Bioluminescence measurement system

The present invention provides a method of increasing the duration of detectable photon emission of a luciferase-luciferin reaction. The method provides a luciferase-luciferin reaction in which photon emission can be detected for up to and including eight hours. A method of the present invention can also be used to detect the presence of luciferase in biological samples. The present invention also provides a composition used in detecting the presence of luciferase in biological samples.

FIELD OF THE INVENTION 
This invention relates generally to the stabilization of luciferase 
catalyzed luminescence. The invention relates as well to compositions and 
methods for improving the lifetimes of the luminescence reaction. This 
invention also includes assay kits for detection of an expressed 
luciferase gene used in reporter-gene techniques. 
BACKGROUND OF THE INVENTION 
Luciferases are found in a wide variety of organisms including fireflies, 
photobacteria, jellyfish and many others. Luciferases are enzymes which 
catalyze the production of light by oxidizing luciferin to oxyluciferin in 
a process known generally as bioluminescence. 
The production of photons by luciferase occurs through a two step reaction 
which consumes luciferin, adenosine triphosphate (ATP) and O.sub.2. In the 
first step, luciferase catalyzes the formation of luciferyl adenylate from 
luciferin and ATP. In this first step pyrophosphate is released and a 
Mg.sup.+2 cofactor or another divalent cation is required for proper 
luciferase function. Upon formation, luciferyl adenylate remains within 
the active site of luciferase. In the next step, luciferase oxidizes 
luciferyl adenylate to an electronically excited oxyluciferin with the 
consumption of oxygen. Light production occurs when the electronically 
excited oxyluciferin decays to the ground state oxyluciferin. The decay 
from the excited state to the ground state occurs with the concomitant 
emission of a photon. The color of the light produced differs with the 
source of the luciferase and appears to be determined by differences in 
the structure of the various luciferases. 
Luciferases have recently become useful in reporter-gene technology. In 
this technique, a reporter gene, such as a luciferase encoding 
polynucleotide is used as an indicator for the transcription and 
translation of a gene in a cellular expression system. The reporter gene 
is operatively linked to a promoter that is recognized by the cellular 
expression system. Other commonly used reporter genes include 
.beta.-galactosidase, and chloramphenicol acetyltransferase (CAT). In a 
typical reporter gene assay, a DNA vector containing the reporter gene is 
transfected into a cell capable of expressing the reporter gene. After a 
sufficient amount of time to allow for the expression of the reporter gene 
has passed, the cellular membrane is disrupted to release the expressed 
gene product. The reagents necessary for the catalytic reaction of the 
reporter gene are then added to the reaction solution and the enzymatic 
activity of the reporter gene is determined. Alternatively, the cell can 
be disrupted in the presence of all reagents necessary for the 
determination of the enzymatic activity of the reporter gene. If a 
.beta.-galactosidase encoding polynucleotide is used as the reporter gene, 
the hydrolysis of a galactoside is determined. If a chloramphenicol 
acetyltransferase encoding polynucleotide is used as the reporter gene, 
the production of an acetylated chloramphenicol is determined. When 
luciferase is used as the reporter gene the photons produced from the 
luciferase-luciferin reaction is measured. 
A major problem in determining expression of a luciferase gene as a 
reporter gene is the short duration of photon production. Typically, 
luciferase catalyzed photon production ceases within a few seconds. Means 
for extending the period of photon production have been eagerly sought. 
Currently a commercially available kit from the Promega Corporation 
(Madison, Wis.) can extend the half-life of luciferase catalyzed photon 
production to roughly five minutes. Nevertheless, for the measurement of 
large numbers of samples, luciferase catalyzed photon production with a 
half-life of only five minutes is not a viable alternative. As used 
herein, half-life is the time it takes for photon production to decrease 
by one half. 
The present invention provides methods and compositions for increasing the 
duration of detectable photon emission of a luciferase-luciferin reaction. 
BRIEF SUMMARY OF THE INVENTION 
In one aspect, the present invention provides a method for increasing the 
duration of detectable photon emission of a luciferase-lucifefin reaction. 
In one embodiment, a reaction mixture containing luciferase, luciferin, 
ATP, and cofactors required for luciferase catalytic activity is mixed 
with a composition containing adenosine monophosphate, a radical scavenger 
and a chelating agent to form an admixture. The photons produced by the 
luciferase-luciferin reaction is then detected by measuring the 
luminescence of the admixture. The luciferase catalyzed photon production 
can be detected for more than five minutes. 
In a preferred embodiment, 100 ml of the admixture contains about 2.8 mg 
luciferin, about 110 mg adenosine triphosphate (ATP), cofactors necessary 
for luciferase catalytic activity, about 2.2 mg adenosine monophosphate 
(AMP), about 385 mg dithiothreitol (DTT), and about 20 mg 
ethylenediaminetetraacetic acid (EDTA). 
In another embodiment, the amount of one of the components of the admixture 
can be varied while the amounts of all other components remain unchanged. 
For example, in 100 ml of the admixture, the amount of luciferin can be 
varied between about 0.2 to about 30 mg while maintaining the amount of 
ATP at about 110 mg, AMP at about 2.2 mg, DTT at about 385 mg and EDTA at 
about 20 mg. Similarly, components can be varied individually as follows: 
for 100 ml of the admixture, the amount of luciferin can be varied from 
about 0.2 to about 30 mg; the amount of ATP can be varied from about 10 to 
about 300 mg; the amount of AMP can be varied from about 0.2 to about 30 
mg; the amount DTT can be varied from about 200 to about 2000 mg; and the 
amount of EDTA can be varied from between about 10 to about 50 mg. 
In another embodiment, the present invention contemplates a method for 
detecting the presence of luciferase in a biological sample. The 
biological sample suspected of containing luciferase is mixed with a 
reaction mixture which contains luciferin, adenosine triphosphate, 
cofactors required for luciferase catalytic activity, adenosine 
monophosphate, dithiothreitol, ethylenediaminetetraacetic acid, 
phenylacetic acid, oxalic acid, and a detergent to form an admixture. The 
photons produced by the luciferase-luciferin reaction are then detected by 
measuring the luminescence of the admixture. 
In a preferred embodiment, the present invention contemplates an admixture 
for detecting the presence of a luciferase in a biological sample. One 
hundred ml of the admixture contains about 2.8 mg luciferin, about 110 mg 
adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg 
dithiothreitol, about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg 
phenylacetic acid, and about 0.85 mg oxalic acid. 
In another embodiment, the amount of one of the components of the admixture 
can be varied while the amounts of all other components remain unchanged. 
For example, in 100 ml of the admixture, the amount of luciferin can be 
varied between about 0.2 to about 30 mg while maintaining the amount of 
ATP at about 110 mg, AMP at about 2.2 mg, DTT at about 385 mg, EDTA at 
about 20 mg, phenylacetic acid at about 4.5 mg, and oxalic acid at about 
0.85 mg. Similarly, components can be varied individually as follows: for 
100 ml of the admixture, the amount of luciferin can be varied from about 
0.2 to about 30 mg; the amount of ATP can be varied from about 10 to about 
300 mg; the amount of AMP can be varied from about 0.2 to about 30 mg; the 
amount DTT can be varied from about 200 to about 2000 mg; the amount of 
EDTA can be varied from about 10 to about 50 mg; the amount of 
phenylacetic acid can be varied from about 1 to about 10 mg; and the 
amount of oxalic acid can be varied from about 0.2 to about 5 mg. 
In another aspect, the present invention contemplates a composition used in 
detecting the presence of luciferase in biological samples by detecting an 
emitted photon from a luciferase-luciferin reaction. One hundred ml of 
this composition contains about 2.8 mg luciferin, about 110 mg adenosine 
triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg 
dithiothreitol, about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg 
phenylacetic acid, about 0.85 mg oxalic acid, and about 4 g. Triton (a 
registered trademark of Union Carbide Chemicals and Plastics Co., Inc.)

DETAILED DESCRIPTION OF THE INVENTION 
Luciferases catalyze the oxidation of luciferin with the concomitant 
emission of photons. The present invention provides compositions and 
methods for increasing the duration of detectable photon emission of a 
luciferase-luciferin reaction. The present invention provides a 
luciferase-luciferin reaction in which light production can be detected 
for more than five minutes. In a preferred embodiment, the photon emission 
of a luciferase-luciferin reaction can be detected for up to and including 
eight hours. In another embodiment, detectable photon emission from a 
luciferase-luciferin reaction is linear for up to eight hours (see FIGS. 
1-5). 
In one embodiment, a reaction mixture containing luciferase, luciferin, 
ATP, and cofactors required for luciferase catalytic activity is mixed 
with a composition containing adenosine monophosphate, a radical scavenger 
and a chelating agent to form an admixture. The photons produced by the 
luciferase-luciferin reaction are then detected by measuring the 
luminescence of the admixture. Luciferase catalyzed photon emission as 
disclosed by the methods and compositions of the present invention can be 
detected for more than five minutes. In a preferred embodiment, photon 
emission can be detected for more than thirty minutes and for up to eight 
hours. In a preferred embodiment, photon emission decays linearly for up 
to eight hours as shown in FIGS. 1-3. 
In a preferred embodiment, the present invention provides a 
luciferase-luciferin reaction admixture. One hundred ml of the admixture 
contains about 2.8 mg luciferin, about 110 mg adenosine triphosphate, 
about 2.2 mg adenosine monophosphate, about 385 mg dithiothreitol, and 
about 20 mg ethylenediaminetetraacetic acid. 
In another embodiment, the amount of one of the components of the admixture 
can be varied while the amounts of all other components remain unchanged. 
For example, in 100 ml of the admixture, the amount of luciferin can be 
varied between about 0.2 to about 30 mg while maintaining the amount of 
ATP at about 110 mg, AMP at about 2.2 mg, DTT at about 385 mg and EDTA at 
about 20 mg. Similarly, components can be varied individually as follows: 
for 100 ml of the admixture, the amount of luciferin can be varied from 
about 0.2 to about 30 mg; the amount of ATP can be varied from about 10 to 
about 300 mg; the amount of AMP can be varied from about 0.2 to about 30 
mg; the amount DTT can be varied from about 200 to about 2000 mg; and the 
amount of EDTA can be varied from between about 10 to about 50 mg. As an 
example, the present invention contemplates an admixture of the following 
composition: 100 ml of the admixture contains about 2.8 mg luciferin, 
about 110 mg adenosine triphosphate, between about 0.2 to about 35 mg 
adenosine monophosphate, about 385 mg dithiothreitol, and about 20 mg 
ethylenediaminetetraacetic acid. Other admixtures in which only one 
component is varied in accordance with the limitations disclosed above are 
contemplated. 
In another aspect, the present invention contemplates a method for 
detecting the presence of luciferase in a biological sample. The 
biological sample suspected of containing luciferase is mixed with a 
reaction mixture which contains luciferin, adenosine triphosphate, 
cofactors required for luciferase catalytic activity, adenosine 
monophosphate, dithiothreitol, ethylenediaminetetraacetic acid, 
phenylacetic acid, oxalic acid, and a detergent to form an admixture. The 
photons produced by the luciferase-luciferin reaction is then detected by 
measuring the luminescence of the admixture. A preferred biological sample 
is a cell that produces luciferase. An exemplary detergent is Triton.RTM. 
N-101 (nonylphenoxypolyethoxyethanol). The present invention provides 
methods and compositions in which the presence of luciferase in the 
biological sample can be detected for more than five minutes by detection 
of the emitted photon. In a preferred embodiment, photon emission can be 
detected for more than thirty minutes and for up to eight hours. In a 
preferred embodiment, photon emission decays linearly for up to eight 
hours as shown in FIGS. 1-3. 
In a preferred embodiment, the present invention contemplates an admixture 
for detecting the presence of luciferase in a biological sample. One 
hundred ml of the admixture contains about 2.8 mg luciferin, about 110 mg 
adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg 
dithiothreitol, about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg 
phenylacetic acid, about 0.85 mg oxalic acid, and a detergent. 
In another embodiment, the amount of one of the components of the admixture 
can be varied while the amounts of all other components remain unchanged. 
For example, in 100 ml of the admixture, the amount of luciferin can be 
varied between about 0.2 to about 30 mg while maintaining the amount of 
ATP at about 110 mg, AMP at about 2.2 mg, DTT at about 385 mg, EDTA at 
about 20 mg, phenylacetic acid at about 4.5 mg, and oxalic acid at about 
0.85 mg. Similarly, components can be varied individually as follows: for 
100 ml of the admixture, the amount of luciferin can be varied from about 
0.2 to about 30 mg; the amount of ATP can be varied from about 10 to about 
300 mg; the amount of AMP can be varied from about 0.2 to about 30 mg; the 
amount DTT can be varied from about 200 to about 2000 mg; the amount of 
EDTA can be varied from about 10 to about 50 mg; the amount of 
phenylacetic acid can be varied from about 1 to about 10 mg; and the 
amount of oxalic acid can be varied from about 0.2 to about 5 mg. 
In another aspect, the present invention contemplates a composition used in 
detecting the presence of luciferase in biological samples by detecting 
emitted photons from a luciferase-luciferin reaction. One hundred ml of 
this composition contains about 2.8 mg luciferin, about 110 mg adenosine 
triphosphate, about 2.2 mg adenosine monophosphate, about 385 mg 
dithiothreitol, about 20 mg ethylenediaminetetraacetic acid, about 4.5 mg 
phenylacetic acid, and about 0.85 mg oxalic acid. 
In a more preferred embodiment, the present invention contemplates a 
reaction mixture for detecting the presence of a luciferase in a 
biological sample, 100 ml of which contains about 2.8 mg luciferin, about 
110 mg adenosine triphosphate, about 2.2 mg adenosine monophosphate, about 
385 mg dithiothreitol, about 20 mg ethylenediaminetetraacetic acid, about 
4.5 mg phenylacetic acid, about 0.85 mg oxalic acid, and a detergent. As 
an example, the present invention contemplates an admixture of the 
following composition: 100 ml of the admixture contains about 2.8 mg 
luciferin, about 110 mg adenosine triphosphate, about 2.2 mg adenosine 
monophosphate, between about 200 to 2000 mg dithiothreitol, about 20 mg 
ethylenediaminetetraacetic acid, about 4.5 mg phenylacetic acid, and about 
0.85 mg oxalic acid. Other admixtures in which only one component is 
varied as discussed above are contemplated. 
As used herein, the term "luciferase" is an enzyme which catalyzes the 
oxidation of luciferin with the concomitant emission of a photon. 
Luciferases can be isolated from biological specimens which produce 
luciferase or from a cell which has been transformed or transfected with a 
polynucleotide encoding for a luciferase. It is within the skill of one of 
ordinary skill in the art to isolate a luciferase from a biological 
specimen that produces luciferase. Similarly, means of transforming or 
transfecting a cell with a polynucleotide that encodes for a luciferase 
are well known in the art. 
One aspect of the invention provides a method for increasing the duration 
of detectable photon emission of a luciferase-luciferin reaction for more 
than thirty minutes by the addition of certain reagents. A large number of 
substances which influence the luciferin-luciferase reaction including 
dithiothreitol, cytidine nucleotides, AMP, pyrophosphate, coenzyme A, 
EDTA, protease inhibitors, and luciferase inhibitors including luciferin 
analogs have been reported. 
Decomposition and inactivation of luciferase decreases the lifetime of the 
luciferase-luciferin reaction by inactivation of luciferase. In reporter 
gene techniques where cell lysates are used, the inactivation of 
luciferase by endogenous proteases present in the cell lysate is a 
particular problem. The addition of protease inhibitors such as 
phenylacetic acid (PAA), oxalic acid, monensine, acetyl phenylalanine, 
leupeptine, ammonium chloride, aprotinin and others prevent the 
degradation of luciferase by endogenous proteases present in the 
biological sample. By slowing down or preventing the inactivation of 
luciferase, luciferase catalyzed light production is lengthened. The above 
list is illustrative only and many other protease inhibitors are well 
known in the art. The use of other protease inhibitors is contemplated by 
the present invention. 
The addition of luciferin analogs and other inhibitors of luciferase 
increase the lifetime of light production by inhibiting luciferase. Care 
must be taken to ensure that the inhibitor binds to luciferase reversibly. 
Analogs that bind irreversibly permanently inhibit luciferase. Exemplary 
reversible inhibitors of luciferase include phenylbenzothiazol, 
2-aminoethanol, benzothiazole, 2-hydroxyphenylbenzothiazole and 
pyrophosphate. 
Adenosine monophosphate (AMP) is a catalytic product of the 
luciferase-luciferin reaction. The addition of AMP promotes the initial 
phase of the luciferase luciferin reaction. As shown in FIG. 1, in the 
absence of AMP, initial photon emission is low and maximum light 
production occurs nearly six hours after the initiation of the 
luciferase-luciferin reaction. In contrast, when the concentration of AMP 
is between about 2.2 and about 35.2 mg/100 ml, maximum light production 
occurs immediately and decreases in a linear manner for more than eight 
hours. FIG. 1 shows the effect of varying the AMP concentration on 
luciferase-luciferin light production. 100 ml of the admixture contained 
110 mg adenosine triphosphate, 385 mg dithiothreitol, 2.8 mg luciferin, 20 
mg ethylenediaminetetraacetic acid, 4 ml of a 10% Triton.RTM. N-101 
(nonylphenoxypolyethoxyethanol) solution in H.sub.2 O, 4.5 mg phenylacetic 
acid, and 0.85 mg oxalic acid in 50 mM 
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH, 7.8. 
The amount of AMP added to 100 ml of the assay solution was varied from 
about 0 to about 35.2 mg. With the addition of about 8.8 mg and about 35.2 
mg of AMP, detectable photon emission was linear between one and eight 
hours. The addition of 2.2 mg of AMP resulted in photon emission which was 
linear for up to eight hours. 
Thiol compounds such as dithiothreitol (DTT), dithioerythritol, glutathione 
and other well known reducing agents are radical scavengers and increase 
the duration of detectable photon emission of a luciferase-luciferin 
reaction. While the mechanism of thiol compound stabilization of the 
luciferase-luciferin reaction is not well understood, these compounds 
probably function by limiting the availability of oxygen necessary in the 
second step of the luciferase-luciferin reaction. At higher 
concentrations, DTT increases emission of photons through an unknown 
mechanism. FIG. 2 shows the effect of varying DTT concentration on 
luciferase-luciferin light production. in FIG. 2, 100 ml of the assay 
solution contained 2.2 mg adenosine monophosphate, 110 mg adenosine 
triphosphate, 2.8 mg luciferin, 20 mg ethylenediaminetetraacetic acid, 4 
ml of a 10% Triton.RTM. N-101 (nonylphenoxypolyethoxyethanol) solution in 
H.sub.2 O, 4.5 mg phenylacetic acid, and 0.85 mg oxalic acid in 50 mM 
N[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH, 7.8. 
The amount of DTT in the admixture varied from 193 to 770 mg. The addition 
of 385 and 770 mg of DTT resulted in detectable photon emission which was 
linear for up than eight hours. When 193 mg of DTT is added, light 
production was reduced and was linear between two and eight hours. 
FIG. 3 shows the effect of varying both DTT and AMP concentration on the 
duration of detectable photon emission from a luciferase-luciferin 
reaction. 100 ml of the admixture contained 110 mg adenosine triphosphate, 
2.8 mg luciferin, 20 mg ethylenediaminetetraacetic acid, 4 ml of a 10% 
Triton.RTM. N-101 (nonylphenoxypolyethoxyethanol) solution in H.sub.2 O, 
4.5 mg phenylacetic acid, and 0.85 mg oxalic acid, in 50 mM 
N[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid](HEPES), pH, 7.8. 
The addition of 385 mg DTT and 2.2 mg AMP resulted in detectable photon 
emission which was linear between 1.75 and 8 hours. The addition of 770 mg 
DTT and 35 mg AMP resulted in detectable photon emission which was linear 
for up to eight hours. 
Chelating agents which bind metal ions can increase the duration of photon 
emission from a luciferase-luciferin reaction by binding Mg.sup.+2 and 
other divalent cations including Ca.sup.+2, Fe.sup.+2, Mn.sup.+2, 
Co.sup.+2, and Zn .sup.+2. In reporter gene techniques, the concentrations 
of divalent cations cannot be rigorously controlled because whole cell 
lysates are used. Lysis of cells expressing a luciferase encoding gene 
release all of the endogenous divalent cations including Mg.sup.+2 and 
Ca.sup.+2 into the luciferase-luciferin reaction admixture. The addition 
of chelating agents effectively removes the released Mg.sup.2+ and 
Ca.sup.+2. Exemplary chelating agents include ethylenediaminetetraacetic 
acid (EDTA), and ethylene glycol-bis (.beta.-aminoethyl ether) 
N,N,N',N',-tetraacetic acid (EGTA). The use of other chelating agents are 
contemplated by the present invention. 
The use of detergents to lyse cells suspected of expressing luciferase is 
well known in the art. Exemplary anionic detergents include the series of 
Triton.RTM. detergents including Triton.RTM. N-101. The substitution of 
other detergents, including both ionic and anionic detergents is within 
the skill of an ordinary artisan. 
The use of reagents to maintain the pH of the luciferase-luciferin reaction 
solution is well known in the art. An exemplary buffering reagent is 
N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES). Many 
other buffering reagents are well known and are commercially available. 
The present invention provides a combination of reagents which increase the 
duration of detectable photon emission from a luciferase-luciferin 
reaction. FIG. 4 shows the effect on the lifetime of the 
luciferase-luciferin reaction in an admixture consisting of luciferase, 
luciferin, ATP, cofactors required for luciferase catalytic activity and 
EDTA (20 mg/100 ml) in 50 mM HEPES and AMP, DTT or coenzyme A. In all of 
the combinations shown in FIG. 4, the detectable photon emission from a 
luciferase-luciferin reaction lasts for at least for eight hours with the 
exception of a solution containing EDTA and 250 .mu.M coenzyme A in which 
photons can be detected for 7 hours. 
The linearity in the decrease of photon emission during the life of a 
luciferase-luciferin reaction is an important aspect of the present 
invention. Because photon emission is linear during the life of the 
luciferase-luciferin reaction, initial luminescence can be calculated 
easily from the luminescence detected at any time during the life of the 
luciferase-luciferin reaction. From the calculated initial luminescence, 
the concentration of the luciferase in the measured sample can be 
determined. For example, in the reporter gene technique, samples suspected 
of expressing luciferase can be measured over a period of many hours and 
the initial concentration of luciferase in the samples can be determined. 
As discussed below in Examples 1 and 2 the present invention allows for the 
measurement of large numbers of samples. Recently, instrumentation for 
measuring photon emission from 96 well microtiter plates became available 
("FopCount Microplate Scintillation and Luminescence Counter" from the 
Packard Instrument Company, Inc. of Downers Grove, Ill.). The light output 
from each 96 well plate takes roughly ten minutes to measure. Thus with a 
linear emission of photons of eight hours, forty eight 96-well plates can 
be easily measured. This results in the measurement of photon emission 
from 4608 individual samples. 
EXAMPLE 1 
Reporter-gene Assay Using a Luciferase cDNA 
The reporter gene technique using a cloned luciferase was used to 
demonstrate the increased duration of the luciferase-luciferin reaction. 
The production of light from transiently transformed human T-cells 
(Jurkat) was measured for eight hours. Jurkat cells in the logarithmic 
phase of growth were transformed by the DEAE-dextran technique and 
incubated in a humidified incubator for 48 hours under assay conditions. 
The plasmid vector contained a luciferase gene under the control of the EF 
promoter. After 48 hours, 100 .mu.l of the cell suspension was mixed with 
100 .mu.l of PBS and 100 .mu.l of a 100 ml solution containing 2.2 mg 
adenosine monophosphate, 110 mg adenosine triphosphate, 385 mg 
dithiothreitol, 2.8 mg luciferin, 20 mg ethylenediaminetetraacetic acid, 4 
ml of a 10% Triton.RTM. N-101 (nonylphenoxypolyethoxethanol) solution in 
H.sub.2 O, 4.5 mg phenylacetic acid, 0.85 mg oxalic acid, and 50 mM 
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid](HEPES), pH 7.8. 
The production of light was then measured for up to eight hours. The "high 
activity" curve (solid line) represents light production from the 
transformation of Jurkat cells with 2 .mu.g of the luciferase containing 
vector at a concentration of 1.times.10.sup.6 cells/mi. The "low activity" 
curve represents light production from the same experiment but with a 
cellular concentration of 50,000 cells/mi. The half life of the "high 
activity" sample was found to be roughly 3.5 hours and the half life of 
the "low activity" curve was found to be roughly 5 hours. 
EXAMPLE 2 
High-capacity Luciferase Reporter Gene Assay. for HIV-Rev-RRE-Interaction 
A transient transfection assay using the DEAE-dextran technique for T-cell 
lines was established to screen for inhibitors of the HIV-rev regulatory 
protein (RRE). Two expression vectors, one for the HIV-1 gene (pEF-cRev) 
and the other for a luciferase reporter gene (pCMV-Luc/RRE) are 
cotransfected into the Jurkat cell line (lymphoblastoid cells). RRE is 
expressed together with Luciferase if a certain level of functional rev 
protein has been accumulated within a cell. The assay mimics the natural 
action of rev. Rev transports unspliced or singly spliced RRE-containing 
mRNA out of the nucleus to the translation machinery in the cytoplasm. If 
a certain threshold level of rev protein accumulates in the nucleus, rev 
binds to RRE containing mRNA, multimerizes and mediates transport of this 
mRNA into a cellular pathway bypassing the cellular splicing machinery. 
This is accomplished by binding of the Rev/RRE complex to a cellular 
factor, translation initiation factor elF-5a, of the pre-ribosomes formed 
within the nucleoli. Inhibitors of any of these steps in the mode of 
action of rev is detectable in this reporter gene assay. 
The assay conditions have been optimized to obtain a stimulation of 
luciferase expression by rev to approx. 20-100 fold over background within 
20 hours of transfection. The basal luciferase expression as determined in 
control transfections with an identical expression vector carrying the rev 
gene in antisense orientation to the promoter (pEF-AScRev) is usually 
below 5% of the rev-induced expression. The toxicity of candidate 
compounds is determined by a parallel transfection assay. In this assay, a 
rev-independent luciferase construct (pCMV-LUC) is cotransfected with the 
rev-expression vector (pEF-cRev) to keep conditions similar to the 
rev-assay. This toxicity assay also identifies compounds which are 
inhibitors of the luciferase enzyme or to transcription factors binding to 
the CMV-promoter. Therefore, the toxicity assay is also capable of acting 
as a specificity control. 
The assay has been adapted to microplate technology and can be automated. 
While detectors to measure multiple samples have been known for some time, 
modern single photon measurement techniques with an extraordinarily high 
sensitivity for detection of bioluminescence signals from microplates came 
to the market recently. The stabilization of the bioluminescence signal 
from a halflife of a few minutes to more than 4 hours allows for the 
automation of the assay by using the newly available microplate single 
photon counters. A cDNA encoding for a luciferase is therefore an ideal 
reporter gene. The use of a luciferase is applicable to many other 
mechanism-based cellular test systems for pharmaceutical, toxicological 
and other screening systems. 
Jurkat T-cell suspension cultures, stock 1722 are split daily with a 
dilution of at least 1:2. Final cell density should not exceed 
6.times.10.sup.5 cells/ml to keep the cells in the logarithmic growth 
phase. Cells can be seeded at a density 0.5.times.10.sup.5 /ml or lower 
for maintaining stock cultures. Culturing in roller bottles (approx. 10 
rpm) is the preferred culture method. 
The following buffers and reagents are used: 
Dulbecco's PBS (phosphate buffered saline) 
DEAE-Dextran (MW ca. 500.000), PHARMACIA #17-0350-00 solved at 10 mg/ml in 
PBS and filtered through 0.2 .mu.m Nalgene filters. 
Assay medium: RPMI 1640 without phenol red supplemented with: 
+10% FCS inactivated 
+1% v/v HEPES buffer (1M) 
+1% Penicillin/Streptomycin, GIBCO 043-05070 
+0.1% Gentamicin, GIBCO 043-05750 
1% Chloroquine (10 mM=5.52 mg/ml in PBS), Sigma #C6628 
Wash medium: RPM11640 without phenol red supplemented with: 
+1% v/v HEPES buffer (1M) 
+1% Penicillin/Streptomycin, GIBCO 043-05070 
+0.1% Gentamicin, GIBCO 043-05750 
DNA preparations, purified by two cycles of CsCl gradient centrifugation 
and stored frozen in aliquots at -20.degree. C. (dissolved at 1 mg/ml in 
H.sub.2 O) 
pCMV-Luc/RRE DNA, #18xx 
pEF-cREV DNA, #21xx 
pEF-AScREV DNA, #22xx 
pCMV-Luc DNA, #3xx 
luciferase-substrate solution: 
2.2 mg AMP (Sigma A1877) 
110 mg ATP (Sigma A5394) 
385 mg Dithiothreitol 
280 .mu.l Luciferin (10 mg/ml in H.sub.2 O), Sigma L5256 dissolved in 100 
ml buffer consisting of: 
5 ml HEPES (1M) 
20 mg EDTA (Titriplex III) 
4 ml Triton.RTM. N-101 (nonylphenoxypolyethoxyethanol) (10% in H.sub.2 O) 
4.5 mg Phenylacetic acid, Sigma No. P4514 
0.85 mg Oxalic acid, Sigma No. 07626 mg adjusted to 7.8 (2N NaOH. ca 1.1 
ml) 
Microplates: Packard CulturPlates 6005180 with adhesive seals 
Cellfilter Falcon #2340 
The assay protocol is as follows: 
Preparation of Dilutions: 
Pure substances CHC (500 .mu.M in DMSO saturated H.sub.2 O)) 1:50 to 1:6400 
final dilution: 
Medium=RPMI 1640 plus additives (see above) 
Predilution: 8 .mu.l sample plus 192 .mu.l assay medium (Microplate) 
Add 50 .mu.l predilution each in parallel positions to 3 white Packard 
CulturPlates (B1 through H12) 
Blank: 50 .mu.l medium (A1 through A12) 
Plates are kept in a CO.sub.2 -cabinet until addition of cells 
Broth Of Actinomycetes at 1:200, Fungi at 1:100 and Bacteria at 1:100 final 
dilutions: 
Predilution: 8 .mu.l sample plus 392 .mu.l assay medium=1:50 (Titertek 
racks), for bacteria and fungi 
8 .mu.l sample plus 792 .mu.l assay medium=1:100 (Titertek racks), for 
actinomycetes 
Add 150 .mu.l of predilution each in 3 parallels to white Packard 
CulturPlates (B1 to H12) 
Blank: 50 .mu.l assay medium (A1 to A12) 
Plates are kept in a CO.sub.2 -cabinet until addition of cells 
Designation of control wells in the primary screen: 
______________________________________ 
A1, A2 plate control 
medium only, no cells 
A3 to A10 
`high` control 
pCMV-Luc/RRE + pEF-cREV 
A11, A12 plate control 
medium only, no cells 
______________________________________ 
Secondary screening (validation) of the positive/toxic hits (1:50 to 1:6400 
final dilution for substances): 
Predilution: 20 .mu.l sample plus 480 .mu.l assay medium (Titertek racks) 
Add 100 .mu.l predilution in duplicates to the wells of rows A (3 to 12) to 
two Packard CulturPlates in parallel 
Add 50 .mu.l of assay medium to all the remaining wells 
Dilute columns 3 to 12 by serially transferring 50 .mu.l and discarding the 
rest after row H. 
Preparation of Cells and Transfection Procedure: 
Cells as described above, are centrifuged (200 g, 10 min) and washed twice 
with prewarmed wash medium. 
Cells are suspended in prewarmed PBS and counted in a hemacytometer; the 
cell density is adjusted to 4.times.10.sup.6 cells/mi. 
DNA-solutions are prepared in prewarmed PBS (2.5 ml/plate, 25 .mu.l/well to 
be tested): 
Add 25 .mu.l DEAE/Dextran pr ml PBS. 
Add DNA: 
For REV/RRE: 0.60 .mu.l/ml pCMV-Luc/RRE #18xx and 1 .mu.l/ml pEF-cREV 
#21xx. 
For negative controls: 0.60 .mu.l/ml pCMV-Luc/RRE #18xx and 1 .mu.l/ml 
AScREV #22xx. 
For toxicity/unspecific inhibition: 0.60 .mu.l/ml pCMV-Luc #3xx and 1 
.mu.l/ml pEF-cREV #21xx. 
For toxicity/handling control: 0.60 .mu.l/ml pCMV-Luc #3xx and 1 .mu.l/ml 
pEF-AScREV #22xx. 
Add an equal volume of cell suspension (4.times.10.sup.6 cells/ml) to each 
of the DNA-solutions. 
The DNA/cell suspension is incubated for 10 min. at 37.degree., shaken 
gently by hand and incubated for another 10 min. 
An equal volume of prewarmed assay medium is added, cells are shaken gently 
by hand and incubated for another 10 min. 
The pelleted cells are resuspended in prewarmed RPMI 1640 plus additives 
(assay medium) and adjusted to a final concentration of 1.times.10.sup.6 
cells/ml. 
Cells are sucked once through a syringe and filtered through a cell filter 
immediately before being dispensed to the plates. 
50 .mu.l of cell suspension is added to each of the respective wells: 
Secondary Jurkat-Rev Assay: 
`High` control and substance dilutions: pCMV-luc/RRE+pEF-cRev in wells 
A1-H1 
Blanks: 50 .mu.l medium in wells A2-D2 
`Low` control: pCMV-Iuc/RRE+pEF-AScRev in wells E2-H2 
Secondary, Jurkat-Toxicity Assay: 
`High` control and substance dilutions: pCMV-luc+pEF-cRev in wells A1-H1 
Blanks: 50 .mu.l medium in wells A2-D2 
`Handling` control: pCMV-luc+pEF-AScRev in wells E2-H2 
Plates axe shaken using a microplate-shaker and stored in the incubator at 
37.degree. C. in a 5% CO.sub.2 atmosphere for 16-24 hours. 
Measurement of Luciferase Activity 
After incubation, 100 .mu.l of luciferase substrate solution, to be 
prepared fleshly every day, is added to each well. 
Plates are shaken using a microplate shaker for a few seconds and sealed 
with an adhesive seal (do NOT use heat-seals). 
Plates are counted in a Packard "TopCount Microplate Scintillation and 
Luminescence Counter" or an equivalent instrument after a count delay of 2 
min. for 0.15 min. per well. The temperature of the counting chamber as 
well as the plate stack should not exceed 22.degree. C., otherwise the 
half life of the light emission can drop below 4 hours. 
The REV-Luc/RRE (`high`-control)- and the Luc (toxicity-control)-background 
corrected valued should be at last 2000 cps, the AsREV-Luc/RRE 
(*low*-control) should not exceed 100 cps. 
Results 
FIGS. 6 and 7 show the results of a large scale compound screening test for 
compounds which inhibit HIV-rev regulatory protein (RRE). Jurkat cells are 
co-transfected with two plasmids, pEF-cRev and either pCMV-Luc/RRE or 
pCMV-Luc. The closed square represents cells which have been transfected 
with pEFc-REV and pCMV-Luc/RRE. The open square represents cells which 
have been transfected with pEF-cREV and pCMV-Luc. If a candidate compound 
inhibits RRE, cells transfected with pEF-cREV and pCMV-Luc/RRE should show 
a decrease in light production from the luciferase-luciferin reaction 
compared to cells transfected with pEF-cREV and pCMV-Luc. If a candidate 
compound does not inhibit RRE, then light production from the 
luciferase-luciferin reaction should remain unchanged whether cells are 
transfected with pCMV-Luc/RRE or pCMV-Luc. 
FIG. 6 shows the effect of Compound A on RRE. The inhibition curves 
obtained from cells co-transfected with pEF-cRev and either pCMV-Luc/RRE 
(closed square) or with pCMV-Luc (open square) is not dramatically 
different. Compound 6 therefore does not appear to be an inhibitor of RRE. 
Alternatively, the toxicity of Compound A may prematurly kill the 
transfected cells before meaningful data can be obtained. 
FIG. 7 shows the effect of Compound B on RRE. The inhibition curves 
obtained from cells co-transfected with pEF-cRev and either pCMV-Luc/RRE 
(closed square) or with pCMV-Luc (open square) is dramatically different. 
There is a significant difference in inhibition of RRE between cells 
transfected with pCMV-Luc/RRE and cells transfected with pCMV-Luc. In 
cells transfected with pCMV-Luc/RRE, about 0.1 .mu.M Compound B results in 
50% inhibition. In cells transfected with pCMV-Luc, Compound B must be 
present in concentrations greater than 10 .mu.M to obtain 50% inhibition 
(data not shown).