KR800001002B1 - Blood test pin - Google Patents

Abstract

The test pin for occult blood detection consist of absorbent materials impregnated with a chromogenic substance, H2O2 and a thiazole [I; R1 = lower alkyl, substituted aralkenyl; R2 = H2; R3 = lower alkyl as a sensitizing agent. Thus, a filter paper impregnated with an emulsion contg. arabic gum, Na lauryl sulfate, cumyl peroxide, etc., dried, and then impregnated with soln. contg. O-toluidine, 2-styryl-4-methyl-thiazole and CHCL3. The paper cuts were glued to a PVC film to give test pin. The test pin could blood concns. 1:1000000 diln.

Description

Occult blood test pin

The present invention relates to a test piece for detecting occult blood in feces containing a thiazole compound as a color source, a hydroperoxide and a sensitizer. More specifically, the present invention provides general formulas as colorants, hydroperoxides and sensitizers.

Wherein R ₁ is alkyl, substituted or unsubstituted aralkenyl, or substituted or unsubstituted aralkyl; R ₂ and R ₃ are each hydrogen or alkyl; and R ₂ and R ₃ are bound to each other to adjacent carbon atoms You may form a benzene ring at the time.)

The present invention relates to a test piece for detecting occult blood in feces, such as urine or feces, containing a thiazole compound (I).

Test specimens according to the invention can be used to detect occult blood in feces (eg, urine, feces), vomiting, gastric contents, or spinal fluid. The bleeding as described above may be caused by cancer, ulcers, hemorrhoids, rash typhus, scurvy, purpura, kidney failure, bladder stones, kidney stones, and the like. Bleeding resulting from such a disease is usually a minor amount and therefore cannot be tested visually or through a microscope.

By the test piece of the present invention, occult blood can be detected with high sensitivity easily and reliably. Thus, the test piece of the present invention is useful for early diagnosis of the disease as described above.

The principle of the occult blood detection method is well known, that is, a method in which oxygen generated by oxidizing hemoglobin in blood as a peroxide as a catalyst is sent to a color source to oxidize the colorant to obtain a colorant, which indicates the presence of occult blood. However, specimens prepared using this principle directly do not give satisfactory results in trace occult blood detection. In addition, the test specimen thus prepared has a drawback that its stability is insufficient during clinical practice.

As a test piece using a sensitizer in addition to the occult blood colorant, a test piece using quinine (Japanese Patent No. 478,815) and a test paper using phenanthridine (US Patent No. 3,853,472) are known. However, the former does not have sufficient sensitivity, and the latter has the drawback that the sensitizer contains a carcinogen.

Sufficient sensitivity is important for the rapid testing of occult blood, and safety must be considered in its handling. As a result of actual clinical trials in the technical field, a concentration of 1: 200,000 or more is required for sensitivity to detect occult blood. In addition, the four seasons physicians pointed out that excessive sensitivity is not desirable for such specimens because false positive dye reactions occur.

In addition to the prior art, the use of pyridine derivatives has recently been proposed as described, for example, in US Pat. No. 3,917,452. Although this proposal is capable of detecting occult blood diluted 1: 1,000,000 times, it has not yet been developed commercially, and thus there are no real advantages in increasing diagnostic sensitivity.

The present inventors have studied extensively to increase the sensitivity of the occult blood test specimen, and found that the thiazole compound (I) as described above has a sensitizing effect of increasing the sensitivity of the occult blood detection application. Completed.

Thus, the main object of the present invention is to provide a test piece for easily and reliably detecting the presence of occult blood.

Another object of the present invention is to provide a simple, quick and convenient method for detecting occult blood in feces or body fluids, in particular urine.

According to the present invention, the following general formula as a sensitizer

Wherein R ₁ is alkyl, substituted or unsubstituted aralkenyl, or substituted or unsubstituted aralkyl; R ₂ and R ₃ are each hydrogen or alkyl; and when R ₂ and R ₃ are bonded to adjacent carbon atoms You may form a benzene ring.)

An occult blood detection test piece containing a thiazole compound (I) represented by is provided.

In the thiazole compound (I), C _1-3 alkyl (eg methyl, ethyl or propyl) is suitable as the alkyl group for R ₁ , R ₂ and R ₃ . As the substituent for R ₁ , substituted or unsubstituted aralkylyl (II) and substituted or unsubstituted aralkyl are suitable as shown in the following formula.

및

And

[Wherein n is 1 or 2; R ′ is hydrogen, halogen (eg oxo, chlorine, cancelled, fluorine), C _1-3 alkyl (eg methyl, ethyl, propyl), or C _1-3 alkoxy (eg methoxy, ethoxy, propoxy )to be.]

Representative examples of thiazole compounds falling within the range of general formula (I) include 2-styryl-4-methyl-thiazole, 2- (p-methoxystyryl) -thiazole and 2- (p-ethoxy Styryl) thiazole, 2- (p-methoxystyryl) -4-methylthiazole, 2-styrylthiazole, 1-phenyl-4-thiazolyl- (2) -butadiene, 2- (p- Methylstyryl) -thiazole, 2- (p-methoxystyryl) -benzothiazole, 2- (p-styryl) -benzothiazole, 2- (p-chlorostyryl) -thiazole, 2 -(p-bromostyryl) -thiazole, 2- (p-fluorostyryl) -thiazole, 2,4-dimethyl-thiazole, 1-phenyl-2- (2-thiazole) ethane and the like Can be mentioned.

Of course, it is clear that all of the compounds falling within the range of the general formula (I) do not have the same degree of sensitization. Thus, the sensitivity of the test piece can be adjusted according to actual requirements. For example, sensitized test pieces are obtained by using the following compounds in a certain order as sensitizers:

2-styryl-4-methyl-thiazole, 2- (p-methoxystyryl) -thiazole, 2- (p-methoxystyryl) -4-methylthiazole> 2-styrylthiazole, 2- (p-methoxystyryl) -thiazole, 1-phenyl-4-thiazolyl- (2) -butadiene, 2- (p-methoxystyryl) -benzothiazole> 2,4-dimethylthia Sol, 1-phenyl-2- (2-thiazole) -ethane, 2- (p-chlorostyryl) -thiazole, (2-styryl) -benzothiazole.

The test piece of the present invention can be prepared, for example, by impregnating an absorbent carrier with the whole reagent required for the color reaction. This impregnation method is suitably in two stages. Initially, the absorbent carrier is impregnated with an emulsion containing a stabilizer of hydroperoxide, emulsifier, tackifier, buffer, color and hydroperoxide. The impregnated material is then dried and impregnated with a solution of sensitizer (I) and color source.

The absorbent carrier thus obtained is dried to prepare a desired test piece. Although the method described above is a suitable method, the order of impregnation of the absorbent carrier due to the impregnation method is not important and can be appropriately changed.

The sensitizer (I) can be used in any of the foregoing, but it is preferable to use the sensitizer in an amount of about 0.5 to 10 times the color source used and the weight.

Examples of the absorbent carrier include filter paper, ion-exchanged cellulose paper, fibers, and those having the same water absorbency.

As hydroperoxides for example cumene hydroperoxide, p-methane hydroperoxide, tetralin hydroperoxide, tert-butylhydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide and di Isopropyl benzene hydroperoxide is mentioned. In addition, other hydroperoxides which can release oxygen in the presence of blood and cause a color reaction which can be detected by oxidizing the color source can be used in the same manner. These hydroperoxides are sufficient to oxidize the color source in the presence of blood to produce a colored material, but are usually used in excess.

Examples of the emulsifier include gum arabic and polyvinyl alcohol, and these are usually used at a concentration of 12.5 to 25% (W / V). In addition, about 5% (W / V) of sodium lauryl sulfate is suitably used as a stabilizer of hydroperoxide.

Gelatin is effective for microencapsulation of the hydroperoxide used. Gelatin is suitably used at a concentration of about 10% (W / V).

Examples of buffers include grate, phosphate, phthalate oxalate, carbonate and organic or inorganic acid-morpholine (or derivatives thereof, such as N-ethylmorpholine). These buffers are used to maintain the pH of the impregnation solution at 5-7, preferably 5-6.

As a color source, benzine, O-tolidine, m-tolidine, dianisidine, 3,3 ', 5,5'-tetramethylbenzidine and diacids in diaminodiphenyl derivatives (e.g. hydrochloric acid, hydrobromic acid, Salts with nitric acid or sulfuric acid) can be exemplified. In addition to this, chloroform extract, guajac, is also useful. Among them, O-tolidine or an N-alkyl derivative thereof is particularly preferable in terms of practical use. In order to easily observe the brightening reaction, a dye such as taratazine may be added as a coloring dye. In preparing the impregnation solution, water, ethanol, chloroform and toluene are used as solvents.

The test piece according to the present invention can detect blood at a concentration diluted 1: 5,00,000 to 1: 1,000,000 times. The sensitivity of the test piece containing no sensitizer can only detect blood at a concentration diluted 1: 10,000 to 1: 30,000 times. Thus, specimens with low sensitivity cannot be used in clinical trials. The test piece of the present invention can measure occult blood in feces or body fluids, especially urine.

The concept of the present invention can be used in compositions and test pieces. The composition can be formulated in a commercial manner suitable for the pharmaceutical art. However, in view of their stability and handling, it is preferable to use this composition in the form of a test piece.

Thiazole compound (I) can be manufactured by following Formula.

(Wherein R ₂ , R ₃ and R ′ each have the same meaning as described above).

That is, compound (I) can be prepared by reacting the corresponding 2-methylthiazole with the corresponding aldehyde while heating in the presence of Lewis acid (eg zinc chloride).

The following examples describe the aspects of the present invention in detail, and the scope of the present invention is not limited by these embodiments.

Example 1

Filter paper (Toyo Filtration No. 131) was impregnated with an emulsion containing the following ingredients:

[First Solution]

25% (W / V) gum arabic 10ml

5% (W / V) sodium lauryl sulfate 2 ml

Cumene Hydroperoxide 1ml

5 ml of 10% (W / V) gelatin-0.2M citrate buffer (pH5.0)

0.5 ml of 1% (W / V) tathrazine / ethanol solution

20 ml of 2M citric acid-N-ethylmorpholine (pH5.5)

The filter paper was removed from this solution and dried at 60-80 ° C. for 5-10 hours. This filter paper was then impregnated with a second solution containing the following ingredients:

[Second solution]

0-Tolidine 50mg

2-styryl-4-methylthiazole 50 mg

10 ml of chloroform

The filter paper was taken out and dried under reduced pressure in the dark for 30 minutes. This filter paper can then be cut to a suitable size and adhered onto a polyvinyl chloride sheet for convenient use. Since there was no difference between the preliminary reaction using human blood and the rat blood, a test reaction was performed using the blood of the rat. Blood was diluted 100 times with water and then diluted with normal urine. When the test piece of the present invention is immersed in blood samples of various dilution concentrations, the color reaction is slightly weak, but can detect a 1: 1,000,000-fold dilution blood. After impregnation, it was judged for 30 seconds. The test piece (yellow) of the present invention was discolored to greenish yellow to greenish blue to the dilution concentration of blood.

The test pieces obtained above showed good results for occult blood, and the test results are shown in the following table, in which Labstix was used for comparison. Labsticks is a commercial true blood detection specimen and uses cumene hydroperoxide, 0-tolidine and a citrate buffer.

TABLE 1

: 양성증색반응. 이 증색반응은 시험편을 시료로 함침시킨 후 30초동안 판정했다. Y=황색, GY=녹황색, G=녹색, BG=청녹색, B=청색.Note: HL / S ^* represents hue-brightness / saturation, respectively, and the color reaction was expressed by Japanese Industrial Standard (JIS) (872-1964). Raptics' color reaction is expressed in terms of standard color chips: Ie-: negative response, ±: extremely weak response, +,

: Positive color reaction. This color reaction was determined for 30 seconds after the test piece was impregnated with the sample. Y = yellow, GY = green yellow, G = green, BG = blue green, B = blue.

As is clear from the above table, the test piece of the present invention can easily detect urine blood loss diluted 1: 400,000 times. The test specimen of the present invention can detect blood diluted 1: 1,000,000 times, but the color is slightly weak.

However, detection of blood diluted 1: 400,000 fold is sufficient for clinical use.

The sensitivity and stability of test pieces containing various sensitizers in clinical trials under various conditions were tested, and the results are shown in the following table.

TABLE 2

Note: Each symbol has the same meaning as defined in Table 1. *, **: The test piece containing these sensitizers was manufactured by the method as described in Example 2.

From the above table, it is clear that the test piece containing the thiazole compound of the present invention has good stability during storage for 1 month at 4 ° C. In particular, test pieces containing 2- (styryl) -4-methylthiazole have good stability. However, the stability of the test piece of the present invention is slightly reduced when it is stored at room temperature.

The stability test of the test piece containing various color source was done, and the result is shown in the following table.

TABLE 3

Note: *, **: Preparation I contains 0-tolidine as the color source and Preparation II contains 3,5,3 ', 5'-detramethylbenzidine. Article II is obtained in the same manner as described above.

The occult blood of the patient contains hemolytic and disease-free non-hemolytic blood, but the ratio of both in urine is not constant. In general, the proportion of hemolytic blood in urine samples increases over time. Therefore, it is meaningful to test the color reaction using urine sample quantum. The test results are shown in the following table.

TABLE 4

Note I: Dilution with distilled water (hemolytic blood)

II: Dilution with isotonic solution

III: Diluted with 1: 100 times of water and then diluted with urine (hemolytic blood)

IV: Dilute with urine (blood free)

Rat blood was washed with isotonic solution to prepare an erythrocyte suspension. The color reaction was judged for 30 or 60 seconds after impregnation of the test piece obtained in Example 1.

From the above table, it can be seen that the test piece of the present invention is more sensitive to hemolytic blood in urine than non-hemolytic blood.

Example 2

As a substitute for 2-styryl-4-methylthiazole of Example 1, 2- (p-methoxystyryl) -thiazole, 2- (p-methoxystyryl) -4-methylthiazole, 2 -Styrylthiazole, 1-phenyl-4-thiazolyl- (2) -butadiene, 2- (p-methylstyryl) -thiazole, 2- (p-methoxystyryl) -benzothiazole, 2 -(Styryl) -benzothiazole, 2- (p-chlorostyryl) -thiazole, 2,4-dimethylthiazole or 1-phenyl-2- (2-thiazole) ethane was used.

The test specimens were prepared by repeating a procedure similar to those described in Example 1, and they showed the same color reaction as those obtained in Example 1.

Example 13

Diethiamino ethylcellulose paper (DE-81, manufactured by whatman Co.) was immersed in the following solution (first solution):

[First Solution]

10% (w / y) polyvinyl alcohol 500 10ml

5% (w / v) sodium lauryl sulfate 4 ml

Cumene Hydroperoxide 3ml

10 ml of 10% albumin-0.2 citrate buffer (pH 5.0)

0.5 ml of 1% (w / v) tatarazine-ethanol aqueous solution

2M tangerine-morpholine mixture (pH5.5) 20 ml

The impregnated paper was taken out and dried at 60-80 ° C. for 5-10 hours and impregnated in the following second solution:

[Second solution]

0-Tolidine 100mg

2- (p-methoxystyryl) -thiazole 50 mg

Toluene 10ml

The impregnated paper was taken out and dried in the dark for 30 minutes under reduced pressure to prepare the desired test piece. This test piece showed the same sensitivity as the test piece of Example 1 for the urine blood test.

Citric acid-N-ethylmorpholine was changed to citric acid-morpholine and the amount of 0-tolidine was increased to 100 mg as in Example 1 to carry out the same procedure to obtain a test piece having high sensitivity.

Claims (1)

In a test piece for detecting latent blood in feces or body fluids, the formula, as a colorant, a hydroperoxide and a sensitizer,

Wherein R ₁ is lower alkyl, substituted or unsubstituted aralkenyl, or substituted or unsubstituted aralkyl, R ₂ and R ₃ are each hydrogen or lower alkyl, and R ₂ and R ₃ are adjacent to each other at an adjacent carbon atom. A benzene ring may be formed at the time of bonding.) A test specimen for occult blood comprising a multi-step or hygroscopic absorbent carrier impregnated with a thiazole compound (I).