US 3001915 A
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3,001,915 DIAGNOSTIC COMPOSITION Dale E. Former, Elkhart, Ind., assignor to Miles Laboratories, Inc., Ellthart, Ind., a corporation of Indiana No Drawing. Filed Dec. 16, 1957, Ser. No. 702,819 3 Claims. (Cl. 195-1035) This invention relates to a diagnostic composition. Particularly, the invention relates to a diagnostic composition for detecting conditions of abnormality in biological fluids. Still more particularly, the invention relates to a diagnostic composition for body fluids which represents a combination of two or more tests performed in a single operation.
The art is familiar with various diagnostic tests and de vices which are operable to aid the physician in diagnosing a human ailment. Such test devices have been completely described hereto-fore and have been found to be of a great value to the practicing physician. Three of such diagnostic compositions or test indicators have been described in Serial No. 514,395, filed June 9, 1955, Serial No. 585,977, filed May 21, 1956, and Serial No. 691,156, filed October 21, 1957, and assigned to the instant assignee. Since the diagnostic composition of this invention relates directly to subject matter of these applications, they are hereby incorporated by reference and made a part hereof.
The instant invention has as its object the description of a combination diagnostic composition whereby two or more tests may be made on a biological fluid simultaneously. A further object of the invention is to furnish to those interested in diagnoses a test indicator capable of performing a plurality of tests upon a biological fluid with a single motion. The advantages of such a combination test device are obvious and readily apparent to those skilled in the art.
Briefly stated, the diagnostic composition to which this invention is directed comprises a bibulous carrier which is impregnated at separate portions thereof with a reagent material which will indicate the presence or absence of a different abnormality of a biological fluid. Thus it is possible, for example, to test urine for glucose, albumin, ketone bodies, pH or any combination of these constituents at one time. The bibulous carrier is preferably in the form of a stick or strip of a material which is ca pable of being saturated with a solution of the reagent material. Strips of filter paper, for example, or other bibulous material which comprises a mass or complex of cellulose fibers may be used. Although the preferred embodiment of this invention contemplates a strip of bibulous material which may be held by the fingers on one hand and dipped into the biological fluid so as to immerse the test portions thereof in said fluid, any other physical embodiment such as a continuous roll or strip of the bibulous carrier may be used.
In order to insure and maintain separation of the components of the various test portions of the diagnostic composition of this invention and to prevent the co-rningling when the composition is being used, it is essential that each test portion be separated from the other by means of a barrier. This barrier may be formed by impregnating a portion of the bibulous carrier between the separate test portions with a solution of a material which is impervious to water. The solvent then evaporates leaving the barrier. It is essential that the barrier be present throughout the cross sectional area of the bibulous carrier. That is to say, the barrier portion must be suificient to completely prevent the co-minglinig of the various reagents used in the separate test portions. A solvent must be chosen which will completely dissolve the water im pervious material and leave a fluid film or layer of such material throughout the barrier portion. It is preferred,
nited States Patent Patented Sept. 2 1961 therefore, to use a rapidly evaporating solvent. It has also been found expedient to color the barrier forming solution for ease in examination for imperfections, etc. Any suitable color forming material may be used for this purpose.
The solvent chosen must be one in which the waterimpervious material is sufficiently soluble and which evap crates at a uniformly rapid rate so as to leave the desired complete barrier. Operable solvents include acetone, methylethylketone, the various hydrocarbon solvents such as benzene, petroleum naphtha, petroleum ether, and the like. The preferred material used for preparing the barrier portion is a 5% solution of ethyl cellulose in acetone to which there is added 50 rug/liter of a coloring agent such as D&C Yellow #11.
As was previously stated, the compositions of this -in vention comprise multiple testing devices for determining abnormalities in biological fluids. For example, the combination diagnostic may be so prepared to test, by a single operation, a biological fluid for the presence or absence of glucose, albumin, ketone bodies "and hydrogen ion concentrations or any combination of two or more of these. Contemplated are the following diagnostic composition combinations Glucose-albumin-ketone bodies-pH Glucose-albumin-ketone bodies Glucose-ketone bodies-pH Glucose-albumin-pH Albumin-ketone bodies-pH Glucose-albumin Glucose-ketone bodies Glucose-pH Albumin-ketone bodies Albumin-pH Ketone bodies-pH The relative positions of the separate test portions may be varied to suit the manufacturing processes desired. For practical purposes of observation of results, however, it is preferred to maintain the relative positions consistent throughout all combinations.
Following are set out in detail formulae and procedures for preparing some of the indicating compositions or reagent materials which may be used in impregnating the separate portions of the bibulous carrier according to the concept of the instant invention. It will be realized by those familiar with the art that other formulae may, be used without departing from the spirit of the instant invention.
GLUCOSE INDICATOR Formula:
Glucose oxidase grams 9.2 Peroxidase do 0.19 O-tolidine do Sodium citrate do Citric acid "}do 11.1 Gelatin do 7.2 Sodium alginate do 2.5 Surface active agent (1% suspension) ml. 41.0 Ethanol ml..- 63.0 FD&C Red #3 mg 250.0
Procedure-The sodium alginate dissolved in ml. of distilled water, the glucose oxidase and the peroxidase dissolved in 48 ml. of water, and the surface active agent were thoroughly mixed. The glucose oxidase had an activity of about 2600 units per gram, a unit being by definition that quantity of enzyme which will cause a rate of oxygen uptake of 10 cubic ml. of oxygen at 30 C. by a solution ofglucose contained in a Warburg flask. The peroxidase used was obtained from horseradish, and
its activity was of the same order of that of the hemoglobin of blood. The surface active agent was Tween- 81, a polyoxyethylene substituted sorbitan mono-oleate.
The gelatin was dissolved in 150 ml. of water and mixed with a solution of the sodium citrate and citric acid in 150 ml. of water. The o-tolidine and the FD&C Red #3 (disodium salt of 9-o-carboxyphenyl-6-hydroxy-2,4,5,7-tetraiodo-3-isoxanthone) was dissolved in the ethanol, and this solution added to the gelatin-butter solution. The two solutions were then mixed and stirred. This final solution was used to impregnate the desired portion of the bibulous carrier and the impregnated carrier was dried.
This indicator, when contacted with a biological fluid containing as little as 0.05 glucose gave a blue color. A solution containing no glucose resulted in no color formation whatsoever.
It is postulated that the glucose oxidase enzyme present in the test portion of the carrier converts the glucose present in the solution being tested to gluconic acid and hydrogen peroxide. The peroxidase then liberates active oxygen from the hydrogen peroxide and it is the reaction between the liberated oxygen and the indicator dye which results in the color change. It is to be seen, therefore, that the glucose indicator may comprise an enzyme which has oxidase activity for glucose plus a material which undergoes a color reaction in the presence of a product which is formed when said enzyme is contacted with glucose.
Other reagent materials useful for indicating the presence or absence of glucose may be used. Instead of the sodium citrate-citric acid butter, any other buffer composition capable of buifering from a pH of 2 to 8, preferably 4 to 8, may be used.
Besides the o-tolidine, other color forming materials such as benzidine and the salts thereof, guaiacols, orcinol, pyrogallol gallic acid, leuco dyes such as leucomalachite, leucophenolphthalein and the like may be used.
The preferred glucose indicator contemplates a glucose oxidase, peroxidase, and a color forming substance oxidizable by hydrogen peroxide in the presence of said peroxidase, particularly o-tolidine.
ALBUMIN INDICATOR Procedure.Thc tetrabromphenol blue was dissolved in the alcohol. The citric acid and sodium citrate were dissolved in the water and the two solutions mixed with stirring. This mixture was used to impregnate a portion of a bibulous carrier and the impregnated carrier then dried. When contacted with a biological fluid containing 0.01% albumin, the yellow-colored test portion turned a light green. No color change was observed in the absence of albumin. 7
In addition to the tetrabromphenol blue, other indicator materials which exhibit protein error may be used. These indicators will, in a solvent containing protein, by their color indicate a higher pH value for such solution than is actually the case. The extent to which the characteristic color change point of the indicator is shifted is some indication of the amount of protein in the solution. In addition to tetrabromphenol blue the ethyl ester of tetrabromophenolphthalein, brom cresol green, dimethylaminoazobenzene and Congo red may be used.
The preferred composition is selected so as to lower the pH of the carrier and the dye to a pH slightly below the pH value at which the dye characteristically undergoes its color change. By proper combination a great number of buffers with which the art is familiar may be used. The preferred composition therefore comprises a dye which exhibits protein error, and a fluid acid reacting material effective to buffer the carrier and said dye at a point adjacent to, but on the acid side of, the pH at which the color change of said dye normally occurs.
KETONE BODY INDICATOR thoroughly mixed with the glycine. The mixture was then placed in a suitable beaker, and there was added, with rapid stirring, the monosodium phosphate solution. The mixture was then cooled by means of an ice bath to room temperature.
The sodium nitroprusside was then dissolved in water and added with stirring to the cooled solution. This final solution was used to impregnate the desired portion of the bibulous carrier. The impregnated carrier is dried. When contacted with a biological fluid containing 0.005% of acetoacetic acid, a lavender color was formed in 30 seconds. No color change was observed in the absence of acetoacetic acid.
The preferred embodiment of the invention contemplates a ketone body indicator which comprises a water soluble nitroprusside, a diacid phosphate, a monoacid phosphate, and aminoacetic acid, said constituents being so adjusted as to have, in solution, a pH within the range of from 6.5 to 7.5.
pH INDICATOR Formula:
Ml. Methyl Red (0.1% solution in alcohol) 1.2 Brom Thymol Blue (0.2% solution) 8.0 Surface active agent (2.0% solution) 0.8 Ethanol (93%) 8.0 Stabilizer (10% solution) 24.0
pH 5 Orange. pH 6 Orange-yellow. pH 7 Yellow-green. pH 8 Green pH 9 Blue Barrier The barrier portion of the bibulous carrier, as was stated above, is prepared by impregnating a portion of the bibulous carrier with a material which is impervious to water in such a fashion as to prevent co-mingling of the various reagent materials described above during use, that is to say, when all of the test portions are simultaneously contacted with a biological fluid under test. The various materials which exhibit the desired properties, solubility in a suitable carrier medium or solvent, and imperviousness to water, include the various cellulose esters such as methyl, ethyl, and propyl cellulose, cellulose nitrate, cellulose acid phthalate, polystyrene,
rosin, parafiin, the various polyethylene glycols, unsaturated esters such as vinyl acetate, vinyl butyl, etc.
Example Strips of a bibulous carrier approximately 2 inches long and one quarter of an inch wide were immersed in the glucose indicator described above so that the indicator impregnated the lower one half inch of the strip. The strips were then dried at room temperature. A barrier portion was then prepared by impregnating a one eighth inch area of the strip immediately above the glucose indicator portion with a 5% solution of ethyl cellulose in acetone containing 5-0 ing/liter of D&C Yellow #11 (2-(2-qninolyl)-1,3-indandione). When the barrier portion was dried the next one half inch was impregnated with the albumin indicator described above, and the strip was dried again. The next one eighth inch of the strip was then impregnated with the barrier solution. After drying the strip, the next one half inch was impregnated with the ketone body indicator and dried again. Then a third one eighth inch barrier portion was prepared and the next one half inch of the strip was impregnated with the pH indicator. The impregnations were carried out by means of hypodermic needles, and when finished, the strip comprises a diagnostic composition for testing biological fluids for glucose, albumin, ketone bodies and hydrogen ion concentrations reading from bottom to top. A sample of urine of pH 7 and containing 0.05% glucose, 0.1% albumin, and 0.005% acetoacetic acid was tested by immersing the strip in the urine so as to completely cover the test portions. The glucose portion turned blue, the albumin test portion green, the ketone body test portion lavender, and the pH indicating portion, a yellow-green within 30 seconds.
Diagnostic compositions for various combinations of the reagent materials described above were prepared and checked with a test solution. The solution used was a normal urine of pH 7.0 which was made up to contain 0.05% glucose, 0.1% albumin, and 0.005% aminoacetic acid. Results obtained are set out in the table below.
It is also contemplated that the reagent strips in accordance with the instant invention may be prepared by a multiple operation which involves impregnating a roll of bibulous carrier with continuous lines of the barrier material and of each of the indicating compositions. After drying the roll is then cut into separate test strips. Various other modifications of the impregnating procedure familiar to the art may be used without departing from the spirit of the invention. i
TABLE I Results Combination 1 Glucose pH Albumin Ketone Bodies G-AK blue. yellow-green lavender.-
to greenishblue. GApH do doyellowgreen. GKpH do lavender.-. Do. AK-nFT yellow-green to -do Do.;
greenish-blue. G-A him: (in G-K do lavenden--- G--pH do Do. AT yellow-green to lavenden...
greenish-blue. A-pl-T (in D0- KpH lavender. D0.
1 Legend: G=glucose; A=albumin; K=ketone bodies.
What is claimed is:
1. A test indicator [for biological fluids which comprises a bibulous strip having at least two test portions separately impregnated with a reagent material capable of detecting abnormality of said biological fluid, each test portion being separated from the other by a barrier portion to prevent co-mingling of said reagent material when contacted by said biological fluid, said barrier portion having been formed by dissolving in a solvent therefor a barrier material selected from the group consisting of ethyl cellulose, polystyrene, rosin and paraffin, impregnating said bibulous strip with said dissolved material so that said material completely saturates the cross-sectional area of said bibulous strip and removing said solvent from said impregnated bibulous strip. 7
2. A test indicator for biological fluids according to claim 1, wherein said solvent is acetone and wherein said barrier material is ethyl cellulose.
3. A test indicator for biological fluids according to claim 1, wherein said barrier portion contains a coloring amount of 2-(2-quinolyl)-1,3-indandione.
References Cited in the file of this patent UNITED STATES PATENTS Yagoda Sept. 13, 1938 Cook Aug. 5, 1952 OTHER REFERENCES