CA1055825A - Calibrator albumin composition - Google Patents
Calibrator albumin compositionInfo
- Publication number
- CA1055825A CA1055825A CA254,123A CA254123A CA1055825A CA 1055825 A CA1055825 A CA 1055825A CA 254123 A CA254123 A CA 254123A CA 1055825 A CA1055825 A CA 1055825A
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- Prior art keywords
- albumin
- standard solution
- analyte
- surfactant
- solution
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT
A standard solution useful in connection with the quantitative colorimetric or spectrophotometric determination of analyte albumin using a specie specific dye-based color reagent is disclosed. The standard solution comprises an aqueous solution of albumin, e.g., bovine, of different specie than the analyte, e.g., human, and a water soluble surfactant containing at least one hydrophobic group of contiguous carbon atoms. The surfactant is of a type and present in an amount such that the standard solution mimics, with respect to spectrophotometric response, an aqueous solution of the analyte when the standard solution and the analyte solution contain the same concentrations of albumin and color reagent.
A standard solution useful in connection with the quantitative colorimetric or spectrophotometric determination of analyte albumin using a specie specific dye-based color reagent is disclosed. The standard solution comprises an aqueous solution of albumin, e.g., bovine, of different specie than the analyte, e.g., human, and a water soluble surfactant containing at least one hydrophobic group of contiguous carbon atoms. The surfactant is of a type and present in an amount such that the standard solution mimics, with respect to spectrophotometric response, an aqueous solution of the analyte when the standard solution and the analyte solution contain the same concentrations of albumin and color reagent.
Description
~101558Z5 The present invention xelates to the analysis of biologic fluids and, more particularly, to the spectrophotometric analysis of biologic fluids using dye binding methods wherein the color imparting dye exhibits species specificity.
Spectrophotometric methods are widely used for determining the concentration of various constituents in biologic fluids such as blood.
The methods usually involve adding a color-forming reagent, generally in an aqueous solution, to the fluid which thereby forms a colored complex having a characteristic wavelength of maximum light absorption with the analyte to be detected. By exposing a sample of the fluid containing the complexed reagent to a light source of the characteristic wavelength of maximum absorption, or maximum differe.nce of absorption with reagent in the blank,and thereafter measuring the degree of light absorption, the content of the analyte in the biologic fluid can be simply determined from '. .
1~55~32~i a calibration graph constructed from absorption measurements made on calibrator solutions containing known concentrations of the analyte. The absorbance of both the sample and the standard are generally read against a blank containing the reagent solu-tion. Of course, to accomplish the determination, a color-forming reagent must be selected which has the ability to form - a colored derivative with the analyte to be detected which derivative is either different in intensity or absorption wave-length characteristics than the color-forming reagent itself.
With respect to the determination of albumin in sera, several general types of dyes are useful as components of color-forming reagents. These include the sulphonphthalein dyes such as bromcresol green, hydroxyphenyl azo dyes such as 2-(hydroxy-phenylazo)-benzoic acid (commonly called HABA), and biuret type reagents. A dye marketed by Pierce Chemical Company of Rockford, Illinois called "SpecTru AB2"* is quite useful. A
particularly advantageous procedure employing the suphonphthalein dyes is described in my United States patent 3,884,637. The use of HABA and "SpecTru AB2"* dyes for determination of albumin has been practiced for a number of years. As to procedures using biuret, these are most frequently accomplished when total prot.ein (albumin plus globulin) is to be determined since biuret forms colored complexes with both of these proteins. When biuret is to be used for just albumin determination, it is necessary to ~irst remove globulin from the sample.
Some albumin-binding dyes exhibit species specificity with respect to albumin. Thus, whereas bromcresol green and ~;~
biuret reagent display little difference in their binding capa-cities with respect to either human or bovine albumin, HABA and "SpecTru" AB2* dyes exhibits a more pronouned capacity to bind * denotes Trade Mark ~ - 2 -.:. ,, :
.. . .
~0~5~S
human albumin than bovine albumin. As a result, when albumin concentration is to be determined in human sera using such a dye, it has heretofore been necessary to also use human albumin as the calibrator. Recently, however, the availability o~ pure albumin of human origin has become irregular, in part because many former sources have provi.ded human albumin potentially carrying hepatitis. On the other hand, suitable bovine albumin i5 generally in good supply.
- 2a -.
.
l(~S58Z5 ~ ccordingly, the prlncipal objective of the present invention resides in providing a solution of one specie of albumin which can be used as a standard in connection with the spectro-photometric determination of another specie of albumin utilizing a species specific dye.
Furthermoxe, there are many instances when it is desir-able to not only determine albumin concentration but also globulin.
These determinations are frequently accomplished by determining albumin with the albumin-binding color reagent and then using biuret reagent to obtain total protein. By difference, the globulin concentration can then be ascertained.
Therefore, a further objective of the present invention is to provide a standard solution having the characteristics recited in the principal object which can be used as the standard for both the determination of alb~nin and the determination of total protein.
Other objects and advantages of the present invention will become apparent as the description proceeds.
Briefly stated, the present invention is based on the discovery that the spectrophotometric response of an aqueous solution containing one species of albumin and a species specific dye-based color reagent can, by addition thereto of a particular type of surfactant, be made to closely mimic the response of an aqueous solution containing the same concentrations of the dye-based color reagent and another spPcie of albumin. As a result, for example, an aqueous solution containing the surfactant, an azo dye color reagent and bovine albuTnin has been found to be useful as a standard in the spectrophotometric procedure for determining human albumin concentration.
Surfactants useful in the present invention as additives for the standard solution are characterized as being water soluble .'~ .
~L~)55~5 and containing a~ least one hyclrophobic group of contiguous carbon atoms. The hydrophobic group contains a chain of at least about six, and preferably at least about nine, contiguous carbon atoms, and while preferably a hydrocarbon, the chain can contain substituen-ts, e.g., alkyl, aryl, halogen, etc. To be water soluble and otherwise useful, and depen~ing on the contiguous carbon atom chain length which can extend up to about 20 carbon atoms, or even higher, e.g., 25, the surfactants also contain hydrophilic groups. In general, the selection of the hydrophilic group must be such that the surfactant is water soluble, or water solubiliz-~ able, at a solution pH of less than about 7, acids pHs being the : most customary for albumin determination.
Further as to the hydrophilic group, useful surfactants can contain non-ionic hydrophilic groups, anionic groups or cationic groups. However, with respect to cationic groups, such as are provided by a quaternary nitrogen atom, it is necessary that the surfactant also contain either a non-ionic hydrophilic group or an anionic group such as provided by a carboxylate or sulphonic acid part. Further with respect to the hydrophilic group of the surfactant, when this group is non-ionic or combined cationic and non-ionic, the non-ionic part is preferably provided by one or more polyethlene oxide chains having at least about six, and ;:.
preferably at least about nine, repeating ethylene oxide units.
And, as a practical matter, the repeating units will generally not extend much above 25. As to anionic hydrophilic groups, the . sulphates, phosphates, and sulfonates are most useful. ~
~ Further with respect to the selection of the water .
~ soluble surfactant the non-ionic and cationic surfactants are generally useful when the dye employed gives greater absorbance per gram albumin per liter with the albumin in the analyte than the albumin selected for the standard. Thus, these surfactants ara most useful when the analyte is human albumin, the standard is bovine x ' ~ ' ' ' ' " ' . ' ' ' ' ~
~SS~25 albumin, and the dye employed is either HABA dye or "SpecTru"
AB2* dye. On the other hand, the anionic sur~actants are most useful when the dye selected gives greater absorbance per gram albumin per liter with the albumin in the standard than it has with respect to the albumin analyte.
Examples of useful surfactants are the following:
E.I~ DuPont product DDN*; a long chain betaine having the structure:
fH3 C12H -N+-CH -COO~
"Ethoquad" 18/25*; an ethoxylatedl hydrocarbon ca-tionic surfactant and having the structure:
fH3 Cl~H3 -N+-(EtO)H
(Et O)nH
"BRIJ" 35*; polyethylene oxide adduct of lauryl alcohol having the structure C12H25O-(Et O)23H
"Bion" NE-9*; polyethylene oxide adduct of p-nonyl phenol having the structure:
9 19 ~ -O-(EtO)gH
Sodium dodecylsulfate having the s-tructure:
C12H25-O-S-O- Na+
O
* denotes Trade Mark ~ _ 5 _ J)~
~)55~Z5 The invention is also directed to a method for the colorimetric or spectrophotometric determination of analyte albumin concentration in a fluid using an albumin specie specific dye-based color reagent. The method entails adding the color reagent to the fluid whereby a colored complex is formed with the analyte albumin, exposing the fluid to a light sourcel measuring the degree of light absorption and determining the concentration of analyte albumin in the fluid by comparison ~ :
of the measured degree of light absorption with the measured degree of light absorption obtained from at least one similarly treated standard solution containing a known concentration of albumin. The invention entails the improvement wherein the albumin, in the standard solution, is of a di~ferent specie than the analyte albumin, and the standard solution is character-ized as comprising an aqueous solution of albumin in known concentration, and a water soluble surfactant containing at least one hydrophobic group containing at least about nine contiguous ~-:
carbon atoms and also containing a hydrophilic group. ~ .
The albumin of the standard solution may be bovine, the analyte albumin is human and the surfactant may be selected from fH3 ~H3 ..
C12H25--I CH2 C18H37--I (EtO)~5_n CH3 (~tO)Hn ~.
C12H250 (Et)23 or C9Hl9 - ~ - O- (EtO)gH
"~ :
. ~~ ........ ".`, - : . .
~L05S~2~
In its broadest aspects, the present invention is useful in connection with the determination of albumin wherein the reayent dye employed exhibits species specificity and wherein it is desired to use a different source of albumin for the standard solution than the albumin in the analyte. Whether the - dye selected has species specificity can be determined by simply obtaining pure samples of the analyte and standard albumin, pre-.
paring aqueous solutions of the two at identical sample concentra-tions, adding identical color-forming reagent compositions to both solutions and then measuring absorbance of each solution at the optimum wavelength. If the measured absorptivity at the same wavelength differs for the serum albumins obtained from the different species or if the rnaximum absorbance is obtained at different wavelengths for the serum albumins from the different species, then the dye exhibi-ts the re~uisite species specificity.
Once it is ascertained that the dye to be used exhibits - species specificity, utilization of the present invention is appropriate. And, to that end, a plurality of solutions are then prepared containing the identical, sample concentrations of the standard albumin and color-forming reagent. Thereafter, one of the above described surfactants is selected and varying concentra-tions of the surfactant are added to the standard solutionsfollowed by absorbance measurements. That concentration which results in a spectrum substantially identical to that of the analyte solutions is then determined to be the appropriate concen~
- tration, forthat surfactant and dye, necessary to obtain the desired mimicing effect.
The following example illustrates the present invention:
A preservative solution was prepared containing the following ingredients in four liters of deionized water:
8.0 gm. sodium azide 25 gm. potassium bromide ~ - 6 -~O~S!3~5 16 gm. sodium fluoride ~ 43.45 gm. phenoxy ethanol ; 20 gm. sodium propionate
Spectrophotometric methods are widely used for determining the concentration of various constituents in biologic fluids such as blood.
The methods usually involve adding a color-forming reagent, generally in an aqueous solution, to the fluid which thereby forms a colored complex having a characteristic wavelength of maximum light absorption with the analyte to be detected. By exposing a sample of the fluid containing the complexed reagent to a light source of the characteristic wavelength of maximum absorption, or maximum differe.nce of absorption with reagent in the blank,and thereafter measuring the degree of light absorption, the content of the analyte in the biologic fluid can be simply determined from '. .
1~55~32~i a calibration graph constructed from absorption measurements made on calibrator solutions containing known concentrations of the analyte. The absorbance of both the sample and the standard are generally read against a blank containing the reagent solu-tion. Of course, to accomplish the determination, a color-forming reagent must be selected which has the ability to form - a colored derivative with the analyte to be detected which derivative is either different in intensity or absorption wave-length characteristics than the color-forming reagent itself.
With respect to the determination of albumin in sera, several general types of dyes are useful as components of color-forming reagents. These include the sulphonphthalein dyes such as bromcresol green, hydroxyphenyl azo dyes such as 2-(hydroxy-phenylazo)-benzoic acid (commonly called HABA), and biuret type reagents. A dye marketed by Pierce Chemical Company of Rockford, Illinois called "SpecTru AB2"* is quite useful. A
particularly advantageous procedure employing the suphonphthalein dyes is described in my United States patent 3,884,637. The use of HABA and "SpecTru AB2"* dyes for determination of albumin has been practiced for a number of years. As to procedures using biuret, these are most frequently accomplished when total prot.ein (albumin plus globulin) is to be determined since biuret forms colored complexes with both of these proteins. When biuret is to be used for just albumin determination, it is necessary to ~irst remove globulin from the sample.
Some albumin-binding dyes exhibit species specificity with respect to albumin. Thus, whereas bromcresol green and ~;~
biuret reagent display little difference in their binding capa-cities with respect to either human or bovine albumin, HABA and "SpecTru" AB2* dyes exhibits a more pronouned capacity to bind * denotes Trade Mark ~ - 2 -.:. ,, :
.. . .
~0~5~S
human albumin than bovine albumin. As a result, when albumin concentration is to be determined in human sera using such a dye, it has heretofore been necessary to also use human albumin as the calibrator. Recently, however, the availability o~ pure albumin of human origin has become irregular, in part because many former sources have provi.ded human albumin potentially carrying hepatitis. On the other hand, suitable bovine albumin i5 generally in good supply.
- 2a -.
.
l(~S58Z5 ~ ccordingly, the prlncipal objective of the present invention resides in providing a solution of one specie of albumin which can be used as a standard in connection with the spectro-photometric determination of another specie of albumin utilizing a species specific dye.
Furthermoxe, there are many instances when it is desir-able to not only determine albumin concentration but also globulin.
These determinations are frequently accomplished by determining albumin with the albumin-binding color reagent and then using biuret reagent to obtain total protein. By difference, the globulin concentration can then be ascertained.
Therefore, a further objective of the present invention is to provide a standard solution having the characteristics recited in the principal object which can be used as the standard for both the determination of alb~nin and the determination of total protein.
Other objects and advantages of the present invention will become apparent as the description proceeds.
Briefly stated, the present invention is based on the discovery that the spectrophotometric response of an aqueous solution containing one species of albumin and a species specific dye-based color reagent can, by addition thereto of a particular type of surfactant, be made to closely mimic the response of an aqueous solution containing the same concentrations of the dye-based color reagent and another spPcie of albumin. As a result, for example, an aqueous solution containing the surfactant, an azo dye color reagent and bovine albuTnin has been found to be useful as a standard in the spectrophotometric procedure for determining human albumin concentration.
Surfactants useful in the present invention as additives for the standard solution are characterized as being water soluble .'~ .
~L~)55~5 and containing a~ least one hyclrophobic group of contiguous carbon atoms. The hydrophobic group contains a chain of at least about six, and preferably at least about nine, contiguous carbon atoms, and while preferably a hydrocarbon, the chain can contain substituen-ts, e.g., alkyl, aryl, halogen, etc. To be water soluble and otherwise useful, and depen~ing on the contiguous carbon atom chain length which can extend up to about 20 carbon atoms, or even higher, e.g., 25, the surfactants also contain hydrophilic groups. In general, the selection of the hydrophilic group must be such that the surfactant is water soluble, or water solubiliz-~ able, at a solution pH of less than about 7, acids pHs being the : most customary for albumin determination.
Further as to the hydrophilic group, useful surfactants can contain non-ionic hydrophilic groups, anionic groups or cationic groups. However, with respect to cationic groups, such as are provided by a quaternary nitrogen atom, it is necessary that the surfactant also contain either a non-ionic hydrophilic group or an anionic group such as provided by a carboxylate or sulphonic acid part. Further with respect to the hydrophilic group of the surfactant, when this group is non-ionic or combined cationic and non-ionic, the non-ionic part is preferably provided by one or more polyethlene oxide chains having at least about six, and ;:.
preferably at least about nine, repeating ethylene oxide units.
And, as a practical matter, the repeating units will generally not extend much above 25. As to anionic hydrophilic groups, the . sulphates, phosphates, and sulfonates are most useful. ~
~ Further with respect to the selection of the water .
~ soluble surfactant the non-ionic and cationic surfactants are generally useful when the dye employed gives greater absorbance per gram albumin per liter with the albumin in the analyte than the albumin selected for the standard. Thus, these surfactants ara most useful when the analyte is human albumin, the standard is bovine x ' ~ ' ' ' ' " ' . ' ' ' ' ~
~SS~25 albumin, and the dye employed is either HABA dye or "SpecTru"
AB2* dye. On the other hand, the anionic sur~actants are most useful when the dye selected gives greater absorbance per gram albumin per liter with the albumin in the standard than it has with respect to the albumin analyte.
Examples of useful surfactants are the following:
E.I~ DuPont product DDN*; a long chain betaine having the structure:
fH3 C12H -N+-CH -COO~
"Ethoquad" 18/25*; an ethoxylatedl hydrocarbon ca-tionic surfactant and having the structure:
fH3 Cl~H3 -N+-(EtO)H
(Et O)nH
"BRIJ" 35*; polyethylene oxide adduct of lauryl alcohol having the structure C12H25O-(Et O)23H
"Bion" NE-9*; polyethylene oxide adduct of p-nonyl phenol having the structure:
9 19 ~ -O-(EtO)gH
Sodium dodecylsulfate having the s-tructure:
C12H25-O-S-O- Na+
O
* denotes Trade Mark ~ _ 5 _ J)~
~)55~Z5 The invention is also directed to a method for the colorimetric or spectrophotometric determination of analyte albumin concentration in a fluid using an albumin specie specific dye-based color reagent. The method entails adding the color reagent to the fluid whereby a colored complex is formed with the analyte albumin, exposing the fluid to a light sourcel measuring the degree of light absorption and determining the concentration of analyte albumin in the fluid by comparison ~ :
of the measured degree of light absorption with the measured degree of light absorption obtained from at least one similarly treated standard solution containing a known concentration of albumin. The invention entails the improvement wherein the albumin, in the standard solution, is of a di~ferent specie than the analyte albumin, and the standard solution is character-ized as comprising an aqueous solution of albumin in known concentration, and a water soluble surfactant containing at least one hydrophobic group containing at least about nine contiguous ~-:
carbon atoms and also containing a hydrophilic group. ~ .
The albumin of the standard solution may be bovine, the analyte albumin is human and the surfactant may be selected from fH3 ~H3 ..
C12H25--I CH2 C18H37--I (EtO)~5_n CH3 (~tO)Hn ~.
C12H250 (Et)23 or C9Hl9 - ~ - O- (EtO)gH
"~ :
. ~~ ........ ".`, - : . .
~L05S~2~
In its broadest aspects, the present invention is useful in connection with the determination of albumin wherein the reayent dye employed exhibits species specificity and wherein it is desired to use a different source of albumin for the standard solution than the albumin in the analyte. Whether the - dye selected has species specificity can be determined by simply obtaining pure samples of the analyte and standard albumin, pre-.
paring aqueous solutions of the two at identical sample concentra-tions, adding identical color-forming reagent compositions to both solutions and then measuring absorbance of each solution at the optimum wavelength. If the measured absorptivity at the same wavelength differs for the serum albumins obtained from the different species or if the rnaximum absorbance is obtained at different wavelengths for the serum albumins from the different species, then the dye exhibi-ts the re~uisite species specificity.
Once it is ascertained that the dye to be used exhibits - species specificity, utilization of the present invention is appropriate. And, to that end, a plurality of solutions are then prepared containing the identical, sample concentrations of the standard albumin and color-forming reagent. Thereafter, one of the above described surfactants is selected and varying concentra-tions of the surfactant are added to the standard solutionsfollowed by absorbance measurements. That concentration which results in a spectrum substantially identical to that of the analyte solutions is then determined to be the appropriate concen~
- tration, forthat surfactant and dye, necessary to obtain the desired mimicing effect.
The following example illustrates the present invention:
A preservative solution was prepared containing the following ingredients in four liters of deionized water:
8.0 gm. sodium azide 25 gm. potassium bromide ~ - 6 -~O~S!3~5 16 gm. sodium fluoride ~ 43.45 gm. phenoxy ethanol ; 20 gm. sodium propionate
2 gm. benzoic acid 2 gm. disodium salt of ethylenediaminetetraacetic acid dihydrate.
About 1/2 liter of the above preservative solution was placed in a one liter flask and 108 grams of bovine albumin added thereto. Then 83.3 ml of DuPont product "DDN"* (25%
active ingredient) was added to the flask and deionized water then added to bring the volume of the modified bovine albumin solution up to one liter.
Two color reagent solutions are then prepared con-taining H~BA and SpecTru AB2* dye, respectively. The solutions contain about 41.5 grams of dye in 850 liters of deionized water containing the following ingredients~
33.3 gm. monoethanol amine 33.3 liter isopropanol 83.2 sodium sulphite anhydrous 23~6 gm. potassium phosphate monobasic anhydrous * denotes Trade Mark - 6a -~5S~
1878 gm. sodium phosphate dibasic hep-tahydrate 2620 ml. formaldehyde (37~ solution) 260 gm. "BION" NE-9*
A concentrated biure-t color reagent solutlon was prepared in one liter of deionized water by dissolving the following reagents therein:
16 gm. cupric sulphate péntahydrate 53 grams sodium potassium tartrate tetrahydrate (Rochelle salt) 5 gm. potassium iodide 85 gm. sodium hydroxide A dilute biuret solution was similarly prepared in four liters of water.
A calibration graph is constructed by making appro-priate dilutions of the modified bovine albumin solution pre-pared above. The solution prepared above acts as a 108 gram/
liter solution of albumin towards either the biuret reagent, HABA, or SpecTru AB2* solutions. As a result, by simply diluting portions of the modified bovine albumin solution, an appropriate calibration graph can be constructed. In making measurements on the calibrator solutions, 0.05 ml. aliquots of the modified bovine albumin solutions are added to individual ; test tubes (13 x 100 mm.) and 3.0 ml. o~ the color reagent solution then added. With respect to HABA and SpecTru AB2*, colorimetric or spectrophotometric measurements are made at 505 nm against a reagent blank set at 0. The blank is prepared by placing 0.05 ml of distilled water and 3.0 ml of the color reagent solution in a similar type of test tube. With respect to dilute biuret reagent, the procedure is the same except that the colorimeter or spectrophotometer is set at 550 nm.
; ~
* denotes Trade Mark , . .
S~25 Once the calibration graph is constructed, albumin concentration and total protein concentration can be readily determined. To accomplish -this, 0.05 ml of human serum sample is placed in a tes-t tube (13 x 100 mm) and 3.0 ml oE either HABA or SpecTru AB2* reagent solution added thereto and the ingredients mixed. The color reaction is substantially instantaneous and, accordingly, the solution color may be read immediately after mixing against a reagent blank as prepared above. The final solution color is stable for at least about 6 hours. The albumin concentration in the human sample can be readily obtained from the calibration graph.
To determine total protein, either a new human serum sample can be used or -the sample used ~or albumin determination can be employed. If the latter procedure is used, the biuret reagent solution should be added within two hours after the addition of the HABA or SpecTru AB2* dye solution. In determining total protein, whether in the unknown sample or for ; calibration purposes, 1.00 ml of the concentrated biuret reagent solution is added to 0.05 ml of the sample. The solution is then allowed to stand for about 30 minutes at room temperature during which time the characteristic lavender color develops.
As mentioned, absorbance is read at 550 nm and total protein in the sample can, as with albumin, be obtained from the calibration graph.
While the present invention has been described in connection with certain specific emboaiments, it is to be under-stood that it is not to be limited to those embodiments. On the contrary, it is intended to cover all alternatives and modifica-tions falling within the spirit and scope of the invention as set forth in the appended claims.
* denotes Trade Mark .
About 1/2 liter of the above preservative solution was placed in a one liter flask and 108 grams of bovine albumin added thereto. Then 83.3 ml of DuPont product "DDN"* (25%
active ingredient) was added to the flask and deionized water then added to bring the volume of the modified bovine albumin solution up to one liter.
Two color reagent solutions are then prepared con-taining H~BA and SpecTru AB2* dye, respectively. The solutions contain about 41.5 grams of dye in 850 liters of deionized water containing the following ingredients~
33.3 gm. monoethanol amine 33.3 liter isopropanol 83.2 sodium sulphite anhydrous 23~6 gm. potassium phosphate monobasic anhydrous * denotes Trade Mark - 6a -~5S~
1878 gm. sodium phosphate dibasic hep-tahydrate 2620 ml. formaldehyde (37~ solution) 260 gm. "BION" NE-9*
A concentrated biure-t color reagent solutlon was prepared in one liter of deionized water by dissolving the following reagents therein:
16 gm. cupric sulphate péntahydrate 53 grams sodium potassium tartrate tetrahydrate (Rochelle salt) 5 gm. potassium iodide 85 gm. sodium hydroxide A dilute biuret solution was similarly prepared in four liters of water.
A calibration graph is constructed by making appro-priate dilutions of the modified bovine albumin solution pre-pared above. The solution prepared above acts as a 108 gram/
liter solution of albumin towards either the biuret reagent, HABA, or SpecTru AB2* solutions. As a result, by simply diluting portions of the modified bovine albumin solution, an appropriate calibration graph can be constructed. In making measurements on the calibrator solutions, 0.05 ml. aliquots of the modified bovine albumin solutions are added to individual ; test tubes (13 x 100 mm.) and 3.0 ml. o~ the color reagent solution then added. With respect to HABA and SpecTru AB2*, colorimetric or spectrophotometric measurements are made at 505 nm against a reagent blank set at 0. The blank is prepared by placing 0.05 ml of distilled water and 3.0 ml of the color reagent solution in a similar type of test tube. With respect to dilute biuret reagent, the procedure is the same except that the colorimeter or spectrophotometer is set at 550 nm.
; ~
* denotes Trade Mark , . .
S~25 Once the calibration graph is constructed, albumin concentration and total protein concentration can be readily determined. To accomplish -this, 0.05 ml of human serum sample is placed in a tes-t tube (13 x 100 mm) and 3.0 ml oE either HABA or SpecTru AB2* reagent solution added thereto and the ingredients mixed. The color reaction is substantially instantaneous and, accordingly, the solution color may be read immediately after mixing against a reagent blank as prepared above. The final solution color is stable for at least about 6 hours. The albumin concentration in the human sample can be readily obtained from the calibration graph.
To determine total protein, either a new human serum sample can be used or -the sample used ~or albumin determination can be employed. If the latter procedure is used, the biuret reagent solution should be added within two hours after the addition of the HABA or SpecTru AB2* dye solution. In determining total protein, whether in the unknown sample or for ; calibration purposes, 1.00 ml of the concentrated biuret reagent solution is added to 0.05 ml of the sample. The solution is then allowed to stand for about 30 minutes at room temperature during which time the characteristic lavender color develops.
As mentioned, absorbance is read at 550 nm and total protein in the sample can, as with albumin, be obtained from the calibration graph.
While the present invention has been described in connection with certain specific emboaiments, it is to be under-stood that it is not to be limited to those embodiments. On the contrary, it is intended to cover all alternatives and modifica-tions falling within the spirit and scope of the invention as set forth in the appended claims.
* denotes Trade Mark .
Claims (11)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A standard solution useful in connection with the quantitative colorimetric or spectrophotometric determination of analyte albumin using an albumin specie specific dye-based color reagent, said standard solution comprising an aqueous solution of albumin in known concentration of different specie than the analyte and a water soluble surfactant containing at least one hydrophobic group containing at least about nine contiguous carbon atoms and also containing a hydrophilic group, said surfactant being of a type and present in an amount such that said standard solution mimics, with respect to spectrophotometric response, an aqueous solution of the analyte when said standard solution and said analyte solution contain the same concentrations of albumin and color reagent.
2. The standard solution of claim 1 wherein the hydrophilic group is an anionic group selected from sulfate, phosphate or sulfonate.
3. The standard solution of claim 1 wherein the hydrophilic group is a mixed cationic/anionic or cationic/non-ionic group.
4. The standard solution of claim 1 wherein the hydrophilic group is a non-ionic group.
5. The standard solution of claim 3 wherein the non-ionic group is a polyethylene oxide chain having at least about nine repeating ethylene oxide units.
- Page 1 of Claims -
- Page 1 of Claims -
6. The standard solution of claim 4 wherein the non-ionic group is a polyethylene oxide chain having at least about nine repeating ethylene oxide units.
7. The standard solution of claim 1 wherein the surfactant is selected from a long chain betaine, an ethoxylated, hydrocarbon cationic surfactant, a polyethylene oxide adduct of lauryl alcohol or p-nonylphenol, or sodium dodecylsulfate.
8. The standard solution of claim 1 wherein the albumin of the standard solution is bovine, the analyte albumin is human and said surfactant is selected from , or
9. The standard solution of claim 8 wherein the surfactant is
10. In a method for the colorimetric or spectro-photometric determination of analyte albumin concentration in a fluid using an albumin specie specific dye-based color reagent comprising adding said color reagent to said fluid whereby a - Page 2 of Claims -colored complex is formed with the analyte albumin, exposing said fluid to a light source, measuring the degree of light absorption and determining the concentration of analyte albumin in said fluid by comparison of said measured degree of light absorption with the measured degree of light absorption obtained from at least one similarly treated standard solution containing a known concentration of albumin, the improvement wherein the albumin in said standard solution is of a different specie than said analyte albumin and said standard solution is as characterized in claim 1.
11. The method of claim 10 wherein the albumin of the standard solution is bovine, the analyte albumin is human and said surfactant is selected from , or - Page 3 of Claims -
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB2496875A GB1538218A (en) | 1975-06-11 | 1975-06-11 | Calibrator compositions |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1055825A true CA1055825A (en) | 1979-06-05 |
Family
ID=10220109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA254,123A Expired CA1055825A (en) | 1975-06-11 | 1976-06-04 | Calibrator albumin composition |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA1055825A (en) |
| GB (1) | GB1538218A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2829531A1 (en) * | 1978-07-05 | 1980-01-24 | Heuck Claus Christian Dr Rer N | METHOD FOR QUANTITATIVE DETERMINATION OF A SERUM PROTEIN IN BLUE SERUM AND PLASMA SAMPLES |
| EP0141879A1 (en) * | 1983-10-18 | 1985-05-22 | Victor A. Bernstam | Surfactant compositions and methods for clarifying and partitioning aqueous lipid-containing specimens |
-
1975
- 1975-06-11 GB GB2496875A patent/GB1538218A/en not_active Expired
-
1976
- 1976-06-04 CA CA254,123A patent/CA1055825A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB1538218A (en) | 1979-01-10 |
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