U.S. patent application number 10/100515 was filed with the patent office on 2002-11-28 for anti-gpe antibodies, their uses, and analytical methods for gpe.
Invention is credited to Batchelor, David Charles, Breier, Bernhard Hermann Heinrich, Thomas, Gregory Brian.
Application Number | 20020177239 10/100515 |
Document ID | / |
Family ID | 23058100 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177239 |
Kind Code |
A1 |
Thomas, Gregory Brian ; et
al. |
November 28, 2002 |
Anti-GPE antibodies, their uses, and analytical methods for GPE
Abstract
GPE antibodies recognize GPE with high specificity. When used in
a radioimmunoassay, they reliably measure the concentration of GPE.
They may be used to extend the half-life of GPE both in vivo and in
vitro, and in methods of purifying the GPE receptor. Two methods of
rpHPLC accurately revolve GPE on the basis of its
hydrophobicity.
Inventors: |
Thomas, Gregory Brian;
(Auckland, NZ) ; Breier, Bernhard Hermann Heinrich;
(Auckland, NZ) ; Batchelor, David Charles;
(Auckland, NZ) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
23058100 |
Appl. No.: |
10/100515 |
Filed: |
March 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60276796 |
Mar 16, 2001 |
|
|
|
Current U.S.
Class: |
436/514 ;
530/388.26 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/44 20130101; C07K 16/18 20130101 |
Class at
Publication: |
436/514 ;
530/388.26 |
International
Class: |
G01N 033/53; C07K
016/40 |
Claims
We claim:
1. An anti-GPE antibody.
2. The antibody of claim 1 having the ability to specifically bind
to GPE at a final titer of 1:600 using .sup.125I-YGPE tracer.
3. The antibody of claim 1 having the ability to bind to GPE in
normal tissue.
4. The antibody of claim 1 having the ability to bind to GPE in
diseased or injured tissue of the central or peripheral nervous
system.
5. The antibody of claim 1 that is the CK5 antibody.
6. An antibody to Bolton and Hunter derivatized GPE.
7. An antibody to Bolton and Hunter derivatized GPE having the
ability to specifically bind to Bolton and Hunter derivatized GPE
at a final titer of 1:18,000 using Bolton and Hunter derivatized
.sup.125I-YGPE tracer.
8. Anti-GPE antiserum.
9. A reverse-phase HPLC method for the detection of GPE in a sample
of body fluid or tissue, comprising: (a) derivatizing the sample
with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, (b) eluting
the sample in a reverse phase HPLC system, and (c) detecting the
GPE by excitation at 250 nm and detection at 395 nm.
10. An rpHPLC assay kit for the practice of the method of claim 9
comprising a GPE standard, 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate and derivatizing buffer, a column, and column running
buffer.
11. A reverse-phase HPLC method for the detection of radioactive
GPE in a sample of body fluid or tissue, comprising: (a) eluting
the sample in a reverse phase HPLC system, and (b) detecting the
GPE by detection of the emitted radioactivity.
12. An rpHPLC assay kit for the practice of the method of claim 11,
comprising a radioactive GPE standard, a column, and column running
buffer.
13. A radioimmunoassay kit for the detection or quantitation of
GPE, comprising the antibody of claim 1, a GPE standard, an assay
buffer, a purified hormone for iodination and a second antibody or
a precipitated antibody.
14. An radioimmunoassay kit for the detection or quantitation of
GPE, comprising the antibody of claim 1, a GPE standard, an assay
buffer, tritiated GPE, and a second antibody or precipitating
antibody.
15. A radioimmunoassay kit for the detection or quantitation of GPE
comprising the antibody of claim 1, a GPE standard, Bolton and
Hunter reagent, a derivatizing buffer, an assay buffer, Bolton and
Hunter derivatized GPE for iodination, and a second antibody or
precipitating antibody.
16. A method of isolating or purifying the GPE receptor from a
sample, comprising: (a) treating the sample with GPE to form a
GPE-(GPE receptor) complex, (b) treating the sample with the
antibody of claim 1 conjugated to a photoactivatable binding group,
(c) photoactivating the binding group to form an antibody-GPE-(GPE
receptor) complex, (d) isolating the complex from the sample, and
(e) releasing the GPE receptor from the complex and isolating or
purifying it.
17. A method of extending the half-life of GPE, or of modulating
the free concentration of GPE, comprising co-administering GPE and
the antibody of claim 1.
18. The method of claim 17 comprising co-administering GPE and the
antibody of claim 1 in vivo.
19. The method of claim 17 comprising co-administering GPE and the
antibody of claim 1 to a mammal.
20. A method of extending the half-life of GPE, or of modulating
the free concentration of GPE, in a mammal, comprising
administering the antibody of claim 1 to the mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the priority under 35 USC 119(e) of
US Provisional Application No. 60/276,796, filed Mar. 16, 2001,
which is incorporated by reference into this application.
FIELD OF THE INVENTION
[0002] This invention relates to anti-GPE antibodies and their
uses, and to analytical methods for GPE.
BACKGROUND TO THE INVENTION
[0003] GPE is the tripeptide glycyl-L-prolyl-L-glutamic acid
(gly-pro-glu). It and the dipeptides glycyl-L-proline (gly-pro, GP)
and L-prolyl-L-glutamic acid (pro-glu, PE) were first disclosed in
EP 366638, which disclosed that GPE is effective as a
neuromodulator and is able to affect the electrical properties of
neurons.
[0004] The applicants have established that GPE has neuroprotective
properties and that it therefore has utility in the prevention or
inhibition of neuronal and glial cell death (WO 95/172904).
[0005] It would be desirable to have antibodies to GPE, both for an
accurate method for the detection of GPE, and for therapeutic uses.
It would also be desirable to have an accurate analytical method
for GPE.
SUMMARY OF THE INVENTION
[0006] In a first aspect, this invention is antibodies against GPE
("anti-GPE antibodies").
[0007] In a second aspect, this invention is radioimmunoassay
methods for the measurement of GPE using the anti-GPE antibodies of
the first aspect of this invention, and kits for the same.
[0008] In a third aspect, this invention is methods of reverse
phase high-performance liquid chromatography ("rpHPLC") that
accurately resolves GPE and related compounds, and kits for the
same.
[0009] In a fourth aspect, this invention is therapeutic methods
for using the anti-GPE antibodies of the first aspect of this
invention.
[0010] In a fifth aspect, this invention is a method of isolating
and purifying the GPE receptor using the anti-GPE antibodies of the
first aspect of this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a radioimmunoassay displacement curve showing
competitive displacement by unlabeled GPE of .sup.125I-labeled YGPE
binding to the CK5 antibody.
[0012] FIG. 2 is a radioimmunoassay displacement curve showing
competitive displacement by unlabeled Bolton and Hunter derivatized
GPE of .sup.125I-labeled Bolton and Hunter derivatized YGPE binding
to the CK5 antibody and lack of cross reactivity with the Bolton
and Hunter derivatized forms of glycine, proline, glutamate,
insulin-like growth factor-1 (IGF-1), gly-pro, and urea.
[0013] FIG. 3 compares the displacement of radio-labeled
.sup.125I-YGPE by GPE with the displacement of .sup.125I-Bolton and
Hunter derivatized GPE by Bolton and Hunter derivatized GPE.
[0014] FIG. 4 shows the measurement of GPE in plasma using the CK-5
antibody following intravenous administration of GPE at 3 mg/kg, 30
mg/kg and 100 mg/kg.
[0015] FIG. 5 is a rpHPLC chromatogram showing the resolution of
GPE in plasma following derivatization by AccQTag.RTM. reagent.
[0016] FIG. 6 is a rpHPLC chromatogram showing detection of GPE in
blood at various times following intravenous administration of 30
mg/kg GPE.
[0017] FIG. 7 is an rpHPLC chromatogram showing the resolution of
tritiated GPE in plasma.
DESCRIPTION OF THE INVENTION
[0018] In a first aspect, this invention is antibodies against GPE
("anti-GPE antibodies"). These anti-GPE antibodies may be prepared
by immunization of animals (e.g. rabbits) with immunogens
containing GPE conjugated to an antigen such as keyhole limpet
hemocyanin, as described in Examples 1, 2, and 6 below. The
polyclonal anti-GPE antibody, which we refer to as CK5 antibody,
specifically recognizes GPE and binds to GPE with high titer. In
preferred embodiments of the invention, the antibodies to GPE are
characterized by the ability to specifically bind to GPE using
.sup.125I-YGPE tracer with a final titer of at least about 1:600.
In embodiments of the invention, the anti-GPE antibodies have the
ability to bind specifically to GPE in normal tissues; or have the
ability to bind specifically to GPE in diseased or injured tissue.
In a most preferred embodiment of the invention, the anti-GPE
antibodies have the ability to bind GPE in diseased or injured
tissue of the central or peripheral nervous system. In another
embodiment of the invention, the anti-GPE antibodies have the
ability to specifically bind to Bolton and Hunter derivatized GPE
using .sup.125I-Bolton and Hunter derivatized GPE tracer with a
final titer of at least about 1:18,000.
[0019] Anti-GPE antibodies find use in determining the
pharmacokinetics and pharmacodynamics of GPE and GPE-related
compounds (GPE analogs); and in assays to determine the
neuroprotective concentration of GPE in blood and CSF required in
the treatment of a disease or in the treatment of injury. In
preferred embodiments of the invention, anti-GPE antibodies find
use in assays to determine the neuroprotective concentration of GPE
in blood and CSF required in the treatment of Parkinson's disease,
multiple sclerosis, Alzheimer's disease, Huntington's disease,
peripheral neuropathy, stroke, cardiac artery bypass graft surgery,
ischemic brain injury, hypoxic brain injury, traumatic brain
injury, and in the treatment of pancreatic disease including type 1
and type 2 diabetes. Further embodiments of the invention provide
methods for the use of anti-GPE antibodies in the in vitro
evaluation of GPE function. Such methods include evaluation of the
effects of in vitro administration of GPE in the presence and in
the absence of anti-GPE antibodies.
[0020] In a second aspect, this invention is a radioimmunoassay
method for the measurement of GPE using the anti-GPE antibodies of
the first aspect of this invention, as described in Example 3
below. The radioimmunoassay method allows for the selective
quantitation of GPE in body fluids (e.g. blood, serum,
cerebrospinal fluid, and urine) and in body tissues. The level of
GPE may be a suitable marker of drug efficacy and/or effective
dosing. In one embodiment of the invention, a radioimmunoassay kit
comprises an anti-GPE antibody, a GPE standard, an assay buffer, a
GPE compound (e.g. YGPE) for iodination, and a second antibody or a
precipitated antibody, for example an antibody precipitated with
polyethylene glycol (PEG). In another embodiment, the kit comprises
an anti-GPE antibody, a GPE standard, an assay buffer, tritiated
GPE, and a second antibody or a precipitated antibody. In a further
embodiment, a radioimmunoassay kit comprises an anti-GPE antibody,
a GPE standard, Bolton and Hunter reagent
(N-succinimidyl-3-[4-hydroxyphenyl] propionate), a derivatizing
buffer, an assay buffer, Bolton and Hunter derivatized GPE (BH-GPE)
for iodination, and a second or precipitating antibody, for example
an antibody precipitated with polyethylene glycol.
[0021] In a third aspect, this invention is methods of reverse
phase high-performance liquid chromatography ("rpHPLC") that
accurately resolves and quantitates GPE and related compounds. Two
methods are described, in Examples 4 and 5, the first using
derivatization of the amino groups with AccQTag.RTM. reagent,
6-aminoquinolyl-N-hydroxysuccinim- idyl carbamate in acetonitrile
and borate buffer, and the second, for the measurement of
radioactive (e.g. tritiated) GPE, using a Hypercarb.RTM. column
with no derivatization, and detection of the radioactivity in the
eluate. The level of GPE may be a suitable marker of drug efficacy
and/or effective dosing. In one embodiment, an rpHPLC assay kit
comprises a GPE standard, 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate and derivatizing buffer, a column, and column running
buffer. In another embodiment, An rpHPLC assay kit comprises a
radioactive GPE standard, a column, and column running buffer.
[0022] In a fourth aspect, this invention is therapeutic methods
using the anti-GPE antibodies of the first aspect of this
invention, as exemplified in Examples 7 and 8. This invention is
methods for the extension of the half-life of GPE in vitro and in
vivo comprising co-administration of GPE with anti-GPE antibodies
effective that a significant fraction of the GPE is bound to the
anti-GPE antibody a significant fraction of the time, thereby
protecting the GPE from degradation, non-specific binding, and
metabolic modification and clearance. In further embodiments of the
invention, antibodies to GPE may be used as non-blocking antibodies
to modulate concentrations of GPE. In particular, antibodies to GPE
may be used as non-blocking antibodies to modulate the free
concentrations of GPE in vivo and in vitro.
[0023] In a fifth aspect, this invention is methods for the
purification of the GPE receptor, comprising the use of the
anti-GPE antibodies of the first aspect of this invention, as
described in Example 9. In these methods, tissues, suspensions and
solutions comprising GPE receptors contact surfaces, substrates,
and solutions comprising GPE, effective to bind the GPE to the GPE
receptors; and such surfaces, substrates, and solutions are
subsequently contacted with anti-GPE antibodies so that the
anti-GPE antibody binding to the GPE that is bound to GPE receptors
is effective to aid in the purification of the GPE receptors.
[0024] The following non-limiting Examples illustrate this
invention. All animal experimental protocols were conducted in
accordance with guidelines approved by the University of Auckland
Animal Ethics Committee.
EXAMPLE 1
Preparation of a Polyclonal Antibody to GPE (the CK5 Antibody), and
Detection of GPE by the CK5 Antibody
[0025] Three New Zealand White rabbits were injected subcutaneously
with 200-300 .mu.g of a peptide-conjugate immunogen (a 1:1 mixture
of GPE conjugated to keyhole limpet hemocyanin (KLH) using
glutaraldehyde (GA) and KYFGGPE conjugated to KLH using GA)
emulsified in Freund's complete adjuvant (primary immunization).
Booster injections with the same immunogen emulsified in Freund's
incomplete adjuvant were given at 3 to 4 weekly intervals. Blood
samples were taken from the marginal ear vein 10 days after each
injection for titer determination, and booster immunizations
continued until a suitable titer was achieved (9 injections). The
rabbits were then anesthetized and killed by terminal
exsanguination. The blood was allowed to clot, then centrifuged,
and the supernatant serum recovered. This serum contains the
polyclonal anti-GPE antibody, which we refer to as CK5 antibody,
and was frozen at--20.degree. C. until ready for use. Since the
presence of other non-GPE related immunologic reactions does not
interfere with the reaction between GPE and its antibody (anti-GPE
antiserum), the polyclonal CK5 antibody did not undergo any further
purification. Characterization of the CK5 antibody was performed
using both double antibody radioimmunoassay and immunohistochemical
techniques.
[0026] The CK5 antibody was characterized using a standard double
antibody radioimmunoassay technique. Tubes containing either 100
.mu.L 0.02 M phosphate buffered saline assay buffer or peptide
(GPE, YGPE, KYFGGPE, or IGF-1) dissolved in assay buffer at various
concentrations were preincubated with CK5 antibody (diluted at
1:600) for 24 h at 4.degree. C. I.sup.125-labeled YGPE (10,000 cpm)
was then added to the tubes. After a further 48 h incubation at
4.degree. C., the bound and free GPE were separated by adding
donkey anti-rabbit serum (1:100). The tubes were incubated with
this second antibody overnight at 4.degree. C. before
centrifugation (3,200 rpm for 30 min), after which the supernatant
was aspirated, and the precipitate counted in a gamma counter. The
results are shown in FIG. 1. One antibody, CK5, was identified.
Under the assay conditions described above, CK5 exhibits 14.7%
specific binding to GPE at a final titer of 1:600, using
I.sup.125-labeled YGPE as the tracer. Unlabeled GPE was able to
displace I.sup.125-labeled YGPE with an ED.sub.50 of approximately
200 ng/tube. Importantly, the CK5 antibody does not cross-react
significantly with the GPE parent molecule, IGF-1.
[0027] The specificity of the CK5 antibody was further confirmed by
Western blot and dot blot analysis. GPE immunoreactivity was
detected when membranes were incubated with CK5 overnight at
4.degree. C.; whereas preabsorption of CK5 overnight with excess
unlabeled GPE completely abolished GPE immunoreactivity. Thus, CK5
is a specific antibody that recognizes and competitively binds
GPE.
EXAMPLE 2
Detection of Bolton and Hunter Derivatized GPE Using the CK5
Antibody
[0028] The CK5 antibody was prepared as described in Example 1, and
characterized using a modified double antibody radioimmunoassay
technique. The CK5 antibody was used at a final dilution of
1:18,000. Tubes containing either 100 .mu.L assay buffer (pH 7.8,
0.05 M sodium phosphate), peptide (GPE, glycine, proline, glutamic
acid, GP, PE, or IGF-1), or urea were derivatized with Bolton and
Hunter reagent, N-succinimidyl-3-[4-hydroxyphenyl] propionate, and
dissolved in assay buffer. The tubes were incubated with CK5
antibody and I.sup.125-labeled Bolton and Hunter derivatized GPE
(15,000 cpm) for 48 h at 4.degree. C. The bound and free GPE were
then separated by adding sheep anti-rabbit gamma globulin (1:100).
The tubes were incubated with this second antibody for 4 h at room
temperature before centrifugation (3,200 rpm for 45 min), after
which the supernatant was tipped off and the precipitate counted in
a gamma counter. The results are shown in FIGS. 2 and 3. Under the
assay conditions described above, CK5 exhibits 50% specific binding
to Bolton and Hunter derivatized GPE at a final titer of 1:18,000,
using I.sup.125 labeled Bolton and Hunter derivatized GPE as the
tracer. Unlabeled Bolton and Hunter derivatized GPE was able to
displace I.sup.125 labeled Bolton and Hunter derivatized GPE with
an ED.sub.50 of approximately 0.01 ng/tube and the minimal
detectable level of GPE was 0.005 ng/tube. The addition of either
rat or human plasma to the standard curve resulted in parallel
displacement. Importantly, CK5 antibody does not cross-react
significantly with Bolton and Hunter derivatized glycine, proline,
glutamic acid, GP, PE, IGF-1, or urea. The assay of differing
volumes of rat plasma (25, 50, 75, 100 .mu.L) containing known
amounts of added GPE resulted in a linear relationship.
[0029] The specificity of CK5 was further confirmed by Western blot
and dot blot analysis. GPE immunoreactivity was detected when
membranes were incubated with CK5 overnight at 4.degree. C.;
whereas preabsorption of CK5 overnight with excess unlabeled GPE
completely abolished GPE immunoreactivity. Thus CK5 is a specific
antibody that recognizes and competitively binds Bolton and Hunter
derivatized GPE with a higher ED.sub.50 than for underivatized GPE.
The ED.sub.50 for modified displacement of Bolton and Hunter
derivatized GPE was 0.0094 ng/tube (antibody diluted at a final
dilution of 1/18,000), whereas the ED.sub.50 for the standard
displacement was 199.8 ng/tube (antibody diluted at a final
dilution of 1/600).
EXAMPLE 3
Radioimmunoassay (RIA) Measurement of GPE in Biological Fluids and
Tissues Using CK5 Antibodies and Bolton and Hunter Derivatized
GPE
[0030] This procedure uses a pre-RIA derivatization of the
GPE-containing sample and comprises three steps: initial
preparation of the sample using a tungstate extraction procedure to
remove large proteins and to prevent overloading of the Bolton and
Hunter reagent with an excess of amino groups; derivatization of
samples and standards with Bolton and Hunter reagent; and a
standard RIA protocol combining the CK5 antibody, .sup.125I-labeled
Bolton and Hunter derivatized GPE as the tracer, and PEG
precipitation.
[0031] Part 1a: Acid tungstate precipitation from blood,
cerebrospinal fluid (CSF), and urine
[0032] Whole blood was collected into collection tubes containing a
metalloprotease inhibitor, for example Sigma protease inhibitor
cocktail, and centrifuged at 3,000.times.g for 15 min at 4.degree.
C. The supernatant (plasma) was transferred into a new tube and
stored at -80.degree. C. until ready for assay. CSF and urine were
collected into collection tubes containing a metalloprotease
inhibitor, for example Sigma protease inhibitor cocktail, and
stored at -80.degree. C. until ready for assay. The samples were
thawed on ice. During-thawing of the samples, 800 .mu.L of 0.04 M
sulfuric acid was added to 1.5 mL micro-centrifuge tubes and
incubated on ice. Aliquots (100 .mu.L) of the samples were
transferred to the micro-centrifuge tubes, and the tubes were
vortexed and incubated on ice for 5 min, after which 100 .mu.L of
10% sodium tungstate was added. The tubes were vortexed and
incubated on ice for 10 min, twice. The tubes were then centrifuged
at 20,000.times.g for 20 min at 4.degree. C., after which 900 .mu.L
of the acid tungstate-treated sample was removed to a new
micro-centrifuge tube and stored at -80.degree. C. Tritiated GPE
was used to determine a recovery level of 90-92% for the extraction
procedure.
[0033] Part 1b: Acid tungstate precipitation from tissue
[0034] All steps were performed on ice to prevent degradation of
GPE. Approximately 50 mg of tissue was accurately weighed in a
micro-centrifuge tube and 5 .mu.L of protease inhibitor and 160
.mu.L of 0.67 N H.sub.2SO.sub.4 added. The sample was homogenized
for 3 min with a micro-centrifuge tube fitting pestle
(approximately 100 strokes) or until a liquid homogenate was
obtained. The pestle was rinsed into the tube with 400 .mu.L of
water using a pipette, and the tube sonicated for 1-5 sec. The
probe was rinsed with 180 .mu.L of water and the rinse added to the
homogenate, then 60 .mu.L of 10% sodium tungstate added, and the
tube vortexed and incubated on ice for 10 min, twice. The tube was
then centrifuged at 20,000.times.g for 20 min at 4.degree. C. and
the supernatant transferred to a new tube. To the pellet was added
100 .mu.L of water, and the pellet was resuspended by vortexing and
sonication for 1-5 sec, again rinsing the probe with 100 .mu.L of
water. The pellet was then centrifuged at 20,000.times.g for 20 min
at 4.degree. C., and the supernatant added to the original
supernatant. Chloroform (100 .mu.L) was added, and the tube was
vortexed and centrifuged at 20,000.times.g for 5 min at 4.degree.
C. One mL of the upper layer was transferred to a new tube, taking
care not to disturb the chloroform layer, and frozen at -80.degree.
C. Tritiated GPE was used to determine a recovery level of 92-94%
for the extraction procedure.
[0035] Part 2: Bolton and Hunter derivatization of samples
[0036] To 100 .mu.L of thawed treated sample was added 100 .mu.L of
0.1 M phosphate buffer, and the mixture vortexed, after which 20
.mu.L of 20 mM Bolton and Hunter reagent was added, and the samples
incubated at room temperature for 4 h. The derivatized samples were
then lyophilized overnight, re-suspended in 100 .mu.L of assay
buffer, and transferred to polypropylene plastic assay tubes
(12.times.75 mm). For the standards, 100 .mu.L of Bolton and Hunter
reagent was added to 1 mL of standard sub-stock containing 640
ng/mL phosphate buffer. The standard was incubated at room
temperature for 4 h. The derivatized standards were lyophilized
overnight, and re-suspended in 1 mL assay buffer.
[0037] Part 3: Radioimmunoassay of Bolton and Hunter derivatized
GPE
[0038] Rabbit CK5 antibody was used at a final dilution of 1:18,000
in assay buffer, and .sup.125I-labeled Bolton and Hunter
derivatized GPE (BH-GPE) was used as tracer at 150,000 cpm/mL in
assay buffer. Sheep anti-rabbit gamma globulin (1% in 0.01 M PBS
with 8% PEG), with 0.05% normal rabbit serum, incubated for 90 min
at 4.degree. C. before use, was the second antibody precipitation
reagent.
[0039] BH-GPE sub-stock containing 640 ng/mL BH-GPE was serially
diluted to concentrations ranging from 640 ng/mL to 0.0002 ng/mL.
Three 100 .mu.L aliquots of each concentration were then
transferred to polypropylene plastic assay tubes (12.times.75 mm).
To each sample was added 100 .mu.L antibody and 100 .mu.L tracer,
and the tubes vortexed. The samples were incubated for 72 h at
4.degree. C., and 1 mL of secondary antibody reagent was added. The
sample was incubated at room temperature for 2 h, then centrifuged
at 3,000.times.g for 45 min at 4.degree. C. The supernatant was
poured off and counted for 1 min in a Cobra Gamma counter (Packard
Biosciences). The results are shown in FIG. 4. The addition of the
CK5 antibody and .sup.125I-Bolton and Hunter derivatized GPE tracer
in a radioimmunoassay allows the specific measurement of GPE plasma
concentrations in samples following intravenous dosing. Using the
CK5 antibody, GPE is detectable in blood following dosing and has a
half -life of approxamately 1-2 min.
EXAMPLE 4
Reverse HPLC Using AccQTag.RTM. Derivatization
[0040] GPE-containing samples were prepared as in Parts 1a and 1b
of Example 3. The samples were derivatized by the Waters
AccQTag.RTM. method, which involves incubation of the sample with
10 mM 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate in
acetonitrile and borate buffer at 55.degree. C. for 10 min and
converts primary and secondary amino groups to fluorescent
derivatives, before being transferred to the HPLC injection vial.
These reaction products were resolved by HPLC and compared to known
amino acid standards. The reverse phase HPLC system consisted of a
Waters 2690 Alliance seperation module, a 300.times.3.9 mm C18
Pico-tag (Waters) column at 37.degree. C., and a Waters 474
fluorescene detector set at 250 nm excitation, 395 nm detection,
gain 100. This was linked to a PC running the Waters
Millennium.sup.32 program (Waters Corporation, Milford Mass.
01757). The mobile phase consisted of three components: component A
was MilliQ water, component B was a buffer made up with 80 mM
sodium acetate, 3 mM triethylamine, 2.7 .mu.M EDTA, brought to pH
6.43 with orthophosporic acid, and component C was acetonitrile.
The mobile phase was run in the gradient shown in Table 1 below
over 112.1 min, at a flow rate of 1.2 mL/min at 37.degree. C.
1TABLE 1 Time (min) % A % B % C Curve 0 49.9 49.9 0.2 6 13 48.7
48.7 2.6 6 27 48.6 48.6 2.8 6 50 48.5 48.5 3 6 75 46 46 8 6 82 45
45 10 6 98 43 43 14 6 108 41.5 41.5 17 11 108.1 40 0 60 11 112.1
49.9 49.9 0.2
[0041] The results are shown in FIGS. 5 and 6. The rpHPLC elution
profile of FIG. 5 shows that GPE elutes with a retention time of
approximately 72 min, and the GPE peak is sharp and resolved and
clearly detectable above control plasma. No GPE was detected in
`unspiked` control plasma. This method has also been repeated with
tritiated GPE, which eluted with the same retention time. The
profiles of FIG. 6 show that rpHPLC allows the specific measurement
of GPE plasma concentrations in samples at different times
following intravenous dosing. Reverse phase HPLC of AccQTag
derivatized GPE-containing samples is a reliable and sensitive
method to detect GPE.
EXAMPLE 5
Reverse Phase HPLC Using A Hypercarb.RTM. Column
[0042] GPE-containing samples were prepared as in Parts 1a and 1b
of Example 3. Samples were thawed on ice before being transferred
to the HPLC injection vial. The reverse phase HPLC system consisted
of a 100.times.4.6 mm Hypercarb 5 .mu.m (Hypersil) column between a
Waters Wisp Autosampler (Waters) and a BioCAD Sprint workstation
(Applied Biosystems) running Version 2.062 of the BioCAD
workstation software and an Advantec fraction collector set to
collect 0.5 mL fractions. Samples were run onto the column in a
mobile phase consisting of 10% methanol, 0. 1% trifluoroacetic acid
in MilliQ water, then eluted using a linear gradient with a mobile
phase consisting of 90% methanol, 0.1% trifluoroacetic acid in
MilliQ water using 0-100% gradient over 25 min as in Table 2 below,
with a flow rate of 1.0 mL/min at room temperature. UV absorbance
detection was set at 220 nm, and 0.5 mL fractions were collected
into 5 mL scintillation vials from time of injection until the end
of the gradient. Scintillation fluid (4 mL) was then added to each
vial, and the samples counted in a 14XX Rack-beta scintillation
counter (Wallac, Perkin Elmer).
2TABLE 2 Time 10% MeOH/ 90% MeOH/ (min) 0.1% TFA 0.1% TFA Event 0
100 0 5 100 0 10 100 0 Injection/fraction start 35 0 100 Fraction
collection stop 40 0 100 41 100 0 45 100 0
[0043] The results are shown in FIG. 7. Tritiated GPE eluted in
fractions 27 and 28. The GPE peak is sharp, resolved, and clearly
detectable. Metabolic products of tritiated GPE (Gly-Pro and
Proline)elute in the void. The method was repeated with "cold"
(non-tritiated) GPE and eluted with the same retention time.
EXAMPLE 6
Polyclonal Antibody Production in Rabbits
[0044] Twelve female New Zealand White rabbits are injected
subcutaneously with 600-1000 .mu.g of peptide-conjugate emulsified
in Freund's complete adjuvant. Three rabbits received a mixture of
300 .mu.of GPE conjugated to KLH using GA and 300 .mu.g KYFGGPE
conjugated to KLH using GA; three rabbits received a mixture of 300
.mu.g GPE conjugated to KLH using GA and 600 .mu.g GPE conjugated
to KLH using diethyl carbodiimide, and six rabbits, primed with
Bacillus Calmette-Guerin [BCG] vaccine, received 1000 .mu.g CGPE
conjugated to a purified protein derivative of tuberculin (Statens
Serum Institut, Denmark) using sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane 1-carboxylate (sulfo-SMCC,
Pierce, Ill., USA). Booster injections emulsified in Freud's
complete adjuvant were given at 3-4 weekly intervals. Blood samples
were taken from the marginal ear vein 10 days after each injection
for titer determination, and regular immunizations continued for up
to 8 months (maximum 10 injections) until a suitable titer was
achieved.
[0045] Characterization of the anti-GPE antibody was performed
using both the double antibody radioimmunoassay technique described
in Examples 1 and 2 above and by immunocytochemistry.
EXAMPLE 7
Passive Immunization Against GPE in Rats
[0046] Following hypoxic-ischemic injury, rats were treated with
either GPE alone or GPE combined with anti-GPE antibodies. Nine
pairs of adult Wistar rats (280-320 g) were prepared under
halothane/O.sub.2 anesthesia. The right side carotid artery was
ligated. To facilitate the intracerebroventricular administration
of treatment, a guide cannula was placed on the dura at stereotaxic
coordinates AP +7.5 mm, R +1.5 mm. The rats were allowed to recover
for 1 h and were then placed in an incubator with humidity 90.+-.5%
and temperature 31.+-.0.5.degree. C. for 1 h before hypoxia. The
oxygen concentration was then reduced and maintained at 6.+-.0.2%
for 10 min. The rats were kept in the incubator for 2 h after
hypoxia and then treated with either 3 .mu.g GPE or 3 .mu.g GPE
plus 25 .mu.L anti-GPE antibodies. A further 6 rats were used as
normal controls. The rats were killed by being deeply anaesthetized
with an overdose of pentobarbital and then transcardially perfused
with normal saline followed by 10% buffered formalin. The brains
were removed and kept in the same fixative for two days before
being processed using a standard paraffin tissue procedure.
[0047] Coronal (8 .mu.m) sections were cut from the striatum,
cerebral cortex and hippocampus, mounted on glass slides and
stained with Thionin and Acid Fuchsin. With the experimenter
blinded to the treatment groups, the histological outcome was
assessed using two levels: at the mid-level of the striatum and the
level where the ventral horn of the hippocampus just appears. Dead
neurons are acidophilic (red) and have contracted nuclei. An
indirect technique was used to determine the extent of cortical
damage; the area of intact cortical tissue in both hemispheres was
measured using an image analyzer (SigmaScan (SPSS Science) Chicago,
Ill.). Brain tissue with selective neuronal death and/or
pan-necrosis was considered to be damaged. The right/left (R/L)
ratio of area of intact cortex was compared between the treatment
groups. Surviving neurons from both sides of the CA1-2 subregions
of the hippocampus were counted from the boundary between CA3 and
CA1-2 and towards CA1-2 for 600 .mu.m. The R/L ratio of surviving
neurons in the CA 1-2 subregions of the hippocampus was compared
between treatment groups. Striatal damage was scored using the
following scoring system: 0, no tissue damage; 1, <5% tissue
damage; 2, <50% tissue damage; 3, >50% tissue damage. Passive
immunization against GPE actively blocks the neuroprotective
effects of GPE, suggesting that following GPE treatment,
neuroprotective effects are specific to GPE action.
EXAMPLE 8
Passive Immunization Against GPE in Lesioned Rats
[0048] Following a lesion with 6-hydroxy dopamine (6-OHDA), rats
were treated with GPE either alone or combined with anti-GPE
antibodies. Eighteen male Wistar rats (50-60 days, 280-310 g) were
used for the study. Under 3% halothane anesthesia, the 6-OHDA (8
.mu.g in 2 .mu.L 0.9% saline containing 1% ascorbic acid) was
administered into the right medial forebrain bundle (MFB) at
stereotaxic coordinates AP +4.7 mm, R +1.6 mm, V -8 mm using a 100
.mu.L Hamilton syringe with a 30 G needle controlled by a
microdialysis infusion pump at an infusion rate of 0.2
.mu.L/minute. The infusion needle was slowly withdrawn 5 minutes
after the infusion. The surgery and procedures for the
intracerebroventricular administration are described in Guan et al.
(1993), The effects of IGF-I treatment after hypoxic-ischemic brain
injury in adult rats, Journal of Cerebral Blood Flow and Metabolism
13:609-616. A 6 mm long, 21 G guide cannula is fixed on the top of
the dura with coordinates of AP +7.5 mm, R +1.5 mm immediately
after the injection of 6-OHDA. Either 3 .mu.g GPE, 3 .mu.g GPE plus
25 .mu.L anti-GPE antibodies, or vehicle was infused into the right
lateral ventricle 2 h after lesion at an infusion rate of 2
.mu.L/min. Rats were then housed in a holding room with free access
to food and water for the next two weeks. The rats were killed by
being deeply anaesthetized with an overdose of pentobarbital and
then transcardially perfused with normal saline followed by 10%
buffered formalin. The brains were removed and kept in the same
fixative for two days before being processed using a standard
paraffin tissue procedure.
[0049] Coronal sections from the striatum and the substantia nigra
compacta (SNc) were cut on a microtome to 8 .mu.m thickness,
mounted on chrome-alum coated slides, and air-dried. For staining,
the sections were deparaffinized, rehydrated, washed with 0.1 M
phosphate buffered saline (PBS), pretreated with 1% H.sub.2O.sub.2
for 20 min, washed with 0.1 M PBS (3.times.5 min), and incubated in
rabbit polyclonal antisera raised against tyrosine hydroxylase
(Protos Biotech, USA) diluted 1:500 with 1% goat serum for 48 h at
4.degree. C. The sections were then washed in PBS (3.times.5 min)
and incubated overnight at room temperature in donkey anti-rabbit
biotinylated secondary antibody (1:200, Amersham Life Science). The
sections were washed again in 0.1 M PBS, incubated in
streptavidin-linked horse radish peroxidase (1:200, Amersham Life
Science) for 3 h, washed again in PBS, and then treated with 0.05%
3,3'-diaminobenzidine tetrahydrochloride and 0.01% H.sub.2O.sub.2
to produce a brown reaction product. The sections were then
dehydrated in a graded alcohol series, cleared in xylene, and
coverslipped with mounting medium.
[0050] With the experimenter blinded to the treatment groups, the
number of tyrosine hydroxylase-positive (TH-positive) neurons on
both sides of the SNc are counted using light microscopic
examination (20.times.magnification) at three representative levels
(AP +4.2 mm, +3.8 mm and +3.4 mm). The average densities of TH
staining on both sides of the SNc are measured using an
image-analyser (Mocha image analysis software). The average density
of TH staining in the striatum is also measured using three
adjacent sections from the middle of the striatum. The average
density from the background reading is also measured. The
difference in average density between the background and TH
staining is calculated and used for data analysis. Right/left (R/L)
ratios of the number of TH-positive neurons and the R/L ratio of
the average density of TH staining from each level of the SNc is
compared between the two treatment groups using two-way ANOVA. The
R/L ratio of the TH staining density from three striatal sections
is averaged and compared between the two groups using the t-test.
Data is presented as mean.+-.SEM. The morphological changes in the
SNc and the striatum are photographed using a Leitz Dialux light
microscope (10.times. and 40.times.magnifications) or a digital
camera and the images processed using Adobe Photoshop.RTM. and
Pagemaker.RTM. software. Passive immunization against GPE actively
blocks the neuroprotective effects of GPE, suggesting that
following GPE treatment neuroprotective effects are specific to GPE
action.
EXAMPLE 9
Purification of the GPE Receptor
[0051] CK5 antibody was resuspended to a final concentration of
1/100 in 0.1M PBS pH 7.8. Sulfosuccinimidyl
2-[m-azido-o-nitrobenzamido]-ethyl-1,3- '-dithiopropionate (SAND)
in DMSO was added to a final concentration of 10 mM, and the
reaction mixture incubated in the dark at 37.degree. C. for 30 min.
Unreacted SAND was removed by dialysis against several changes of
0.1M PBS, pH 7.8. The CK5-SAND complex was then stored at
-80.degree. C. until used. Fresh frozen brain slices (60 .mu.m
thick) or cells grown in 80 cm cell culture dishes were briefly
exposed to 100 .mu.M GPE or vehicle in 0. 1M PBS, excess unbound
GPE was then washed off with three washes of PBS, and the samples
incubated in the dark for 1 h with CK5-SAND complex to enable the
antibody to bind to the GPE, which is bound to its receptor.
Photoactivation by 3-5 bright camera flashes resulted in
crosslinking of the CK5-SAND complex to the GPE receptor. The
cells/tissues were then solubilized in 1% Triton in 25 mM HEPES, pH
7.6; and CK5-SAND-receptor immunocomplexes were then purified using
a HiTrap Protein G Column (Amersham Pharmacia Biotech) following
the manufacturer's instructions. 2-Mercaptoethanol was then added
to the sample extract to cleave the crosslinker; and the separated
CK5 antibody and GPE receptor were resolved by two dimensional
electrophoresis before blotting to PVDF membranes and staining with
Coomassie blue.
[0052] GPE and vehicle treated extractions were compared and
potential receptor bands identified. These bands were excised and
sequenced using a gas-phase Sequencer (model 470A, Applied
Biosystems) following the manufacturer's instructions or by MS/MS
analysis.
[0053] All documents cited throughout this application are
incorporated by reference into this application. Those persons
skilled in the art will appreciate that the present invention is
described by way of example only and is not intended to be limited
to the specific experimental details given. Numerous changes and
variations can be made without departing from the scope of the
invention, and all such changes and variations are intended to be
within the scope of the following claims and their equivalents.
* * * * *