SOLUTION D'ETALONNAGE CONTENANT DE L'ALBUMINE

CALIBRATOR ALBUMIN COMPOSITION

Abrégé anglais

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.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
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 -

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
<IMG>
<IMG>
, or <IMG>
9. The standard solution of claim 8 wherein the
surfactant is
<IMG>
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
<IMG> <IMG>
, or <IMG>
- Page 3 of Claims -

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.
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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
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~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~
CH3
"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 _
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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
- 5A -
"~ :
. ~~ ........ ".`,
- : . .

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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
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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
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~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
.