Note: Descriptions are shown in the official language in which they were submitted.
CA 02255855 1998-11-19
,
METHOD FOR ELIMINATING HEMOGLOBIN-INTERFERENCES
WHEN DETERMINING ALBUMIN
Description
The present invention concerns a method using optical measurements
for assaying albumin in a sample containing free hemoglobin.
It is well known that the assay albumin in serum and plasma (for
instance employing the bromocresol green procedure) is interfered with to
such extent by hemoglobin (Hb) and compounds analogous to hemoglobin
that spuriously raised albumin contents are evinced. The cause is a reaction
of hemoglobin with the dye, whereby Hb is quantitatively determined as
albumin (Doumas et al, Clin. Chim. Acta 31, 87-96 (1971)).
The European patent document 0,268,025 B1 points out that it is
impossible to remedy Hb interference in the albumin assay by a conventional
2-point measurement. However said document suggests determining a
correction factor from the relation between degree of hemolysis and
measurement errors, and to use said factor (following separate determination
of degree of hemolysis) to correct by calculation the spurious albumin result
of a given sample.
Jay & Provasek (Clip. Chem. 39/9, 1804-1810, 1993) describe that in
their laboratory and for each detectable hemolytic sample they separately
determine this sample's Hb content and, using a computer program, they
correct by calculation the (spurious) result of the analyte determination. The
magnitude of the correction is then stated in the analytical results.
Another approach to correct the Hb interference with albumin is
described in the German Offenlegungsschrift 4,427,492 A1. Therein a
separate determination of degree of hemolysis is eliminated because it can
be derived from a reaction preliminary to the main reaction. When using the
so-called ratePius method, the test result from the main reaction is corrected
by calculation using an ascertained relation between degree of hemolysis and
interference.
The previously described procedures eliminating Hb interference in
albumin assays generally require mathematically correcting the analytical
CA 02255855 1998-11-19
2
results by a quantity corresponding to the Hb content. This Hb content or the
degree of hemolysis is ascertained therein either by a separate measurement
(for instance by multi-wavelength analysis or by an independent procedure)
or by a preliminary reaction, a two-reagent assay always being necessary.
However at present albumin is frequently assayed as a one-point
measurement using a single reagent, the color reaction being started by
mixing sample and reagent (for instance the albumin reagent from Boehringer
Mannheim GmbH). Aside easy handling, such an assay also offers the
advantage of low cost. However eliminating the Hb interference is complex:
it entails either a single reagent and separately determining the Hb content
or
a two-reagent assay and avoidance of separate determinations of the Hb
content. In every instance the analytical result obtained then must be
corrected by calculation.
Moreover, when testing samples of serum or plasma containing
~ hemoglobin, the main reaction (albumin with the testing reagent) will be
~, superposed on an interfering reaction (hemoglobin with the testing reagent)
and on a decrease of the hemoglobin absorption signal caused by the
interfering reaction. Lastly the absorption spectra both from the main
reaction
and from the interfering reaction differ as a function of particular albumin
assay reagents.
Now it was surprisingly found that the effect of the interfering reaction
on the main-reaction test results depends on the combination of test
wavelengths and also on the reaction time.
Accordingly eliminating the hemoglobin interference when assaying
albumin is highly complex because the spurious signal is not a typical
interference signal that might be eliminated by a plain bichromatic
measurement or by simply measuring the value of a dummy sample. Rather
the interfering substance, that is native, crosslinked, polymerized or
recombinantly prepared hemoglobin does directly react with the reagent.
Accordingly test wavelengths must be sought at which this interference
reaction can be definitively distinguished from the main reaction proper.
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This problem has been solved by changing the combination of test
wavelengths, that is the main test wavelength and the secondary test
wavelength of a bichromatic assay. Now it was surprisingly found that when
changing the combinations of wavelengths, assay errors caused by free
hemoglobin in the sample can be substantially suppressed (see Figs. 1, 3, 4,
5, 7, 8, 10 and 11 ). At such combinations of wavelengths that as a rule will
be outside the absorption maximum of the assay reagents, the measurement
signal from a sample containing hemoglobin will change in time-dependent
manner whereas the measurement signal from a sample substantially free of
hemoglobin will be essentially constant.
However such an effect is not observed in the wavelength combination
conventionally used in assaying albumin, that is for a main wavelength of 600
nm and a side wavelength of 700 nm. In such a case a large assay error will
be found in a sample containing hemoglobin (Figs. 2, 6 and 9).
~ Accordingly one object of the present invention is a method for
~, assaying albumin in a sample free of hemoglobin by optically measuring at
least two wavelengths, characterized in that
(a) for the assay an adequately strong measurement signal for
albumin is present at a first test wavelength,
(b) at a second test wavelength (i) no albumin measurement signal or
(ii) a comparatively smaller albumin measurement signal than for the first
wavelength is present, and
(c) a relatively large time-dependent interference signal is present in
the first and second test wavelengths which is caused by a reaction of
hemoglobin with the assay reagent used for albumin assay.
The method of the invention uses a combination of test wavelengths
wherein a first test wavelength results in an albumin measurement signal
sufficiently high for determination. Be it borne in mind that the first test
wavelength need not be at the absorption maximum of the assay reagents
employed, instead even preferably being outside the absorption maximum.
The second test wavelength evinces an albumin measurement signal which
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is substantially smaller than the measurement signal of the first wavelength.
Preferably the measurement signal of the first test wavelength is at least 20
mE and more preferably at least 50 mE higher than that of the second test
wavelength.
Furthermore a comparatively equal interference signal is assumed
being present at the first and the second test wavelengths. When a single-
point measurement is involved, preferably the measurement point shall be
' selected in such a way that the interference signal caused by the hemoglobin
reacting with the assay reagent shall be equally or approximately equally
large in the first and in the second test wavelengths.
As regards a multiple-point measurement, for instance a two-point
measurement (two-reagent assay measuring the dummy sample value and
the analyte value), the measurement points preferably are selected in such
a way that, while taking into account the dilution factor from the pipetting
, volumes of the individual reagents, an equally or approximately equally
large
'~ interference signal shall be obtained at the measurement times, for
instance
at the first and second measurement times. Preferably the multiple-point
assay is carried out as a terminal-point measurement, even though kinetic
measurements also are feasible.
In the method of the invention, the albumin content is determined
optically using a dye reaction. Preferably the albumin content shall be
determined using the bromocresol green or bromocresol purple procedure.
Experiments carried out to-date allowed ascertaining three preferred
combinations of test wavelengths for the assay of albumin using the
bromocresol green procedure and a succinate buffer. In a first preferred
embodiment the assay has been carried out at a first test wavelength of 500
to 600 nm, more preferably 560 to 580 nm, especially 570 t 5 nm and in
particular about 570 nm, and at a second test wavelength of 540 to 552 nm,
especially of 546 t 5 nm and in particular about 546 nm.
In a second preferred embodiment of the present invention, a first test
wavelength of 640 to 680 nm, preferably 660 t 5 nm and in particular about
CA 02255855 1998-11-19
660 nm and a second test wavelength of 470 to 490 nm, preferably 480 t 5
nm and in particular about 480 nm are used.
In a third preferred embodiment of the present invention, a first testing
wavelength of 620 to 640 nm, preferably of 630 t 5 nm and in particular of
5 630 nm is used, and a second test wavelength of 590 to 610 nm, preferably
of 600 t 5 nm and in particular of about 600 nm is used.
Preferably a bromocresol green procedure using a citrate buffer uses
' a first test wavelength of 560 to 580 nm, especially 570 t 5 nm and in
particular about 570 nm and a second test wavelength of 490 to 520 nm,
especially of 505 t 5 nm and in particular about 505 nm.
When using other assay reagents and/or other buffers, the expert will
be able by means of simple assays, for instance by analyzing diode-array
spectra and time-dependent absorption measurements, to identify further
suitable combinations of wavelengths.
, Surprisingly, when using such combinations of wavelengths and
measuring a sample containing hemoglobin, an at least substantially
uncontaminated-value of albumin concentration in the sample will be
obtained, said value no longer requiring subsequent correction by calculation.
The method of the invention allows albumin recovery in the range of 100 t 10
% and preferably 100 t 5 %, especially with albumin values in the lower
medical decision range of about 3.5 g/dl.
The optimal reaction time of the method of the invention can be
ascertained by simple preliminary tests from an absorption/time plot showing
the reaction; in general it is within the range of 0.2 to 10 min. As regards
the
assay of albumin using the bromocresol green procedure in a succinate buffer
for a sample containing hemolytic hemoglobin, it was found that when testing
at the wavelength combination (a), the measurement signal remains
substantially constant over a measurement time of 1 - 10 min, whereby
measurement may be carried out at arbitrary times within this range or, as
called for, also subsequent to it.
A one-point measurement is carried out on a sample containing a
CA 02255855 1998-11-19
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crosslinked hemoglobin, for instance diaspirin-crosslinked (DCL) hemoglobin
as a blood substitute using a single reagent for the wavelength-combination
(a) preferably following a reaction time of 3-10 min, especially 5-7 min and
in
particular about 6 min. As regards the wavelength combination (b) on the
other hand the measurement is preferably carried out after a reaction time of
1 - 4 min, preferably 2-3 min and in particular about 2.5 min following start
of
the reaction.
As regards a sample containing recombinantly prepared hemoglobin
as the blood substitute, a single-point measurement using a singe reagent is
carried out using the wavelength combination (a) preferably following a
reaction time of 1-3 min, especially 1-2 min and in particular about 1.5 min.
As regards the wavelength combination (b), a single-point measurement is
preferably taken after a reaction time of 1-4 min.
When determining albumin using the bromocresol green procedure and
, a citrate buffer of a sample containing DCL hemoglobin, the analyte-value
'~ assay is carried out using a single reagent preferably after 20 to 90 sec
and
especially 30 to 60 s and when using a two-reagent assay preferably 0.2 to
5 min following addition of the reagent 2 (starting reagent).
Again two preferred combinations of test wavelengths were
ascertained for assaying albumin using the bromocresol purple procedure and
they substantially eliminate interference caused by free hemoglobin in the
sample. In a first preferred embodiment the assay is carried out at a first
test
wavelength of 560 to 580 nm, preferably 570 t 5 nm and in particular about
570 nm and at a second test wavelength of 490 to 520, preferably 505 t 5 nm
and in particular about 505 nm.
A second preferred embodiment of the present invention uses a first
test wavelength of 560 to 580 nm, preferably 570 t 5 nm and in particular
about 570 nm and a second test wavelength of 540 to 552, preferably 546 t
5 nm and in particular about 546 nm.
As regards a sample containing a crosslinked hemoglobin, for instance
DCL hemoglobin as the blood substitute, the measurement of the analyte
CA 02255855 1998-11-19
7
value in a two-reagent assay is carried out preferably over 0.2 to 8 min, in
particular about 5 min, following addition of the reagent 2 (starting
reagent).
The measurement of the dummy sample is carried out at the first wavelength
combination (570/546 nm) preferably over 1 to 4 min, especially at about 2.5
min following addition of reagent 1 to the sample. In the second wavelength
combination, the measurement of the dummy sample is carried out preferably
over 0.2 to 4 min, especially 0.2 to 1 min and in particular about 0.5 min
following addition of reagent 1 to the sample.
However the critical feature of the method of the invention is the novel
combination of the main and test wavelengths. Still the measurement at
different optimal measurement times for different kinds of Hb may be used to
further raise the accuracy of measurement.
In this manner it is possible for the first time, and without having to
additionally determine the content of hemoglobin or the degree of hemolysis
, and without having to carry out subsequent corrective calculations for
'~ analytical results affected by hemoglobin, to determine the accurate
content
of albumin in a single measurement step, that is in the form of a 1-point
measurement. The invention offers the further decisive advantage that it is
possible to determine the accurate albumin values also using a single
reagent. This feature allows substantial advantages regarding handling and
prices.
Furthermore a 2-reagent assay can be used, even though not
mandatory. However a 2-reagent assay also offers the advantage within the
scope of the present invention to eliminate as desired the determination of
the
degree of hemolysis together with subsequent correction by calculation of the
spurious albumin values caused by the hemoglobin.
Conventionally serum or plasma samples are used as the samples
containing hemoglobin. It must be noted in this respect that the terminology
"free hemoglobin" in the sense of the present invention relates both to
hemolytic samples and to those samples to which was added a compound
analogous to hemoglobin as a blood substitute, for instance a modified,
CA 02255855 1998-11-19
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polymerized and/or crosslinked derivative of human or bovine hemoglobin, for
instance diaspirin-crosslinked (DCL) hemoglobin or also a recombinantly
prepared hemoglobin.
Furthermore, the method of the invention is preferabling carried out in
automated analysis. An example of suitable automated analytical equipment
is the Boehringer Mannheim/Hitachi 717 analyzer.
The invention is elucidated below by illustrations and examples.
Fig.1 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the present invention using a single reagent at a
combination of 546/570 nm test wavelengths (succinate buffer) in the
presence of crosslinked hemoglobin (DCL-Hb),
Fig. 2 is the plot of the time-dependent measurement signal in the
assay of albumin in the manner of the state of the art using a single reagent
at a 700/600 nm combination of test wavelengths (succinate buffer) in the
, presence of crosslinked hemoglobin (DCL-Hb),
Fig. 3 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the present invention using a single reagent at a
546/570 nm combination of test wavelengths (succinate buffer) in the
presence of recombinantly prepared hemoglobin, and
Fig. 4 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the present invention using a single reagent at a
546/570 nm combination of test wavelengths (succinate buffer) in the
presence of hemolytic hemoglobin,
Fig. 5 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the present invention using a single reagent at a
480/660 nm combination of test wavelengths (succinate buffer) in the
presence of DCL hemoglobin,
Fig. 6 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the state of the art using a single reagent at a
700/600 nm combination of test wavelengths (citrate buffer) in the presence
of DCL hemoglobin,
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Fig. 7 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the present invention using a single reagent at a
505/570 nm combination of test wavelengths (citrate buffer) in the presence
of DCL hemoglobin,
Fig. 8 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the present invention using a two-reagent assay
at a 505/570 nm combination of test wavelengths (citrate buffer) in the
presence of DCL hemoglobin,
Fig. 9 is a plot of the time-dependent measurement signal in the assay
of albumin in the manner of the state of the art using the bromocresol purple
procedure with a two-reagent assay at a 700/600 nm combination of test
wavelengths in the presence of DCL hemoglobin,
Fig. 10 is a plot of the time-dependent measurement signal in the
assay of albumin in the manner of the present invention using the
, bromocresol purple procedure with a two-reagent assay at a 505/570 nm
combination of test wavelengths in the presence of DCL hemoglobin,
Fig. 11 is a plot of the time-dependent measurement signal in the
assay of albumin in the manner of the present invention using the
bromocresol purple procedure with a two-reagent assay in the presence of
DCL hemoglobin,
Fig. 12 shows a diode array spectrum for the assay of albumin using
a single reagent (succinate buffer) and
Fig. 13 shows a diode array spectrum for the assay of albumin using
a single reagent (citrate buffer).
EXAMPLE 1
Albumin was assayed using a single reagent according to the
bromocresol green procedure (Doumas et al, Clin. Chim. Acta 31, 1971, 87-
96) in a succinate buffer. The reagent was:
succinate buffer 75 mmole/Itr, pH 4.2; 0.15 mmole/Itr bromocresol
green.
The assay was carried out as follows:
CA 02255855 1998-11-19
350 NI reagent was added to 3 NI of sample and thereupon the time-
dependent measurement signal (mE) was determined.
The assay was carried out in a Boehringer/Hitachi 717 analyzer using
the 700/600 nm combination of test wavelengths (state of the art) and
5 546/570 nm or 480/660 nm combination of test wavelengths (invention).
The time-dependent measurement signal for samples without
hemoglobin, with 1,000 mg/dl hemoglobin and with 2,000 mg/dl hemoglobin
in the form of crosslinked hemoglobin (DCL-Hb) is shown in Fig. 1 for the
546/570 nm combination of test wavelengths and in Fig. 2 for the 700/600 nm
10 combination of test wavelengths.
It will be observed that with respect to the 546/570 nm combination of
test wavelengths, a drop in the measurement signal was found as the reaction
time increased for the samples containing hemoglobin, whereas the
measurement signal for the sample without hemoglobin remained essentially
, constant. The preferred reaction time at which the measurement signal
'~ corresponds to the signal of an Hb-free sample is about 5-7 min. Fig. 2
shows that when using a 700/600 nm combination of test wavelengths, both
the signals from the Hb-free sample and those of the Hb-containing samples
remain essentially constant.
Table 1 lists the percentage recovery of the albumin content in samples
with different hemoglobin contents. As regards the 700/600 nm combination
of test wavelengths of the state of the art (measurement after a reaction time
of 80 s), marked spuriousness of the assay value was noted as the
hemoglobin content rose. As regards the 546/570 nm combination of test
wavelengths of the invention (measurement following a reaction time of 340
s) and the 480/660 nm combination of the test wavelengths of the invention
(measurement following a reaction time of 140 s), on the other hand, a
recovery of 100 t 2 % independent of the hemoglobin content of the sample
was observed.
Fig. 3 shows the time-dependent measurement signal for samples
without Hb and also with 1,000 or 2,000 mg/dl recombinantly prepared
CA 02255855 1998-11-19
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hemoglobin for the 546/570 nm combination of test wavelengths. The
preferred reaction time is about 1- 2 min.
Fig. 4 shows the time-dependent measurement signal for samples
without Hb and for those with 1,000 mg/dl hemolytic hemoglobin.
Fig. 5 shows the time-dependent measurement signal for samples
without Hb as well as with 1,000 mg/dl or 2,000 mg/dl Hb for the 480/660 nm
combination of test wavelengths of the invention. The preferred reaction time
at which the measurement signal from the DCL-Hb containing sample
corresponds to the signal from an Hb-free sample is about 1-3 min.
With respect to samples containing hemolytic hemoglobin or
recombinantly prepared hemoglobin, good elimination of interference also
could be achieved using the 480/660 nm combination of test wavelengths of
the invention.
EXAMPLE 2
, Albumin was assayed using a single reagent according to the
~ bromocresol green procedure in a citrate buffer (95 mmoles/I, pH 4.1; 0.11
mmoles/l bromocresol green; detergent).
The assay was carried out as described in Example 1. The assay took
place using 700/600 nm and 505/570 nm combinations of test wavelengths
(state of the art and invention resp.) and a measuring time of 80 s (state of
the
art) and of 40-60 s, preferably 50 s, following start of reaction (invention).
For the 700/600 nm combination of test wavelengths, the time-
dependent measurement signals for samples without hemoglobin, with 1,000
mg/dl hemoglobin and with 2,000 mg/dl hemoglobin in the form of DCL
hemoglobin are shown in Fig. 6 and for the 505/570 nm combination of test
wavelengths in Fig. 7.
These Figures show that when using the 505/570 nm combination of
test wavelengths of the invention, as the reaction time increases, the
measurement signal decreases in the samples containing hemoglobin,
whereas the measurement signal of the sample without hemoglobin remains
essentially constant. The preferred reaction time at which the measurement
CA 02255855 1998-11-19
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signal corresponds to the signal of an Hb-free sample is about 20-90 s.
Table 2 shows the percentage recovery of the albumin content in
samples having different contents of hemoglobin. As regards the 505/570 nm
combination of test wavelengths of the invention (measurement taken after
a reaction time of 60 s), a recovery of 100 t 5 % independent of the sample's
hemoglobin content was achieved, whereas with respect to the 700/600 nm
combination of test wavelengths of the state of the art, strongly spurious
assay values were observed with increasing hemoglobin contents.
In samples containing hemolytic hemoglobin or recombinantly prepared
hemoglobin also good interference suppression could be achieved when
using the combination of test wavelengths of the invention.
EXAMPLE 3:
Albumin was assayed by the bromocresol green procedure in a citrate
buffer using a two-reagents assay. The reagents were as follows:
, Reagent 1: 95 mmole/I citrate buffer, pH 4.1; detergent
Reagent 2: 95 mmole/I citrate buffer, pH 4.1; 0.66 mmole/I
bromocresol green; detergent
The assay was carried out as follows:
250 pl of reagent 1 were added to 3 NI of sample and following about
4.5 min the dummy sample value E~ was measured. Thereupon 50 NI of
reagent 2 were added and 0.5 min thereafter the analyte measurement value
E2 was taken. Also, during the entire time the time-dependent measurement
signal was recorded. The assay took place in a Boehringer/Hitachi 717
analyzer using the 505/570 nm combination of test wavelengths of the
invention. The control used was the single reagent cited in Example 2 at the
700/600 nm combination of test wavelengths of the state of the art.
Fig. 8 shows the time-dependent measurement signals for samples
without hemoglobin, with 1,000 mg/dl hemoglobin and with 2,000 mg/dl
hemoglobin in the form of DCL hemoglobin for the 505/570 nm combination
of test wavelengths of the invention.
It may be observed that a decrease in the measurement signal was
CA 02255855 1998-11-19
13
found in the hemoglobin-containing samples at a 505/570 nm combination of
test wavelengths. The preferred measurement times are selected in such
manner that the spurious signal caused by the hemoglobin is equally large,
while taking into account the dilution factor due to the pipetting volumes.
Accordingly and preferably E~ (dummy sample value) is measured about 4.5
min following addition of reagent 1 to the sample and E2 (analyte value) is
measured about 0.5 min following addition of reagent 2.
Table 3 shows the percentage recovery of albumin contents in samples
having different hemoglobin contents. A recovery of 100 t 2 % was achieved
independently of hemoglobin content in the 505/570 nm combination of test
wavelengths of the invention, whereas strongly spurious measurement values
were observed in the 700/600 nm combination of the test wavelengths of the
state of the art.
In samples containing hemolytic hemoglobin or recombinantly prepared
, hemoglobin, good interference suppression also could be achieved using the
combination of wavelengths of the invention.
EXAMPLE 4
Albumin was assayed by menas of the bromocresol purple procedure
using a two-reagent test and two-point measurement. The following reagents
were used:
Reagent 1: 100 mmole/I acetate buffer, pH 5.3; detergent
Reagent 2: 100 mmole/I acetate buffer, pH 5.3; detergent, 526
mmole/I bromocresol purple.
The assay was carried out as follows:
250 NI of reagent 1 were added to 3 NI of sample and about 2.5 min
later the dummy sample value E~ was measured. Thereupon 150 pl of
reagent 2 were added and 5 min later the analyte value E2 was measured.
Moreover the time-dependent measurement signal was determined during all
this time.
The assay was carried out in a Boehringer-Mannheim/Hitachi 717
analyzer using the 700/600 nm combination of test wavelengths of the state
CA 02255855 1998-11-19
14
of the art and 505/570 or 546/570 nm combinations of the invention.
The time-dependent measurement signal for samples without
hemoglobin, with 1,000 mg/dl hemoglobin and with 2,000 mg/dl hemoglobin
in the form of DCL hemoglobin is shown for the 700/600 nm combination of
test wavelengths in Fig. 9, for the 505/570 nm combination of test
wavelengths in Fig. 10 and for the 546/570 nm combination of test
wavelengths in Fig. 11.
It may be noted that with respect to the 700/600 nm combination of test
wavelengths, the hemoglobin-caused rise in signal strength is larger following
addition of reagent 2 than before. This feature indicates that the hemoglobin
itself participates in the reaction with bromocresol purple and that as a
result
the assay of albumin is interfered with. The measurement signals from the
hemoglobin-containing samples as well as that from the sample without
hemoglobin however remain nearly constant both before and after addition of
, reagent 2.
On the other hand a drop of the measurement signals from the
hemoglobin-containing samples can be observed at the 505/570 nm
combination of test wavelengths of the invention, whereas the signal from the
sample lacking hemoglobin remains substantially constant. Accordingly the
preferred reaction times must be selected in such manner in order to
circumvent interference that, while taking into account the dilution factor,
the
hemoglobin-caused interference signal prior to addition of reagent 2 is as
large as, or approximately as large as, the interference signal caused by
hemoglobin after addition of reagent 2. Therefore E~ in Example 4 is
measured about 2.5 min (505/570 nm) or 0.5 min (546/570 nm) following
addition of reagent 1 to the sample and E2 is measured at both combinations
of wavelengths about 5 min after addition of reagent 2.
Table 4 shows the percentage recovery of the albumin content in
samples having different albumin contents. With respect to the 505/570 nm
and 546/570 nm combination of test wavelengths of the invention, a recovery
of 100 t 4 % independent of the sample's hemoglobin content was achieved,
CA 02255855 1998-11-19
whereas with increasing hemoglobin content a strongly spurious
measurement value was found using the 700/600 nm combination of test
wavelengths of the state of the art.
Good interference suppression also was achieved with samples
5 containing hemolytic hemoglobin or recombinantly prepared hemoglobin when
using the combinations of test wavelengths of the invention.
EXAMPLE 5
Figs. 12 and 13 show wavelength-dependent spectra of diode arrays
used in the assay of albumin by means of the bromocresol green procedure
10 employing a single reagent in a succinate or citrate buffer. Sample 1 is a
control with 0.9 % NaCI. Sample 2 is serum + NaCI and sample 3 is serum
+ DCL-hemoglobin. In each case the reaction time was 90 s.
Fig. 12 shows that a further 600/630 nm combination of test
wavelengths is suitable besides the preferred 546/570 or 480/660 nm
15 , combination of test wavelengths cited in Example 1.
CA 02255855 2004-O1-14
16
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CA 02255855 2004-O1-14
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