U.S. patent application number 11/473573 was filed with the patent office on 2006-10-26 for assaying apparatus, kit, and method for lipids and associated enzymes.
Invention is credited to Leena Chakravarty, Beth E. Drees, Michael J. Mostert, Paul O. Neilsen, Glenn D. Prestwich.
Application Number | 20060240497 11/473573 |
Document ID | / |
Family ID | 25537744 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060240497 |
Kind Code |
A1 |
Drees; Beth E. ; et
al. |
October 26, 2006 |
Assaying apparatus, kit, and method for lipids and associated
enzymes
Abstract
Lipid assay method, kit, and apparatus involving exposure of a
protein, having a lipid recognition motif that interacts with a
target lipid and a competing lipid, to a solution containing the
competing lipid, and determining whether the target lipid is
present in the solution. The target lipid has a stronger affinity
to the lipid recognition motif than does the competing lipid. The
lipid recognition motif is preferably a pleckstrin homology (PH)
domain, with the target lipid being a phosphoinositide. The assay
determines activity of a lipid kinase, the target lipid being a
phosphorylation product of a reaction between the lipid kinase and
a substrate lipid. The assay can be a cancer screening method for
detection of cancer cells, where detection of certain levels of a
PI(3,4,5)P3 target lipid is an indicator of a cancer cell.
Inventors: |
Drees; Beth E.; (Park City,
UT) ; Prestwich; Glenn D.; (Salt Lake City, UT)
; Neilsen; Paul O.; (Draper, UT) ; Chakravarty;
Leena; (Sandy, UT) ; Mostert; Michael J.;
(Salt lake City, UT) |
Correspondence
Address: |
JENNIFER M MCCALLUM, PH D, ESQ;THE MCCALLUM LAW FIRM, LLC
685 BRIGGS STREET
PO BOX 929
ERIE
CO
80516
US
|
Family ID: |
25537744 |
Appl. No.: |
11/473573 |
Filed: |
June 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09991933 |
Nov 26, 2001 |
7067269 |
|
|
11473573 |
Jun 22, 2006 |
|
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Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
G01N 33/573 20130101;
G01N 33/574 20130101; G01N 33/92 20130101 |
Class at
Publication: |
435/007.92 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A lipid assay comprising; a) a target lipid that is a
phosphorylation product of a reaction between a phosphoinositide
kinase and a substrate phosphoinositide lipid; b) a predetermined
amount of a competing lipid that is labeled by a non-radioactive
signal; and c) a protein having a phosphoinositide lipid
recognition motif that interacts with said target lipid and said
competing lipid.
2. The lipid assay of claim 1, which further comprises a multi-well
assay plate.
3. The lipid assay of claim 2, wherein said multi-well assay plate
includes said competing lipid immunobilized in wells of said
multi-well assay plate.
4. The lipid assay of claim 1, which further comprises primary and
secondary antibodies.
5. The lipid assay of claim 1, wherein said target lipid is a
component of a sample selected from the group consisting of a
tissue sample, a blood sample, and a serum sample.
6. The lipid assay of claim 1, wherein said assay is selected from
the group consisting of an amplified luminescence proximity
homogenous assay (ALPHA), a fluorogenic assay, and an enzyme linked
immunosorbent assay (ELISA).
7. The lipid assay of claim 1, wherein said target lipid is a
phosphoinositide.
8. The lipid assay of claim 1, wherein detection of said signal is
automated.
9. The lipid assay of claim 1, wherein said assay comprises a
kit.
10. The lipid assay of claim 1, wherein said protein is selected
from the group consisting of an anti-PI(3,4,5)P.sub.3 antibody, an
anti-PI(3)P antibody, a lipid recognition protein with specificity
for PI(3,4,5)P.sub.3, and a lipid recognition protein with
specificity for PI(3)P.
11. The lipid assay of claim 1, wherein said protein contains an
affinity tag fusion with a PH domain.
12. The lipid assay of claim 1, wherein detection of said target
lipid is indicative of the presence of a cancer cell.
13. The lipid assay of claim 1, wherein said assay determines
activity of a lipid kinase, the target lipid being a product of a
reaction between said lipid kinase and a substrate lipid.
14. The lipid assay of claim 13, wherein said lipid kinase is PI
3-K and said target lipid is a product of PI 3-K.
15. The lipid assay of claim 14, wherein said product of PI 3-K is
PI(3,4,5)P.sub.3 or PI(3)P.
16. A lipid assay, comprising; a) a target lipid; b) a competing
lipid; and c) a protein having a lipid recognition motif that
interacts with said target lipid and said competing lipid.
17. The lipid assay of claim 16, wherein the competing lipid emits
a signal.
18. The lipid assay of claim 17, wherein detection of said signal
is automated.
19. The lipid assay of claim 16, wherein said assay comprises a
kit.
20. The lipid assay of claim 16, which further comprises primary
and secondary antibodies.
21. The lipid assay of claim 16, wherein said assay is selected
from the group consisting of an amplified luminescence proximity
homogenous assay (ALPHA), a fluorogenic assay, and an enzyme linked
immunosorbent assay (ELISA).
22. The lipid assay of claim 16, wherein said target lipid is a
component of a sample selected from the group consisting of a
tissue sample, a blood sample, and a serum sample.
23. The lipid assay of claim 16, which further comprises a
multi-well assay plate.
24. The lipid assay of claim 23, wherein said multi-well assay
plate includes said competing lipid immunobilized in wells of said
multi-well assay plate.
25. The lipid assay of claim 16, wherein said protein is selected
from the group consisting of an anti-PI(3,4,5)P3 antibody, an
anti-PI(3)P antibody, a lipid recognition protein with specificity
for PI(3,4,5)P3, and a lipid recognition protein with specificity
for PI(3)P.
26. The lipid assay of claim 16, wherein said protein contains an
affinity tag fusion with a PH domain.
27. The lipid assay of claim 16, wherein said target lipid is a
phosphoinositide.
28. The lipid assay of claim 16, wherein detection of said target
lipid is indicative of the presence of a cancer cell.
29. A lipid assay comprising; a) a sample putatively containing
3,4,5-trisphosphate; b) biotin labeled 3,4,5-trisphosphate; and c)
a protein having a pleckstrin homology domain to a phosphoinositide
that interacts with said target lipid and said competing lipid.
30. A method comprising; exposing a test solution containing an
unknown amount of a target lipid, wherein said target lipid is a
product of a reaction between a kinase that is added to said test
solution and a substrate lipid that is added to said test solution,
to a solution containing a protein having a lipid recognition motif
that interacts with said target lipid and a predetermined amount of
a competing target lipid, thereby allowing for quantification of
the kinase activity.
31. A lipid assay for the detection of a phosphoinositide target
lipid comprising; a) a Grp1 protein having a pleckstrin homology
domain to a phosphoinositide; and b) a predetermined amount of a
competing lipid that is labeled by a non-radioactive signal;
wherein the target lipid has a stronger affinity to the pleckstrin
homology domain than does the competing lipid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a divisional of U.S. patent application
Ser. No. 09/991,933, filed Nov. 26, 2001.
FIELD OF THE INVENTION
[0002] The present invention relates to enzymes that phosphorylate
an inositol lipid, including phosphatidylinositol (4,5)
bisphosphate (PI(4,5)P.sub.2) and phosphoinositol (PdtIns) in
general, at the D-3 position of the inositol ring. More
particularly, the present invention relates to detection methods,
kits, and apparatuses for detection of such enzymes.
BACKGROUND OF THE INVENTION
[0003] Phosphoinositides are key lipid second messengers in
cellular signaling, with phosphatidylinositol (PI) dependent
signaling pathways playing central roles in the regulation of many
cellular processes. Disruption of these pathways is common to many
disease states, including inflammation, diabetes, cardiovascular
disease, and cancer. Because the activity of PI second messengers
is determined by their phosphorylation state, the enzymes that act
to modify these lipids are central to the correct execution of PI
dependent signaling pathways.
[0004] In particular, phosphatidylinositol 3-kinase (PI 3-K) is
important in pathways mediating cell proliferation, survival,
differentiation and motility. Inhibitors of PI 3-K have been used
to confirm the cellular functions of PI 3-K, but thus far, such
inhibitors have not been deemed suitable for therapeutic uses
because of problems such as toxicity and low selectivity. The PI
3-K family of heterodimeric lipid kinases is known primarily for
its involvement in the phosphorylation of inositol lipids via
transfer of the .gamma. phosphate of ATP to the D-3 position of the
inositol ring of PI, PI(4)P, and PI(4,5)P.sub.2 giving rise to
PI(3)P, PI(3,4)P.sub.2, and PI(3,4,5)P.sub.3 respectively. FIG. 1
shows an overview of these phosphoinositide metabolic pathways.
PI(4,5)P.sub.2 is a minor component of the plasma membrane's inner
leaflet, and is part of a second messenger system that transduces
many hormone signals. When not effected by PI 3-K, the
PI(4,5)P.sub.2 pathway includes a receptor with seven transmembrane
segments, a heterotrimeric G-protein, and a specific protein kinase
phopholipase C (PLC). Ligand binding to the receptor activates the
G protein, G.sub.q, whose membrane-anchored a subunit in complex
with GTP diffuses laterally along the plasma membrane to activate
the membrane-bound PLC. As shown in FIG. 1, the activated PLC
catalyzes the hydrolysis of PI(4,5)P.sub.2 at its glycero-phospho
bond, yielding inositol-1,4,5-trisphosphate (Ins(1,4,5)P.sub.3 and
diacylglycerol (DG).
[0005] PI 3-kinase can be activated by tyrosine kinase receptors in
response to growth factor stimulation. As discussed above, PI
3-kinase is then involved in catalyzing the formation of
PI(3,4,5)P.sub.3 via phosphorylation of its substrate
(PI(4,5)P.sub.2. By increasing cellular levels of PI(3,4,5)P.sub.3,
PI 3-K induces the formation of defined molecular complexes that
act in signal transduction pathways. Notably, PI 3-K activity
suppresses apoptosis and promotes cell survival through activation
of its downstream target, PKB/Akt. PI(3,4,5)P.sub.3 signaling is
regulated by its formation and by its conversion into
PI(4,5)P.sub.2. The lipid phosphatases PTEN and SHIP are two
enzymes that both act to decrease the cellular levels of
PI(3,4,5)P.sub.3 by conversion either to PI(4,5)P.sub.2 or
PI(3,4)P.sub.2.
[0006] There is considerable evidence that the activity of PI 3-K
and the regulation of the level of its lipid products, in
particular PI(3,4,5)P.sub.3, is often defective in tumorigenesis,
as reported in D. Roymans et al., Phosphatidylinositol 3-kinases in
Tumor Progression, 268 Eur. J. Biochem. 487 (2001). PI 3-K activity
and elevated PI(3,4,5)P.sub.3 levels appear to contribute to cancer
progression via constitutive activation of PKB/Akt, as reported in
T. Franke et al., PI3K: Downstream AKTion Blocks Apoptosis, 88 Cell
437 (1997). Activated PKB/Akt provides a cell survival signal that
blocks apoptosis and promotes survival following growth factor
withdrawal or detachment from the cellular matrix. W. Phillips et
al., Increased Levels of Phosphatidylinositol 3-kinase Activity in
Colorectal Tumors, 83 Cancer 41 (1998) establish findings that
elevated PI 3-K levels have been observed in some cancers. Further,
experiments have indicated that cellular transformation is PI 3-K
dependent. D. Roymans et al., 268 Eur. J. Biochem. 487, A. Klippel
et al., Activation of Phosphatid linositol 3-kinase is Sufficient
for Cell Cycle Entry and Promotes Cellular Changes Characteristic
of Oncogenic Transformation, 18 Mol. Cell Biol. 5699 (1998). The
gene encoding the catalytic subunit of PI 3-K, PIK3CA, is an
oncogene which is amplified in ovarian and cervical cancers. In
addition, mutations that affect the regulation of PI 3-K signaling
also contribute to tumorigenesis. PTEN is a tumor suppressor that
is deleted or mutated in many cancer types. By converting
PI(3,4,5)P.sub.3 to PI(4,5)P.sub.2, PTEN acts as a negative
regulator of PKB/Akt activation by PI 3-K. Loss of PTEN activity
results in abnormal activation of PKB/Akt and suppression of
apoptosis. The lipid phosphatase SHIP also acts as a negative
regulator PKB/Akt activity. Ablation of SHIP in transgenic mice
leads to chronic hyperplasia, and loss of SHIP activity is one
characteristic of chronic myelogenous leukemia, providing
additional evidence linking the loss of regulation of
PI(3,4,5)P.sub.3 levels with an abnormal proliferative state.
[0007] From the above discussion, it is clear that there is a need
in the art for assaying methods and assay kits for measurement of
either PI 3-K activity or presence of phosphoinositide products of
PI 3-K activity in tissues, as such have the potential to become
powerful molecular diagnostic tools. In addition, it is clear that
there is a need in the art for assay platforms that are developed
for measurement of P13-K activity in clinical samples, and for use
in vitro assaying methods for novel PI 3-K inhibitors.
SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to meet the
above-described needs and others. Specifically, it is an object of
the present invention to provide a lipid assay method. The method
includes the step of first exposing a protein, having a lipid
recognition motif that interacts with a target lipid and a
competing lipid, to a solution containing the competing lipid. The
method further includes the step of determining whether the target
lipid is present in the solution. According to the method, the
target lipid has a stronger affinity to the lipid recognition motif
than does the competing lipid.
[0009] According to one embodiment of the invention, the protein is
Grp1, and the lipid recognition motif is a pleckstrin homology (PH)
domain. Consequently, the target lipid is a phosphoinositide in
this embodiment. In such a case, the Grp1 protein preferably
contains a glutathionine-S-transferase fusion with the PH
domain.
[0010] The assay determines activity of a lipid kinase, the target
lipid being a phosphorylation product of a reaction between the
lipid kinase and a substrate lipid. According to one embodiment of
the invention, the lipid kinase is PI 3-kinase, and the target
lipid is PI(3,4,5)P.sub.3. In a most preferred embodiment of the
invention, the lipid assay is a cancer screening method for
detection of cancer cells, and detection of certain levels of the
PI(3,4,5)P.sub.3 target lipid is an indicator of a cancer cell.
[0011] The assay can be any of a number of assay types, but is
preferably a plate-based assay. Examples include an enzyme linked
immunosorbent assay (ELISA), an amplified luminescence proximity
homogenous assay (ALPHA), and a fluorogenic assay.
[0012] In the embodiment where the assay is an ELISA assay, prior
to exposing the protein having a lipid recognition motif to a
target lipid and a competing lipid, a substrate of the assay plate
can be coated with the competing lipid. Preferably, the coating
step includes coating a streptavidin-coated substrate with the
competing lipid.
[0013] Additional competing and noncompeting lipids can also be
present in the solution, enabling the assay method of the present
invention to be used with complex solutions including bodily
tissues, fluids, and plasma.
[0014] The present invention is further directed to a lipid assay
kit, which includes a target lipid, a competing lipid, and a
protein that has a lipid recognition motif that interacts with the
target lipid and the competing lipid. The target lipid has a
stronger affinity to the lipid recognition motif than the competing
lipid.
[0015] The assay kit can further include a multi-well assay plate.
The multi-well assay plate preferably includes the competing lipid
immobilized in wells of the multi-well assay plate.
[0016] The lipid assay kit can further include primary and
secondary antibodies. As mentioned with respect to the assay
method, additional competing and noncompeting lipids can also be
present in the solution, enabling the assay method of the present
invention to be used with complex solutions including bodily
tissues, fluids, and plasma.
[0017] According to one embodiment of the invention, the protein is
Grp1, and the lipid recognition motif is a pleckstrin homology (PH)
domain. Clearly, however, the invention is easily adaptable to the
use of other lipid receptor proteins or monoclonal antibodies. For
example, various antibodies and lipid receptor proteins that are
specific for PI(3,4,5)P.sub.3 or PI(3)P can be used in accordance
with the invention. The protein preferably contains an affinity tag
fusion with the lipid recognition motif. Examples of affinity tags
include glutathionine-S-transferase, myc, or FLAG, etc. fused with
a lipid recognition motif such as the 1 PH domain.
[0018] The target lipid is preferably a phosphoinositide. The assay
kit is most preferably used for determination of activity of a
lipid kinase. Thus, the target lipid would be a phosphorylation
product of a reaction between the lipid kinase and a substrate
lipid. The lipid kinase is preferably a PI 3-kinase, and the target
lipid is preferably PI(3,4,5)P.sub.3. The lipid assay is most
preferably a cancer screening method for detection of cancer cells,
and detection of a predetermined level of the PI(3,4,5)P.sub.3
target lipid is an indicator of a cancer cell.
[0019] The assay can be any of a number of assay types, but is
preferably a plate-based assay. Examples include an enzyme linked
immunosorbent assay (ELISA), an amplified luminescence proximity
homogenous assay (ALPHA), and a fluorogenic assay.
[0020] In the embodiment where the assay is an ELISA assay, prior
to exposing the protein having a lipid recognition motif to a
target lipid and a competing lipid, a substrate of the assay plate
can be coated with the competing lipid. Preferably, the coating
step includes coating a streptavidin-coated substrate with the
competing lipid. Most preferably, the protein, target protein, and
competing protein are not radiolabeled reagents.
[0021] The present invention is also directed to a multi-well plate
for a lipid assay, which includes a lipid that is immobilized in
wells of the multi-well assay plate. Another aspect of the
invention is a method of making such a multi-well plate. The lipid
can be immobilized in wells of the multi-well assay plate via, for
example, a streptavidin-coating on a substrate of the wells. The
multi-well plate is preferably used in an assay for determining
activity of a lipid kinase. Most preferably, the lipid is a
non-radiolabeled derivative of PI(3,4,5)P.sub.3 such as, for
example, biotinylated-PI(3,4,5)P.sub.3.
[0022] Additional objects, advantages and novel features of the
invention will be set forth in the description which follows or may
be learned by those skilled in the art through reading these
materials or practicing the invention. The objects and advantages
of the invention may be achieved through the means recited in the
attached claims.
[0023] To achieve these stated and other objects, the present
invention may be embodied and described as set forth in the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The accompanying drawings illustrate the present invention
and are a part of the specification. Together with the following
description, the drawings demonstrate and explain the principles of
the present invention.
[0025] FIG. 1 shows a diagram of phosphoinositide interconversions,
including the phosphorylation of PI(4,5)P.sub.2 to PI(3,4,5)P.sub.3
using PI 3-K.
[0026] FIG. 2 shows a graphical representation of results of
absorbance measurements representing the binding of the LRP
detector protein to increasing amounts of immobilized
PI(3,4,5)P.sub.3 in an ELISA assay.
[0027] FIG. 3 shows a graphical representation of the results of a
PI(4,5)P.sub.2:PI(3,4,5)P.sub.3 competition ELISA assay.
[0028] FIG. 4 shows a graphical representation of an ELISA
quantitation of substrate conversion to PI(3,4,5)P.sub.3 and the
effect of the addition of inhibitor.
[0029] FIG. 5 shows a graphical representation of the results of a
competition ELISA assay between PI(4,5)P.sub.2 and PI(3,4,5)P.sub.3
for LRP binding.
[0030] FIG. 6 shows a graphical representation of the results of in
vitro detection of PI 3-kinase activity in an ELISA assay.
[0031] FIG. 7 shows a graphical representation of a
PI(3,4,5)P.sub.3 ALPHA binding assay.
[0032] FIG. 8 shows a graphical representation of a
PI(3,4,5)P.sub.3 ALPHA competition assay.
[0033] FIG. 9 shows a graphical representation of the ALPHA
competition profiles of increasing PI(3,4,5)P.sub.3 in a background
of PI(4,5)P.sub.2, and of a standard curve of increasing
PI(3,4,5)P.sub.3 competitor alone.
[0034] FIG. 10 shows a graphical representation of the results of
an ALPHA test of kinase activity using purified, recombinant PI
3-K.alpha. enzyme.
[0035] FIG. 11 shows a graphical representation of the results of
an ALPHA test of kinase activity in vitro, in the presence of
inhibitor.
DETAILED DESCRIPTION
[0036] Using the drawings, the preferred embodiments of the present
invention will now be explained. The present invention is directed
to the use of lipid recognition proteins (LRP's) as detectors of
target PIP.sub.ns, in a convenient assay platform system that can
be readily used in the industry.
[0037] LRP's that are used in accordance with the present invention
can be recombinant proteins expressed as fusions of lipid
recognition domains that are present in cellular proteins. The
domains of the cellular proteins that interact with a lipid are
fused to an affinity tag, such as glutathione-S-transferase (GST),
myc, or FLAG, for example. The proteins having such domains are
typically involved with such cellular functions as phosphorylation
of lipids, or are adaptor proteins that assist in forming complexes
with the cellular membrane to allow the cell membrane to interact
with lipid structures. The domains from these proteins that allow
the proteins to perform such functions can be extracted from the
naturally occurring proteins, or prepared through recombinant
methods, and then fused to GST to form an LRP.
[0038] For example, a group of proteins possess a certain type of
pleckstrin homology (PH) domain which interacts specifically with
the second messenger PI(3,4,5)P.sub.3. Examples of these proteins
include serine/threonine-specific protein kinases, PKB and PDK1,
Bruton's tyrosine kinase BTK, the adaptor proteins DAPP1 and Bab1,
as well as the ADP Ribosylation Factor (ARF), GTPase activating
protein (GAP) centaurin-.alpha., and the ARF guanine nucleotide
exchange factor Grp1.
[0039] The PH domain is well known to those skilled in the art.
Furthermore, different PH domains often exhibit specificity for
different phosphoinosides. See Dowler et al., 2000 Biochem J.
351:19-31, which is incorporated herein by reference. Such domains
are typically of .about.100 residues, and are found in over 70
other known proteins. All PH domains are predicted to fold into a
similar 3-dimensional structure, and may mediate protein-lipid
interactions, protein-protein interactions, or both. Polypeptides
with PH domains of determined tertiary structure include plecktrin,
spectrin, dynamin, and phospholipase C-.gamma.. The percentage of
amino acid identity is poor between PH domains in general. However,
there are certain positions that show high levels of residue type
conservation. The residues thought to be required for high affinity
interaction with PI(3,4,5)P.sub.3 lie in the PI(3,4,5)P.sub.3
Binding Motif (PPBM) near the N-terminal end of the PH domain. A
single position near the C-terminal end of the PH domain shows
complete identity throughout the domain family. Secondary structure
predictions indicate that residues 450-530 of PDK1, for example,
(positions 1-80) are likely to contain regions of .beta.-sheet,
while the residues between 531-550 (positions 80-100) are likely to
form an extended .alpha.-helix, a prediction that is consistent
with the known structures of other PH domains.
[0040] The molecular basis by which certain PH domains are able to
interact with PI(3,4,5)P.sub.3 has not been established
definitively. However, reports have indicated that six conserved
residues that lie at the N-terminal region of the PH domain in a
K--X--Sm--X.sub.6-11--R/K--X--R-Hyd-Hyd motif (where X is any amino
acid, Sm is a small amino acid and Hyd is a hydrophobic amino
acid), appear to correlate with high affinity binding of
PI(3,4,5)P.sub.3. In fact, most if not all specific
PI(3,4,5)P.sub.3 binding proteins identified possess this PPBM. The
term PPBM is also known to those skilled in the art. The term
"small amino acid" includes glycine, alanine, threoninie, and
serine. An aspartate or proline amino acid residue may
alternatively be present at the position in the motif where a small
amino acid is preferred. The term "hydrophobic amino acid" includes
tyrosine, leucine, isoleucine, tryptophan and phenylalanine. A
glutamine amino acid residue may alternatively be present at the
first position where a hydrophobic amino acid residue is preferred.
A glutamine, asparagine or histidine amino acid residue may be
present at a position where a lysine or arginine residue is
preferred. It is strongly preferred that an acidic or hydrophobic
residue is not present at a position where a lysine or arginine
residue is preferred, or at the position in the motif where a small
amino acid is preferred. It is preferred that the PH domain has at
least five of the six specified residues of the PPBM. It is
somewhat preferred that the PH domain has both hydrophobic amino
acids of the motif and/or the first lysine (K) residue of the
motif. It is preferred that the PH domain also has a tryptophan
residue at the position equivalent to position 280 of TAPP1.
[0041] In a broad sense, the present invention involves methods,
assay kits and apparatuses that use a specific LRP as a detection
reagent for specific lipids in an enzyme assay for lipid
metabolism. More particularly, the present invention involves the
use of Grp1 as a cellular protein as a probe that interacts
specifically with specific lipids in such assays. For example, the
Grp1 protein includes a PH domain. While PH domains vary in the
specificity of their interactions with various PIP.sub.ns, the Grp1
PH domain exhibits a strong preference for interaction with
PI(3,4,5)P.sub.3. Thus, in a narrow sense, the present invention
involves methods, assay kits and apparatuses that use a recombinant
LRP containing a GST fusion with the Grp1 PH domain as a lipid
detection probe.
Enzyme Linked Immunosorbent Assay (ELISA)
[0042] In order to readily distinguish PI(3,4,5)P.sub.3 from other
PIPs, a derivative such as biotinylated diC.sub.6 PI(3,4,5)P.sub.3
is immobilized in the wells of streptavidin-coated assay
plates.
[0043] As an example, a 96-well plate can be used, although the
present embodiment of the invention is clearly adaptable for a
variety of assay plate sizes and formats. Initial experiments
establish a range of LRP detector protein and biotinylated
PI(3,4,5)P.sub.3 where detection of the target phosphoinositide is
optimized in an assay tray format. For example, in a standard curve
binding procedure, a 96-well streptavin-coated assay plate marketed
as StreptaWell.RTM. (Roche) is coated with increasing amounts of
biotinylated PI(3,4,5)P.sub.3 per well. The coated wells are then
blocked for an hour at room temperature using 100 .mu.l of
Stabilguard.RTM. (SurMedics) per well. The samples are then
incubated with 10 pmol of LRP in a 100 .mu.l volume per well.
Several washes are then performed. Next, 100 .mu.l of a 1:1000
dilution of an anti-GST HRP-conjugated antibody, provided by Sigma,
is added to each well. The GST HRP-conjugated antibody is provided
as a reagent that interacts with the GST portion of the lipid
recognition protein, and hence allows for subsequent calorimetric
detection. After one hour of incubation at room temperature, the
plates are washed and 100 .mu.l of 3', 3', 5',
5'-tetramethylbenzidine (TMB) substrate solution as a development
reagent, obtainable from Sigma, is added to each well.
[0044] Following color development, the reaction is stopped by the
addition of 100 .mu.l 0.5 M H.sub.2SO.sub.4. Absorbance at 450 nm
is measured. The results of the absorbance measurements
representing the binding of the LRP detector protein to increasing
amounts of immobilized PI(3,4,5)P.sub.3 are shown in FIG. 2. As
shown, the absorbance (450 nm) of samples having 10-fold
incremental pmol amounts of biotinylated PI(3,4,5)P.sub.3 are
compared with a control, a sample containing no LRP detector
protein, and a sample containing no biotinylated
PI(3,4,5)P.sub.3.
[0045] From the above findings, a competition procedure can be
performed using similar methodology. Assay plates such as the
StreptaWell.RTM. microtiter plates used in the previously discussed
optimization process are prepared by coating the wells with 10 pmol
of biotinylated PI(3,4,5)P.sub.3 per well. In a separate incubation
apparatus, 10 pmol of LRP is preincubated with increasing amounts
of closely related derivative PIPs. For example, the LRP is
preincubated with either diC.sub.8 PI(3,4,5)P.sub.3 or diC.sub.8
PI(3,4,5)P.sub.2, prior to binding to the biotinylated
PI(3,4,5)P.sub.3 coated surface of the assay plate wells.
[0046] The results of the competition procedure can be determined
by, for example, measuring absorbance (450 nm) for each of
PI(4,5)P.sub.2 and PI(3,4,5)P.sub.3 at various pmol increments of
competing PIP. Results of an example procedure are shown in FIG. 3.
As shown in the graph of FIG. 3, PI(4,5)P.sub.2 is able to compete
weakly for binding, but PI(3,4,5)P.sub.3 is a stronger competitor.
The difference in competitiveness is particularly evident at lower
levels of PIP, and the difference clearly exemplifies the ease of
which the assay can distinguish between the two
phosphoinositides.
[0047] Results from a second example further demonstrate the
ability of the ELISA format for detection of PI 3-K activity.
Recombinant PI 3-K .alpha. enzyme is incubated with 70 pmoles
diC.sub.8 PI(4,5)P.sub.2 substrate for one hour. Enzyme activity is
stopped and the reaction mixtures are pre-incubated with LRP, then
tested in a competition binding assay, in the above example. FIG. 4
shows quantitation of substrate conversion to PI(3,4,5)P.sub.3 and
the effect of the addition of 50 .mu.M LY29004, a compound which
acts as a PI 3-K inhibitor.
[0048] A standard curve in which increasing amounts of
PI(3,4,5)P.sub.3 competitor is added to the assay is run alongside
the enzyme reactions. The degree of PI(4,5)P.sub.2 conversion to
PI(3,4,5)P.sub.3 is then estimated by comparing the values obtained
for the reaction mixtures in the competition ELISA. After a one
hour incubation with the enzyme, about 50 pmoles of
PI(3,4,5)P.sub.3 are generated, indicating that most of the
PI(4,5)P.sub.2 substrate has been converted. The presence of
LY29004 blocks the enzyme activity to the extent that only a few
picomoles of PI(4,5)P.sub.2 is converted to PI(3,4,5)P.sub.3. These
results confirm the suitability of the ELISA format for
determination of PI 3-K activity. These results also exhibit the
suitability of this approach in an ELISA-based PI 3-K assay kit
which is of use to individual researchers, and overcomes the
shortcomings of the radioactive labeling and separation methods
which are used in the related art.
[0049] Additional tests reveal that the assay of the present
invention can be used to distinguish mixtures of lipids containing
different ratios of PI(4,5)P.sub.2 and PI(3,4,5)P.sub.3. For
example, a competition experiment involves the preincubation of 10
pmol of LRP with 10 pmol of total lipid containing different
mixtures of PI(4,5)P.sub.2 and PI(3,4,5)P.sub.3 prior to binding
these compounds to biotinylated PI(3,4,5)P.sub.3 coated plates. The
ability of the detector protein to distinguish between differences
in the amount of PI(3,4,5)P.sub.3 that is present can be determined
by measuring absorbance (450 nm) for each of numerous samples. As
shown in FIG. 4, some of the competition mixture samples included
percentages of PI(3,4,5)P.sub.3:PI(4,5)P2 of 0:100, 10:90, 20:80,
30:70, 40:60, 50:50, 100:0, and samples that omitted competing
PIP.sub.n lipids, LRPs, biotinylated PI(3,4,5)P.sub.3, and
horseradish peroxidase (HRP), conjugated secondary antibody. The
results in FIG. 4 show that the ability of the LRP detector protein
to distinguish between differences in the amount of
PI(3,4,5)P.sub.3 that is present appears to be more sensitive when
PI(3,4,5)P.sub.3 is present at less than 50% of the lipid
mixture.
[0050] Most experiments testing for lipid kinase activity exemplify
actual cellular conditions in vivo where the level of substrate
conversion is approximately 5 to 15%. Because the ability of the
LRP detector protein to distinguish between differences in the
amount of PI(3,4,5)P.sub.3 that is present appears to be more
sensitive when PI(3,4,5)P.sub.3 is present at less than 50% of the
lipid mixture, the assay of the present invention is well suited
for detecting differences in the range for which tests are commonly
performed. Further, the discovery of the LRP's ability to
distinguish between various phosphoinisotides at various ratios
exhibits the ability of the assay to be detect PI 3-K activity in
vitro, as next discussed.
In vitro ELISA Assay
[0051] To perform an assay for detection of PI 3-K activity in
vitro, cell lysate containing active recombinant PI 3-K is mixed
with 20 .mu.g diC.sub.8 PI(4,5)P.sub.2 in a reaction buffer
containing 2.5 mM MgCl.sub.2, 5 mM HEPES, pH 7.0, and 25 .mu.M ATP.
The PI 3-K inhibitor wortmannin (200 nM) is added to some reactions
for purposes of comparison with reactions not containing inhibitor.
Reaction mixes are incubated at room temperature for 1 hour. Lipids
are extracted from the reaction mixture, dried down, and
re-suspended in water. The re-suspended samples are incubated with
10 pmol LRP and tested in the competitive ELISA. Results of this
example are shown in FIG. 5. As shown, absorbance (450 nm) is
remarkably lower after one hour of incubation for samples without
PI 3-K inhibitor. Thus, an increase in competition for LRP binding
is seen after the PI(4,5)P.sub.2 incubation with the enzyme.
Competition is decreased in samples containing wortmannin,
presumably due to inhibition of kinase activity and less conversion
of PI(4,5)P.sub.2 to PI(3,4,5)P.sub.3. These results establish the
feasibility of using LRPs as detection reagents in vitro for
determination of PI 3-K activity as modulated by kinase
inhibitors.
[0052] The above embodiments of the present invention demonstrate a
novel plate-based assay such as an ELISA assay for detection of
PI(3,4,5)P.sub.3 using a recombinant LRP, GST-Grp1-PH as a
detection device. The novelty of the LRP specificity for
PI(3,4,5)P.sub.3 in a competition ELISA is also demonstrated, as
well as the detection of PI 3-K activity in vitro, and the
inhibition of kinase activity by the inhibitor wortmannin using
this approach. Of course, a variety of kinase inhibitors and
potential inhibitors can be applied to the in vitro assay.
Amplified Luminescence Proximity Homogeneous Assay (ALPHA) Using
LRP Detector Protein
[0053] The principles of the present invention can also be applied
to other assay methods, such as the type using AlphaScreen.RTM.
reagents and the Fusion Alpha Universal Microplate Analyzer from
Packard Bioscience.RTM.. The AlphaScreen.RTM. system detects
emission shifts due to reactions involving oxygen singlets. More
particularly, the system uses photosensitive donor beads which
convert ambient oxygen to a singlet state upon illumination at 680
nm. If an acceptor bead is in close proximity to the donor bead,
due to a biological interaction, the diffusion of singlet oxygen
activates chemiluminscent receptors and fluorescent acceptor
molecules on the bead, resulting in an emission shift from 520 to
620 nm.
[0054] According to the principles of the present invention,
streptavidin-coated donor beads and acceptor beads coated with
anti-GST are incubated with biotinylated PI(3,4,5)P.sub.3 and the
LRP detector protein before analysis. In order to establish the
lipid to protein ratio that gives the best results, a standard
curve PI(3,4,5)P.sub.3 binding experiment is first performed.
[0055] As an example of the binding experiment, one pmol of LRP is
incubated with increasing amounts of biotinylated PI(3,4,5)P.sub.3
in a 25 .mu.l reaction volume. Streptavidin-coated donor beads and
anti-GST antibody acceptor beads (5 .mu.l each, 20 .mu.g/ml final
concentration) are then added to the reaction mix. Following a 2
hour incubation, luminescence is measured. The measurement can be
made using the AlphaScreen.RTM. mode on a Fusion Universal
Microplate Analyzer (Packard Bioscience). Results are shown in FIG.
6, with luminescence measured according to increasing pmol amounts
of biotinylated PI(3,4,5)P.sub.3 added to the LRP. As shown in FIG.
6, an increase in luminescence with the addition of increasing
amounts of biotinylated PI(3,4,5)P.sub.3 to the reaction is
observed.
[0056] A competition assay is then performed, the results of which
are shown in FIG. 7. In the competition assay, the LRP is
preincubated with increasing pmol amounts of PI(3,4,5)P.sub.3. As
an example, LRP (1 pmol) is preincubated with increasing amounts of
competing PI(3,4,5)P.sub.3 for 1.5 hours. The LRP is then added to
1 pmol biotinylated PI(3,4,5)P.sub.3, and 5 .mu.l donor and
acceptor beads. Luminescence is measured following an additional 2
hours of incubation. As shown in FIG. 8, luminescence decreases as
the interaction of biotinylated PI(3,4,5)P.sub.3 on the donor beads
and LRP on the acceptor beads is competitively displaced by
unlabeled PI(3,4,5)P.sub.3.
[0057] As another example, using an ALPHA assay technique the
luminescent assay for detection of changes in PI(3,4,5)P.sub.3
levels in a background of PI(4,5)P.sub.2 is tested to mimic the
substrate conversion which occurs in an enzymatic assay. The
results of this test reveal that increasing PI(3,4,5)P.sub.3 in a
background of PI(4,5)P.sub.2 yields a competition profile which is
virtually identical to that created by a standard curve of
increasing PI(3,4,5)P.sub.3 competitor alone, as shown in FIG. 9.
Thus, comparison of values obtained from enzymatic reactions can be
compared to a standard curve of increasing PI(3,4,5)P.sub.3 to
allow determination of the level of substrate conversion which has
occurred. To allow for greater ease of performing this analysis,
the data is converted to a logarithmic scale.
[0058] Another example tests the ability and sensitivity of the
ALPHA assay for detection of PI 3-K activity. FIG. 10 shows the
results of a test of kinase activity using purified, recombinant PI
3-K.alpha. enzyme. 10 pmols of PI(4,5)P.sub.2 substrate is
incubated with varying amounts of PI 3-K.alpha. in the presence and
absence of 50 .mu.M LY29004 for one hour. The resulting lipid
mixtures are used to compete for the interaction of LRP coated
donor beads with PI(3,4,5)P.sub.3 coated acceptor beads in the
ALPHA assay. Conversion of PI(4,5)P.sub.2 to PI(3,4,5)P.sub.3 is
evident in the increased competition and decreased luminescent
signal in samples containing PI 3-K.alpha.. The addition of ten
times more enzyme results in almost complete substrate conversion
to PI(3,4,5)P.sub.3. The activity of the enzyme is inhibited at
least ten-fold by the presence of LY29004.
[0059] The results from a similar experiment are shown in FIG. 11.
In this case, NIH 3T3 cells are used as the source of PI 3-K. Cells
are stimulated with AMOUNT insulin to activate PI 3-K. Cells are
then lysed, and PI 3-K is recovered from the cell lysate by
immunoprecipitation using a polyclonal antibody against the p85
subunit. Aliquots of immunoprecipitated enzyme are incubated with
10 pmoles PI(4,5)P.sub.2 substrate for one hour, with 50 mM LY29004
or AMOUNT wortmannin added to some samples. The resulting lipid
mixtures are used to compete for the interaction of LRP coated
donor beads with PI(3,4,5)P.sub.3 coated acceptor beads in the
ALPHA assay. In the absence of insulin stimulation, there is no
measurable enzyme activity, while enzyme isolated from
insulin-stimulated cells is able to convert most of the substrate
to PI(3,4,5)P.sub.3. The addition of LY29004 or wortmannin inhibits
enzyme activity, considerably reducing the amount of substrate
conversion.
[0060] These results validate the ALPHA assay as a sensitive
measure of PI 3-K activity using a LRP as a detection reagent. We
have demonstrated that our assay is suitable for both in vitro
tests of enzyme activity and for determining the relative activity
of PI 3-K isolated directly from cells.
[0061] The ALPHA assay format is an example of yet another assay
method that can be used in accordance with the principles of the
present invention. The ALPHA format has the advantages of high
specificity and sensitivity, and requires significantly less
protein and lipid reagents than the ELISA assay format.
[0062] Thus, the ALPHA assay can be performed using LRP-coated
acceptor beads and PI(3,4,5)P.sub.3-coated donor beads, and in
competition experiments in which free PI(3,4,5)P.sub.3 is shown to
compete for interaction of the donor and acceptor beads. Using the
principles set forth regarding ELISA assays, the ALPHA assay can be
used to perform competition assays involving different amounts of
PI(3,4,5)P.sub.3 and other PIP.sub.nS to establish a degree of
specificity of PI(3,4,5)P.sub.3 as a competitor, and to perform in
vitro assays and screening panels of PI 3-K inhibitors.
Fluorogenic Assay Using LRPs
[0063] Further, the principles of the present invention can be
readily applied to other assay formats for detection of lipids or
associated enzyme activity using the LRP as a detection device. For
example, a fluorogenic assay method would be suited for using an
LRP as a detection reagent. Like the luminescence assay described
above using the ALPHA format, a fluorogenic assay has the advantage
of being easy to perform, with no requirement for washing steps,
and minimal mixing and detection steps, which makes these assay
formats ideal for adaptation to high throughput (HTS) systems.
[0064] The components involved in a fluorogenic assay method
include a florophoreconjugated PI(3,4,5)P.sub.3, and a
quencher-labeled LRP. Interaction between the LRP and the labeled
PI(3,4,5)P.sub.3 results in fluorescence quenching, while reduced
binding in the presence of competitor results in the restoration of
a fluorescent signal. As examples of a fluorescent label for
PI(3,4,5)P.sub.3, BODIPY.TM. or FITC.TM., both produced by Echelon
Research Laboratories, Inc. First, working concentrations of the
labeled PI(3,4,5)P.sub.3 suitable for detection are determined by
making serial dilutions in, for example, a microtiter plate.
Fluorescence is then measured using a plate reader such as a Gemini
Fluorescence.TM. plate reader made by Molecular Devices. Tests
using a BODIPY-FL-PI(3,4,5)P.sub.3 indicate that a range of 1 to 20
pmol is sufficient for good detection. The LRP can be conjugated
to, for example, Dabcyl-SE
[4-((4-(dimethylamino)-phenyl)azo)benzoic acid, succinimidyl
ester], a non-fluorescent molecule that absorbs in the range at
which FITC and BODIPY fluoresce, and is produced by Molecular
Probes. Dabcyl-SE is an amine-reactive probe for conjugation to
proteins. QSY-7, a higher efficiency quencher, may be used as
well.
[0065] Once ideal parameters for lipid and protein labeling are
established, and the amounts suitable for use in this assay are
determined, performing the assay is simply performed as follows.
The quencher-LRP is preincubated with a mixture of unlabeled
competitor lipids for approximately 1 hour. Then this mixture is
added to the fluorescent-PI(3,4,5)P.sub.3 in the wells of, for
example, a 96-well microtiter plate, although a variety of plate
sizes may be incorporated into the method. After 1-2 h of
incubation, fluorescence is measured using a plate reader. The
ability of the Dabcyl-LRP to interact with
BODIPY-FL-PI(3,4,5)P.sub.3 reveals a decrease in fluorescent signal
upon addition of increasing amounts of quencher-labeled protein.
Furthermore, the addition of excess unlabeled PI(3,4,5)P.sub.3
results in an increase in fluorescence, as LRP binding to the
BODIPY-PI(3,4,5)P.sub.3 is competitively displaced.
[0066] Results from this fluorogenic essay method reveal that the
assay is simple and rapid to perform, and ideal for adaptation to
high throughput screening applications. Consequently, this type of
assay is particularly advantageous to the drug discovery industry,
for example.
Automation of the Assays
[0067] In addition to the above-described advantages of the assay
methods of the present invention, the non-radioactive assay of
using an LRP as a lipid detection reagent for assaying enzyme
activity readily lends itself to automation. Assay platforms used
in the non-radioactive assay can be used, for example, with an
automatic analyzer such as the Fusion Universal Microplate Analyzer
by Packard Bioscience. Such a device is easily integrated with
automated systems for plate stacking, liquid handling and
cell-based assays. Other automation can be applied to the process
in order to increase the assay process rate, including a plate
washer and harvester and plate scintillation counter, such as
Orca/Biomek.RTM. instrumentation.
Detection of PI 3-K Activity in Biological Samples
[0068] The determination of PI 3-K activity and PI(3,4,5)P.sub.3
levels in biological samples can also be determined using the
plate-based assays described above. Although the mixture of lipids
extracted from cells or tissues is complex, the principles of the
present invention are adaptable to tissue or cellular extractions.
However, because endogenous lipids are poorly soluble in aqueous
solution, lipid extraction should be followed by rehydration and
formation of micelles when necessary. Techniques for such
extraction, rehydration, and micelle formation are disclosed by I.
M. Bird, Analysis of Cellular Phosphoinositides and
Phosphoinositols by Extraction and Simple Analytical Procedures, 27
Methods Mol. Biol. 227 (1994).
[0069] The present inventors have determined for the first time
that increases in PI(3,4,5)P.sub.3 levels occur in cells following
growth factor stimulation and that PI(3,4,5)P.sub.3 levels are
elevated in some types of cancers. As mentioned above, a variety of
published reports link PI 3-K activity or loss of PTEN activity to
cancers. It is expected by the inventors that PI(3,4,5)P.sub.3
levels are likely to be elevated in at least cervical, prostate,
ovarian, lung, and colon cancer. Using lipids or PI 3-K extracted
from these and other types of biological samples, the plate-based
assays using LRPs are able to detect changes in PI 3-K activity in
cells and tissues. Immunodetection using anti-PI(3,4,5)P.sub.3
antibody can be performed concurrently to confirm increases in
cytoplasmic PI(3,4,5)P.sub.3. In addition, changes in
PI(3,4,5)P.sub.3 and PI(4,5)P.sub.2 ratios can be biochemically
verified using lipid extractions and fractionation through thin
layer chromatography.
[0070] Further, the principles of the present invention may be
applied in a clinical assay setting. A clinical assay can be
performed for PI 3-K activity that is suitable for analysis of
small, less invasive clinical samples, such as blood, pap smears,
and needle biopsies. As an example of this type of clinical
application of the plate-based assay using LRPs, known amounts of
PI(3,4,5)P.sub.3 are added to samples of blood or serum prior to
lipid isolation and analysis. Construction of a calibration curve
showing PI(3,4,5)P.sub.3 competition in the assay is then
performed, and a measure of assay sensitivity to changes in
PI(3,4,5)P.sub.3 in these types of samples is then determined.
Based on these determinations, the assay can be applied to samples
of cells or biological fluid for direct detection of lipid without
performing a lipid extraction.
Assay Kits and Apparatuses
[0071] Another embodiment of the present invention involves a rapid
plate-based assay kit for PI 3-K activity, including lipids,
plates, and detection reagents. Such kits will satisfy particular
needs from clinical or research laboratory scientists, for example.
A rapid plate-based assay apparatus can be used for HTS drug
discovery efforts in the pharmaceutical industry, for example.
PI 3-K Assay Kits
[0072] As discussed above, the assay formats discussed above are
partly designed to allow detection of PI 3-K activity in vitro and
for determining PI(3,4,5)P.sub.3 levels in cellular samples. A
scientist may be interested in determining whether PI 3-K is
activated in a tumor cell line or in response to treatment of a
cell with a particular stimulus. Kits for performing the assays of
the present invention allow in vitro determination of PI 3-K
activity on PI(4,5)P.sub.2 substrate using an enzyme that has been
isolated from experimental samples by immunoprecipitation.
ELISA-based assay kits designed for these applications can contain
immobilized PI(3,4,5)P.sub.3 in large plates such as 96-and
384-well plates, GST-LRP or antibody detection reagents, and the
appropriate primary and secondary antibodies. Synthetic PIP.sub.ns
are also supplied as controls and as substrates for in vitro kinase
assays.
[0073] A fluorogenic assay kit for use by laboratory research can
include Dabcyl-LRP conjugate, fluorescently labeled
PI(3,4,5)P.sub.3, and synthetic PIP.sub.ns as controls and
substrates. A benchtop PI(3,4,5)P.sub.3/PI 3-K detection kit using
these assay formats can also be provided for use in in vitro assays
of PI 3-K activity. In a clinical setting, the assay is designed
for accurate determination of PI(3,4,5)P.sub.3 levels in tissue,
blood, and serum samples. A kit designed for use in a clinical
setting can use either an ELISA or fluorogenic format, and would be
similar to that designed for use in a research lab.
[0074] Preparation of lipids from clinical samples may require
extraction and desiccation, then rehydration. Alternatively, direct
determination of lipids without extraction of the lipids can be
performed. Lipids isolated from PI(3,4,5)P.sub.3-enriched samples
and samples lacking PI(3,4,5)P.sub.3 can be included as controls,
along with synthetic PI(3,4,5)P.sub.3 and other PIP.sub.nS.
b) Apparatuses Involving a PI 3-K Assay Adapted to an HTS
Platform
[0075] PI 3-K is an important target for anti-cancer drug
development, and there is a need in the pharmaceutical industry for
new methods of screening for PI 3-K inhibitors. In-vitro assays of
the present invention are advantageous for the discovery of
potential drugs targeting PI 3-K. Present methods involve costly
and cumbersome radioactive extractions, while the present assay
formats provide a less expensive, simple, and non-radioactive
alternative.
[0076] The present assays for use in drug discovery are convertible
to a HTS format. The amplified luminescence homogeneous proximity
assay (ALPHA) and the fluorogenic assay formats are particularly
well-suited for HTS applications. Automation of assay platforms can
be performed using an automated liquid handling system interfaced
to a Fusion Universal Microplate Analyzer.TM. made by Packard
Bioscience. The system includes a multi-well pipettor/washer
integrated with a plate-handling robot for highly accurate and
simultaneous delivery of microvolumes of liquids in large plates
such as a 96- and 384-well format. Transfer to the analyzer, sample
analysis, and data collection is also be automated and computer
controlled.
[0077] It will be appreciated that the present invention is not
limited to any of the exact constructions that have been described
above and illustrated in the accompanying drawings, and that
various modifications and changes can be made without departing
from the scope and spirit thereof. It is intended that the scope of
the invention only be limited by the appended claims.
[0078] The preceding description has been presented only to
illustrate and describe the invention. It is not intended to be
exhaustive or to limit the invention to any precise form disclosed.
Many modifications and variations are possible in light of the
above teaching.
[0079] The preferred embodiment was chosen and described in order
to best explain the principles of the invention and its practical
application. The preceding description is intended to enable others
skilled in the art to best utilize the invention in various
embodiments and with various modifications as are suited to the
particular use contemplated. It is intended that the scope of the
invention be defined by the following claims.
* * * * *