U.S. patent application number 11/608943 was filed with the patent office on 2007-07-05 for chromogenic tests in the presence of cells.
Invention is credited to Thomas Nikolaus, Hans-Juergen Rode, Manfred Watzele.
Application Number | 20070154975 11/608943 |
Document ID | / |
Family ID | 35686510 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070154975 |
Kind Code |
A1 |
Watzele; Manfred ; et
al. |
July 5, 2007 |
CHROMOGENIC TESTS IN THE PRESENCE OF CELLS
Abstract
The present invention concerns a method for the colorimetric
measurement of enzyme activities comprising the following steps:
preparing a cell suspension or a cell lysate containing cell
components adding a chromogenic substrate at the start of the
enzyme reaction adding a first additional substance to stop the
enzyme reaction colorimetric measurement wherein a second
additional substance which reduces the light scattering and/or
absorption caused by the cells or the cell components is added
during or after termination of the enzyme reaction.
Inventors: |
Watzele; Manfred; (Weilheim,
DE) ; Nikolaus; Thomas; (Muenchen, DE) ; Rode;
Hans-Juergen; (Sandhausen, DE) |
Correspondence
Address: |
ROCHE DIAGNOSTICS OPERATIONS INC.
9115 Hague Road
Indianapolis
IN
46250-0457
US
|
Family ID: |
35686510 |
Appl. No.: |
11/608943 |
Filed: |
December 11, 2006 |
Current U.S.
Class: |
435/23 |
Current CPC
Class: |
G01N 33/52 20130101;
C12Q 1/00 20130101; G01N 21/78 20130101; C12Q 1/25 20130101; G01N
21/82 20130101 |
Class at
Publication: |
435/023 |
International
Class: |
C12Q 1/37 20060101
C12Q001/37 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2005 |
EP |
05027062.8 |
Claims
1. A method for colorimetric measurement of an enzyme reaction
comprising the steps of: preparing a cell suspension or a cell
lysate comprising cell components, adding a chromogenic substrate
at the start of the enzyme reaction, adding a substance which
reduces light scattering and/or absorption caused by the cells or
the cell components, adding a reagent to stop the enzyme reaction,
and conduction a colorimetric measurement wherein said substance is
added before, during or after termination of the enzyme
reaction.
2. The method according to claim 1 wherein said substance adjusts
the refractive index of the cell suspension to that of the cells,
or said substance adjusts the refractive index of the cell lysate
to that of the cell components.
3. The method according to claim 1 wherein said substance removes
water from the cells or cell components due to its molar
concentration and by means of osmotic pressure.
4. The method according to claim 1 wherein said substance dissolves
a membrane of the cell.
5. The method according to claim 4 wherein said substance is a
protein or a peptide.
6. The method according to claim 4 wherein said substance is a
detergent.
7. A kit for colorimetric measurement of an enzyme reaction in a
cell suspension or cell lysate containing cell components, the kit
comprising: a chromogenic substrate, a substance which, when added
to the cell suspension or cell lysate, reduces light scattering
and/or absorption caused by the cells or the cell components, and a
reagent to stop the enzyme reaction.
8. The kit according to claim 7 wherein said substance adjusts the
refractive index of the cell suspension or cell lysate to that of
the cells or cell components.
9. The kit according to claim 7 wherein said substance dissolves a
membrane of the cell.
Description
RELATED APPLICATIONS
[0001] This application claims priority to European application EP
05027062.8 filed Dec. 12, 2005.
FIELD OF THE INVENTION
[0002] The present invention originates from the field of
colorimetric measurement of enzyme activities in cellular
suspensions or lysates. In particular the present invention
concerns a method for measuring enzyme activities in which the
turbidity of the sample caused by cell components is minimized by
adding suitable additives.
BACKGROUND
[0003] If one wants to examine the effect of reagents, active
substances or other influences on an organism, these experiments
are often carried out in cell culture in in vitro models. The
various enzymatic activities of cells yield information on their
condition and physiological properties.
[0004] Often attempts are made to detect the activation of genes by
means of so-called reporter gene tests. In these tests the gene for
a certain detection enzyme (=reporter gene) is placed behind a
promoter whose activation one wishes to detect. Secreted enzymes
such as for example the secretory alkaline phosphatase, the
activity of which is then tested with the enzyme substrate
4-nitrophenyl phosphate, can also be used for this as reporter
genes. This substrate is converted by this enzyme into the product
4-nitrophenol which absorbs light at a wavelength of 405 nm.
[0005] If one wishes to genetically modify cells, one has to
firstly transport the appropriate plasmid DNA through the plasma
membrane and then into the cell nucleus with the aid of suitable
methods. The methods used for this such lipofection must be
optimized in each case for the relevant cell line. Plasmids
containing reporter genera such as the secretory alkaline
phosphatase are also frequently used in these cases in which the
aim is to optimize the transfectability of different cells. The
success of the transfection can then be checked on the basis of the
measured enzyme activity.
[0006] Different methods are also very frequently used to measure
the cytotoxic properties of a substance. A number of tests for
determining cytotoxicity may be found in the literature.
[0007] Many of these tests are based on the property of cytotoxic
substances to damage the cell membrane. Enzymes which can be
detected in the cell culture supernatant by an enzymatic test are
released from cells whose plasma membrane has been damaged. The
amount of released enzymes is proportional to the number of damaged
cells. Such enzyme release tests have been described for
glutamate-oxalocetate transaminase, for glutamate pyruvate
transaminase, for arginosuccinate lyase and for alkaline and acid
phosphatase (Masanet, J., Gomez-Lechon, M. J., and Castell, J. V.,
Toxic. in Vitro 2 (1988) 275-282; Martin, Angela and Clynes,
Martin, In Vitro Cell Dev. Biol. 27A (1991) 183-184). The release
of alkaline phosphatase from human embryonic fibroblasts caused by
lymphocytes was for example tested in this manner (Szekeres, Julia,
Pasca, A. S. and Pejtsik, B., J. Immun. Meth. 40 (1981) 151-154).
However, lactate dehydrogenase is used most frequently for enzyme
release tests since the aforementioned enzymes are often difficult
to determine due to their small amount in many cells. In contrast
to other enzymes lactate dehydrogenase is a very stable cytoplasmic
enzyme which is present in all cells. It is very rapidly released
from cells with a damaged plasma membrane and can be easily
detected in the culture supernatant (Decker, Thomas and
Lohmann-Matthes, Marie-Luise, J. Immun. Meth. 15 (1988) 61-69;
Korzeniewski, Carol and Callewaert, Denis M., J. Immun. Meth.
64(1983) 313-320).
[0008] In this test NAD.sup.+ is reduced in a first step to
NADH/H.sup.+ by the LDH-catalysed conversion of lactate to
pyruvate. In the second step a second enzyme (=catalyst diaphorase)
transfers the H/H.sup.+ from NADH/H.sup.+ to the tetrazolium salt
INT (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium
chloride) which is reduced in this process to the formazan (FIG.
1). The formazan dye is water-soluble and has a broad absorption
maximum at 500 nm, while the substrate INT does not absorb at this
wavelength (FIG. 2).
[0009] In other methods for measuring cytotoxicity fluorescent
substrates such as resazurin are used instead of colorimetric
enzyme substrates to measure LDH (Cytotox-One.TM. assay from the
Promega Company, Madison, Wis.) or glucose-6-phosphate
dehydrogenase (Vybrant Assay from the Molecular Probes Company from
Eugene, Oreg.) in a similar reaction mixture. In other methods
released glyceraldehyde-3-phosphate dehydrogenase is used to
synthesize ATP by a coupled enzyme reaction. The ATP that is formed
in this reaction is used in a bioluminescence reaction with
luciferin and luciferase in which a measurable light signal is
generated (Corey, Michael J., et al., J. Immun. Meth. 207 (1997)
43-51).
[0010] In addition to the tests for measuring a destroyed plasma
membrane, the physiological activity and proliferation rate of
cells is often also determined by means of their ability to reduce
certain colour substrates. Colour substrates are used for this
which change their colour when they are reduced in such a manner
that only the product absorbs light of a certain wavelength which
is then measured. Known reagents for this are for example
tetrazolium salts such as MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT
(sodium
3'-[1-phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitr-
o)-benzylsulfonic acid hydrate) and WST-1 which are offered by
Roche Applied Science (Mannheim, Germany) under the names Cell
Proliferation Kit I (MTT), Cell Proliferation Kit II (XTT) and Cell
Proliferation Reagent WST-1.
[0011] In addition to the tetrazolium salts, the blue dye resazurin
is also used which is converted on reduction into the red strongly
fluorescent dye resazurin which can be measured colorimetrically as
well as fluorimetrically (Lancaster et al., U.S. Pat. No.
5,501,959).
[0012] WO 2003/089635 describes a combination of reagents for
determining released LDH with the substrates resazurin or MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphopheny-
l)-2H-tetrazolium, internal salt). The reduced form of MTS is
measured colorimetrically and the reduced form of resazurin is
measured colorimetrically or preferably fluorimetrically, In the
case of this combination of reagents it is also suggested that a
stop reagent should be added which stops the reaction. A soap or a
detergent or a strong base such as NaOH is suggested as the stop
reagent. A 3% SDS solution is recommended as the soap or detergent.
A person skilled in the art is very familiar with the
protein-denaturing properties of SDS where the reaction is stopped
due to the denaturation of LDH. NaOH stops the reaction by changing
the pH. However, only a very few known detergents or soaps result
in a reaction stop as shown by the examples given below. The
teaching in WO 2003/089635 would thus be to use such detergents or
soaps which would result in a reaction stop i.e. which have
protein-denaturing properties. Furthermore, with regard to the
soaps or detergents stated in this patent no indication is given
that they can be used to reduce light scattering or absorption
caused by cells and thus to increase the sensitivity of
colorimetric measurements.
[0013] In all methods in which an enzyme activity is detected
colorimetrically in the presence of cells, the problem arises
especially in the case of high cell counts that the light is
scattered and absorbed by the cells in the entire wavelength range
of visible light. Thus for example in the method described by
Decker and Lohmann-Mattes for measuring released lactate
dehydrogenase it is pointed out that in the case of high cell
counts it is better to transfer the cell-free supernatants from the
cell culture plate into new reaction vessels because of the
observed high absorption of the cells and to measure the enzymatic
activity in these reaction vessels. This procedure requires a
centrifugation of the culture plates and a careful removal of the
culture supernatant. These are additional working steps which make
it more difficult to use the method for high throughput analysis
for example with the aid of appropriate robots. In addition the
cells may be damaged by the centrifugation or the removal of the
supernatant thus releasing cytoplasmic enzymes which can interfere
with the test result.
[0014] In other methods in which a fluorimetric or a
chemiluminescent measurement is carried out it is not possible to
work with the widely used colorimetric measurement instruments
especially for standard microtitre plates, the so-called ELISA
readers, which considerably restricts the use of these substrates.
In the case of chemiluminescent substrates the signal is often
stable for only a relatively short period which requires a rapid
measurement directly after the end of the test.
[0015] In the method published in WO 2003/089635 for determining
released LDH using the substrates resozurin or MTS, these
substrates are also described for colorimetric measurements. NaOH
or SDS is claimed as a stop reagent. However, it must be noted that
the colorimetric measurement of reduced resazurin only allows a low
sensitivity. The use of NaOH or SDS with tetrazolium salts such as
MTS is disadvantageous since NaOH leads to a precipitation of
reduced tetrazolium salts (see example 2 of this application). Also
SDS is not preferred since in this case tetrazolium salts are
surprisingly bleached out relatively rapidly (see example 2 of this
application).
SUMMARY OF THE INVENTION
[0016] Hence the present invention concerns a method for the
colorimetric measurement of enzyme activities comprising the
following steps: [0017] preparing a cell suspension or a cell
lysate containing cell components [0018] adding a chromogenic
substrate at the start of the enzyme reaction [0019] adding a first
additional substance to stop the enzyme reaction [0020]
colorimetric measurement which is characterized according to the
invention in that a second additional substance which reduces the
light scattering and/or absorption caused by the cells or the cell
components is added during or after termination of the enzyme
reaction.
[0021] This can for example be achieved by means of the fact that
the second additional substance is a substance which adjusts the
refractive index of the suspension or of the lysates to that of the
cells or cell components.
[0022] Alternatively this can be achieved by means of the fact that
the second additional substance is a substance which, due to it
molar concentration, removes water from the cells or cell
components by osmotic pressure.
[0023] This can preferably also be achieved by means of the fact
that the second additional substance dissolves the cell membrane.
Such substances can for example be various types of detergents and
also certain proteins or peptides.
[0024] In addition the present invention also encompasses kits for
the colorimetric measurement of enzyme reactions in cell
suspensions or cell lysates containing cell components comprising
[0025] a chromogenic substrate [0026] a first substance to stop the
enzyme reaction [0027] a second substance which, when added,
reduces the light scattering and/or absorption caused by the cells
or the cell components.
[0028] The second substance can for example according to the
invention adjust the refractive index of the suspension or of the
cell lysate to that of the cells or cell components.
[0029] Alternatively a second additional substance can, due to its
molar concentration, remove water from the cells or cell components
by osmotic pressure.
[0030] The second additional substance can preferably dissolve cell
membranes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1: In the first step lactate dehydrogenase (LDH)
released into the medium reduces NAD.sup.+ to NADH+H.sup.+ by
oxidizing lactate to pyruvate. In the second enzymatic reaction 2H
from NADH+H.sup.+ are transferred to the tetrazolium salt INT
(2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium
chloride).
[0032] FIG. 2: Absorption spectrum of the working solution from the
cytotoxicity detection kit (LDH). The reaction mixture from the
cytotoxicity detection kit (LDH) was added to RPMI 1640 medium
containing 1% bovine serum albumin (BSA) and the absorption
spectrum was measured in the absence ( . . . ) and presence (-) of
LDH.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention is based on a method for the
colorimetric measurement of enzyme activities in the presence of
cells, characterized in that one or more substances are added to
cells which minimize the light scattering and absorption caused by
the cells.
[0034] This is essentially achieved by a method comprising the
following steps [0035] preparing a cell suspension or a cell lysate
containing cell components [0036] adding a chromogenic substrate at
the start of the enzyme reaction [0037] adding a first additional
substance to stop the enzyme reaction [0038] colorimetric
measurement wherein a second additional substance which reduces the
light scattering and/or absorption caused by the cells or the cell
components is added during or after termination of the enzyme
reaction
[0039] Usually if the cell density is high, a cell-free supernatant
has to be firstly formed by centrifugation which, after it has been
removed, can then be measured. The invention described here avoids
these additional working steps by adding reagents to the cells
during or at the end of the test which minimize light scattering
and absorption caused by the cells. This can for example be
achieved by adjusting the refractive index of the cells to that of
the test medium and thus reducing or minimizing the light
scattering. This can for example be achieved by adding glycerol or
ethylene glycol up to a maximum final concentration of 70%.
[0040] In alternative embodiments the volume of the cells and thus
their absorption can be reduced by adding substances such as salts
at high molar concentrations which remove water from the cells due
to the high osmotic pressure of the medium. As a result the volume
of the cells decreases which increases the transparency of the
sample to be tested. The addition of at least 0.3 M NaCl has for
example been proven to be sufficient in this connection.
[0041] The light scattering and absorption of cells is preferably
reduced by permeabilization of the cell membrane. This can for
example be carried out by lytic enzymes such as lipases or
mellitin.
[0042] Detergents are, however, also preferably suitable for
permeabilization. A person skilled in the art knows a large variety
of different detergents. A distinction is made between ionic,
non-ionic and zwitterionic detergents. Among the ionic detergents a
further distinction is made between anionic detergents such as SDS
(sodium dodecyl sulphate), N-laurylsarcosine or sodium cholate and
cationic detergents such as cetyltrimethylammonium bromide (CTAB)
or dodecyltrimethylammonium bromide (DTAB). Examples of non-ionic
detergents are known to a person skilled in the art under the names
Triton.RTM.X-100 (octylphenoxypolyethoxyethanol), Nonidet P40 or
TWEEN.RTM.20 (polyoxyethylene(20)sorbitan monolaurate). Common
zwitterionic reagents are for example CHAPS
([3-3-cholamidopropyl)dimethylammonium]-1-propane sulfonic acid) or
Zwittergent.RTM.3-12(n-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonic
acid.
[0043] Surprisingly the cationic detergents CTAB and DTAB increased
the absorption of the reduced INT substrate which resulted in an
increase in the sensitivity of the test. In addition the signals
with these detergents were very stable over a period of 5 days
whereas the signal decreased considerably with pure PBS buffer or
with the non-ionic detergent Triton X100, and the anionic detergent
SDS caused a rapid loss in absorption.
[0044] All detergents were added at a maximum final concentration
of 3% (v/v) and preferably up to 1% (v/v).
[0045] If the cell membrane is permeabilized in order to reduce the
light scattering and absorption of the cells, car must be taken
that substances released by this step do not affect the reaction.
Thus for example enzymes could be released which produce additional
reaction product or further convert the reaction product so that it
is no longer measurable. Thus for some enzymatic reactions it makes
sense to stop the reaction at the same time as permeabilizing the
cells. Suitable measures for this depend on the reaction which is
to be measured.
[0046] Suitable measures for terminating enzymatic reactions can
for example be the addition of specific enzyme inhibitors or a
change in the reaction conditions such that the reaction is
terminated. In many cases a change in the pH of the reaction medium
is suitable for stopping the reaction. In the cases studied here in
the examples NaOH and preferably HCl was used to change the pH. HCl
was also suggested as a stop reagent for LDH in the Cytotoxicity
Detection Kit (LDH) (Roche Applied Science Cat. No. 11644793001).
In their publication Decker and Lohmann-Matthes (Decker, Thomas and
Lohmann-Matthes, Marie-Luise, J. Immun. Meth. 15 (1988) 61-69)
propose a 15 mM sodium oxamate solution as an inhibitor to stop
LDH.
[0047] Furthermore, enzymatic reactions can be stopped by adding
protein-denaturing substances such as urea or by adding
proteases.
[0048] If these substance, whose addition minimizes the turbidity
of the sample caused by cell components, are already added at the
start of the reaction or during the reaction then it is necessary
that these substances do not influence the enzyme reaction as
such.
[0049] The present invention also concerns the use of certain
substances for the colorimetric measurement of enzyme activities in
the presence of cells or cell components characterized in that
these substances reduce light scattering and absorption caused by
cells or cell components and in doing so do not affect the enzyme
activities.
[0050] The proteins described above such as lipases or mellitin
have proven to be suitable for such a use.
[0051] However, the use of appropriate detergents is particularly
preferred such as N-laurylsarcosine, sodium cholate,
cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium
bromide (DTAB), Triton.RTM.X-100 (octylphenoxypoly-ethoxyethanol),
Nonidet P40, TWEEN.RTM.20 (polyoxyethylene(20)sorbitan
monolaurate), CHAPS
([3-3-cholamidopropyl)dimethylammonium]-1-propanesulfonic acid) and
Zwittergent.RTM.3-12
(n-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonic acid). All
said detergents are used according to the invention up to a maximum
final concentration of 3% (v/v) and preferably up to a maximum
final concentration of 1% (v/v).
[0052] The present invention also concerns reagent kits that enable
the method according to the invention to be carried out. Reagent
kits in the sense of the invention can for example be composed of
one or more substrates of which at least one changes its colour
after the enzymatic conversion such that either the substrate or
the product of this reaction can be specifically measured at a
certain wavelength. These substrates can be a component of the
reagent kit either in a common vessel or, if it ensures a higher
stability, in different vessels.
[0053] Hence the invention also concerns a kit for the colorimetric
measurement of enzyme reactions in cell suspensions or cell lysates
containing cell components comprising [0054] a chromogenic
substrate [0055] a first substance to stop the enzyme reaction
[0056] a second substance which, when added, reduces the light
scattering and/or absorption caused by the cells or the cell
components.
[0057] The second additional substance can for example be a
substance which adjusts the refractive index of the suspension or
of the lysate to that of the cells or cell components.
[0058] Alternatively the second additional substance can be a
substance which, due to its molar concentration, removes water from
the cells or cell components by osmotic pressure.
[0059] The second addition substance can preferably be a substance
which dissolves the cell membrane. Such substances can for example
be various types of detergents such as those that have been
described in connection with the method according to the invention.
Alternatively the permeabilizing substances can be appropriate
proteins or peptides such as lipases or mellitin.
[0060] Additional typical components of a reagent kit are solutions
which contain buffer substances and salts optimized for the
respective enzyme reaction. These solutions are usually added to
the enzyme reaction. These solutions can either be offered
separately or in mixtures with other components of the reagent kit.
Many enzymes require coenzymes which can also be a component of the
reagent kit in a separately filled or common formulation with other
enzymes.
[0061] In many cases an enzyme reaction to be measured is coupled
with other chemical or enzymatic reactions. In this process either
additional substrate is then generated for the reaction to be
measured or the reaction product is converted further. An example
of this is to be found in Corey, Michael J., et al., J. Immun.
Meth. 207 (1997) 43-51 in which the released
glyceraldehyde-3-phosphate dehydrogenase is used to synthesize ATP
via a coupled enzyme reaction. The ATP that is formed in this
reaction is used in a bioluminescence reaction with luciferin and
luciferase to generate a measurable light signal. Hence reagent
kits for such coupled reactions expediently contain other enzymes,
as well as coenzymes, reaction buffers and substrates. In addition
the reagent kit in the sense of the invention contains at least one
substance which minimizes light scattering and absorption caused by
the cells and in doing so does not affect the enzyme activity. This
substance can be present in a separate bottle or for example be a
component of a reaction buffer. In cases in which a cytoplasmic
enzyme is measured and a permeabilization of the cell membrane is
used to minimize light scattering and absorption caused by the
cells, the reagent kit advantageously additionally contains a
substance which stops the chromogenic reaction. This component can
be in a common formulation with another component of the reagent
kit such as the substance for permeabilizing the cell membrane or
be a separate component of the reagent kit.
[0062] The invention is further elucidated by the following
examples, publications and figures the protective scope of which
results from the patent claims. The described procedures are to be
understood as examples which still describe the object of the
invention even after modifications.
SPECIFIC EMBODIMENTS
Example 1
Measurement of the Absorption of Cells after Treatment with
Different Reagents
[0063] The human suspension cell line U 937 was cultured in RPMI
1640 medium (Sigma Company) containing 10% foetal calf serum and 2
mM glutamine. The cells were than washed in fresh RPMI medium and
adjusted to 25,000, 50,000 and 100,000 cells. 100 .mu.l. 100 .mu.l
thereof was in each case transferred to a 96-well standard
microtitre plate. 50 .mu.l RPMI medium containing the appropriate
reagents was added by pipette to each of the cell solutions.
[0064] The absorption of each well was measured at a wavelength of
690 nm after 5 min and 75 min incubation.
[0065] The following table shows that the absorption of the cells
increases with an increasing cell count. In addition it is clear
that all reagents lower the absorption compared to the pure medium
but the optimal concentrations to be used still have to be
determined. TABLE-US-00001 Absorption Absorption after 5 after
Reagent Cell count min 75 min Triton X100 final con- 25 000 cells
0.001 0.001 centration 1% (v/v) 50 000 cells 0.004 0.004 100 000
cells 0.008 0.008 SDS final concentration 25 000 cells 0.002 0.003
1% (v/v) 50 000 cells 0.006 0.006 (state of the art) 100 000 cells
0.010 0.010 CTAB final concentration 25 000 cells 0.006 0.003 1%
(v/v) 50 000 cells 0.031 0.014 100 000 cells 0.025 0.018 DTAB final
concentration 25 000 cells 0.005 0.006 1% (v/v) 50 000 cells 0.012
0.011 100 000 cells 0.024 0.021 N-laurylsarcosine final 25 000
cells 0.001 0.002 concentration 1% (v/v) 50 000 cells 0.003 0.004
100 000 cells 0.012 0.013 RPMI (negative control) 25 000 cells 0.01
0.011 50 000 cells 0.018 0.018 100 000 cells 0.044 0.048
Example 2
Termination of the LDH Reaction with Various Stop Reagents
[0066] For this experiment lysates of U 937 cells were firstly
prepared as a source of LDH. For this purpose 6.times.10.sup.5
cells were suspended in 1 ml distilled water and treated with
ultrasound until all cells were lysed. Subsequently the lysate was
centrifuged for 10 min at 200.times.g and the supernatant free of
cell residues was used for further experiments.
[0067] Cell free lysates of 5000 cells in each case were used in an
LDH test containing components from the Cytotoxicity Detection Kit
(LDH) (Roche Applied Science Cat. No. 11644793).
[0068] As a digression from the said kit, a solution consisting of
2 mM INT, 90 mM L-lactate, 100 mM Tris, pH 8.5 without detergent
was used as the dye solution.
[0069] After a reaction time of 5 min, different stop reagents were
added to these mixtures and the absorption was monitored at 492 nm
(the absorption maximum of reduced INT). TABLE-US-00002 Absorption
0 Absorption 30 Absorption 120 min after min after min after adding
stop adding stop adding stop Stop reagent reagent reagent reagent
RPMI medium 0.113 0.309 0.852 (negative control) SDS final 0.113
0.113 0.105 concentration 3% (v/v) (state of the art) 15 mM sodium
0.113 0.127 0.179 oxamate 15 mM sodium 0.113 0.127 0.191 oxamate +
Triton X100 final concen- tration 1% (v/v)
[0070] The table shows that SDS as well as sodium oxamate stop the
LDH reaction. However, the termination with the oxamate
concentration used is not 100% successful as is the case for SDS.
An optimization of the oxamate concentration would be necessary in
this case. The example with the combination of oxamate with Triton
X100 shows that this inhibitor is also active in the presence of
this detergent. When using SDS a slight decrease in absorption is
observed which indicates an instability of the substrate in
combination with SDS.
[0071] In another experiment HCl and NaOH were used as a stop
reagent.
[0072] For this purpose cell-free lysates each of 1000 cells were
used in an LDH test with the components from the Cytotoxicity
Detection Kit (LDH) containing the modified dye solution. After a
reaction time of 30 min different stop reagents were added to these
mixtures and the absorption was monitored at 492 nm (the absorption
maximum of reduced INT). TABLE-US-00003 Absorption 0 Absorption 30
Absorption 120 min after min after min after adding stop adding
stop adding stop Stop reagent reagent reagent reagent RPMI medium
0.280 0.448 0.962 0.2 M HCl 0.275 0.271 0.261 0.2 M HCl + 0.268
0.269 0.261 Triton X100 final concen- tration 1% (v/v)
[0073] In this case it is clear that HCl is suitable stop reagent
for the LDH reaction. The combination of HCl with Triton X100 leads
in this case to the same result.
[0074] The addition of NaOH at a final concentration of 0.2 M
results in an immediate precipitation of the dye.
Example 3
Measurement of Different Amounts of Released LDH in the Presence of
High Amounts of Cells
[0075] Cell-free lysates of 50,000, 10,000 and 1000 U 937 cells
were incubated at room temperature in volumes of 50 .mu.l in the
wells of a standard microtitre plate for 0, 5 and 30 minutes in a
LDH test containing 100 .mu.l reaction solution from the
Cytotoxicity Detection Kit (LDH) with a modified dye solution. The
reaction was terminated after 5 or 30 minutes incubation time with
6.5 .mu.l 25% HCl. Subsequently 50 .mu.l containing 150,000 U937
cells was added to each of these mixtures and at the same time 50
.mu.l of various detergent solutions was added.
[0076] Subsequently measurement were carried out at a wavelength of
492 nm after 10 minutes and after 5 days. The blank values of a
control mixture in which the reaction was immediately stopped after
0 minutes were in each case subtracted from the measured values.
TABLE-US-00004 Absorption after 5 min Absorption after 30 min
measurement 10 min after measurement 10 min after Amount of lysate
terminating the reaction terminating the reaction Reagent (cell
count) (measurement after 5 days) (measurement after 5 days) PBS
(phosphate buffer 50 000 cells 0.048 (0.024) 0.88 (0.119)
containing salts) 10 000 cells 0.000 (0.000) 0.067 (0.033)
(negative control) 1 000 cells 0.000 (0.000) 0.000 (0.000) 0 cells
0.000 (0.000) 0.000 (0.000) Triton X100 final 50 000 cells 0.101
(0.093) 0.338 (0.150) concentration 1% 10 000 cells 0.038 (0.035)
0.078 (0.065) (v/v) 1 000 cells 0.003 (0.001) 0.006 (0.001) 0 cells
0.000 (0.000) 0.000 (0.000) SDS final 50 000 cells 0.093 (0.008)
0.364 (0.021) concentration 1% 10 000 cells 0.011 (0.000) 0.078
(0.000) (v/v) (state of the art) 1 000 cells 0.000 (0.000) 0.007
(0.000) 0 cells 0.000 (0.000) 0.000 (0.000) CTAB final 50 000 cells
0.095 (0.085) 0.72 (0.63) concentration 1% 10 000 cells 0.036
(0.021) 0.084 (0.077) (v/v) 1 000 cells 0.001 (0.002) 0.010 (0.004)
0 cells 0.000 (0.000) 0.000 (0.000) DTAB final 50 000 cells 0.88
(0.082) 0.70 (0.99) concentration 1% 10 000 cells 0.034 (0.026)
0.075 (0.069) (v/v) 1 000 cells 0.002 (0.002) 0.008 (0.005) 0 cells
0.000 (0.000) 0.000 (0.000)
[0077] It can be seen that LDH can already be detected in the
lysate of 10,000 cells in the presence of 150,000 whole cells after
5 minutes reaction time by adding the detergents whereas this is
not possible when using pure PBS buffer without detergent. After 30
minutes reaction time the LDH from 1000 cells can already be
detected in this background in the detergent-treated samples
whereas only 10,000 cells are detectable without detergent. This is
due to the fact that a higher background absorption caused by the
150,000 cells is present in the samples that were not treated with
detergent.
[0078] It was surprisingly ascertained that cationic detergents
such as CTAB and DTAB kept the measured values for this colour
substrate almost stable over 5 days whereas SDS in this case had a
destabilizing effect.
* * * * *