes), oxidizing agents, buffers, etc., do not have to be specially selected for the present invention, but can be selected from existing ones. Indicators (dyes), oxidizing agents, buffers, etc. used in reagent compositions for detecting occult blood can be used without any problems.

That is, the indicator (dye) may be any substance that is oxidized with a color change in the presence of blood components hemoglobin, myoglobin, or their decomposition products, such as anilines, phenols, etc. 0-}luidine, p-toluidine, 0-phinyl diamine, N,N'
- dimethyl-p-phenylenediamine, N,N'-
Ethyl-p-7 22-diamine, p-anidine, dianisidine, 〇-tolidine, 0-cresol, m-
Examples include cresol, p-cresol, α-na7tol, β-naphthol, catechol, quaiacol, birogallol, and the like.

Typical examples of the oxidizing agent include organic idronoso oxide, and specific examples thereof include cumene, dipropylene, and diisopropylbenzene. Oxide, paramenthano-idoronoκ-oxyP, 2,5
-dimethylhexane-2,5-dihydroperoxide and the like.

In addition, hydro-J-oxy-P is generally unstable, and in particular, it often decomposes itself or alters other substances when it comes into contact with other substances, so it is usually used as
For example, it is used in a state where it is isolated from other substances by making microcapsules with an encapsulating substance, etc. Examples of encapsulating substances used for this purpose include gelatin, arginine, carrageenan, gum arabic,
Examples include proteins such as casein and albumin, and polysaccharides.

However, it goes without saying that the encapsulating substance is not particularly limited to these, and the method of isolation from other substances is not limited to so-called microencapsulation.

Examples of the activator used in the present invention to increase sensitivity include acridine, acridine hydrochloride, 2-methoxyacridine, 7,8-penzoquinoline, quinoline, 6-methoxyquinoline, and 4,6-dimethylquinoline. ,
6-methylquinoline, 7-methylquinoline, 2.6-1
Examples include methylquinoline and 2-methylquinoline.

Examples of buffering agents include buffering agents that maintain the pH within a range of 4 to 7, such as tartaric acid, phosphoric acid, phthalic acid, citric acid, and acetate. It goes without saying that the concentration should be appropriately selected and used depending on the dye used.

The novel reagent composition of the present invention can be used in various forms. That is, each reagent constituting the composition is dissolved in water, an appropriate organic solvent, or a mixed solvent thereof to form an impregnating liquid, and a K-absorbing carrier is immersed in the impregnating liquid to impregnate the reagents and then dried. As mentioned above, the material may be cut into a suitable size to form a disk, or fixed on a suitable support to form a stick.

Examples of the absorbent carrier include, without exception, absorbent carriers commonly used for this type of purpose, such as paper, cellulose, chemical fibers, synthetic resin woven fabrics, and nonwoven fabrics.

Examples of the support for holding the absorbent carrier include glass fiber, polyvinyl chloride, Teflon, polystyrene, polyvinyl acetal, acetylcellulose, and nitrocellulose. Examples include sheets of synthetic polymer compounds such as hynylidene ytrvtx and boridelopyrene, and cardboard coated with these.

There are no particular restrictions on the size, dimensions, etc., of these absorbent carriers and supports, and it is sufficient to use a size or dimension K that is similar to those normally used for this type of purpose.

Also, regarding the method of adhering the absorbent carrier to the support, any method normally used in this field, such as using an adhesive or adhesive tape, may be used. No method required.

Examples are shown below, but the present invention is not limited to these Examples K in any way.

"Example" Example 1. (1) Preparation of sample urine 1) Preparation of isotonic urine While measuring the osmotic pressure with an osmometer (manufactured by FISKE), add sodium chloride, IJum or distilled water to dale urine.
A solution isotonic with 9% physiological saline was prepared.

11) Preparation of sample urine 10 tttl volumetric flask K blood was mixed with 0. 1 d, and the isotonic urine prepared in i) was added to make a total volume of 10 ml.

This was prepared using the above-mentioned isotonic urine so that the blood concentration was diluted 20,000 times, and this was used as a non-hemolyzed specimen.

In addition, for hemolyzed samples, 0.1 g of blood was mixed with isotonic urine to make a total volume of 1 aw.
/! This was prepared according to the method for preparing a non-hemolyzed specimen, except that the whole volume was diluted with distilled water instead of diluted with distilled water.

(21 Preparation of impregnating solution [Part I] 600 d of 30% aqueous gum arabic solution was heated to 60°C, 40 t of cumenehyde α-oxide was added to it, and 0.5 M citric acid buffer containing 5% K gelatin was added. Liquid (pH
5) After adding 300 liters of waste and keeping it at 60°C, it was cooled with water and a 0.0-liter solution containing 12 saponin (chasaponin) was added. 5
300 xtl of M citrate buffer (pH 5) was added to prepare the first solution.

[Part ■Liquid]

6t of o-tolidine and 0.52 of 6-methoxyquinoline in chloroform II! K was dissolved to prepare the liquid No. 1.

(3) Preparation of test piece A chromatography paper was immersed in the first liquid to impregnate it with the test liquid, and then placed in a dryer and dried at 60 to 80°C. Then,
This was immersed in the liquid No. 1, further impregnated with the test liquid, and then dried again. The test paper prepared in this way was
It was cut into a square with a W angle and attached to a polyvinyl chloride sheet (6×8 an ) K using a double-sided tape to make a test piece.

(4) Measurement of degree of coloration A test piece was momentarily immersed in sample urine, and coloration was measured every 10 seconds using a colorimeter (manufactured by Minolta, CR-121).

In addition, the coloring situation is the sensory chromaticity (L.a.b.
) is the standard, and the degree of coloration is determined by the sensory chromaticity difference (Δ
Eab). The results are shown in Table 1.

Comparative example 1. The impregnating liquid was prepared and the test piece was prepared in exactly the same manner as in Example 1, except that the liquid I was obtained by removing saponin from the liquid I in Example 1. The chromaticity was measured. The results are also shown in Table 1.

Table 1 The numerical values are the chromaticity difference (ΔE
ab).

As is clear from Table 1, when saponin was added (Example 1), the difference in chromaticity difference between hemolyzed and non-hemolyzed samples was greater than when saponin was added (Comparative Example 1). small,
Furthermore, it can be seen that the chromaticity difference values of the hemolyzed specimens are generally high.

In other words, with conventional test strips, there was a large color difference between hemolyzed and non-hemolyzed samples, but the addition of saponin promoted color development in non-hemolyzed samples and improved the color difference between hemolyzed and hemolyzed samples. , it can be seen that the K judgment is more clearly K than ever before.

Example 2. In Example 1K, even when chasaponin was replaced with digitonin, exactly the same results were obtained.

Example 3. In Example 1, even when chasaponin was replaced with tigonine, exactly the same results were obtained.

Example 4. In Example 1, even when chasaponin was replaced with melittin K, exactly the same results were obtained.

Example 5. In Example 1, even when chasaponin was replaced with gramicidin, exactly the same results were obtained.

Example 6. In Example 1, 0.52% of 6-methylquinoline was used instead of 0.52% of 6-methoxyquinoline when preparing the liquid (1).
452 was used, the impregnating solution and the test piece were prepared in the same manner as in Example 1, and the degree of coloration was measured in the same manner as in Example 1. The results were obtained.

Example 2 In Example 1, instead of using 0.52 of K,6-methoxyquinoline when preparing the liquid No. 1, 0.555 of acridine was used.
The impregnating solution was prepared and the test piece was prepared in the same manner as in Example 1, except for using '. Similar results were obtained.

"Effects of the Invention" As described above, the present invention has high sensitivity and accuracy, and significantly reduces the decrease in color sensitivity when measuring non-hemolyzed specimens, which was inevitable with conventional reagent compositions for detecting occult blood. Point K
This is an invention that has remarkable effects, and is an extremely significant invention that will contribute greatly to the industry.

Claims (3)

[Claims] (1) An indicator that is oxidized with a color change due to the presence of complementary blood molecules present in the blood, an oxidizing agent effective for oxidizing the indicator, a buffer that maintains the pH in the range of 4 to 7, and sensitivity. A reagent composition for detecting occult blood in body fluids and body excreta, comprising an activator for increasing hemolytic activity and cytolysin with high hemolytic activity. (2) The composition according to claim (1), wherein the cytolysin with high hemolytic activity is saponin. (3) Saponins include dichitonin, solanine, sarsasaponin, osladin, timosaponin, dioscin, ditonin, ammonin, sapotoxin, cammonin, lily of the valley saponins, smilonin, tigonin, tocorosapotoxin, trilin, trilarin, yukkonin, chasaponins, camellia Claim (2) which is saponin, panakirone, sapinosaponin, spinach saponin, esculin
).