CA1043677A - Stable colored reference standard for enzymatic determination - Google Patents
Stable colored reference standard for enzymatic determinationInfo
- Publication number
- CA1043677A CA1043677A CA236,982A CA236982A CA1043677A CA 1043677 A CA1043677 A CA 1043677A CA 236982 A CA236982 A CA 236982A CA 1043677 A CA1043677 A CA 1043677A
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- Canada
- Prior art keywords
- weight
- solution
- nitrophenyl
- reference standard
- iodophenyl
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
Abstract
A B S T R A C T
A stable, colored reference standard suitable for use in the deter-mination of enzymatic reactions capable of reducing the colorless 2-(p-iodo-phenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride to the red-colored 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is described. The refer-ence standard of this invention is an aqueous solution containing, in speci-fied amount,1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazaan; serum albumin; an N,N'-dimethylformamide or a dimethylsulfoxide solvent; and iso-propyl alcohol. An inert bulking agent may be added to the aqueous colored standard and the solution obtained may then be lyophilized. The lyophilized form of the colored reference standard is stable for at least six months at 4°C. and 24°C. and can be easily reconstituted with water. The novel colored reference standard solution has an absorbence maximum at 500 nanometers.
Enzymatic determinations in which the colored reference standard of this in-vention may be used include the determination of serum lactate dehydrogenase, creatine phosphokinase, glucose-6-phosphate dehydrogenase and the like.
A stable, colored reference standard suitable for use in the deter-mination of enzymatic reactions capable of reducing the colorless 2-(p-iodo-phenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride to the red-colored 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is described. The refer-ence standard of this invention is an aqueous solution containing, in speci-fied amount,1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazaan; serum albumin; an N,N'-dimethylformamide or a dimethylsulfoxide solvent; and iso-propyl alcohol. An inert bulking agent may be added to the aqueous colored standard and the solution obtained may then be lyophilized. The lyophilized form of the colored reference standard is stable for at least six months at 4°C. and 24°C. and can be easily reconstituted with water. The novel colored reference standard solution has an absorbence maximum at 500 nanometers.
Enzymatic determinations in which the colored reference standard of this in-vention may be used include the determination of serum lactate dehydrogenase, creatine phosphokinase, glucose-6-phosphate dehydrogenase and the like.
Description
:
This invention relates to a stable, colored reference standard suitable for use in the determination of enzymatic reactions capable of re-ducing the colorless 2-(p^iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride to the red-colored l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenyl-formazan.
Methods for the quantitative determination of certain enzymes are based on the production of NADH (reduced nicotinamide adenine dinucleotide) -~
or NADPH (reduced nicotinamide adenine dinucleotide phosphate) as a result of the enzymatic reaction on a substrate; the NADH or NADPH in turn retuces colorless 2-tp-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoliwm chloride ~ ~commonly known as INT) in the presence of an electron carrier such as phen-¦ azine methosulfate, to l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan I tcommonly known as INT formazan), which is bright red and can be measured spectrophotometrically. Concentrations of enzyme are tetermined by simul-taneously running the test reaction on a reference sample containing known quantities of the enzyme and comparing spectrophotometric readings of the unknown against the reference sample.
Represcntative enzymatic determinations in which the above-described general methot is used include the following: Babson, A.L. and Phillips, G.E., "A Rapid Colorimetric Assay for Serum Lactic DehYdro~enase", i`
Clin. Chim, Acta 12, 210-215 (19653; Van Der Veen et al., "Isoenzvmes of Creatine Phosphokinase in Tissue Extracts and in Normal ant Patholo~ical Sera", Clin. Chim, Acta 13, 312-316(1966); and Nachlas, M.M., et al., "The Determination of Lactic Dehydrogenase with a Tetrazolium Salt", Anal.
Biochem., 1, 3I7-326 (1960). ;
. ~ , -The accuracy of results using aforementioned methods for deter-mining enzymatic activities depends, to a large extent, on the preparation ~j ànd stability of the~reference sample containing the known quantity of ~ enzyme against which results with the test sample are compared. Furthermore, -; 30 these enzymatic test methods involve double work, since in every instance, d~ -. .
9~
1~ ' ' .
-1~436~7 the test itself must be run on the reference sample, as well as on the un-known sample. Such dupli ~tion requires extensive checking in order to insure that inaccuracies have not occurred.
Thus, the need for and the advantages of a stable colored reference standart for use in enzymatic determinations without simultanéous running of a control are obvious. However, such a product is not commercially available.
According to the present invention, there is provided a colored reference standard for use in diagnostic determinations involving enzymatic reactions in which l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is produced, which comprises an aqueous solution of:
A. From about 0.001% to about 0.020% by weight, based on the weight of the total solution, of l-tp-iodophenyl)-5-~p-nitrophenyl)-3-phenylformazan;
B. From about 0.01% to about 0.10% by weight of serum albumin;
C. Prom about 2% to about 10% by weight of solvent selected from the group consisting of N,N' dimethylformamide and dimethylsulfoxide; and D. From about 2% to about 10% by weight of isopropyl alcohol;
said aqueous color standard solution having an absorbence maximum at S00 ~-nanometers, From 5% to 20% by weight of an inert bulking agent may be added to the aqueous colored reference standard solution, which may then be lyophilized, The lyophilized product is easily reconstituted with water. The ~queous colored rePerence standard solution has an absorbence maximum at 500 nano-meters and is suitable for use in the determination of certain enzyme activities, such as serum lactate dehydrogenase, creatine phorphokinase, glu-cose-6-phosphate dehydrogenase and like.
The lyophilized form of the colored reference standard of this invention is stable for at least six months at 4C. and 24C. Furthermore, the lyophilized form of the reference standard of this invention may be reconstituted with water to form a clear solution which can be utilized, without difficulty, as a reference standard in enzymatic determinations.
This invention relates to a stable, colored reference standard suitable for use in the determination of enzymatic reactions capable of re-ducing the colorless 2-(p^iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride to the red-colored l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenyl-formazan.
Methods for the quantitative determination of certain enzymes are based on the production of NADH (reduced nicotinamide adenine dinucleotide) -~
or NADPH (reduced nicotinamide adenine dinucleotide phosphate) as a result of the enzymatic reaction on a substrate; the NADH or NADPH in turn retuces colorless 2-tp-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoliwm chloride ~ ~commonly known as INT) in the presence of an electron carrier such as phen-¦ azine methosulfate, to l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan I tcommonly known as INT formazan), which is bright red and can be measured spectrophotometrically. Concentrations of enzyme are tetermined by simul-taneously running the test reaction on a reference sample containing known quantities of the enzyme and comparing spectrophotometric readings of the unknown against the reference sample.
Represcntative enzymatic determinations in which the above-described general methot is used include the following: Babson, A.L. and Phillips, G.E., "A Rapid Colorimetric Assay for Serum Lactic DehYdro~enase", i`
Clin. Chim, Acta 12, 210-215 (19653; Van Der Veen et al., "Isoenzvmes of Creatine Phosphokinase in Tissue Extracts and in Normal ant Patholo~ical Sera", Clin. Chim, Acta 13, 312-316(1966); and Nachlas, M.M., et al., "The Determination of Lactic Dehydrogenase with a Tetrazolium Salt", Anal.
Biochem., 1, 3I7-326 (1960). ;
. ~ , -The accuracy of results using aforementioned methods for deter-mining enzymatic activities depends, to a large extent, on the preparation ~j ànd stability of the~reference sample containing the known quantity of ~ enzyme against which results with the test sample are compared. Furthermore, -; 30 these enzymatic test methods involve double work, since in every instance, d~ -. .
9~
1~ ' ' .
-1~436~7 the test itself must be run on the reference sample, as well as on the un-known sample. Such dupli ~tion requires extensive checking in order to insure that inaccuracies have not occurred.
Thus, the need for and the advantages of a stable colored reference standart for use in enzymatic determinations without simultanéous running of a control are obvious. However, such a product is not commercially available.
According to the present invention, there is provided a colored reference standard for use in diagnostic determinations involving enzymatic reactions in which l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is produced, which comprises an aqueous solution of:
A. From about 0.001% to about 0.020% by weight, based on the weight of the total solution, of l-tp-iodophenyl)-5-~p-nitrophenyl)-3-phenylformazan;
B. From about 0.01% to about 0.10% by weight of serum albumin;
C. Prom about 2% to about 10% by weight of solvent selected from the group consisting of N,N' dimethylformamide and dimethylsulfoxide; and D. From about 2% to about 10% by weight of isopropyl alcohol;
said aqueous color standard solution having an absorbence maximum at S00 ~-nanometers, From 5% to 20% by weight of an inert bulking agent may be added to the aqueous colored reference standard solution, which may then be lyophilized, The lyophilized product is easily reconstituted with water. The ~queous colored rePerence standard solution has an absorbence maximum at 500 nano-meters and is suitable for use in the determination of certain enzyme activities, such as serum lactate dehydrogenase, creatine phorphokinase, glu-cose-6-phosphate dehydrogenase and like.
The lyophilized form of the colored reference standard of this invention is stable for at least six months at 4C. and 24C. Furthermore, the lyophilized form of the reference standard of this invention may be reconstituted with water to form a clear solution which can be utilized, without difficulty, as a reference standard in enzymatic determinations.
-2- ~ -.. . . , : .. . ~. , : . . . . . .
1~36~7 The l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan in the colored reference standard of this invention (INT formazan) is commercially available (National Biochemicals Corp., Cleveland, Ohio) and has the follow-ing formula:
I ~ N=N-C=N-NH ~ No2 ~' .
INT formazan is frequently encountered in microbiological identification techniques, particularly for the examination of foods and pathological specimens where bacteria, if present, reduce the colorless 2-(p-iodophenyl)-
1~36~7 The l-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan in the colored reference standard of this invention (INT formazan) is commercially available (National Biochemicals Corp., Cleveland, Ohio) and has the follow-ing formula:
I ~ N=N-C=N-NH ~ No2 ~' .
INT formazan is frequently encountered in microbiological identification techniques, particularly for the examination of foods and pathological specimens where bacteria, if present, reduce the colorless 2-(p-iodophenyl)-
3-(p-nitrophenyl)-5-phenyltetrazolium chloride ~INT) which has the formula:
N ~ ~ I
N N
~3 N02 to the brightly colored INT formazan. Additionally, certain enzymatic re-actions will cause the reduction of INT to INT formazan and it is in the determination of these enzymes that the colored reference standard of the invention finds utility, The solvents which have been found to improve the solubility characteristics of INT formazan and to permit easy reconstitution with water after lyophilization are N,N'-dimethylformamide and dimethylsulfoxide. The addition of isopropyl alcohol also aids in this solubilizing process. It is of considerable importance that these solvents do not interfere with the .. . .
. .
1~3~77 absorbence maximum of the aqueous colored reference standard.
The stable, colored reference standard of this invention also con-tains serum albumin. Bovine serum albumin is preferred. This material is believed to aid in stabilizing bcth the lyophilized and aqueous forms of the reference standard.
Suitable inert bulking agents for use in the reference standard of this invention include natural and synthetic gums such as gum arabic, sodium alginate, extract of Irish moss, carboxymethyl cellulose, polyvinyl pyrrolidinone and the like; sugars such as sorbitol, mannitol, sucrose and the like; and carbohydrates such as starch, dextran and the like. The pre-ferred bulking agent is a dextran having a molecular weight of approximately 10,000.
A preferred colored reference standard may be prepared according to the practice of this invention by forming an aqueous solution, suitable for lyophilization, containing 0.005% to 0.015% by weight, based on the weight of the total solution of l-tp-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan; from 0.02% to 0.075% by weight of bovine serum albumin; from 8% to 14% by weight of dextran having a molecular weight of approximately 2~ 10,000; 2% to 10% by weight of N,N'-dimethylformamide; and from 2% to 10%
by weight of isopropyl alcohol. ;
A particularly preferred colored reference standard, according to i the teaching of this invention, contains, in an aqueous solution, 0.0123%
by weight, based on the weight of the total solution, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan; 0.0563% by weight of bovine serum albumin;
9% by weight dextran having a molecular weight of approximately 10,000; 2.5%
by weight of N,N',dimethylformamide and 7.5% by weight of isopropyl alcohol.
The absorbence of the aqueous colored reference standard of this solution at 500 nanometers is approximately 1.34.
3~ The lyophilized form of the colored reference standard of this
N ~ ~ I
N N
~3 N02 to the brightly colored INT formazan. Additionally, certain enzymatic re-actions will cause the reduction of INT to INT formazan and it is in the determination of these enzymes that the colored reference standard of the invention finds utility, The solvents which have been found to improve the solubility characteristics of INT formazan and to permit easy reconstitution with water after lyophilization are N,N'-dimethylformamide and dimethylsulfoxide. The addition of isopropyl alcohol also aids in this solubilizing process. It is of considerable importance that these solvents do not interfere with the .. . .
. .
1~3~77 absorbence maximum of the aqueous colored reference standard.
The stable, colored reference standard of this invention also con-tains serum albumin. Bovine serum albumin is preferred. This material is believed to aid in stabilizing bcth the lyophilized and aqueous forms of the reference standard.
Suitable inert bulking agents for use in the reference standard of this invention include natural and synthetic gums such as gum arabic, sodium alginate, extract of Irish moss, carboxymethyl cellulose, polyvinyl pyrrolidinone and the like; sugars such as sorbitol, mannitol, sucrose and the like; and carbohydrates such as starch, dextran and the like. The pre-ferred bulking agent is a dextran having a molecular weight of approximately 10,000.
A preferred colored reference standard may be prepared according to the practice of this invention by forming an aqueous solution, suitable for lyophilization, containing 0.005% to 0.015% by weight, based on the weight of the total solution of l-tp-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan; from 0.02% to 0.075% by weight of bovine serum albumin; from 8% to 14% by weight of dextran having a molecular weight of approximately 2~ 10,000; 2% to 10% by weight of N,N'-dimethylformamide; and from 2% to 10%
by weight of isopropyl alcohol. ;
A particularly preferred colored reference standard, according to i the teaching of this invention, contains, in an aqueous solution, 0.0123%
by weight, based on the weight of the total solution, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan; 0.0563% by weight of bovine serum albumin;
9% by weight dextran having a molecular weight of approximately 10,000; 2.5%
by weight of N,N',dimethylformamide and 7.5% by weight of isopropyl alcohol.
The absorbence of the aqueous colored reference standard of this solution at 500 nanometers is approximately 1.34.
3~ The lyophilized form of the colored reference standard of this
-4-~ 3677 invention which, as stated a6OYe, has-been found to be stable for at least 6 months at temperatures of 4C and 24C, when reconstituted with water o~
0.1 N hydrochloric acid, is easily handled ~or dilutions which may be required to construct standard curves for fixed-point colorimetric enzy-matic assays. Reconstituted colored reference standard solutions are stable for eight hours at room temperature, and for at least 48 hours at 4C.
In order to prepare the stable colored reference standard of this invention, the various ingredients named above must be added one to the other in a particular fashion to obtain a product having the necessary stability and solubility to permit reconstitution after lyophilization. Such charac-teristics are achieved by a process which comprises:
A. Completely dissolving from about 0.001% to about 0.020% by weight, based on the weight of the total solution, l-(p-iodophenyl~-5-(p-nitrophenyl~-3-phenylformazan, in from about 2% to about 10% by weight of solvent selected from the group consisting of N,N'-dimethylformamide and dimethylsulfoxide;
B. Adding from about 2% to about 10% by weight of isopropyl alcohol to (A.) slowly, and mixing thoroughly;
CO Dissolving from about 0.01% to about 0.10% by weig~t of serum albumin in water;
D. Adding solution ~B.~ to solution ~C.) slowly, with stirring. In the preferred embodiment of this invention, where lyophilization is desired, the inert bulking agent is dissolved in water along with the serum albumin. The final solution obtained in dispensed in aliquots and subjected to the usual lyophilization procedures.
For use in enzymatic determinations, the a6Ove described 1YG-philized form of the colored reference standard is reconstituted with water or O.lN hydrochloric acid. The colored reference standard solution serves as a standard of known activity against which the unknown test samples are read in order to determine the activity of enzyme in the unknown. The colored reference standard s~ e invention may be diluted, if desir-ed, for construction of curves for the fixed point colorimetric enzymatic assays.
Thus, there is provided a colored reference standard which has -excellent stability in the lyophilized state and which, upon reconstitution, is easily handled in currently used enzymatic determinations.
To further illustrate the present invention, the following examples are given:
EXAMPLE I
Preparation of the Stable Colored Reference Standard Solution A. 12.3 mg of INT formazan is dissolved in 2.5 ml of N,N'-dimethyl-formamide. To this solution 7.5 ml of reagent grade isopropanol is added and mixed thoroughly.
B. 9.0 g of dextran having a molecular weight of approximately 10,000 and 56.3 mg of bovine serum albumin are dissolved in 90 ml of purified water.
C. Solution A is added slowly to Solution B with stirring. The combined solution obtained is dispensed in 4.0 ml aliquots into 10 ml vials, `
frozen and lyophilized for 36 hours with a 30C. set-point. The vials are capped under dry nitrogen. Each vial is reconstituted with 10 ml of 0.1 N -~
hydrochloric acid and the absorbence at 500 nanometers is 1.340 ~ 0.010 which `~-~
represents 540 International Units of lactate dehydrogenase activity at 37C;
or 420 International Units of creatine phosphokinase activity at 30C.
EXAMPLE II
Colorimetric Assay for Serum Lactate Dehydrogenase Reagents for the assay are prepared as follows:
Color Reagent: 50 mg of INT is dissolved in about 15 ml of water withprolonged agitation until complete~dissolution is obtained. 125 mg of nicotinamide adenine dinucleotîde is added and dissolved, follo~ed by the addition of 12.5 mg phenazine methosulfate. The solution is transferred with washings immediately to a low actinic 25-ml volumetric flask and diluted with 1()43~77 water to the mark.
A Buffer Reagent: 1.0 g of ethoxylated oleyl alcohol (Lipal 10-OA, ....
Drew Chemical Co., Boonton, N.J.) is dissolved in 10 ml of water by heating to about 95C. The solution is diluted with water to 50 ml and 12.1 g of Tris is added. The pH is adjusted to 8.2 with 3 N HCL and then diluted to lOO ml.
Substrate Reagent: (0.1 M L~+)lactate) 5.0 ml of a 20% solution of L(+) lactic acid is added to about 50 ml of water. The pH is adjusted to
0.1 N hydrochloric acid, is easily handled ~or dilutions which may be required to construct standard curves for fixed-point colorimetric enzy-matic assays. Reconstituted colored reference standard solutions are stable for eight hours at room temperature, and for at least 48 hours at 4C.
In order to prepare the stable colored reference standard of this invention, the various ingredients named above must be added one to the other in a particular fashion to obtain a product having the necessary stability and solubility to permit reconstitution after lyophilization. Such charac-teristics are achieved by a process which comprises:
A. Completely dissolving from about 0.001% to about 0.020% by weight, based on the weight of the total solution, l-(p-iodophenyl~-5-(p-nitrophenyl~-3-phenylformazan, in from about 2% to about 10% by weight of solvent selected from the group consisting of N,N'-dimethylformamide and dimethylsulfoxide;
B. Adding from about 2% to about 10% by weight of isopropyl alcohol to (A.) slowly, and mixing thoroughly;
CO Dissolving from about 0.01% to about 0.10% by weig~t of serum albumin in water;
D. Adding solution ~B.~ to solution ~C.) slowly, with stirring. In the preferred embodiment of this invention, where lyophilization is desired, the inert bulking agent is dissolved in water along with the serum albumin. The final solution obtained in dispensed in aliquots and subjected to the usual lyophilization procedures.
For use in enzymatic determinations, the a6Ove described 1YG-philized form of the colored reference standard is reconstituted with water or O.lN hydrochloric acid. The colored reference standard solution serves as a standard of known activity against which the unknown test samples are read in order to determine the activity of enzyme in the unknown. The colored reference standard s~ e invention may be diluted, if desir-ed, for construction of curves for the fixed point colorimetric enzymatic assays.
Thus, there is provided a colored reference standard which has -excellent stability in the lyophilized state and which, upon reconstitution, is easily handled in currently used enzymatic determinations.
To further illustrate the present invention, the following examples are given:
EXAMPLE I
Preparation of the Stable Colored Reference Standard Solution A. 12.3 mg of INT formazan is dissolved in 2.5 ml of N,N'-dimethyl-formamide. To this solution 7.5 ml of reagent grade isopropanol is added and mixed thoroughly.
B. 9.0 g of dextran having a molecular weight of approximately 10,000 and 56.3 mg of bovine serum albumin are dissolved in 90 ml of purified water.
C. Solution A is added slowly to Solution B with stirring. The combined solution obtained is dispensed in 4.0 ml aliquots into 10 ml vials, `
frozen and lyophilized for 36 hours with a 30C. set-point. The vials are capped under dry nitrogen. Each vial is reconstituted with 10 ml of 0.1 N -~
hydrochloric acid and the absorbence at 500 nanometers is 1.340 ~ 0.010 which `~-~
represents 540 International Units of lactate dehydrogenase activity at 37C;
or 420 International Units of creatine phosphokinase activity at 30C.
EXAMPLE II
Colorimetric Assay for Serum Lactate Dehydrogenase Reagents for the assay are prepared as follows:
Color Reagent: 50 mg of INT is dissolved in about 15 ml of water withprolonged agitation until complete~dissolution is obtained. 125 mg of nicotinamide adenine dinucleotîde is added and dissolved, follo~ed by the addition of 12.5 mg phenazine methosulfate. The solution is transferred with washings immediately to a low actinic 25-ml volumetric flask and diluted with 1()43~77 water to the mark.
A Buffer Reagent: 1.0 g of ethoxylated oleyl alcohol (Lipal 10-OA, ....
Drew Chemical Co., Boonton, N.J.) is dissolved in 10 ml of water by heating to about 95C. The solution is diluted with water to 50 ml and 12.1 g of Tris is added. The pH is adjusted to 8.2 with 3 N HCL and then diluted to lOO ml.
Substrate Reagent: (0.1 M L~+)lactate) 5.0 ml of a 20% solution of L(+) lactic acid is added to about 50 ml of water. The pH is adjusted to
5.5 with 1 N NaOH and diluted to 120 ml with water. The solution is saturated wi~h a few drops of chloroform.
Control Reagent: 0.2 g of potassium oxalate and 0.2 g of ethy-lenediaminetetraacetic acid, disodium dihydrate, are dissolved in 100 ml of water. The above preparations are described in Babson et al. Clin Chim~ Acta 2: 210-215 (1965).
Procedure: 0.1 ml of serum and 0.2 ml of buffer reagent are .
pipetted in each of two tubes. 0.5 ml of substrate is added to one tube and 0.5 ml of control reagent is added to the other tube. Both tubes are mixed and warmed to 37C. At precisely timed intervals, 0.2 ml of color reagent is added to both tubes, and the contents are mixed and immediately returned to the water bath. Exactly five minutes after adding color reagent, 5 ml of 0.1 N HCl is added to both tubes and the contents mixed. The difference in absorbence between the control tube and the serum sample tube, determined at 500 nm within 20 minutes is 0.67. This absorbence is compared with the colored reference standard of Example I which has an absorbence of 1.340 +
0.010 equivalent to 540 International Units of lactate dehydrogenase activity.
The lactate dehydrogenase activity in the serum is calculated to be 270 Inter-national Units at 37C.
~, ~,. ,7~e ~0, .. . . . .
Control Reagent: 0.2 g of potassium oxalate and 0.2 g of ethy-lenediaminetetraacetic acid, disodium dihydrate, are dissolved in 100 ml of water. The above preparations are described in Babson et al. Clin Chim~ Acta 2: 210-215 (1965).
Procedure: 0.1 ml of serum and 0.2 ml of buffer reagent are .
pipetted in each of two tubes. 0.5 ml of substrate is added to one tube and 0.5 ml of control reagent is added to the other tube. Both tubes are mixed and warmed to 37C. At precisely timed intervals, 0.2 ml of color reagent is added to both tubes, and the contents are mixed and immediately returned to the water bath. Exactly five minutes after adding color reagent, 5 ml of 0.1 N HCl is added to both tubes and the contents mixed. The difference in absorbence between the control tube and the serum sample tube, determined at 500 nm within 20 minutes is 0.67. This absorbence is compared with the colored reference standard of Example I which has an absorbence of 1.340 +
0.010 equivalent to 540 International Units of lactate dehydrogenase activity.
The lactate dehydrogenase activity in the serum is calculated to be 270 Inter-national Units at 37C.
~, ~,. ,7~e ~0, .. . . . .
Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A colored reference standard for use in diagnostic determinations involving enzymatic reactions in which 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is produced, which comprises an aqueous solution of:
A. From about 0.001% to about 0.020% by weight, based on the weight of the total solution, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan;
B. From about 0.01% to about 0.10% by weight of serum albumin;
C. From about 2% to about 10% by weight of solvent selected from the group consisting of N,N'-dimethylformamide and dimethylsulfoxide; and D. From about 2% to about 10% by weight of isopropyl alcohol;
said aqueous color standard solution having an absorbence maximum at 500 nanometers.
A. From about 0.001% to about 0.020% by weight, based on the weight of the total solution, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan;
B. From about 0.01% to about 0.10% by weight of serum albumin;
C. From about 2% to about 10% by weight of solvent selected from the group consisting of N,N'-dimethylformamide and dimethylsulfoxide; and D. From about 2% to about 10% by weight of isopropyl alcohol;
said aqueous color standard solution having an absorbence maximum at 500 nanometers.
2. A colored reference standard according to Claim 1 wherein, there is additionally present from about 5% to about 20% by weight of an inert bulking agent in the aqueous solution; and wherein said aqueous solution con-taining said bulking agent is lyophilized.
3. A colored reference standard according to Claim 1 comprising an aqueous solution of:
A. From about 0.005% to about 0.015% by weight, based on the weight of the total solution, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan;
B. From about 0.02% to about 0.075% by weight of serum albumin;
C. From about 2% to about 10% by weight of N,N'-dimethylformamide;
D. From about 2% to about 10% by weight of isopropyl alcohol; and, additionally, E. From about 8% to about 14% by weight of dextran having a molecular weight of approximately 10,000.
A. From about 0.005% to about 0.015% by weight, based on the weight of the total solution, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan;
B. From about 0.02% to about 0.075% by weight of serum albumin;
C. From about 2% to about 10% by weight of N,N'-dimethylformamide;
D. From about 2% to about 10% by weight of isopropyl alcohol; and, additionally, E. From about 8% to about 14% by weight of dextran having a molecular weight of approximately 10,000.
4. A colored reference standard according to Claim 3 which comprises:
A. About 0.0123% by weight, based on the weight of the total solu-tion, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan;
B. About 0.0563% by weight of bovine serum albumin;
C. About 2.5% by weight of N,N'-dimethylformamide;
D. About 7.5% by weight of isopropyl alcohol; and E. About 9% by weight of dextran having a molecular weight of approximately 10,000;
said aqueous color standard solution having an absorbence at 500 nanometers equivalent to approximately 1.34.
A. About 0.0123% by weight, based on the weight of the total solu-tion, of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan;
B. About 0.0563% by weight of bovine serum albumin;
C. About 2.5% by weight of N,N'-dimethylformamide;
D. About 7.5% by weight of isopropyl alcohol; and E. About 9% by weight of dextran having a molecular weight of approximately 10,000;
said aqueous color standard solution having an absorbence at 500 nanometers equivalent to approximately 1.34.
5. A colored reference standard according to Claim 3 or 4 wherein the aqueous solution is lyophilized.
6. A process for preparing a colored reference standard for use in diagnostic determinations involving enzymatic reactions in which 1-(p-iodo-phenyl)-5-(p-nitrophenyl)-3-phenylformazan is produced, which comprises A. Completely dissolving from about 0.001% to about 0.020% by weight, based on the weight of the total solution, 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan, in from about 2% to about 10% by weight of solvent selected from the group consisting of N,N'-dimethylformamide and dimethylsulfoxide;
B. Adding from about 2% to about 10% by weight of isopropyl alcohol to (A.) slowly, and mixing thoroughly;
C. Dissolving from about 0.01% to about 0.10% by weight of serum albumin in water;
D. Adding solution (B.) to solution (C.) slowly, with stirring.
B. Adding from about 2% to about 10% by weight of isopropyl alcohol to (A.) slowly, and mixing thoroughly;
C. Dissolving from about 0.01% to about 0.10% by weight of serum albumin in water;
D. Adding solution (B.) to solution (C.) slowly, with stirring.
7. A process according to Claim 6, wherein, in Step C., from about 5% to about 20% by weight of inert bulking agent is dissolved, in addition to the serum albumin, in the water; wherein, in Step D., solution (B.) is added slowly with stirring to solution (C.) containing the bulking agent and the serum albumin; and wherein, in an additional step, solution (D.) is lyophilized.
8. A process according to Claim 6 wherein, in Step (A.) from about 0.005% to about 0.015% by weight of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is dissolved in from about 2% to about 10% by weight of N,N'-dimethylformamide; and in Step C, from about 0.02% to about 0.075% by weight of serum albumin in addition to from about 8% to about 14% by weight of a dextran bulking agent having a molecular weight of approximately 10,000, are present.
9. A process according to Claim 8, wherein, in Step (A.), about 0.0123% by weight of 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan is dissolved in 2.5% by weight of N,N'-dimethylformamide; in Step B, about 7.5%
by weight of isopropyl alcohol is added; and in Step C, 9% by weight of dextran having a molecular weight of approximately 10,000 and 0.0563% by weight of bovine serum albumin are dissolved in water.
by weight of isopropyl alcohol is added; and in Step C, 9% by weight of dextran having a molecular weight of approximately 10,000 and 0.0563% by weight of bovine serum albumin are dissolved in water.
10. A process according to Claim 8 or 9, wherein, in an additional step, the final solution is lyophilized.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US51226174A | 1974-10-04 | 1974-10-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1043677A true CA1043677A (en) | 1978-12-05 |
Family
ID=24038352
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA236,982A Expired CA1043677A (en) | 1974-10-04 | 1975-10-03 | Stable colored reference standard for enzymatic determination |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPS557239B2 (en) |
| CA (1) | CA1043677A (en) |
| CH (1) | CH617268A5 (en) |
| DE (1) | DE2537499A1 (en) |
| DK (1) | DK442775A (en) |
| FR (1) | FR2287036A1 (en) |
| GB (1) | GB1468494A (en) |
| IT (1) | IT1043092B (en) |
| NO (1) | NO753268L (en) |
| SE (1) | SE408232B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3048662A1 (en) * | 1980-12-23 | 1982-07-22 | Boehringer Mannheim Gmbh, 6800 Mannheim | STABILIZED PREPARATION OF TETRAZOLIUM SALTS |
| JPS59219270A (en) * | 1983-05-30 | 1984-12-10 | Wako Pure Chem Ind Ltd | Method and reagent for stabilization of tetrazolium salt with cyclodextrin |
| CN110726833B (en) * | 2018-07-17 | 2024-01-09 | 上海瀚联诊断科技有限公司 | Blood glucose quality control liquid preparation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1322951A (en) * | 1969-10-29 | 1973-07-11 | Warner Lambert Co | Process and agent for the determination of glucose |
-
1975
- 1975-08-22 DE DE19752537499 patent/DE2537499A1/en not_active Withdrawn
- 1975-09-05 FR FR7527274A patent/FR2287036A1/en active Granted
- 1975-09-15 GB GB3774775A patent/GB1468494A/en not_active Expired
- 1975-09-25 NO NO753268A patent/NO753268L/no unknown
- 1975-10-01 DK DK442775A patent/DK442775A/en unknown
- 1975-10-02 JP JP11829675A patent/JPS557239B2/ja not_active Expired
- 1975-10-03 IT IT27920/75A patent/IT1043092B/en active
- 1975-10-03 SE SE7511133A patent/SE408232B/en unknown
- 1975-10-03 CA CA236,982A patent/CA1043677A/en not_active Expired
- 1975-10-03 CH CH1285975A patent/CH617268A5/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| IT1043092B (en) | 1980-02-20 |
| CH617268A5 (en) | 1980-05-14 |
| SE7511133L (en) | 1976-04-05 |
| DE2537499A1 (en) | 1976-04-15 |
| SE408232B (en) | 1979-05-21 |
| DK442775A (en) | 1976-04-05 |
| FR2287036A1 (en) | 1976-04-30 |
| GB1468494A (en) | 1977-03-30 |
| JPS5162091A (en) | 1976-05-29 |
| NO753268L (en) | 1976-04-06 |
| JPS557239B2 (en) | 1980-02-23 |
| FR2287036B1 (en) | 1978-04-07 |
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