U.S. patent application number 10/323669 was filed with the patent office on 2003-05-15 for luminescence detection workstation.
Invention is credited to Atwood, John, DeSimas, Bruce E. II, Gambini, Michael R., Lakatos, Edward, Levi, Jeff, Metal, Israel, Sabak, George, Voyta, John C., Wang, Yongdong.
Application Number | 20030092194 10/323669 |
Document ID | / |
Family ID | 22510606 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030092194 |
Kind Code |
A1 |
Gambini, Michael R. ; et
al. |
May 15, 2003 |
Luminescence detection workstation
Abstract
A luminescence detecting apparatus and method for analyzing
luminescent samples is disclosed. Luminescent samples are placed in
a plurality of sample wells in a tray, and the tray is placed in a
visible-light impervious chamber containing a charge coupled device
camera. The samples may be injected in the wells, and the samples
may be injected with buffers and reagents, by an injector. In the
chamber, light from the luminescent samples pass through a
collimator, a Fresnel field lens, a filter, and a camera lens,
whereupon a focused image is created by the optics on the
charge-coupled device (CCD) camera. The use of a Fresnel field
lens, in combination with a collimator and filter, reduces
crosstalk between samples below the level attainable by the prior
art. Preferred embodiments of the luminescence detecting apparatus
and method disclosed include central processing control of all
operations, multiple wavelength filter wheel, and robot handling of
samples and reagents. Preferred embodiments of processing software
integrated with the invention include elements for mechanical
alignment, outlier shaving, edge detection and masking,
manipulation of multiple integration times to expand the dynamic
range, crosstalk correction, dark subtraction interpolation and
drift correction, multi-component analysis applications
specifically tailored for luminescence, and uniformity
correction.
Inventors: |
Gambini, Michael R.;
(Bolton, MA) ; Voyta, John C.; (Sudbury, MA)
; Atwood, John; (Redding, CT) ; DeSimas, Bruce E.
II; (Danville, CA) ; Lakatos, Edward; (Bethel,
CT) ; Levi, Jeff; (Trumbull, CT) ; Metal,
Israel; (Flushing, NY) ; Sabak, George;
(Monroe, CT) ; Wang, Yongdong; (Wilton,
CT) |
Correspondence
Address: |
Supervisor, Patent Prosecution Services
PIPER RUDNICK LLP
1200 Nineteenth Street, N.W.
Washington
DC
20036-2412
US
|
Family ID: |
22510606 |
Appl. No.: |
10/323669 |
Filed: |
December 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10323669 |
Dec 20, 2002 |
|
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|
09621961 |
Jul 21, 2000 |
|
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6518068 |
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60144891 |
Jul 21, 1999 |
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Current U.S.
Class: |
436/172 ; 422/52;
422/82.08 |
Current CPC
Class: |
G01N 2333/90241
20130101; G01N 21/31 20130101; G01N 33/581 20130101; G01N 21/15
20130101; G01N 21/274 20130101; G01N 21/6452 20130101; G01N 21/6486
20130101; G01N 21/276 20130101; G01N 21/763 20130101; Y10T
436/115831 20150115; G01N 21/6456 20130101; G01N 2021/6471
20130101; G01N 21/76 20130101 |
Class at
Publication: |
436/172 ; 422/52;
422/82.08 |
International
Class: |
G01N 021/76 |
Claims
What is claimed is:
1. A luminometer for analyzing a plurality of luminescent samples,
comprising: a visible light-impervious chamber containing: a charge
coupled device (CCD) camera; a shuttle for supporting a plate
comprising a plurality of wells, each said well for containing a
single one of said plurality of luminescent samples; a collimator,
positioned between said sample tray and said CCD camera; a Fresnel
lens, positioned between said collimator and said CCD camera; and a
camera lens positioned between said Fresnel lens and said CCD
camera.
2. The luminometer of claim 1, wherein each of said plurality of
luminescent samples is one of a bioluminescent material or a
chemiluminescent material.
3. The luminometer of claim 1, further comprising a central
processing unit for controlling the analysis of said plurality of
luminescent samples.
4. The luminometer of claim 1, further comprising an injector for
placing liquid reagents in said plurality of luminescent samples in
said plurality of wells.
5. The luminometer of claim 4, wherein said injector places a
reagent required for luminescence in said plurality of wells.
6. The luminometer of claim 1, further comprising a robot.
7. The luminometer of claim 1, wherein said chamber is temperature
controlled.
8. The luminometer of claim 1, further comprising a filter, located
between said Fresnel lens and said CCD camera.
9. The luminometer of claim 8, wherein said filter includes filter
elements of varying wavelength.
10. The luminometer of claim 1, further comprising a defogger which
prevents condensation on said Fresnel lens.
11. A method for analyzing a plurality of luminescent samples in a
luminometer, comprising the steps of: placing said plurality of
luminescent samples in a respective plurality of sample wells in a
tray; placing said tray in a visible light-impervious chamber
containing a charge coupled device camera; positioning a collimator
between said tray and said CCD camera; positioning a Fresnel lens
between said collimator and said CCD camera; and positioning a
camera lens between said Fresnel lens and said CCD camera.
12. The method of claim 11, additionally comprising the step of
positioning a filter between said Fresnel lens and said camera
lens.
13. The method of claim 12, wherein said filter includes filter
elements of varying wavelength.
Description
[0001] This application claims the benefit from Provisional
Application Serial No. 60/144,891, filed Jul. 21, 1999. The
entirety of that provisional application is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to the field of apparatus and methods
for detecting and quantifying light emissions, and more
particularly, to detecting and quantifying light emitted from
luminescent-based assays. Still more particularly, this invention
pertains to apparatus and methods for detecting and quantifying
luminescence such as bioluminescence and/or chemiluminescence from
luminescent assays as an indicator of the presence or amount of a
target compound. Preferred embodiments of the invention include as
an imaging device a charge coupled device (CCD) camera and a
computer for analyzing data collected by the imaging device.
Further preferred embodiments include the capacity for use in high
throughput screening (HTS) applications, and provide for robot
handling of assay plates.
[0004] 2. Description of the Related Art
[0005] The analysis of the luminescence of a substance, and
specifically the analysis of either bioluminescence (BL) or
chemiluminescence (CL), is becoming an increasingly useful method
of making quantitative determinations of a variety of luminescent
analytes.
[0006] Recently, methods have been introduced that utilize
luminescence detection for quantitatively analyzing analytes in an
immunoassay protocol. Such luminescence immunoassays (LIA) offer
the potential of combining the reaction specificity of
immunospecific antibodies or hybridizing nucleic acid sequences and
similar specific ligands with the high sensitivity available
through light detection. Traditionally, radioactive reagents have
been used for such purposes, and the specificity and sensitivity of
LIA reagents is generally comparable to those employing traditional
radiolabelling. However, LIA is the preferred analytical method for
many applications, owing to the nontoxic nature of LIA reagents and
the longer shelf lives of LIA reagents relative to radioactive
reagents.
[0007] Among other luminescent reagents, chemiluminescent compounds
such as 1,2-dioxetanes, developed by Tropix, Inc. and other stable
chemiluminescent molecules, such as xanthan esters and the like,
are in commercial use. These compounds are triggered to release
light through decomposition triggered by an agent, frequently an
enzyme such as alkaline phosphatase, which is present only in the
presence, or specific absence, of the target compound. The
detection of light emission is a qualitative indication, and the
amount of light emitted can be quantified as an indicator of the
amount of triggering agent, and therefore target compound, present.
Other well known luminescent compounds can be used as well.
[0008] Luminescent release may sometimes be enhanced by the
presence of an enhancement agent that amplifies or increases the
amount of light released. This can be achieved by using agents
which sequester the luminescent reagents in a microenvironment
which reduces suppression of light emission. Much biological work
is done, perforce, in aqueous media. Water typically suppresses
light emission. By providing compounds, such as water soluble
polymeric onium salts (ammonium, phosphonium, sulfonium, etc.)
small regions where water is excluded that may sequester the light
emitting compound may be provided.
[0009] The majority of instrumentation used to monitor light
emitting reactions (luminometers) use one or more photomultiplier
tubes (PMTs) to detect the photons emitted. These are designed to
detect light at the low light levels associated with luminescent
reactions. The rate at which a PMT based microplate luminometer can
measure signal from all wells of the plate is limited by the number
of PMTs used. Most microplate luminometers have only one PMT so a
384 well plate requires four times longer than is required to read
a 96-well plate.
[0010] The nature of biological research dictates that numerous
samples be assayed concurrently, e.g., for reaction of a
chemiluminescent substrate with an enzyme. This is particularly
true in gene screening and drug discovery, where thousands of
samples varying by concentration, composition, media, etc. must be
tested. This requires that multiple samples be reacted
simultaneously, and screened for luminescence. However, there is a
need for high speed processing, as the chemiluminescence or
bioluminescence may diminish with time. Simultaneously screening
multiple samples results in improved data collection times, which
subsequently permits faster data analysis, and contingent improved
reliability of the analyzed data.
[0011] In order for each specific sample analyte's luminescence to
be analyzed with the desired degree of accuracy, the light emission
from each sample must be isolated from the samples being analyzed
concurrently. In such circumstances, stray light from external
light sources or adjacent samples, even when those light levels are
low, can be problematic. Conventional assays, particularly those
employing high throughput screening (HTS) use microplates, plastic
trays provided with multiple wells, as separate reaction chambers
to accommodate the many samples to be tested. Plates currently in
use include 96- and 384-well plates. In response to the increasing
demand for HTS speed and miniaturization, plates having 1,536 wells
are being introduced. An especially difficult impediment to
accurate luminescence analysis is the inadvertent detection of
light in sample wells adjacent to wells with high signal intensity.
This phenomenon of light measurement interference by adjacent
samples is termed `crosstalk` and can lead to assignment of
erroneous values to samples in the adjacent wells if the signal in
those wells is actually weak.
[0012] Some previously proposed luminometers include those
described in U.S. Pat. No. 4,772,453; U.S. Pat. No. 4,366,118; and
European Patent No. EP 0025350. U.S. Pat. No. 4,772,453 describes a
luminometer having a fixed photodetector positioned above a
platform carrying a plurality of sample cells. Each cell is
positioned in turn under an aperture through which light from the
sample is directed to the photodetector. U.S. Pat. No. 4,366,118
describes a luminometer in which light emitted from a linear array
of samples is detected laterally instead of above the sample.
Finally, EP 0025350 describes a luminometer in which light emitted
through the bottom of a sample well is detected by a movable
photodetector array positioned underneath the wells.
[0013] Further refinements of luminometers have been proposed in
which a liquid injection system for initiating the luminescence
reaction just prior to detection is employed, as disclosed in EP
0025350. Also, a temperature control mechanism has been proposed
for use in a luminometer in U.S. Pat. No. 4,099,920. Control of the
temperature of luminescent samples may be important, for example,
when it is desired to incubate the samples at an elevated
temperature.
[0014] A variety of light detection systems for HTS applications
are available in the market. These include the LEADseeker.TM. from
Amersham/Pharmacia, the ViewLux.TM. offered by PerkinElmer and
CLIPR.TM. from Molecular Devices. These devices are all expensive,
large dimensioned (floorbased models), exhibit only limited
compatibility with robotic devices for plate preparation and
loading, have a limited dynamic range, and/or use optical detection
methods which do not reduce, or account for, crosstalk. The optical
systems used are typically complex teleconcentric glass lens
systems, which may provide a distorted view of wells at the edges
of the plates, and the systems are frequently expensive, costing in
excess of $200,000.00. Perhaps the most popular detection apparatus
is the TopCount.TM., a PMT-based detection system from Packard.
Although the TopCount.TM. device has a desirable dynamic range, it
is not capable of reading 1,536 well plates, and it does not image
the whole plate simultaneously.
[0015] Crosstalk from adjacent samples remains a significant
obstacle to the development of improved luminescence analysis in
imaging-based systems. This can be appreciated as a phenomenon of
simple optics, where luminescent samples produce stray light which
can interfere with the light from adjacent samples. Furthermore,
the development of luminometers capable of detecting and analyzing
samples with extremely low light levels are particularly vulnerable
to crosstalk interference.
SUMMARY OF THE INVENTION
[0016] In order to meet the above-identified needs that are
unsatisfied by the prior art, it is a principal object and purpose
of the present invention to provide a luminescence detecting
apparatus that will permit the analysis of luminescent samples. It
is a further object of the present invention to provide a
luminescence detecting apparatus capable of simultaneously
analyzing a large number of luminescent samples. In a preferred
embodiment of the present invention, a luminescence detecting
apparatus is provided that simultaneously analyzes multiple samples
held in wells, where the well plates contain as many as 1,536
wells. The present invention further includes robot handling of the
multiple well trays during analysis.
[0017] It is yet another object of the present invention to provide
a luminescence detecting apparatus capable of analyzing low light
level luminescent samples, while minimizing crosstalk from adjacent
samples, including and especially minimizing crosstalk from
adjacent samples with higher light level output than the sample to
be analyzed.
[0018] The apparatus of this invention employs a Fresnel lens
arrangement, with a vertical collimator above the well plate, with
dimensions to match the number of wells. Thus, a 1,536-well plate
will employ a dark collimator above the plate with 1,536 cells in
registry with the wells of the plate. Fixed above the collimator is
a Fresnel lens, which refracts the light such that the view above
the lens appears to be looking straight down into each well,
regardless of its position on the plate, even at the edges.
[0019] Above the Fresnel lens is a CCD camera arranged so as to
take the image of the entire plate at one time, viewing through a
35 mm wide angle lens, to give whole plate imaging on a rapid
basis. Between the CCD and Fresnel/collimator is a filter,
typically arrayed on a filter wheel, disposed at an angle to the
lens. The filter is selected to permit the passage of the specific
wavelength of the light emitted, and reflect or absorb all others.
Several filters may be provided on the wheel, to permit sequential
detection of light emitted from multiple reagents emitting light at
different wavelengths.
[0020] The samples are fed to the optical detection platform
through a loading device designed to work well with robotic and
automated preparation systems. The well-plate, with reaction
mixture already provided, is placed on a shuttle by a human, or
preferably, robot. Alignment of the plate on the shuttle may be
relatively coarse, notwithstanding the requirement for tight
tolerances to match the collimator grid array. As the shuttle
leaves the loading position, a resilient means urges the plate into
strict conformal alignment. The shuttle positions the plate under
an overhead injection bar, which may accommodate up to sixteen
wells in a column at one time. If not previously added, a
triggering agent or luminescent reagent is added to the sample
wells, and the plate indexes forward to load the next column of
wells across the plate. The shuttle then advances through a door
into the sample chamber, and the plate is aligned with the
collimator and the Fresnel lens. Since many reactions proceed
better, or only, at elevated temperatures, the sample chamber is
insulated, and provided with heating means, for heating the air in
or provided to the chamber. In order to maintain temperature in the
chamber close to room temperature and to accurately control
temperature, the chamber may also be provided with a heat
exchanger.
[0021] The light emission from the entire multiple well plate is
imaged at once, with subsequent imaging through a different filter
if multiple wavelengths are employed. The signal obtained is
processed to further reduce crosstalk reduced by the collimator and
the presence and amount of luminescence is quickly detected and
calculated by a personal computer using automated software. Data is
then reported as intensity per well or further analyzed relative to
specific assay standards.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0023] FIG. 1 is a cross section of a preferred embodiment of a
luminescence detecting apparatus according to the present
invention;
[0024] FIG. 2 is a detailed cross section of the optics of a
luminescence detecting apparatus according to the present
invention.
[0025] FIG. 3 is a cross-sectional view of the plate transport
system of the invention.
[0026] FIG. 4 is a perspective illustration of the injector arm
assembly of the invention.
[0027] FIG. 5 is an exploded view of the filter wheel assembly.
[0028] FIG. 6 is a cross-sectional view of the optical housing.
[0029] FIG. 6A is a plan view of a robotic mechanism of the
invention.
[0030] FIG. 7 is a flow chart illustration of the processing method
of the invention.
[0031] FIGS. 8-15 are illustrations of the results obtained using
the invention in Examples 1-10, respectively.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0032] Referring now to the drawings, wherein like reference
numerals designate identical or corresponding parts throughout the
several views, and more particularly to FIG. 1 thereof, a preferred
embodiment of the luminescence detecting apparatus of the present
invention uses a shuttle or tray to carry a micro plate (plate) 10
comprising a plurality of sample wells 20 which may in the
preferred embodiment number as many as 1,536 or more. Persons of
ordinary skill in the relevant art will recognize that the number
of sample wells 20 is limited only by the physical dimensions and
optical characteristics of the luminometer elements, and not by the
technology of the present invention. The sample wells 20 may be
filled with analyte manually, or robotically prior to delivery to
the inventive apparatus. Agents necessary for chemiluminescence may
be filled automatically via the injector 30, to which analyte is
supplied through an array of supply tubes 40 or prior to placing
the plate on the tray. Typically, the sample wells will contain
chemiluminescent reagents. These reagents emit light at intensities
proportional to the concentration of analyte in the sample. This
light can be very low intensity and requires an instrument with
sufficient sensitivity to achieve the desired detection limits.
[0033] The operation of injector 30 is controlled by central
processor 50, which in the preferred embodiment may control the
operation of all elements of the luminometer of the present
invention. Data collection, analysis and presentation may also be
controlled by processor 50. Further in a preferred embodiment of
the present invention, the injector 30 may also be used to add
buffer solutions to the analytes and also to add reagents that
enable "glow" and/or "flash" luminescence imaging, that is
sustained or brief, intense emission, respectively, all under
control of central processor 50.
[0034] After the analytes are placed in the sample wells 20, plate
10 is placed in sample chamber 55, which is located in optical
chamber 60 at a fixed focal distance from and directly under the
charge-coupled device (CCD) camera 70, in order to permit the CCD
camera to image the luminescent sample accurately. The sample
chamber 55 is preferably capable of precise temperature control, as
many luminescent reagents and specific luminescent reactions are
temperature dependent. Temperature control is provided by central
processor 50, which can vary the temperature for each individual
sample plate 10, as central processor 50 controls the movement and
injection of the sample wells 20 in each sample tray 10. In a
preferred embodiment of the luminometer of the present invention,
central processor 50 also controls an industrial robot (not shown)
which performs the activities involving analyte handling in the
luminometer of the present invention.
[0035] With the plate 10 placed in the sample chamber 55, the
optics 80 deliver the image of the complete microplate 10 as a
single image to the CCD camera 70.
[0036] Although the operation of the luminometer of this invention
is an integral, continuous practice, and all elements of the
luminometer cooperate together to provide precise, accurate and
reliable data, the invention may be more easily understood by
reference to three separate, integrated systems, the optics system,
the mechanical system and the processing system. Each is discussed
in turn, with a discussion of examples of the operation as a whole
to follow.
Optics System
[0037] Turning now to FIG. 2, the optics 80 are shown in further
detail. Luminescent emission 100 from the analyte in plate well 20
located in the plate 10 travels first through dark collimator 110,
which permits only parallel and semi-parallel light rays to exit
the sample wells 20 for eventual imaging by the CCD camera 70. The
effect of collimation assists with the prevention of stray light
from the sample wells 20 and with the elimination of crosstalk
between luminescent samples. The collimator 110 may be sealably
engaged, or in close proximity, to the sample tray 10, to enhance
the restriction of stray light from the samples. Each well 10 is in
strict registration and alignment with a corresponding grid opening
in collimator 110. From the collimator 110, the luminescent
radiation passes through a Fresnel field lens 120, which focuses
the light toward filter 130. In a preferred embodiment of the
present invention, the collimator 110 and Fresnel field lens 120
are packaged in a cassette that can be changed by the user. Such an
equipment change may be necessitated by varying optical
characteristics of different analytes and different well
distributions in plates.
[0038] The use of a Fresnel field lens is preferable to alternative
optical devices for several reasons. Initially, improvements in
design and materials have capitalized on the superior optical
capabilities of the Fresnel lens, while virtually eliminating its
once inherent limitations. Today, many Fresnel lenses are made of
molded plastic, creating an almost flawless surface with very
little scatter light. The elimination of scatter light is an
important element of eliminating crosstalk between adjacent samples
in the luminometer of the present invention. Furthermore, improved
types of plastics commonly employed in the manufacturing of Fresnel
lenses and other optical devices have optical qualities equivalent
to ground glass lenses.
[0039] Using high tech processes such as computer-controlled
diamond turning, complex aspheric surfaces can be cut into a long
lasting mold for casting Fresnel lenses. In this manner, Fresnel
lenses can be manufactured to produce the precise optical imaging
effect that is most efficient for a charge coupled device camera,
as in the present invention. Also, Fresnel lenses offer an
advantage over conventional lenses in that they can be molded flat
and very thin. Because of the shape of the Fresnel lens, it can
easily be integrated directly into the housing of the luminometer,
enhancing the light-tight properties necessary for accurate imaging
of low light samples. Furthermore, Fresnel lenses are much less
expensive than comparable conventional glass lenses.
[0040] As with any other lens, the total beam spread from a Fresnel
lens depends on the size of the source in relation to the focal
length of the lens. Smaller sources, such as luminescent assay
samples, and longer focal lengths produce more compact beams. Since
there are practical limitations to minimizing the geometry and
dimensions of the optics 80 in the luminometer of the present
invention, the use of Fresnel field lens 120 provides the greatest
opportunity for fine-tuned optics. The emissions from plate 10 pass
through lens 120, and are refracted such that the image obtained at
CCD 70 appears to look directly downward into all wells, even
laterally displaced (edge) ones. This feature is typically called
"telecentric."
[0041] Further in a preferred embodiment of the present invention,
the filter 130 may be configured on a wheel, wherein different
filter elements may occupy different portions of the wheel,
depending on the luminescent characteristics of the sample being
analyzed. Filter 130 is preferably inclined at an angle of
20.degree.-30.degree. relative to the CCD, so that stray reflected
light is reflected outside the field of view. Specifically, the
filter wheel 130 permits the selection of different wavelength
ranges, which not only permit high quality imaging, but may be used
to separate the emissions of different reagents emitting at
different wavelengths. Again, the filter wheel 130 is controlled by
central processor 50, in coordination with central processor 50's
control of the individual sample wells 20 in the sample plate 10.
In many assays, such as those addressed in pending U.S. patent
application Ser. No. 08/579,787, incorporated by reference herein,
multiple luminescent reagents, which emit at different wavelengths,
are employed in a single well. Using multiple filters, each can be
imaged in turn, and the true concentration can be calculated from
the data set resulting using pre-stored calibration factors. Filter
130 is preferably provided with an infrared (IR) filter operating
in conjunction with the selected bandpass, or as an independent
element. Applicants have discovered that stray IR radiation,
resulting from the plate phosphorescence, resulting in abnormally
high backgrounds. An IR filter suppresses this.
[0042] From the filter wheel 130, the sample-emitted light passes
through camera lens 140, which in the preferred embodiment is a
large aperture, low distortion, camera lens. Camera lens 140
focuses the image of the sample on the CCD chip 70. In the
preferred embodiment of the present invention, CCD camera 70 is a
cooled, low noise, high resolution device. The lens is preferably a
35 mm wide angle lens with a low light level (F 1.4) large aperture
character. Magnification of 3-6, preferably about 5.5, is
preferred. In preferred embodiments CCD camera 70 is provided with
an anti-blooming CCD chip, to enhance dynamic range, which is about
10.sup.5 in the claimed invention, referred to as the NorthStar.TM.
luminometer. Blooming occurs when a single pixel is overloaded with
light and its photoelectrons overflow the CCD device well capacity,
obliterating surrounding pixels. Further in the preferred
embodiment of the luminometer of the present invention, the
selected CCD camera includes a liquid cooled thermoelectric
(Peltier) device providing cooling of the CCD to approximately
-35.degree. C., and the CCD has 1280.times.1024 pixels, each of
which are 16 .mu.m square, producing a total active area of 20.5
mm.times.16.4 mm. The quantum efficiency averages 15% over the
range from 450 nanometers to 800 nm. The output is digitized to 16
bit precision and pixels can be "binned" to reduce electronic
noise.
[0043] By using the features disclosed herein, the luminometer of
the present invention has a spatial resolution capable of providing
high quality imaging of high density sample trays. The noise
performance and CCD temperature are designed to provide the desired
detection limit.
Mechanics
[0044] The mechanical systems of the luminometer workstation of
this invention are designed to achieve automated, high throughput
precise delivery of microplates in registration with a collimator
110 so as to be read by the CCD Camera 70. To this end, as shown in
FIG. 3, a cross-section of the inventive luminometer shuttle 200
translates from a load position 202, where plates 10 are loaded on
to the shuttle, preferably by a robotic device such as robot arm,
and the shuttle 200 then translates towards sample chamber 55, to
read position 203. Shuttle 200 is caused to translate by a
conventional stepper motor (not pictured). As shuttle 200 advances
toward sample chamber 55, it may stop underneath injector 30.
Injector 30 is more fully illustrated below in FIG. 4. Referring
still to FIG. 3, injector 30 delivers fluid reagents drawn from
reservoir 204. Syringe pump 205 draws the fluid reagents from
reservoir 204, and pumps the fluid to the injector tubes 40. Two
way valve 206 controls the passage of the fluid drawn by syringe
pump 205 from reservoir 204 and pumped by syringe pump 205 to the
supply tubes 40. In actual practice, there are as many injector
tubes 40 as injection ports being used, and multiple syringe pumps
205 are also used. As will be shown below in FIG. 4, injector 30
has up to sixteen injection ports 302. The plates used in
conjunction with the luminometer when injection is used are
typically prepared with up to sixteen wells in a column. As the
shuttle 200 advances plate 10 underneath injector 30, shuttle 200
stops so that the first column 208 of wells is directly aligned
under injector 30. Precise amounts of analyte are delivered to the
first set of wells, and shuttle 200 indexes forward one column, so
as to inject reagent into the second column of wells 210. This
process is repeated until all wells are filled. Thereafter, shuttle
200 advances forward into sample chamber 55 through hinged door
212. In the alternative, door 212 may be a guillotine door or
similar type of closing mechanism. The wells of plate 10 are then
read in sample chamber 55. Upon completion of reading, shuttle 200
translates back to load position 202.
[0045] Before shuttle 200 advances to the injection bar, it may be
necessary to fully prime the tube with fluid, so as to provide for
precise delivery into the plate. Trough 304 swings out from its
storage position parallel to the direction of travel of shuttle
200, shown by an arrow, to a position directly underlying the
injector 30, perpendicular to the direction of travel. Fluid in the
injector and tubes 204 are delivered into trough 304, and removed
by suction. Trough 304 then returns to its rest position, parallel
to, and away from, the direction of travel of the shuttle 200, when
the shuttle is moved toward the sample chamber 55. On its return
trip to load position 202, locator 214 on shuttle 200 is engaged by
cam 216. Locator 214 is mounted on a resilient means, such that
when engaged by cam 216, the locator 214 recesses away from plate
10. This permits removal of plate 10, and delivery from a robotic
arm or other source of a fresh plate 10, without the requirement of
precise location. As shuttle 200 moves away from load 202, locator
214 is urged forward, firmly locating plate 10 in place. Plate 10
is held against shoulder 217 by the resilient urging of locator
214.
[0046] It is important that each plate be precisely identified, so
that results are correlated with the correct test samples. In most
HTS laboratories, most microplates are labeled with a unique "bar
code." The label is often placed on the surface perpendicular to
the plane of the plate itself. To permit precise identification of
each plate, a bar code reader 218 is mounted on the luminometer
housing generally indicated at 299 and directly above the door 212,
for example on an arm or flange 220. Bar code reader 218 is focused
on a mirror 222 which in turn permits reading directly off the
front or leading edge of plate 10 as it approaches on shuttle 200.
Thus, before each plate arrives in the sample chamber, its identity
has been precisely recorded in processor 50, and the results
obtained can be correlated therewith. Persons of ordinary skill in
the art will recognize that a variety of configurations of
alignment and placement of both bar code reader 218 and mirror 222
will result in the desired identification.
[0047] As more clearly shown in FIG. 4, injector 30 may be
precisely located by operation of actuator wheel 306, provided with
positions corresponding to the total number of wells on the plates
being assayed. Similarly, the vertical position, to account for the
different thicknesses of the plate, may be controlled by wheel 308.
Given the simple translation movement of shuttle 200, and the
precise locating and identification of each plate carried, rapid
cycling of micro-plate test plates into and out of sample chamber
55 can be effected.
[0048] As described above in connection with the optics system of
the invention, a filter is provided which includes or reflects
passage of light other than light falling within the selected
wavelength of the luminescent emitter in use. The filter assembly
is illustrated in exploded format in FIG. 5. Filter frame 502 is
supported by arm 504 which is connected to the hub of the filter
wheel 506. Multiple different filters may be provided on a single
wheel. The filter itself, 508, is securely mounted on the frame and
held there by cover 510, which is secured to frame 502 by grommets,
screws or other holding devices 512. As noted, filter wheel is
positioned so as to hold filter 508 in frame 502 at in incline with
respect to collimator 110, of about 22.degree. nominally, so as to
direct any reflections outside the field of view. Light passes
through the filter opening 514, in alignment with camera lens 140
and CCD camera 70. As further noted above, filter 508 preferably
includes an infrared block, either as a component of the filter
itself, or as a component provided in addition to the filter for
the measured light. An IR block is of value to prevent infrared
emissions caused by extraneous radiation from altering the image
received by the CCD camera.
[0049] Optical chamber 60 is more fully illustrated in FIG. 6. As
shown, optical chamber 60 is bounded by optical housing 602 in
which fits sample housing 604. When a plate 10 is loaded into
optical chamber 60, the plate is secured in sample housing 604
which is positioned in registry with collimator 110, over which is
provided Fresnel lens 120. While many luminescent assays can be
provided at ambient temperatures, some require elevated
temperatures. The luminometer of this device is provided with a
sample chamber in which the sample housing 604 carries insulation
606 which, in a preferred embodiment is polyurethane foam, and
heater element 608 to raise the temperature in the sample chamber
55 above ambient temperature, up to about 42.degree. C.
[0050] There is a tendency, even at ambient conditions, for
condensation to collect on the surface of the Fresnel lens 120, as
a result of moisture coming from the filled wells of plate 10. The
defogger 610 directs a stream of air heated just a few degrees,
preferably about 2-3.degree. degrees, above ambient conditions, or
above the temperature of the chamber if the chamber is above
ambient conditions, across the surface of the Fresnel lens 120,
effectively preventing condensation. Mounted at the top of the
interior of optical chamber 60 is filter motor 610 which drives
filter wheel 612, on which may be mounted filters 614 of varying
wavelength, for filtering undesirable wavelengths prior to imaging.
Of course, a region is provided, indicated at 616, in the optical
housing 602 of the optical chamber 60 for light to be directed onto
the CCD camera after passing through the filter 614. The dimensions
of optical chamber 60 are exaggerated in FIG. 6 to illustrate the
relationship between the optical chamber 60 and the filter wheel
612, and defogger 610. In practice, the filter is located inside
the optical chamber 60, and outside the sample housing 604 but
alternate locations are possible while still achieving the desired
function.
[0051] In FIG. 6A, a plan view of a novel robotic mechanism 616 is
displayed in a preferred embodiment of the present invention, which
provides capacity for use in high throughput screening (HTS)
applications. Referring to FIG. 6A, the operation is as follows:
robot plate stacks 620, 622, 624, 626, and 628 each can be filled
with multiple sample plates 10, arranged in a vertical stack. In
the preferred embodiment of FIG. 6A, robot plate stack 628 is
designated as the discard stack. The remaining robot plate stacks
620, 622, 624, and 626 can be programmed in order of delivery by
software controlled by processor 50 (not shown). In order to load
or pick plates from any of these stacks, robot arm 630 moves
vertically and rotationally to the desired robot plate stack, under
control of the software programmed in processor 50.
[0052] When commanded by processor 50, transport 200 of the
instrument will move the sample plate 10 from load position 202 to
the Read position 203, and return it to load position 202 when
imaging is complete. In the embodiment of the invention shown in
FIG. 6A, the elapsed time between moving the sample plate 10 from
load position 202 to the read position 203, and returning it to
load position 202 is typically 30-120 seconds, including imaging
time.
[0053] Staging positions 632 and 634 are located at 45 degree
positions relative to the position of robot arm 630. In one
embodiment, while imaging is in process, the robot arm 630 can
place a sample plate 10 at staging position 632, in preparation for
placing the sample plate 10 in load position 202. When the imaging
is complete, the robot can move the read plate from load position
202 to staging position 634, then load the plate from staging
position 632 to load position 202, and while the sample plate 10 is
being imaged, the robot can move the plate from staging position
634 to the discard stack 628, and place a new sample plate 10 at
staging position 632. In practice, the staging positions are at
approximately the same level as the load position, so movement is
very quick. In the preferred embodiment, the robot arm 630 can do
the time consuming moves to any of robot plate stacks 620, 622,
624, and 626 while imaging is going on, rather than in series with
imaging.
[0054] With the staging positions 632 and 634, the cycle time for a
single sample plate 10 is 2 moves from/to staging areas (3 seconds
each), plus 2 transport moves IN/OUT to read position 203 (3
secondss each), plus the integration time (image exposure) time
(typically 60 seconds), for a total cycle time of 72 seconds.
Without using staging positions 632 and 634, the time would be 2
moves to stacks (30 seconds each), plus 2 transports (3 seconds
each), plus the integration time (typically 60 seconds) for a total
of 126 seconds. As described in the preferred embodiment of the
robotic mechanism 616, the use of staging positions 632 and 634
decreases cycle time by 43%.
Processing
[0055] As set forth above, the mechanical and optical systems of
the luminometer workstation of the invention are designed to
provide precise, quantified luminescent values in an HTS
environment, taking advantage of the use of a Fresnel
lens/collimator assembly to permit single image viewing by the CCD
camera, and subsequent analysis. The collimator, the lens and the
camera together combine to reduce cross-talk experienced in prior
art attempts. The signals obtained are further processed, as
illustrated in FIG. 7, through software loaded onto processor 50,
or other convenient method, to further refine the values
obtained.
[0056] Prior to processing image data collected through the
integrated mechanical and optical systems of the invention herein
described, the integrated processing component of the invention
must first control the mechanical alignment of those integrated
mechanical and optical systems for reliable data collection. This
process is conducted under control of the processor 50. To conduct
an alignment test, the luminescence detection of the present
invention measures the light emitted from four test sample wells,
called hot wells, of a test plate. In a preferred embodiment, the
hot wells are located near each corner of the sample tray used for
the alignment testing. The adjacent well crosstalk from each of the
four hot wells is analyzed, and the values are compared. When the
collimator is aligned precisely over the sample well tray, the
crosstalk values will be symmetrical for the four hot wells. The
software of the present invention flags any errors detected, such
as incorrect number of test sample wells, incorrect intensity, or
incorrect location. After the detection of no errors or after the
correction of detected and flagged errors, the software of the
present invention performs a symmetry calculation to determine
precise alignment of the sample well tray, collimator, Fresnel lens
and CCD camera assembly. In a known embodiment of the invention,
known software techniques are employed to perform the symmetry
calculation process by performing the following steps:
[0057] 1. Extract the hot well and vertical and horizontal adjacent
well intensities;
[0058] 2. Calculate the averages of the horizontal and vertical
adjacent well intensities separately for each hot well;
[0059] 3. Calculate the differences between the actual adjacent
intensity vs. the average for each of the horizontal and vertical
directions;
[0060] 4. Normalize the differences by the hot well intensity to
convert to a percentage intensity value;
[0061] 5. Find the worst case absolute value of the differences and
display that as the overall misalignment;
[0062] 6. Calculate the average X-direction (horizontal)
misalignment by averaging the four adjacent wells to the right
(horizontal direction) of the hot wells;
[0063] 7. Calculate the average Y-direction (vertical) misalignment
by averaging the four adjacent wells to the top (vertical
direction) of the hot wells;
[0064] 8. Calculate the rotational misalignment by averaging the
left side hot well vertical adjacent wells at the top of the hot
wells, and subtracting that from the average of the right side hot
well vertical adjacent wells, thereby indicating any tilt in
adjacent well values.
[0065] In step A, three actual images for each filter/emitter are
taken. A.sub.1 is a precursor image, A.sub.2 is the full
integration time image, and A.sub.3 is post-cursor image. The
precursor and post-cursor images are taken to avoid the problem of
pixel saturation and to extend the detection dynamic range. The
precursor and post-cursor images refer to reduced integration time
images, which should not contain multiple saturated pixels. If more
than six pixels of the full integration time image are saturated,
the pre- and post-cursor images are averaged together to form the
actual data for that well area. In the absence of six pixel
saturation, the full integration time image is used.
[0066] In order to clearly isolate and read each pixel, in step B,
each image is subjected to edge detection and masking, a processing
step whereby the edge of each well or corresponding light image is
identified, or annotated, to set off and clearly separate each well
region of interest, as disclosed in U.S. patent application Ser.
No. 09/351,660, incorporated herein by reference. Again, edge
detection and masking is performed for each of B.sub.1, B.sub.2 and
B.sub.3, referring to the pre-cursor, full integration time image
and post-cursor images, respectively. The images are then subjected
to "outlier" correction, correcting or "shaving" outliers and
anomalies. In this process, the pixels within the region of
interest are examined to identify "outliers"--those that are in
gross disagreement with their neighbors, in terms of light
intensity detected, and if the intensity of a given pixel or small
pixel area is significantly different than neighboring pixels or
pixel areas, then the average of the surrounding pixels or areas is
used to replace erroneous data. This can be due to random
radiation, such as that caused by cosmic rays. In this process,
this type of intensity is corrected.
[0067] Subsequently, in step C, each image C.sub.1, C.sub.2 and
C.sub.3 is subjected to dark subtraction, subtracting the dark
background, so as to obtain average pixel values within each
mask-defined region of interest. The subtraction is done on a
well-by-well basis from stored libraries which are updated
periodically.
[0068] Specifically, the dark subtraction is conducted to correct
for the fact that even in the absence of light, CCD cameras can
output low level pixel or bin values. This value includes the
electronic bias voltage, which is invariant of position and
integration time, and the "dark current," which may vary by
position, and is proportional to integration time and to the
temperature of the CCD. The CCD may also have faulty pixels that
are always high level or saturated regardless of light input.
[0069] The processing software of the invention subtracts this
background image or data from the real sample well image data in
step C. As persons of ordinary skill in the relevant art will
recognize, it is known to take a "dark" image immediately before or
after a real image, imaging for the same integration time in both
cases, and subtracting the "dark" image data from the real image
data. In the preferred embodiment of the invention, "dark" image
data is collected intermittently, preferably at specific time
intervals. The initial "dark" image background data is collected at
startup, and then typically at four hour intervals during image
processing operations.
[0070] Because the background image has an integration
time-invariant component and an integration time-variant component,
data is collected for each sample well at minimum integration time
and at maximum integration time, and a "slope/intercept" line is
calculated between the two data points, using known data analysis
techniques. This calculation permits data interpolation for any
integration time between the minimum and maximum, and also permits
data extrapolation for integration times below or beyond the
minimum and maximum integration times.
[0071] In a preferred embodiment of the invention, a CCD camera is
employed that has two separate "dark" current functions, caused by
the CCD output amplifier. Operation of the amplifier generates heat
and necessarily creates background "dark" image data. In the
preferred embodiment, for integration times of less than 10
seconds, the amplifier operates continuously, whereas for
integration times of more than 10 seconds, the amplifier remains
off until immediately prior to the read operation. The
"slope/intercept" line calculated for integration times of more
than 10 seconds will then necessarily have a lower slope than a
"slope/intercept" line calculated for integration times of less
than 10 seconds. In step C, the processing software element allows
separate collection and least squares regression for both the 0 to
10 second integration time region a processor 50, the "dark"
background image data is stored separately for each individual
AOI.
[0072] "Dark" current and bias can also vary over time. The
processing software element corrects for this effect by comparing
the integration time normalized (using the regression line
technique described above) "dark reference" pixel values (outside
the imaging field-described above), that were taken when the "dark"
background images were taken, versus the "dark reference" pixel
values taken while real sample well images are being taken. The
difference between the values is then subtracted or added, as
applicable, as a global number, to the "dark" background data. This
corrects for bias drift and also for global CCD temperature
drift.
[0073] As mentioned, all of the above "dark background"
interpolation/subtraction of step C is done on a well by well
basis.
[0074] At step D, if pixel saturation has occurred such that the
average of the pre-cursor and post-cursor image must be used, the
image data is multiplied by the reciprocal of the percentage
represented by the pre-cursor images (e.g., 3%).
[0075] In step E, the well data is corrected for uniformity
variations using a calibration file that is the reciprocal of the
system response to a perfectly uniform input illumination.
[0076] In step F, the cross-talk correction is effected by
processing the data as a whole and preparing a final image in much
the same fashion as reconstruction of three dimensional images from
a two dimensional data array is practiced.
[0077] Specifically in a preferred embodiment of step F, the
impulse response function (IRF) is collected for all 96 wells of
the 96 well plate type. This is done by filling one particular well
in a given plate with a high intensity luminescent source, imaging
the plate, and analyzing all of the wells in the plate for their
response to the one high intensity well. The IRF is collected for
all of the wells individually by repeating the process for every
different well location desired for the complete data set. For 384
plate types, 96 sampling areas are selected, and data for the wells
in between the selected sampled areas are interpolated in two
dimensions. In the preferred embodiment, the 96 sampling areas
comprise every second row and every second column, starting at the
outside and working toward the center. Because in the 384 well
plates the number of rows and columns is even, the two center rows
and the two center columns are interpolated. The reflections in a
384 well plate are also modeled, and used to predict and
interpolate reflections for the missing input data. Further in the
preferred embodiment, all wells are normalized to the well with the
highest intensity.
[0078] Subsequently in step F, the two-dimensional array of well
IRF values for each well are "unfolded" into a one-dimensional
column array, and the two-dimensional arrays of IRF values for
other wells are added as subsequent columns, as shown in Chart 1
following:
1CHART 1 Unfolded Data Into Column 1 IRF for IRF for IRF for A1 B1
C1 A1 A1 A1 Etc B1 B1 B1 C1 C1 C1 D1 D1 D1 E1 E1 E1 F1 F1 F1 G1 G1
G1 H1 H1 H1 A2 A2 A2 B2 B2 B2 C2 C2 C2 Etc Etc Etc
[0079] The unfolded matrix, which has the form of an N.times.N
matrix, where N=the number of wells to be corrected, comprises a
full characterization of the instrument crosstalk, including
reflection factors. This unfolded matrix is then inverted, using
known matrix inversion techniques, and used as a correction to
matrix multiply a one-dimensional matrix unfolded from real assay
data. This arithmetic process may be shown as matrix algebra:
[true source distribution].times.[system IRF]=[instrument
output]
[0080] solving for [true source distribution] produces
[true source distribution]={1/[system IRF]}.times.[instrument
output]
[0081] Subsequently, the calculated well intensities resulting from
the above processing are calibrated to an absolute parameter of
interest, such as the concentration of a known reporter enzyme.
This calibration is conducted through a normalization process
producing any of a variety of calibration curves, which will be
familiar to those of ordinary skill in the relevant art.
[0082] In optional step G, the processed image information is
subjected to any necessary post adjustment processing, for
appropriate correlation with the materials tested. Specifically, in
a preferred embodiment, the processing software of the present
invention is capable of performing multi-component analysis. The
basic problem is to calculate separately the concentration of a
single reagent in a single sample containing other different
reagents. Typically, the reagents used with the invention are
formulated so as to emit over different, but perhaps overlapping,
spectrums. As earlier described with respect to the integrated
optical element, the first step of separating the light from
multiple reagents is accomplished by optical bandpass filters,
which are designed to maximize the sensitivity of the target
reagent emission, while minimizing the sensitivity to other
non-target reagent emission. In the present embodiment of the
invention, there is one optical filter for each target reagent
emission spectrum.
[0083] Since optical filters are interference devices, their
bandpass characteristics vary, dependent on the angle of incidence
of the emission to be filtered. The angle of incidence will be
unique for each well because each well's specific location is
unique relative to the optical filter. Accordingly, all
calculations and filter coeficients must be unique per sample well.
The multi-component calibration is performed as follows:
[0084] Prior to the real multiplexed (multiple reagent) samples,
standards containing only a single reagent in each well are imaged
and analyzed. These standards will produce a set of coefficients to
be used collectively as multi-component coefficients for each
optical filter, for each well. For a given optical filter, the
target reagent for that filter should produce the highest output.
The other reagents may also have spectra in the filter's bandpass,
and will produce smaller outputs, which are a measure of the
overlap of those nontarget reagent spectra into the filter signal.
For example, the filter's output for the target reagent might be
850, and the filter's output for the other 2 reagents might be 100
and 50, respectively. If the 3 reagents were added together in a
single well, the total output would be 1000, and the proportions
would be 850:100:50. These coefficients are measured for each well
location and filter separately, which gives a complete set of
coefficients for simultaneous equations. This will allow a solution
for any combination of concentrations of reagent in one sample
well. Further in the preferred embodiment, these coefficients will
also be normalized by the total intensity read in the "total
emission" filter, so that the calculation will result in the same
intensity as the instrument would measure if only a single reagent
was measured by the "total emission" filter. This calculation may
be shown as follows for a simple case of blue and green reagents
(abbreviated as R in the calculations), and blue and green and
total emission filters (abbreviated as F in the calculations):
[0085] Let A=(output of the instrument for blue R thru the blue
F)/(output of instrument for blue R thru total emission F);
[0086] Let B=(output of the instrument for green R thru the blue
F)/(output of instrument for green R thru total emission F);
[0087] Let C=(output of the instrument for blue R thru the green
F)/(output of instrument for blue R thru total emission F);
[0088] Let D=(output of the instrument for green R thru the green
F)/(output of instrument for green R thru total emission F);
[0089] These coefficients are measured for each well prior to
running a multi-color run. Then for a multi-reagent/color run,
(output of the instrument for the blue F)=A.times.(true intensity
of blue R)+B.times.(intensity of green R);
and
(output of the instrument for the green F)=C.times.(true intensity
of blue R)+D.times.(intensity of green R)
[0090] These 2 simultaneous equations are then solved for the true
intensity of the blue and green reagents by the processing
software, under control of processor 50.
[0091] Further in step G, the raw output of the instrument for each
filter is normalized for integration time before solving the
equations. The resulting intensities could then be calibrated as
concentration by use of standards as described in the previous
section.
[0092] Finally, in step H, the analyzed data is presented in a
user-acceptable format, again controlled by processor 50.
[0093] The invention may be further understood by reference to
examples of assays practiced in HTS format, demonstrating the
dynamic range and flexibility of the NorthStar.TM. luminometer.
EXAMPLES
Example 1
Purified cAMP Quantitation
[0094] cAMP standards were serial diluted and added to a 96-well
assay plate with alkaline phosphatase conjugated cAMP and
anti-cAMP. Plates were processed with the cAMP-Screen.TM. protocol
and imaged for 1 minute on the NorthStar.TM. 30 minutes after
addition of CSPD.RTM./Sapphire-II.T- M.. A sensitivity of 0.06 pM
of purified cAMP is achieved with cAMP-Screen.TM. on the
NorthStar.TM. workstation. The results are depicted in FIG. 8.
Example 2
cAMP Induction in Adrenergic .beta.2 Receptor-Expressing C2
Cells
[0095] Adrenergic .beta.2 Receptor-expressing C2 cells were plated
in a 96-well plate (10,000 cells/well) and stimulated with
isoproterenol for 10 minutes. cAMP production was quantitated in
cell lysates using the cAMP-Screen.TM. assay. The assay plate was
imaged for 1 minute on the NorthStar.TM., 30 minutes after addition
of CSPD.RTM./Sapphire-II.TM.. Increasing cAMP levels were detected
on the NorthStar.TM. from the stimulated adrenergic receptor. The
results are depicted in FIG. 9.
Example 3
Luc-Screen.TM. Reporter Gene Assay in 96-, 384- and 1,536-Well
Format
[0096] pCRE-Luc-Transfected cells were seeded in 96-, 384- and
1,536-well plates, incubated for 20 hours with forskolin, and
assayed with the Luc-Screen.TM. system. PCRE-Luc contains the
luciferase reporter gene under the control of a cAMP response
element (CRE). Forskolin induces intracellular cAMP production
through the irreversible activation of adenylate cyclase. All plate
formats demonstrate comparable forskolin-induced cAMP levels. The
results are depicted in FIG. 10.
Example 4
Forskolin Induction of pCRE-Luc Transfected NIH-3T3 Cells
[0097] pCRE-Luc-Transfected cells were seeded in a 96-well plate.
Four random wells were induced for 17 hours with 1 mM forskolin and
the entire plate was assayed with the Luc-Screen.TM. system. The
results are shown in FIG. 11.
Example 5
Dual-Light.RTM. Quantitation of Luciferase &
.beta.-Galactosidase Reporter Enzymes
[0098] NIH/3T3 cells were co-transfected with pCRE-Luc and
p.beta.gal-Control, and seeded into a 96-well microplate
(2.times.10.sup.4 cells/well). Cells were incubated with forskolin
for 17 hours. Modified Dual-Light.RTM. Buffer A was added to cells
and incubated for 10 minutes. Modified Dual Light.RTM. Buffer B was
injected and luciferase-catalyzed light emission was measured
immediately. Thirty minutes later, Accelerator-II was added, and
then .beta.-galactosidase-ca- talyzed light emission was
quantitated on the NorthStar.TM. HTS workstation. Quantitation is
shown graphically in FIG. 12.
Example 6
Normalized Fold Induction of Luciferase Reporter
[0099] Fold induction of luciferase activity was calculated
following normalization to .beta.-galactosidase activity. The
Dual-Light.RTM. assay enables the use of a control reporter for
normalization, or to monitor non-specific effects on gene
expression. This is depicted in FIG. 13.
Example 7
Effect of BAPTA-AM on Antagonist Activity
[0100] CHO-Aeq-5HT2B cells were loaded with coelenterazine h +/-0.5
.mu.M BAPTA-AM for 4 hours. The antagonist methysergide was added
to the charged cells for 30 minutes. 1 .mu.M agonist a-Me-5HT was
injected, and the emitted light was integrated for 20 seconds on
the NorthStar.TM. system. The reported IC50 for methysergide (0.6
nM) is unchanged in the presence of BAPTA-AM. The data obtained
appears in FIG. 14.
Example 8
Effect of BAPTA-AM on Peptide Agonist Stimulated of the Orexin 2
Receptor
[0101] CHO-Aeq-OX2-A2 cells (Euroscreen) were loaded with
coelenterazine h +/-0.6 .mu.M BAPTA-AM for 4 hours. The peptide
agonist Orexin B was injected into the wells, and the emitted light
was integrated for 20 seconds on the NorthStar.TM.. Using this
assay on the NorthStar.TM. system, the reported EC50 for Orexin B
(0.75 nM) is unchanged in the presence of BAPTA-AM. This is shown
in FIG. 15.
[0102] This invention has been described generically, by reference
to specific embodiments and by example. Unless so indicated, no
embodiment or example is intended to be limiting. Alternatives will
occur to those of ordinary skill in the art without the exercise of
inventive skill, and within the scope of the claims set forth
below.
* * * * *