Note: Descriptions are shown in the official language in which they were submitted.
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Description
Method for the rapid screening of anal.
Technical Field
This invention relates to a method for the rapid screening of
large numbers of analytes, including the rapid screening of chemical
compounds in liquid form for use as potential drugs.
Background Art
Currently the identification of potential compound candidates for
use as drugs is achieved by screening programmes and/or rational drug
design. Whereas the ab initio drug design concept originally offered
wide expectations of success, this approach has not proved successful in
practice, principally due to a lack of a sufficiently clear relationship
between molecular structure and receptor sites.
Accordingly, compound screening is still the technology of
1 ~ choice for the rapid identification and selection of lead compounds as
candidate drugs. Various methods of high throughput screeninU ~HTS)
are currently used in the screening of compounds as potential drug
candidates. However, the speed and cost-effectiveness of HTS is limited
by the unavailability of equipment which can simultaneously handle
large numbers of compounds in liquid form.
In current HTS, the microtitre plate plays a central role being the
standard device in which assays are performed. The microtitre plate
has determined the design of liquid handling equipment such as
programmable liquid handling work stations and has also led to the
development of microtitre plate peripherals for filtration, washing,
reading and other operations involved in HTS.
To accommodate ever increasing high throughput requirements,
micre>titre plate-based equipment has been integrated with robotic
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7 _
manipulators. The central role of the microtitre plate and the
continuously increasing throughput requirements has led to ever larger
and more complex HTS-modules. Such complex modules include robot
rails to access wider surfaces, automatic and larger microtitre plate
incubators and storage devices for disposables. Additionally, complex
operations scheduling programmes and integration software are
required for optimal management of all of the hardware components.
The central role of the microtitre plate also determines other
aspects of a typical screening process such as the chemical library
aspects. Traditionally, compounds are synthesised, stored and
catalogued at central chemical repositories from which new compounds
are dispatched upon request from biological assay laboratories in- or
outside an organisation. Often, each new compound has to be weighed
and dissolved prior to each new assay. Therefore, it is essential that the
compounds can be individually accessed and routinely retrieved.
Driven by the latest HTS-system layouts and capacities, efforts are
being made to adapt the physical storage conditions of compounds to
the microtitre plate format. Hence, the development of
dimethylsulfoxide-( DMSO ) dissolved stock compound stock solutions
which are stored in microtitre plate format and whereby each
compound is addressable through its location.
The basis of the HTS-infrastructure is still the 96-well standard
microtitre plate and the majority of screening systems developed to date
have been developed for use with this format.
However, higher density microtitre plates ( e.g. 384-, 864-, 1536
and 9600 well plates ) are incompatible with most equipment designed
for the 96-well plate. As screening formats are likely to change
considerably in the future. screening systems must have sufficient
flexibility to meet this challenge.
The pace of HTS development to date has been determined by the
development of fast liquid handlin~~ systems capable of handling small
volumes of liquid to allow for the miniaturisation of assays in
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microtitre plate formats of which the number of wells has been scaled
up dramatically in order to cope with ever increasing numbers of
compounds to be screened. However, as the number of wells increases,
the volume capacity of the wells decreases dramatically. Therefore it is
obvious that this trend is becoming a self-limiting one because of the
physical limitations imposed for example by biochemical equilibria
relating to tissue culture in general where the control of pH, carbon
dioxide exchange, humidity and temperature are of the utmost
importance and which parameters are very difficult to control in small
volumes of the type employed in high density microtitre plates.
One problem with the use of high density microtitre plate
formats is that it is not possible to readily achieve serial dilution where
a dose response curve is required. For example, it is not possible to
carry out a serial dilution in the wells as such, so that the serial dilution
must be carried out externally of the wells. However, even when the
serial dilution is carried out in this way, one still has the problem of the
liquid handling aspect of the screening process. Thus, for serial
dilution one is currently effectively confined to the use of the 96-well
microtitre plate. Also, even when the handling of compounds is under
robotic control, the number of compounds that can be handled at any
given time is typically 8 or 12 with a maximum of 96.
We are unaware of any system to date which can simultaneously
process large numbers of analytes for screening 100 or more and even
up to 1000 analytes simultaneously, hereinafter referred to as a larger
number of analytes.
There is a need therefore for a method for the simultaneous
screening of large numbers of analytes and which method obviates the
difficulties and limitations of current HTS systems.
There is also a need for a method for the simultaneous
manipulation of large numbers of analvtes with means for simplifying
identification and retrieval of the analvtes l~or HTS.
CA 02342627 2001-03-O1
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.
Disclosure of Invention
The invention provides a method for the rapid screening of
analytes, comprising the steps of
a) disposing a plurality of analytes to be screened within
individually identifiable containers for storage;
b) simultaneously applying said plurality of analytes onto one or
more solid supports) such that said analytes are directly
applied from said containers to the or each solid support and
remain isolated from one another;
c) contacting said analyte-carrying solid supports) with targets
provided in a semi-solid or liquid medium, whereby said
analytes are released from the solid supports) to the targets;
and
d) measuring analyte-target interactions.
The method according to the invention allows for the
manipulation of thousands of different analytes simultaneously.
Step a) of the method according to the invention ensures that the
analytes are transferred from the containers to the solid supports) in
such a manner as to maintain the transferred contents of each container
separate from those of each other container:
The individually identifiable containers are preferably selected
from tubes, including capillary tubes, pens, including plotter pens, and
print heads or any container allowing for the storage and direct
application of an analyte from the container to' a given solid support.
Further, preferably, the individually identifiable containers are an
array of capillary tubes each of which is identifiable according to its
position within the array, and wherein transfer of the analytes to the
solid supports) occurs by dispensing thereof through the open ends of
the capillary tubes.
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J
Especially preferred arrays are individual containers disposed in
concentric or spiral arrays.
Transfer of the analytes to the solid support can be achieved by
dispensing said analytes from the open ends of the capilliary tubes to
the solid support with or without direct contact between the capilliary
tubes and the solid support.
The analytes can be transferred to the solid support in variable
amounts. Thus, by varying the drop size transferred one can achieve a
serial dilution, if required.
Simultaneous transfer of analyte from the capiiliaries can be
achieved by applying a stimulus such as a change of pressure using a
piezoelectric element. Alternatively, one can use high frequency
conditions to break a liquid column into droplets which are dispensed to
the solid support, as required. The droplet size will typically be
I S nanolitre or picolitre in volume.
Thus, one can achieve an assay format in accordance with the
invention where the analyte is not pipetted in as is currently the norm
but is applied directly from its individual container to the assay
medium.
It will be appreciated that the individually identifiable containers
described herein provide a means of storing stock compounds which
can be accessed and used as required. Thus, the analyte application
units can consist of individual identifiable containers assembled in
addressable compartments that can be retrieved automatically as a
whole and from which the analytes can be applied directly to a solid
support.
For example, a preferred embodiment of the identifiable
container is a capilliary tube and stock compounds in solution are taken
up in a plurality of~ such capilliary tubes by capiIliary action.
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In this way unlimited numbers of capillaries can be filled without
special energy requirements. Capillary tubes filled with stock solutions
of compounds can be stored at desired temperatures and conditions.
The method according to the invention allows for the
simultaneous mass application of analytes onto a solid support. For
example, as described in greater detail herein one can achieve
simultaneous mass application of equal volumes of 10,000 or more
compounds from an array of individual containers such as capilliaries
to a solid phase. The amount of compound delivered can be determined
by the contact time of the capilliaries with the solid support. The
analyte containers can also suitably be individual addressable plotter
pen-like devices allowing simultaneous drawing of parallel lines of
analyte on a solid support of choice.
After compound delivery the compounds, which can be disposed
as a pattern of discrete spots or lines, are left to dry on the solid phase.
In one embodiment, the solid support is of a substantially flat,
disc-, rectangular- or square- shape.
The solid support can comprise a material which allows for
spontaneous release of the analyte(s) when applied thereto.
Alternatively, the solid support can comprise a material which
allows for controlled release of the analyte(s) when applied thereto.
In each case, the material can be said semi-solid medium.
Preferably, when each analyte is applied to the solid support it
diffuses thereon so as to produce a concentration gradient.
In this way one can achieve a serial dilution of analyte if a dose
response curve for a candidate drug is required. rather than a simple
positive or negative (ves/nol result.
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Utilisation of retarded analyte diffusion in a semi-solid medium
or matrix further eliminates the need for physical separation as in the
case of the wells of a microtitre plate and the necessity of serial dilution
of analytes, when such is required as a concentration gradient will be
established by passive diffusion.
Preferably the surface of the solid support onto which the
analytes are applied is selected from polymers, ceramics, metals,
cellulose and glass.
Further, preferably, said semi-solid medium is disposed on a
carrier.
In another embodiment, the solid support is in the form of a
flexible film or tape onto which the target-containing semi-solid
medium is applied, whereby the method can be automated using a
system of rollers to progress the flexible film or tape through the
various steps of the method.
In this embodiment, the carrier can be covered by a further layer
of film or tape and is thereby sandwiched between the solid support and
the covering layer.
Furthermore, the solid support or covering layer (if present) can
be provided with a track for the recordal of information regarding the
applied analytes, whereby the information can be read and processed
simultaneously with the measurement of analyte-target interactions in
an automated process.
In a further embodiment, the solid support is itself a detector or
forms part of a detector.
In this embodiment, the solid support is preferably selected from
a SiO, wafer, a char~7e-coupled device and a photo<yraphic film.
CA 02342627 2001-03-O1
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The surface of the solid support can be coated with a membrane, a
molecular monolayer, a cellular monolayer or a Langmuir-Blodgett film.
All of these coatings can be used to control the release of analytes
when applied thereto.
In another embodiment, the solid support is itself an information
carrier which carries information in electronic, magnetic or digitised
form.
In an alternative embodiment, the surface of the solid support is
reflective. For example, the surface can be the reflective surface of a
compact disc.
The method according to the invention can further comprise the
step of copying said compact disc to a writable compact disc.
In another embodiment, the semi-solid medium comprises a
substance which provides a semi-solid or viscous liquid environment
allowing controlled release of said analytes to said target.
Preferably, the substance which provides a semi-solid or viscous
liquid environment is selected from gelatin, polysaccharides such as
agar and agarose, and polymers such as methylcellulose and
polyacrylamide or a so-called intelligent material. Such substances can
also be used to control the release of the analytes when applied thereto.
So-called intelligent materials are natural and synthetic polymer
gels that are undergo phase transitions and critical phenomena, for
example phase transitions accompanied by a reversible, discontinuous
volume change as large as several hundred times, in response to
infinitesimal changes in environmental conditions.
Examples of so-called intelligent materials are polymeric gel-type
materials, more particularly hydrogels that can take up a fluid and
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CA 02342627 2001-03-O1
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9
subsequently release that fluid in response to a chemical or physical
stimulus or trigger. An example of a chemical stimulus is a change of
pH or ionic or solvent composition and an example of a physical
stimulus is light of a particular wave-length or a laser beam, a change of
temperature or a small electric field.
For example, a gel containing N-isopropylacrylamide (main
constituent) and the light-sensitive chromophore, the trisodium salt of
copper undergoes phase transitions induced by visible light (Suzuki, A.
and Tanaka, T (Nature ( 1990); 346, 345-34 ~.
A range of suitable thermo-sensitive polymers is described by
Snowden, M.J. et al. (Chemistry & Industry (July, 1996); p.p. 531-534.
Other suitable gels are those sold under the Trade Mark THERA
GEL marketed by Gel Sciences Inc., Boston, M.A., U.S.A.
In a further embodiment, steps b) and c) are carried out
simultaneously.
In a still further embodiment, each analyte is applied to a single
solid support.
In this embodiment, the solid support is preferably of a rod shape
or a spherical shape.
Further, preferably each analyte-bearing solid support is contacted
in step b) with a target in a separate compartment of a multi-
compartmented apparatus, more especially said compartments are an
arrangement of mini-wells in said apparatus.
In another preferred embodiment, the analyte containers are small
inert solid supports onto the surfaces of which analytes have been ~'
applied. Dipping of the solid supports into a liquid phase or semi-solid
phase results in time-dependent release of analyte from the solid
support into the liquid or semi-solid phase. In this way, dilutions of
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-
minute amounts of analytes in liquid or semi-solid phase can be
obtained without the use of liquid handling devices. The final
concentration of analyte released into the liquid or semi-solid phase is
determined by the contact time between the analyte-carrying solid
5 suppport and the liquid- or semi-solid phase.
The analytes for rapid screening in the method according to the
invention are preferably selected from chemical compounds, antigens,
antibodies, DNA-probes, cells and beads and liposomes carrying an
analyte of interest.
10 Further, preferably, the analytes, when applied to the solid
support, are dissolved in an organic or inorganic solvent.
Suitably, the solvent includes a so-called intelligent material
responsive to a chemical or physical parameter such that each analyte,
following application to the solid support and drying, liquifies in
response to said chemical or physical parameter.
The analyte in a preferred embodiment is a chemical compound
for screening as a potential drug candidate.
Preferably, the targets are selected from prokaryotic cells,
eukaryotic cells, viruses, molecules, receptors, beads, and combinations
thereof.
As used herein compounds) means any synthetic, semi-synthetic
or naturally-occuring compound or combination thereof.
In one embodiment the targets are cells equipped with reporter
functions.
In a preferred embodiment the targets are mammalian cells
equipped with single or multiple reporter construct(s). The activity
and/or expression of the reporter genes is dependent on the impact of
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the specific experimental conditions, for example the in virrn effect of
compounds released from the solid support to a semi-solid phase.
The incubation unit used in this embodiment will typically be an
incubator of the type that is generally used for tissue culture.
The detection unit used to measure the compound-cell interaction
will suitably consist of an inverted microscope (e.g. Zeiss Axiovert
100} coupled to a video camera (e.g, dage-MTI CCD72E} and
computer system (e.g. PC 300 Mhz Pentium) with KS400 basic
software and the graphic KS400 option. The inverted microscope can
be further equipped with a scanning stage modified to fit all culture
plate configurations ( from 6-well up to 9600 well formats j as well as
other formats of the type described herein and equipped with a stepper
motor with a resolution of 17,600 steps pen revolution. The KS400
software package provides easy to use menus for entering
configuration, calibration and assay parameters or data analysis
specifications.
Other detection means include the use of computer-assisted image
analysers.
In the event that adherent cells are used as a target, these cells are
grown confluently either covering the surface of the solid support or
the surface of small beads which are then homogeneously suspended in
the semi-solid phase.
Still further, preferably, said analyte-target interactions are
measured using one or more of the following methods: microscopic,
colorimetric, fluorometric, luminometric, densitometric, isotopic, and
physical measurements.
For example, one can use a combination of microscopy and
fluorescence. Accordingl~~. the microscope can be configured for
epifluorescence fitted with a stepper motor and a camera system.
Image acquisition and interpretation can be PC controlled.
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It will be appreciated that the method according to the invention
provides for the coupling of the principle of compound diffusion in
semi-solid media containing experimental biological systems to image
microscopy systems. The invention also provides for visualising and
determining the effect of compounds on prokaryotic and eukaryotic cell
function. This visualisation can be achieved with genetically engineered
cells embedded in a semi-solid medium and expressing single or
multiple reporter genes of which the expression products) is/are an
intracellular fluorophore(s) of specific excitation and emission
wavelengths matching wavelength characteristics of a high density
fluorescence image microscopy system.
By way of example, the method according to the invention
typically comprises the steps of:
a) direct application of compounds to be screened as potential
drug candidates onto a solid support;
b) layering of a semi-solid medium containing targets of interest
over the compound carrying support;
c J diffusion of compounds into the semi-solid medium;
d) incubation;
e) visualisation and registration of the compound-target
Interaction.
By way of example if one wishes to screen potential drugs or
drug combinations for use in the treatment of individuals infected with
the HIV virus, having full-blown AIDS or ARC, then suitable targets
for use in accordance wish the invention are genetically engineered
green t~luorescent protein (GFP)-expressing MT4 cells containing the
long term no repeat ( LTR) promotor (pLTR-EGFP-C 1 ) and deposited
at the BCCM on August ?0, 1998, under ,accession No. LMBP3879.
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pLTR-EGFP-C I {~777bp) is based on pEGFP-C I (Clontech). In
pEGFP-C 1 expression of the enchanced green fluorescent protein
(EGFP) is controlled by the strong human cytomegalovirus immediate
early promotor (589bp). In pLTR-EGFP-C 1 the CMV promotor is
replaced by the HIV-1 long terminal repeat LTR (652bp) containing the
highly inducible promotor in the U, region.
Such cells when infected with HIV become fluorescent due to
activation of LTR and expression of GFP. If a candidate drug inhibits
HIV, for example by inhibiting the viral reverse transcriptase, protease
or integrase fluorescence is reduced or totally inhibited and one
observes progressively darkened regions in the area of the compound-
target interaction.
Step b) of the method according to the invention will typically
involve maintaining the anaiytes and the targets under appropriate assay
1 ~ conditions and for a sufficient period of time for the release of the
analytes from the solid support and the diffusion of said analytes into
the liquid or semi-solid phase containing the targets of interest.
Maintaining appropriate assay conditions will include maintaining
correct temperature, osmolality, pH, tonicity and the like. Theses
conditions are further determined by the nature of the targets.
Typically, with mammalian cells as a target, the temperature can
range from about 4°C to about 50°C and pH can range from about
6.~
to about 7.5. Osmolality and tonicity will be chosen in a manner so that
optimal cell-analyte interactions can be obtained. The only limitation on
physical conditions is that the conditions used do not adversely affect
cell viability nor interfere with analyte target interactions.
It will be appreciated that the method according to the invention
can be carried out in a fully integrated system.
The method according to the invention facilitates the use of a data
management unit. Such a unit suitable comprises chemical compound
library data with compound identification. history tracking and
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software technology allowing accessing and identification of compounds
within the warehouse installation at all times.
In another preferred embodiment, analyze application, analyte
target interaction, incubation, detection and data interpretation are
linked in such a way that the entire screening method according to the
invention is a fully automated method from start to finish. One typical
example of an automated method of rapid screening of compounds
according to the invention comprises the steps of:
1. Application of compounds by means of capillary delivery
producing either discrete spots or discrete lines of compounds onto the
surface of a carrier, such as a transparent film or information carrier
used in the audio-casette industry;
2. Drying the surface of the carrier;
3. Layering of cells embedded in a semi-solid matrix of constant
thickness over the surface of a transparent film;
4. Contacting the surface of the carrier containing spotted
compounds or discrete compound lines and the surface of the film
serving as the semi-solid matrix carrier;
~. Winding of the contacted film surfaces;
6. Incubation of the wound film;
7. Unwinding of the film and exposure to a detection and
information reading unit;
8. Continuous reading of exposed film by means, for example, of
a fluorescence microscope image analysis system; and
2~ 9. Data analysis.
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Outer surfaces (non-compound carrying or which are not in
contact with the semi-solid matrix) can contain digital information
regarding the identity of the applied compounds which is read
simultaneously during sample analysis as described in step 8. In this
J way, biological information and compound information can be read and
processed at the same time.
Brief Description of the Drawings
Fig. 1 illustrates the principle of compound diffusion in a semi-
solid matrix as described in Example 1 at a cell density of 10E5/ml;
Fig. 2 illustrates the principle of compound diffusion in a semi-
solid matrix as described in Example i at a cell density of 10E6/ml;
Fig. 3 illustrates the principle of compound diffusion in a semi-
solid matrix as described in Example 1 at a cell density of 10E7/ml;
Fig. 4 is a capillary tube holder device (8 x 12) as described in
I S Example 2;
Fig. ~ shows the fluorescence observed with GFP (green
fluorescent protein) expressing MT4-cells (LTR (long term no repeat)
promotor) in RPMI (Rosemount Park Memorial Institute) medium
without phenol red, 10% FCS (fetal calf serum) and 1 % Pen-Strep
(penicillin-streptomycin) in the absence (a) and in the presence of HIV-
1 (b) as described in Example 3;
Figs. 6(a) and (b) show the fluorescence observed with GFP
expressing MT4-cells (LTR promotor) in semi-solid phase (RPMI
medium without phenol red, 10% FCS, 1 % Pen-Strep, agar 0.34 %) in
2~ the absence (a) and in the presence of HIV- I (b) as described in
Example 3;
Figs. 7(a) - ( a 1 show the tluorescence observed with GFP-
expressina MT4-cells ~ LTR promotori in RPMI medium ( without
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phenol red and supplemented with 10% FCS, 1 % Pen-Strep) in the
absence (a) and in the presence of HIV-1 (b) and in the presence of
HIV-1 and the reverse transcriptase inhibitors: AZT (c), 3TC (d) and
Loviride (e) at a final concentration of 2.5 p.M in a total volume of 20
~,l as described in Example 3;
Figs. 8(a) - (e) show the fluorescence observed with GFP-
expressing MT4-cells (LTR promotor) in semi-solid phase 0.34% agar
in RPMI medium (RPMI medium without phenol red, supplemented
with I O% FCS and 1 % Pen-Strep) in the absence (a) and in the presence
of HIV- t (b) and in the presence of HIV- I and the reverse transcriptase
inhibitors: AZT (c), 3TC (d) and Loviride (e) spotted ( 1 ~tl of a stock
solution) onto a surface of a solid support at a concentration producing
a 2.5 p,M end concentration, assuming complete diffusion of the
compounds in 20 pl semi-solid phase as described in Example 3;
Fig. 9 shows the fluorescence observed with HIV-1 infected
MT4-cells in semi-solid phase (0.34% agar in RPMI medium without
phenol red, supplemented with 10% FCS and 1 % Pen-Strep j when
HIV-1 infected cells were admixed with a solid support onto which 1 ~tl
of 2.5 ~.M, 250 nM and ''.~ nM of reverse transcriptase inhibitors were
applied. left to dry and kept for one week at 4°C prior to use as
described in Example 3;
Fig. 10 shows the fluorescence observed with HIV-1 infected
MT4-cells in medium (RPMI without phenol red, supplemented with
10% FCS and 1% Pen-Strep) when HIV-1 infected cells were added in
medium to the wells of a 384-well tissue culture plate containing 1 ~tl of
2.5 pM, 250 nM and 2.5 nM of the reverse transcriptase inhibitors
AZT, 3TC and Loviride. respectively as described in Example 3;
Fig. 1 1 is a schematic representation of a solid support carrying
spotted compounds in the method according to the invention:
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Fig. 12 illustrates schematically how the distance between
capillaries in an array can be varied and adapted to the specific
requirements of the method according to the invention;
Fig. I3 is a diffusion pattern of calcein in a semi-solid phase on
a solid support in accordance with the invention;
Fig. I4 illustrates the principle of automated on-line mega-
throughput screening using the method according to the invention;
Fig. I S is a schematic representation with exploded detail of
capilliary tube sorting means;
Figs. lb(a) - (c) show the movement of the capilliary tubes of
Fig. 15 to a microtitre plate for filling;
Fig. 17 illustrates the filling of the capilliary tubes of Fig. 15;
Fig. 18 shows a screw device used to reduce the spacing between
the capilliary tubes of Fig. 1 ~;
I S Fig. 19 shows the individual capilliary tubes of Fig. 15 being
formed into a spiral array;
Fig. 20 illustrates means by which the liquid in the capilliary
tubes of Fig. 15 is released onto a solid support;
Fig. 2I illustrates a sample deposition pattern;
Fig. 22 is a plan view of a solid support with analytes spotted
thereon; and
Fig. 23 illustrates the application of a semi-solid phase to the
solid support.
Modes for Carrvine out the Invention
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18
The invention will be further illustrated by the following
Examples:
Example 1
Principle of compound diffusion and interaction with cells embedded in
a semi-solid medium
Calcein, a cell viability marker was dissolved at a concentration
of 5 mM in dimethyl sulfoxide (DMSO). A glass capillary tube with a
total volume capacity of 0.51 was dipped into the calcein solution and
filled by capillary action. The tip of the capillary tube was then
contacted with a polystyrene surface in a such a way that a small drop
of calcein solution was delivered from the capillary tube to the plastic
surface. After drying of the drop, 20~.I of a cell suspension in semi-
solid medium (MT4 cells suspended in RPMI (Rosemount Park
Memorial Institute) 1640 medium, without phenol red, supplemented
with 10% FCS (fetal calf serum), 1 % Pen-Strep (penicillin-
streptomycin) and containing 0.34% agar) was layered over the dried
calcein spot. After an incubation time of 2 hours at 37°C (humidified
atmosphere and 5 % carbon dioxide) the diffusion of the calcein into
the semi-solid phase was observed by means of fluorescence
microscopy and visualisation of the fluorescence produced by the
embedded MT4 cells. The method of drop delivery, drying and
layering of semi-solid matrix containing increasing densities of
embedded MT4 cells is illustrated in Figs. 1,2 and 3.
It follows from these results that the distance over which
diffusion of calcein takes place in a semi-solid matrix of constant
density is also determined by the number of embedded cells.
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Example 2
Princit~le of high density compound application and diffusion in a semi
solid matrix
A bundle of capillaries filled with calcein and arranged {8 x 12)
in a holder device as depicted in Fig. 4 was contacted with a surface of
polystyrene so that a drop of calcein was delivered simultaneously from
each capillary to the polystyrene surface. The holder device is
indicated generally at 10 and comprises capillary tubes 1 I mounted in
plates 12, 13 for maintaining the capillary tubes 11 in the desired
relationship with respect to each other. Following drying, a
homogeneous suspension of MT4 cells in RPMI 1640 medium
supplemented with 10% FCS, 1 % Pen-Strep and 0.34% agar was
layered over the spots. After an incubation period of 2 hours
(humidified atmosphere, 5% carbon dioxide} it was found that for each
IS of the spots the distance of diffusion of the calcein in the semi-solid
matrix was reflected by the fluorescence of the embedded MT4 cells.
Example 3
Compound target interaction' Effect of anti-HIV compounds on the
fluorescence of GFP-expressing MT4 cells (LTR~romotor) in the
presence of HIV- I in a semi-solid' phase
Three compounds of well known activity against HIV-1 (AZT,
3TC and Loviride) were spotted (+/- 1 ~tl from stock in a capillary tube
as hereinbefore described) onto the bottom surface of the wells of a
transparent 384-well polystyrene tissue culture plate. The compounds in
the wells were left to dry and stored at 4°C.
One week later, MT4 cells were collected from tissue culture
flasks and suspended at 10E7/ml in RPMI medium. This cell suspension
was further equally divided into four tubes. These tubes were then
centrifuged at 450 g, for 10 min. To the cell pellets obtained after
centrifugation of two oI~ these tubes, 200 ~tl HIV suspension in RPMI
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medium were added for a period of 2 hours at 37°C. The other two
tubes were treated in the same way, except that no virus was added.
After an incubation time of 2 hours, agar solution (39°C) was
added to
one tube containing cells and HIV and to one tube only containing cells
at a final concentration of 0.34%. Then, 20 p.l of the cell suspension in
agar and 20 ~.l of the cell/virus-agar suspension were added to the
different wells of a 384-well plate containing the spotted compounds as
set forth above.
To the cell pellets of the remaining two tubes, 200 ~tl medium
and 200 ~tl virus containing medium were added respectively. After an
incubation period of 2 hours at 37°C, the final volume was corrected
(made equal to the final volume of the agar composition) and 20 pl of
the non-infected and infected cell suspensions were added to the wells
of the 384-well culture plate with spotted compounds as described
above.
Ipl volumes of compounds were added to the wells immediately
before the HIV-infected cells were added. Total assay volume was
201.
After a 3-day incubation period, the fluorescence of the GFP-
expressing MT4 cells was evaluated by fluorescence microscopy and
plate reading. The results are summarized in Figs. 5-10. These data
show that following the application of compounds onto a solid support
(well of a 384-well microtitre plate) and after storage for 1 week at
4°C, the activity (protective effect of these compounds against HIV-1
infection) did not differ from the situation where compound diffusion
takes place in a liquid phase.
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21
Furthermore, the results obtained show that the concentration-
dependent effects of RT inhibitors on HIV-1 infection as reflected by
GFP-expressing MT4-cells are observed in semi-solid phase and that
the nature of the compound and its effectiveness for HIV-1 RT
inhibition is not affected by the use of a semi-solid medium.
*****~:
The invention will be further illustrated by the following
description of embodiments thereof given by way of example only with
reference to the accompanying drawings.
Referring to Fig. 11, there is indicated a solid support 20
carrying compounds 21 which have been spotted thereon from an
array of 196 bundles of capillary tubess (not shown) each carrying 110
capillary tubes so that 21,560 compounds are spotted onto the solid
support 20.
Fig. 12 illustrates how the distance between the capillaries in an
array can be varied ( 1.414 mm, 1 mm, 2.236 mm, 2 mm, 3.623 mm, 3
mm) to meet the specific reduirements of a screening method in
accordance with the invention.
Fig. 13 is a diffusion pattern of calcein in a semi-solid phase on a
solid support. Calcein was spotted on a polystyrene surface using a
capillary compound holder device as depicted in Fig. 4 with capillary
tubes arranged at a centre to centre distance of 2 mm. The density of
overlying cells, suspended in the semi-solid phase (RPMI 1640 medium,
10% FCS, 1 % Pen-Strep, 0.34% agar) was 10E7 cells (MT4)/ml.
Detection was carried out by fluorescence microscopy after a two hour
incubation period.
Fig. 14 is a schematic representation of an automated method for
the rapid screening of compounds in accordance with the invention. An
information carrier 30 in the form of a film or tape and with
s0 compounds to be screened applied as discrete spots or lines on its
CA 02342627 2001-03-O1
WO 00/14540 ~~ PCT/IB98/01399
surface 31 is brought into contact with surface 32 of an information
carrier 33, which is also a film or tape, bearing targets of interest
embedded in a semi-solid matrix. The respective carriers 30, 33 are
then wound with their surfaces 31 and 32 in contact and incubated in a
temperature, humidity and carbon dioxide controlled environment,
such that the compounds are released to the surface 32 of the carrier
33. The carriers are then unwound and the carrier 33 is then passed to
an analysis - and information reading unit indicated generally at 34.
In the following Figs. 15-23 like parts are denoted by the same
reference numerals.
Referring to Fig. 1 ~, there is indicated generally at 40, apparatus
for feeding capillary tubes 41 from a supply thereof to a conveyor belt
42 with regularly spaced-apart transverse grooves 43. The capillary
tubes 41 enter a channel 44 which can accommodate a single layer of
capillary tubes 41 in response to the anti-clockwise movement of a belt
45 and are delivered one at a time to the grooves 43 as the conveyor
belt 42 moves in a clockwise direction. The capillary tubes 41 travel
along the channel 44 through the combined effects of gravity and the
belt 45. Each time a Groove 43 is positioned at end 46 a capillary tube
41 is delivered thereto.
Referring to Figs. 16a-c and Fig. 17, the steps involved in
transferring the capillary tubes 41 to a microtitre plate 47 for filling
are illustrated. Capillary tubes 41 are lifted from the belt 42 by a
clamping device indicated generally at 4$. The clamping device 48
comprises two elongate members 49 which clamp a plurality of tubes
41 therebetween by applying a sideways force to the tubes. In this way,
the capillary tubes 41 are transferred from the conveyer belt 42 to the
microtitre plate 47 for filling. The spacing of the grooves 43 on the
belt 42 is the same as the spacing of the wells ~0 in the microtitre plate
~7. The clampinU device ~8 lifts and transports the tubes =11 in groups
of 12 corresponding to the number of wells 50 in one row of the
microtitre plate ~7. Durin~l the transporting step, the clamping device
CA 02342627 2001-03-O1
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48 is rotated through 90° so that one end of the capillary tubes 41 is
lowered into the wells ~0 of the microtitre plate 47.
The clamping device 48 and the capillary tubes 41 are free to
move along a vertical axis to allow the tubes 41 to be lowered into the
wells 50 of the microtitre plate 47. The capillary tubes 41 are held in
the wells 50 for a period of time sufficient to allow liquid in the
respective wells 50 to be drawn into the tubes 41 by capillary action.
Once the given filling time has elapsed, the tubes 41 are withdrawn and
the clamping device 48 is rotated through 90° to again assume a
horizontal orientation.
Following filling of the capillary tubes 41, said tubes are
transferred to a screw - or worm device 51 which is used to reduce the
spacing between the capillary tubes 41 as shown in Fig. 18. The
capillary tubes 41 are delivered to the screw device 51 at end 52.
Thread 53 of the screw device S I has a varying pitch, being larger at
end 52 than at end 54. As the screw device 51 turns, the capillary tubes
41 advance along its length in the direction of the arrow and due to the
changing pitch also come closer together. At the end 54 tubes 41 are
only separated by wall 5~ of the thread 53.
Referring to Fig. 19, the capillary tubes 41 are discharged from
the screw device 51 onto a tape 56 which has a layer of adhesive to
which the tubes 41 are stuck with their sides abutting. The tape 56
advances at the same rate as the tubes 41 leave the screw device 51.
The tape 56 is then progressively rolled up into a tightly packed spiral
array as shown at 57.
Referring to Fig. 20, there is indicated generally at 60, apparatus
for releasing the liquid in the capillary tubes 41 defining the array 57
to the surface of a solid support 6l and which is movable relative
thereto. The apparatus 60 comprises a housing 6? which is adapted to
receive said array ~7. The housing 62 is connected to an air pump 63
and by creating an area of positive pressure relative to the exterior of
CA 02342627 2001-03-O1
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the housing 62 forces the liquid out of the tubes 41 onto the surface of
the solid support 61, when required.
Fig. 21 shows the array 57 of capillary tubes 41 disposed above
the solid support 61 following application of droplets of liquid analytes
from the tubes 41 and the pattern of discrete spots 64 disposed on said
solid support 61.
Fig. 22 is a plan view of the solid support 61 showing the spiral
arrangement of the discrete spots 64 of the applied liquid analyte.
Referring to Fig. 23, there is illustrated a device indicated
generally at 70, for applying target cells in a semi-solid phase 71 to
surface 72 of the solid support 61 following drying of the liquid
analytes. Device 70 comprises an arm 73 and a printing head 74. The
solid support 61 is free to rotate and the device 70 is free to move in
the z plane and x or y planes, so that the printing head 74 is located by
a combination of motion of the arm 73 and the solid support 61.
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BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCMT""/
- LMBP-COLLECTION
Page t of Form BCCMr"'/LMBP/BP/4/98-16 Receipt in the case of an original
deposit
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for
= the Purposes of Petant Procedure
°
Receipt in the case of en original deposit Issued pursuant to Rule 7.1 by the
° International Depository Authority BCCM'""/LMBP Identified at the
bottom of next page
a
lnternatlonel Form BCCMT'"/LMBP/BPI4/98-16
a
I
To : Name of the depositor : TIBOTEC N.V.
°
Address : Institute for Antiviral Research
Generaal De Wittelaan L11 B3
B-2800 Mechelen
Belgium
I. Identification of the microorganism:
I. t Identification reference given by the depositor:
pLTR-EGFP-C 1
1.2 Accession number given by the International Depository Authority:
°
LMBP3879
CA 02342627 2001-03-O1
WO 00/14540 PCT/IB98/01399
26
BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCMT'"'/
LMBP-COLLECTION
Page 2 of Form BCCMr'"/LMBP/BP/4/98-16 Receipt in the case of an original
deposit
Il. Scientific description and/or proposed taxonomic designation
The microorganism identified under 1 above was accompanied by:
(mark with a cross the applicable box(es)):
~ a scientific description
D a proposed taxonomic designation
III. Receipt and acceptance
This International Depositary Authority accepts the microorganism identified
under 1
above, which was received by it on (date of original deposit) : August 20, 3
998
IV. International Depositary Authority
Belgian Coordinated Collections of Microorganisms (BCCMT"')
Laboratorium voor Moleculaire Biologie - Plasmidencollectie (LMBP)
Universiteit Gent
K.L. Ledeganckstraat 35
B-9000 Gent, Belgium
Signaturels) of persons) having the power to represent the International
Depositary
Authority or of authorized official(s):
~y~t.
Date . August Zfi, 1998 Martine Vanhoucke
BCCM~'/LMBP curator
CA 02342627 2001-03-O1
N WO 00/14540 ,~,~ t'CT/IB98/Q1399
BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCMT""I
LMBP-COLLECTION
- Page 1 of Form BCCMT'''/LMBP/BP/9/98-16 Viability statement
' Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for
the Purposes of Patent Procedure
Viability statement issued pursuant to Rule 10.2 by the International
Depositary
o Authority BCCMT""ILMBP Identified on the following page
Jnternational Form BCCM'""ILMBPIBPl9198-16
a
To : Party to whom the viability statement is Issued:
Name : Chris Roelant
Address : TIBOTEC N.V.
Institute for Antiviral Research
o Generaa! De Wittelaan L11 B3
B-2800 Mechelen
Belgium
L Depositor:
1.1 Name : TIBOTEC N.V.
1.2 Address : Institute for Antiviral Research
- Generaal De Wittelaan L11 B3
B-2800 Mechelen
Belgium
II. Identification of the microorganism:
11.1 Accession number given by the International Depositary Authority:
LMBP3879
_ IL2 Date of the original deposit (or where a new deposit or a transfer has
been
made, the most recent relevant date) : August 20, 1998
= III. Viability statement.
The viability of the microorganism identified under II above was tested on
0
a : August 25. 1998
' (Give date. In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to
the most recent
viability test).
' On that date, the said microorganism was: (mark the applicable box with a
cross)
viable
a o no longer viable
CA 02342627 2001-03-O1
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BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCMT"Y
LMBP-COLLECTION
Page 2 of Form BCCM"''/LMBP/BP/9198-16 Viability statement
IV. Conditions under which the viability test has been pertormed:
(Fill in if the information has been requested and if the results of the test
were negative}.
V. International Depositary Authority
Belgian Coordinated Collections of Microorganisms (BCCMT"")
Laboratorium voor Moleculaire Biologie - Plasmldencollectie (LMBP)
Universiteit Gent
K.L. Ledeganckstraat 35
B-9000 Gent, Belgium
Signatures) of persons) having the power to represent the International
Depositary
Autho~ty or of authorized officiai(s):
---,
/
Date : August 26, 1998 , ' Martine Vanhoucke
BCCMt'-'/LMBP curator
