Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-INOSITOLPHOSPHOGLYCAN MONOCLONAL ANTIBODIES
Field of the Invention
The present invention relates to antibodies capable of
specifically binding to inositolphosphoglycans (IPGs), to
methods for making these antibodies and their diagnostic
and therapeutic uses, in particular in assays.
eackaround of the Invention
Many of the actions of growth factors on cells are
thought to be mediated by a family of inositol
phosphoglycan (IPG) second messengers (Rademacher et al,
1994). It is thought that the source of IPGs is a °free~~
form of glycosyl phosphatidylinvsitol (GPI) situated in
cell membranes. IPGs are thought to be released by the
action of phosphatidylinositol-specific phospholipases
following ligation of growth factors to receptors on the
cell surface. There is evidence that IPGs mediate the
action of a large number of growth factors including
insulin, nerve growth factor, hepatocyte growth factor,
insulin-like growth factor I (IGF-I), fibroblast growth
factor, transforming growth factor (3, the action of IL-2
on H-cells and T-cells, ACTH signalling of adrenocortical
cells, IgE, FSH and hCG stimulation of granulosa cells,
thyrotropin stimulation of thyroid cells, cell
proliferation in the early developing ear and rat mammary
gland.
Soluble IPG fractions have been obtained from a variety
of animal tissues including rat tissues (liver, kidney,
muscle brain, adipose, heart) and bovine liver. IPG
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2
biological activity has also been detected in malaria
parasitized red blood cells (RBC) and mycobacteria. We
have divided the family of IPG second messengers into
distinct A and P-type subfamilies on the basis of their
biological activities. In the rat, release of the A and
P-type mediators has been shown to be tissue-specific
(Kunjara et al, 1995).
There are some references in the prior art to the
production of polyclonal antibodies having anti-IPG
specificity (see Romero et al, 1990; Huang et al, 1993;
Nestler et al, 1991; Represa et al, 1991). In these
papers, a single polyclonal antibody is described which
was raised against the GPI-anchor of the variant surface
protein (VSG) of Trypanosoma brucei. The authors were
forced to use this cross-reacting antigen for obtaining
anti-IPG antibodies as soluble IPGs had not been purified
and characterised, and so were not available for raising
specific anti-IPG antibodies directly. The polyclonal
antibodies produced using this immunisation protocol
react also with the GPI-anchor moieties of GPI-anchor
containing proteins. The papers report that this
polyclonal antibody inhibits the action of insulin on
human placental steroidogenesis, inhibits the action of
insulin on muscle cells, inhibits the action of insulin
on rat diaphragm and inhibits the action of nerve growth
factor on chick cochleovestibular ganglia (CVG).
Summary of the Invention
The present invention relates to the production of
polyclonal and monoclonal antibodies specific for P and
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3
A-type inositolphosphoglycans (IPGs), and in particular
to the first production of monoclonal antibodies capable
of specifically binding to these substances. The present
invention also relates to the uses of anti-IPG
antibodies, especially in the diagnosis of diabetes and
pre-eclampsia.
In the field of diabetes, a central problem concerns the
identification of patients at risk of developing type I
diabetes. This condition typically arises between the
ages of 5 and 11 and is caused by a patient s immune
system attacking ~i-cells in the pancreas, destroying
these cells and causing inflammation. If patients at
risk of developing type I diabetes could be identified
before this attack takes place, it opens up the
possibility of ameliorating or preventing the attack by
shutting down the pancreas during the period of risk by
providing the patients with insulin or an alternative
treatment for diabetes. After the period of risk is
over, e.g, after puberty, this treatment could cease, and
the patient s pancreas allowed to resume making insulin.
Currently, there is no reliable method of identifying
such patients.
As for pre-eclampsia, at present there is no simple assay
for the diagnosis of this condition which affects 10-12~
of all pregnancies, causing maternal endothelial
dysfunction and problems with activation of the clotting
system, increased vascular permeability and ischaemia in
maternal organs secondary to vasoconstriction.
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Accordingly, in a first aspect. the present invention
provides a hybridoma cell line which produces anti-
inositolphosphoglycan (IPG) monoclonal antibodies,
wherein the cell line is selected from hybridoma cell
lines 2F7, 2D1 and 5H6 deposited at European Collection
of Cell Cultures (ECACC) under accession numbers
98051201, 98031212 and 98030901.
In a further aspect, the present invention provides anti-
IPG monoclonal antibodies as obtainable from the above
hybridoma cell lines or which are capable of binding to
an epitope of an IPG which is bound by a monoclonal
antibody produced by one or more of the deposited cell
lines. In particular, the antibodies were produced
following immunisation with A-type IPGs as obtainable
from rat liver and yet bind to P-type IPGs as obtainable
from human placenta. This surprising result indicates
that the antibodies recognise a common epitope on these
IPGs. Thus, in a preferred embodiment, the present
invention provides antibodies capable of binding to the
same or substantially the same epitope of a P or A-type
IPG as one of the above monoclonal antibodies.
Preferably, the antibodies of the invention do not bind
to the common reactive determinant (CRD) of GPI anchored
proteins. Experiments demonstrating this are set out
below in example 6.
In some embodiments, the antibodies are neutralising
antibodies and can be used as antagonists to reduce or
inhibit one or more of the biological properties of the P
or A-type IPGs.
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In further aspects, the present invention provides the
above antibodies for use in a method of medical treatment
and pharmaceutical compositions comprising the
antibodies.
5
In a further aspect, the present invention provides the
use of the above antibodies for the preparation of a
composition for use in a diagnostic assay. In preferred
embodiments, the assays are for the~diagnosis of pre-
eclampsia or diabetes. In a preferred embodiment, the
use is for the diagnosis of patients at risk of
developing type I diabetes.
In a preferred embodiment, the present invention provides
a method for diagnosing patients at risk of developing
type I diabetes, the method comprising:
(a) contacting a biological sample from the patient
with an antibody capable of specifically binding P and/or
A-type IPGs; and,
(b) determining the binding of IPGs in the sample
to the anti-IPG antibody.
Optionally, the method comprises the further step of
administering insulin and/or P-type IPGs and/or A-type
IPGs to a patient shown in the assay to have or be at
risk of developing type I diabetes, e.g. for the period
of risk.
In an alternative embodiment, the present invention
provides a method for diagnosing pre-eclampsia in a
patient, the method comprising:
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(a) contacting a biological sample from the patient
with one of the above anti-IPG antibodies; and,
(b) determining the binding of IPGs in the sample
to the anti-IPG antibodies.
Optionally, the method comprises the further step of
administering a P-type IPG antagonist to a patient shown
in the assay to have or be at risk of developing pre-
eclampsia.
Exemplary assay protocols are described in more detail
below. Typically, the binding of IPGs in the sample to
the anti-IPG antibody is compared to the amount of
binding that takes place in standards, e.g. from a non-
diabetic patient, or from a person known to have a
particular type of diabetes, e.g a patient known to have
developed type I diabetes or have pre-eclampsia.
In one embodiment, the assay can be carried out by
providing the patient with a carbohydrate load (e. g.
glucose) and measuring the IPG response following the
load over time. The IPG profiles or levels determined
from a patient sample can then be compared with the
profiles or levels obtained from standards to make the
diagnosis. Conveniently, the antibody is selected from
the group of deposited monoclonal anti-IPG antibodies, or
antibodies prepared using the methods described herein.
In a further aspect, the present invention provides the
use.of an antibody described herein in the
immunopurification of P or A-type IPGs.
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In a further aspect, the present invention provides a
method of producing antibodies specific for P and/or A-
type /PGs, the method comprising immunising an animal
with one or more soluble P or A-type IPGs to elicit an
antibody response, wherein the IPGs are not conjugated to
an immunogenic carrier.
The inventors have surprisingly found that despite the
fact that IPGs are small molecules and carbohydrates, it
is possible to raise antibodies by immunising animals
with them. Further, the fact that conjugation of the
IPGs to a carrier is not required means that epitopes on
the IPGs are not blocked or altered by the conjugation
reaction, allowing antibodies to be raised to a full
complement of epitopes on the IPGs. The method is
applicable to the production of both polyclonal and
monoclonal antibodies, as is demonstrated by the examples
below. Preferably, the animal is immunised via an
intraperitoneal route using a soluble form of the IPG.
Embodiments of the present invention will now be
described by way of example and not by limitation with
reference to the accompanying drawings.
Brief Description of the Drawincs
Figure 1 shows a dose-response curve of absorbance
plotted against IPG concentration using anti-IPG
antibodies in a Sandwich ELISA test.
Figure 2 .shows how IPG levels in human serum vary with
time after an oral load of glucose (75g) in a control
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patient without a familiar history of type I diabetes.
Figure 3 shows how IPG levels in human serum vary with
time after an oral load of glucose (75g) in a patient
with a familiar history of type I diabetes
Figure 4 shows how IPG levels in human serum vary with
time after an oral load of glucose (75g) in a patient
with carbohydrate intolerance.
Figures 5 and 6 show investigations into the properties
of monoclonal antibody 2D1 in chick cochleovestibular
ganglia (CVG) culture.
Figure 7 shows the results of experiments comparing the
binding specificity of monoclonal antibody 5H6, a
polyclonal antibody raised against the IPGs and a
polyclonal antibody raised against the cross reactive
determinant (CRD) common to GPI anchored proteins.
Figure 8 shows the results of a PDH phosphatase assay
demonstrating that monoclonal antibody 5H6 is capable of
inhibiting the action of P-type IPG from rat liver.
Figure 9 shows the results of a pre-eclampsia ELISA assay
employing anti-IPG antibodies of the invention to measure
urine IPG levels.
Figure 10 shows the results of a blinded pre-eclampsia
assay using anti-IPG monoclonal antibody 2D1 to measure
urine IPG levels.
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Figure 11 shows the relationship between the IPG assay in
pre-eclamptic urine using anti-IPG monoclonal antibody
2D1 and platelet counts.
Figure 12 shows the relationship between the IPG assay in
pre-eclamptic urine using anti-IPG monoclonal antibody
2D1 and plasma AST activity.
Detailed Description
IPGs and 2PG AnaloQUee
Studies have shown that A-type mediators modulate the
activity of a number of insulin-dependent enzymes such as
cAMP dependent protein kinase (inhibits), adenylate
cyclase (inhibits) and cAMP phospho-diesterases
(stimulates). In contrast, P-type mediators modulate the
activity of insulin-dependent enzymes such as pyruvate
dehydrogenase phosphatase (stimulates), glycogen synthase
phosphatase (stimulates) and cAMP dependent protein
kinase (inhibits). The A-type mediators mimic the
lipogenic activity of insulin on adipocytes, whereas the
P-type mediators mimic the glycogenic activity of insulin
on muscle. Both A and P-type mediators are mitogenic
when added to fibroblasts in serum free media. The
ability of the mediators to stimulate fibroblast
proliferation is enhanced if the cells are transfected
with the EGF-receptor. A-type mediators can stimulate
cell proliferation in the CVG.
Soluble IPG fractions having A-type and P-type activity
have been obtained from a variety of animal tissues
including rat tissues (liver, kidney, muscle brain,
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adipose, heart) and bovine liver. A and P-type IPG
biological activity has also been detected in human liver
and placenta, malaria parasitized RHC and mycobacteria.
The ability of a polyclonal cross-reacting anti-
s inositolglycan antibody to inhibit insulin action on
human placental cytotrophoblasts and HC3H1 myocytes or
bovine-derived IPG action on rat diaphragm and chick
cochleovestibular ganglia suggests cross-species
conservation of many structural features. However, it is
10 important to note that although the prior art includes
these reports of A and P-type IPG activity in some
biological fractions, the purification or
characterisation of the agents responsible for the
activity was not disclosed until it was reported.in the
references below.
A-type substances are cyclitol-containing carbohydrates,
also containing Znz+ ion and optionally phosphate and
having the properties of regulating lipogenic activity
and inhibiting cAMP dependent protein kinase. They may
also inhibit adenylate cyclase, be mitogenic when added
to EGF-transfected fibroblasts in serum free medium, and
stimulate lipogenesis in adipocytes.
P-type substances are cyclitol-containing carbohydrates,
also containing Mn2+ and/or Zn2+ ions and optionally
phosphate and having the properties of regulating
glycogen metabolism and activating pyruvate dehydrogenase
phosphatase. They may also stimulate the activity of
3Q glycogen synthase phosphatase, be mitogenic when added to
fibroblasts in serum free medium, and stimulate pyruvate
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dehydrogenase phosphatase.
Methods for obtaining A-type and P-type IPGs are set out
in detail in Caro et al, 1997, and in W098/11116 and
W098/11117. Methods for obtaining the free GPI
precursors of the A and P-type IPGs are set out below.
Glycolipids were partially purified from rat liver
membranes following the method described in Mato et al
(1987), with minor modifications (Varela-Nieto et al,
1993). The membrane fraction was obtained by sequential
centrifugation from 300 g of material. Total lipids were
obtained by chloroform/methanol extraction followed by
the removal of nonpolar lipids. GPI was separated from
other phospholipids by sequential acid/base silica gel
G60 t.l.c. (see below). Lipids were extracted from the
t.l.c. plate with methanol, dried, applied to a Sep-Pak
C18 cartridge, eluted with methanol, and dried. GPI was
spotted onto the origin of a silica gel G60 t.l.c. plate
which was developed twice in an acidic solvent system
(chloroform: acetone: methanol: acetic acid: water
(50:20:10:10:5, by volume)] and in a basic solvent system
. (chloroform:methanol:ammonium hydroxide: water
(45:45:3:5:10, by volume)]. Alternatively, GPI molecules
were further resolved by double-dimension t.l.c. as
described in Clemente et al (1995) and Avila et al (1992)
or by using high-performance (HP)-t.l.c. plates developed
in chloroform: methanol: ammonium hydroxide: water
(40:45:3:5:15, by volume) as described in Gaulton (1991).
30. Lipid migration was calibrated in parallel with
phospholipid standards that were detected by staining
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with iodine. In order to generate free IPGs from the
purified GPI, the GPI was incubated with 1 unit of PI-PLC
from Bacillus thuringiensis for 3 hours according to the
manufacturer s instructions. At the end of the
incubation, the reaction was terminated by loading the
sample onto a Sep-Pak C18 cartridge. The water eluate
(5m1) was dried.
Antibodies
Antibodies capable of specifically binding to P and A-
type /PGs are disclosed herein. These antibodies can be
modified using techniques which are standard in the art.
Antibodies similar to those exemplified for the first
time here can also be produced using the teaching herein
in conjunction with known methods. These methods of
producing antibodies include immunising a mammal (e. g.
mouse, rat, rabbit, horse, goat, sheep or monkey) with
the IPG or a fragment thereof. Antibodies may be
obtained from immunised animals using any of a variety of
techniques known in the art, and screened, preferably
using binding of antibody to antigen of interest.
Isolation of antibodies and/or antibody-producing cells
from an animal may be accompanied by a step of
sacrificing the animal.
As an alternative or supplement to immunising a mammal
with an IPG, an antibody specific for an IPG may be
obtained from a recombinantly produced library of
expressed immunoglobulin variable domains, e.g. using
lambda bacteriophage or filamentous bacteriophage which
display functional immunoglobulin binding domains on
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their surfaces; for instance see W092/01047. The library
may be naive, that is constructed from sequences obtained
from an organism which has not been immunised with any of
the IPGs (or fragments), or may be one constructed using
sequences obtained from an organism which has been
exposed to the antigen of interest.
The term ~~monoclonal antibody~~ as used herein refers to
an antibody obtained from a substantially homogenous
population of antibodies, i.e. the individual antibodies
comprising the population are identical apart from
possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies can be
produced by the method first described by Kohler and
Milstein, Nature, 256:495, 1975 or may be made by
recombinant methods, see Cabilly et al, US Patent No.
4,816,567, or Mage and Lamoyi in Monoclonal Antibody
Production Techniques and Applications, pages 79-97,
Marcel Dekker Inc, New York, 1987.
In the hybridoma method, a mouse or other appropriate
host animal is immunised with the antigen by
subcutaneous, intraperitoneal, or intramuscular routes to
elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the
IPG used for immunisation. Alternatively, lymphocytes
may be immunised in vitro. Lymphocytes then are fused
with myeloma cells using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell, see
Goding, Monoclonal Antibodies: Principles and Practice,
pp. 59-103 (Academic Press, 1986). Immunisation with
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soluble IPG and via the intraperitoneal route shown in
the examples was surprisingly effective in producing
antibodies specific for IPGs.
The hybridoma cells thus prepared can be seeded and grown
in a suitable culture medium that preferably contains one
or more substances that inhibit the growth or survival of
the unfused, parental myeloma cells. For example, if the
parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient
cells.
Preferred myeloma cells are those that fuse efficiently,
support stable high level expression of antibody by the
selected antibody producing cells, and are sensitive to a
medium such as HAT medium.
Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed
against the IPGs. Preferably, the binding specificity is
determined by enzyme-linked immunoabsorbance assay
(ELISA). The monoclonal antibodies of the invention are
those that specifically bind to either or both P and A-
type IPGs.
The epitope bound by the antibodies can be mapped using
synthetic compounds known to bind to one of the deposited
antibodies, e.g. to see whether an antibody has
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substantially the same or the same binding specificity as
the deposited monoclonal antibodies 2F7, 2D1 or 5H6. This
can also be carried out in competitive studies to
determine whether a given anti-IPG antibody competes with
5 one of the deposited antibodies for a particular IPG
epitope. Thus, the present invention includes antibodies
which are capable of binding to an IPG epitope bound by
an exemplified antibody. Preferably, this epitope is
present on both rat liver A-type IPG and human placenta
10 P-type IPG. The exemplified monoclonal raised using rat
liver A-type IPGs are surprisingly effective in assays
for the diagnosis of pre-eclampsia, as shown in the
examples below.
15 In a pref erred embodiment of the invention, the
monoclonal antibody will have an affinity which is
greater than micromolar or greater affinity (i.e. an
affinity greater than 10-6 mol) as determined, for
example, by Scatchard analysis, see Munson & Pollard,
Anal. Hiochem., 107:220, 1980.
After hybridoma cells are identified that produce
neutralising antibodies of the desired specificity and
affinity, the clones can be subcloned by limiting
dilution procedures and grown by standard methods.
Suitable culture media for this purpose include
Dulbecco~s Modified Eagle s Medium or RPM1-1640 medium.
In addition, the hybridoma cells may be grown in vivo as
ascites tumours in an animal.
The monoclonal antibodies secreted by the subclones are
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suitably separated from the culture medium, ascites
fluid, or serum by conventional immunoglobulin
purification procedures such as, for example, protein A-
Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
Nucleic acid encoding the monoclonal antibodies of the
invention is readily isolated and sequenced using
procedures well known in the art, e.g. by using
I0 oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains
of murine antibodies. The hybridoma cells of the
invention are a preferred source of nucleic acid encoding
the antibodies or fragments thereof. Once isolated, the
nucleic acid is ligated into expression or cloning
vectors, which are then transfected into host cells,
which can be cultured so that the monoclonal antibodies
are produced in the recombinant host cell culture.
Hybridomas capable of producing antibody with desired
binding characteristics are within the scope of the
present invention, as are host cells containing nucleic
acid encoding antibodies (including antibody fragments)
and capable of their expression. The invention also
provides methods of production of the antibodies
including growing a cell capable of producing the
antibody under conditions in which the antibody is
produced, and preferably secreted.
Antibodies according to the present invention may be
modified in a number of ways. Indeed the term ~antibody~~
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should be construed as covering any binding substance
having a binding domain with the required specificity.
Thus, the invention covers antibody fragments,
derivatives, functional equivalents and homologues of
antibodies, including synthetic molecules and molecules
whose shape mimics that of an antibody enabling it to
bind an antigen or epitope, here a P or A-type
inositolphosphoglycan.
Examples of antibody fragments, capable of binding an
antigen or other binding partner, are the Fab fragment
consisting of the VL, VH, C1 and CH1 domains; the Fd
fragment consisting of the VH and CH1 domains; the Fv
fragment consisting of the VL and VH domains of a single
arm of an antibody; the dAb fragment which consists of a
VH domain; isolated CDR regions arid F(ab~)2 fragments. a
bivalent fragment including two Fab fragments linked by a
disulphide bridge at the hinge region. Single chain Fv
fragments are also included.
A hybridoma producing a monoclonal antibody according to
the present invention may be subject to genetic mutation
or other changes. It will further be understood by those
skilled in the art that a monoclonal antibody can be
subjected to the techniques of recombinant DNA technology
to produce other antibodies, humanised antibodies or
chimeric molecules which retain the specificity of the
original antibody. Such techniques may involve
introducing DNA encoding the immunoglobulin variable
region, or the complementarity determining regions
(CDRs), of an antibody to the constant regions, or
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constant regions plus framework regions, of a different
immunoglobulin. See, for instance, EP-A-0184187, GH-A-
2188638 or EP-A-0239400. Cloning and expression of
chimeric antibodies are described in EP-A-0120694 and EP-
A-0125023.
Pharmaceutical Compositions
The antibodies of the invention can be formulated in
pharmaceutical compositions. An example of this is
employing antibodies specific for P-type IPG in the
treatment of pre-eclampsia, a condition associated with
elevated levels of P-type IPGs. Protocols for the
treatment of pre-eclampsia using P-type IPG antagonists
are described in copending W098/10791.
The pharmaceutical compositions may comprise, in addition
to one or more of the antibodies, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or
other materials well known to those skilled in the art.
Such materials should be non-toxic and should not
interfere with the efficacy of the active ingredient.
The precise nature of the carrier or other material may
depend on the route of administration, e.g, oral,
intravenous, cutaneous or subcutaneous, nasal,
intramuscular, or intraperitoneal routes.
Pharmaceutical compositions for oral administration may
be in tablet, capsule, powder or liquid form. A tablet
may include a solid carrier such as gelatin or an
adjuvant. Liquid pharmaceutical compositions generally
include a liquid carrier such as water, petroleum, animal
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or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other
saccharide solution or glycols such as ethylene glycol,
propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active
ingredient will be in the form of a parenterally
acceptable aqueous solution which is pyrogen-free and has
suitable pH, isotonicity and stability. Those of
relevant skill in the art are well able to prepare
suitable solutions using, for example, isotonic vehicles
such as sodium chloride injection, Ringer s injection,
lactated Ringer s injection. Preservatives, stabilisers,
buffers, antioxidants and/or other additives may be
included, as required.
Preferably, the pharmaceutically useful compound
according to the present invention is given to an
individual in a "prophylactically effective amount~~ or a
~~therapeutically effective amount~~ (as the case may be;
although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual.
Typically, this will be to cause a therapeutically useful
effect in the patient, e.g. using the antibodies to
antagonise one or more of the biological activities of P
and/or A-type IPGs to a beneficial extent. The actual
amount of the antibodies administered., and rate and time-
course of administration, will depend on the nature and
severity of the.condition being treated. Prescription of
treatment, e.g. decisions on dosage etc, is within the
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responsibility of general practitioners and other medical
doctors, and typically takes account of the disorder to
be treated, the condition of the individual patient, the
site of delivery, the method of administration and other
5 factors known to practitioners. ExamniA~ ~f t~,o
techniques and protocols mentioned above can be found in
Remington~s Pharmaceutical Sciences, 16th edition, Oslo.
A. (ed), 1980.
10 Hy way of example, depending on the type and severity of
the IPG related condition, the composition can be
administered to provide an initial dose of about 0.01 to
20 mg, more preferably 0.02 to 10 mg, of antibody/kg of
patient weight. As mentioned above, other dosing
15 regimens and the determination of appropriate amount of
the antibodies for inclusion in the compositions can be
readily determined by those skilled in the art.
Inanunoa save
20 The antibodies described above can be employed in the
diagnostic aspects of the invention in a wide variety of
different assay formats. The antibodies can be used as
binding agents capable of specifically binding to IPGs or
as developing agents for determining the fraction of
binding sites of a binding agent occupied by analyte
after exposure to a test sample.
In some instances, employing the antibodies, particularly
as developing agents in assays, involves tagging them
with a label or reporter molecule which can directly or
indirectly generate detectable, and preferably
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measurable, signals. The linkage of reporter molecules
may be directly or indirectly, covalently, e.g. via a
peptide bond or non-covalently. Linkage via a peptide
bond may be as a result of recombinant expression of a
gene fusion encoding antibody and reporter molecule. Any
method known in the art for separately conjugating the
antibody to the detectable moiety may be employed,
including those methods described by Hunter et al, Nature
144:945, 1962; David et al, Biochemistry 13:1014, 1974;
Pain et al, J. Immunol. Meth. 40:219, 1981; and Nygren, J
Histochem. and Cytochem. 30:407, 1982.
One favoured mode is by covalent linkage of each antibody
with an individual fluorochrome, phosphor or laser dye
with spectrally isolated absorption or emission
characteristics. Suitable fluorochromes include
fluorescein, rhodamine, luciferin, phycoerythrin and
Texas Red. Suitable chromogenic dyes include
diaminobenzidine. Other detectable labels include
radioactive isotopic labels, such as 3H, 14C, 'ap~ 3sS~ ~asl~
or 99mTC, and enzyme labels such as alkaline phosphatase,
p-galactosidase or horseradish peroxidase, which catalyze
reactions leading to detectable reaction products and can
provide amplification of signal.
Other reporters include macromolecular colloidal
particles or particulate material such as latex beads
that are coloured, magnetic or paramagnetic, and
biologically or chemically active agents that can
directly or indirectly cause detectable signals to be
visually observed, electronically detected or otherwise
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recorded. These molecules may be enzymes which catalyze
reactions that develop or change colour or cause changes
in electrical properties, for example. They may be
molecularly excitable, such that electronic transitions
between energy states result in characteristic spectral
absorptions or emissions. They may include chemical
entities used in conjunction with biosensors.
Alternatively or additionally, the antibodies can be
employed as binding agents, relying on the fact that the
antibodies are capable of specifically binding the IPGs
in preference to other substances present in the sample.
Conveniently, the binding agents) are immobilised on
solid support, e.g. at defined locations, to make them
easy to manipulate during the assay. This can be
achieved using techniques well known in the art such as
physisorption or chemisorption, e.g. employing
biotin/avidin or biotin/streptavidin to chemically link
the antibodies to the solid support. Generally, the
sample is contacted with the binding agents) under
appropriate conditions so that P and A-type IPGs present
in the sample can bind to the binding agent(s). The
fractional occupancy of the binding sites of the binding
agents) can then be determined using a developing agent
or agents.
As mentioned above, the developing agents are labelled
(e.g. with radioactive, fluorescent or enzyme labels) so
that they can be detected using techniques well known in
the art. Thus, radioactive labels can be detected using
a scintillation counter or other radiation counting
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23
device, fluorescent labels using a laser and confocal
microscope, and enzyme labels by the action of an enzyme
label on a substrate, typically to produce a colour
change. The developing agents) can be used in a
competitive method in which the developing agent competes
with the analyte for occupied binding sites of the
binding agent, or non-competitive method, in which the
labelled developing agent binds analyte bound by the
binding agent or to occupied binding sites. Both methods
provide an indication of the fraction of the binding
sites occupied by the analyte, and hence the
concentration of the analyte in the sample, e.g, by
comparison with standards obtained using samples
containing known concentrations of the analyte.
Diagnostic assays can be carried out with a biological
sample from a patient. These samples can be used
directly or in some instances may require treatment prior
to carrying out the assay, e.g. to remove potentially
20 interfering substances in the sample. Examples of
suitable biological samples are blood, urine, sweat,
tissue or serum.
In one embodiment, the present invention concerns a
25 method a diagnosing patients at risk of developing type I
diabetes, the method comprises the steps of:
(a) contacting a biological sample obtained from
the patient with a solid support having immobilised
thereon antibodies capable of specifically binding to P
and/or A-type IPGs;
(b) contacting the solid support with a labelled
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developing agent capable of binding to unoccupied binding
sites of the antibodies, bound IPGs or occupied binding
sites of the antibodies; and,
(c) detecting the label of the developing agent
specifically binding in step (b) to obtain a value
representative of the concentration of the IPGs in the
sample.
In a further embodiment, the present invention provides a
method for diagnosing pre-eclampsia in a patient, the
method comprising:
(a) contacting a biological sample from the patient
with an anti-IPG antibody of any one of claims 2 to 5;
and,
(b) determining the binding of IPGs in the sample
to the anti-IPG antibody.
Typically, the binding of IPGs in the sample to the anti-
IPG antibody is compared to the amount of binding that
takes place in controls, e.g. from a non-diabetic, or
from a person known to have a particular type of
diabetes, e.g a patient known to have developed type I
diabetes or pre-eclamptic standards. In the protocols
exemplified below, this is assessed by initially
providing the patient with a glucose load and measuring
the IPG response following the glucose load over time:
The IPG profiles or levels determined from a sample can
then be compared with the profiles or levels obtained
from standards to make the diagnosis.
Thus, preferably, the method includes the further step of
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correlating the value representative of the concentration
of the IPGs in the sample to values obtained from known
standards to determine whether the patient is at risk
from developing type I diabetes or has pre-eclampsia.
5
The antibodies of the present invention may be employed
in any known assay method, such as competitive binding
assays, direct and indirect sandwich assays, and
immunoprecipitation assays, see Zola, Monoclonal
10 Antibodies: A Manual of Techniques, pp 147-158 (CRC
Press, Inc, 1987).
Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or
15 epitope, of the IPG to be detected. In a sandwich assay,
the test sample analyte is bound by a first antibody
which is immobilised on a solid support, and thereafter a
second antibody binds to the analyte, thus forming an
insoluble three part complex. The second antibody may
20 itself be labelled with a detectable moiety (direct
sandwich assays) or may be measured using an anti-
immunoglobulin antibody that is labelled with a
detectable moiety (indirect sandwich assay). For
example, one type of sandwich assay is an ELISA assay, in
25 which case the detectable moiety is an enzyme.
The antibodies of the invention also are useful for in
vivo imaging, wherein an antibody labelled with a
detectable moiety for example radioisotope and is
administered to a host, preferably into the bloodstream,
and the presence and location of the labelled antibody in
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26
the host is assayed. This antibody may be labelled with
any moiety that is detectable in a host, whether by
nuclear magnetic resonance, radiology, or other detection
means known in the art.
Affinitv Purification
The antibodies of the invention also are useful as
affinity purification agents. In this process, the
antibodies are immobilised on a suitable support, such as
Sephadex~ resin or filter paper, using methods well known
in the art. The immobilized antibody then is contacted
with a sample containing the IPGs to be purified, and
thereafter the support is washed with a suitable solvent
that will remove substantially all the material in the
sample except the IPGs, which is bound to the immobilised
antibody. Finally, the support is washed with another
suitable solvent, such as glycine buffer, pH 3-5, that
will release the IPGs from the antibody. This method can
also be used to separate a given IPG family member from a
mixture of IPGs by using an antibody capable of
specifically binding that IPG in preference to other
family members.
Example 1
Production of Polyclonal and Monoclonal Antibodies
Against Inositolphosphoglycans (IPGa)
Inositolphosphoglycan (soluble form) obtained by PI-PLC
treatment of GPI purified from rat liver by sequential
Thin Layer Chromatography (TLC) was used to immunize New
Zealand rabbits and Halb/c mice as described below.
Alternatively, human IPGs could be obtained using the
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methods described in Caro et al, 1997.
Rabbit Immunisation Procedure
Two New Zealand rabbits were anaesthetized and then
immunised with 750ug of IPG (soluble form) mixed in 1m1
of PHS with 1m1 of complete Freund~s adjuvant (CFA). The
antigen-adjuvant emulsion was administered 1.5m1 by
intradermal (id) injection and 0.5m1 by intramuscular
(im) injection.
After one month, this protocol was repeated except that
incomplete Freund~s adjuvant (IFA) was used, and 1.5m1 by
administered by subcutaneous (sc) injection and 0.5m1 by
intramuscular (im) injection. This was repeated again on
days 60, 90, 120 and 150.
Mouse Immunisation Procedure
Four female Balb/c 6 weeks old mice were immunised with
60pg of IPG (soluble form) in 250u1 of PHS with 250u1 of
CFA. The antigen-adjuvant emulsion was injected by
intraperitoneal (ip) injection.
After 21 days, the injection was repeated except that IFA
was used. On days 42 and 63 all the animals were
injected ip with IFA. On day 84, the best responder was
injected 100u1 PHS containing 60ug of IPG intravenous
(iv) and 100u1 PBS containing 60ug of IPG (ip). After 87
days, splenocytes from best responder were fused to
myleoma cells using conventional techniques. Monitor
test bleeds were realized regularly.
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Example 2
Indirect ELISA.Assay
The following indirect ELISA protocol was used to screen
the monoclonal antibodies.
Add 100u1/well in all the steps.
(1) Add IPG diluted 1:800 in PBS in a F96 Polysorp Nunc-
Immuno plate. Incubate at least 7 days at 4°C.
(2) Wash with PBS three times.
(3) Add a blocking reagent for ELISA (Boehringer
Mannheim) in distilled water (1:10 v/v) for 2 hours at
room temperature.
(4) Wash with PBS-Tween 20 (0.1%) three times.
(5) Add a purified monoclonal and/or a polyclonal
antibody (diluted between 1:12.5 and 1:3200 in PBS),
overnight at 4°C.
(6) wash with PBS-Tween 20 (0.1%) three times.
(7) Add an anti-mouse IgM, biotinylated whole antibody
(from goat) (Amersham) in case of the previous addition
of a monoclonal antibody, or add an anti-rabbit Ig,
biotinylated species-specific whole antibody (from
donkey) (Amersham), in case of previous addition of a
polyclonal antibody, both of them diluted 1:1000 in PBS,
1 h 30 min at room temperature.
(8) Wash with PBS-Tween 20 (0.1%) three times.
(9) Add a streptavidin-biotinylated horseradish
peroxidase complex (Amersham) diluted 1:500 in PBS, 1 h
min at room temperature.
(10) Wash with PBS three times.
30 (11) Add 2,2-Azino-di-(3-ethylbenzthiazoline sulfonate
(6)) diammonium salt crystals (ARTS) to buffer for ABTS
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(Boehringer Mannheim): Buffer for AHTS is added to
distilled water (1:10 v/v). 1mg of ARTS is added to lml
of diluted buffer for ABTS.
(12) Read the absorbance in a Multiskan Plus P 2.01 using
a 405 nm filter in 5-15 min.
Comments:
Solvents used are phosphate-buffered saline (PHS) pH 7.2
or PBS-Tween 20 (0.1~).
Step 1: IPG (soluble form) is used in an indirect ELISA
(initial concentration approximately 50 uM). Working
dilutions are normally between 1:400 and 1:800.
Several incubation times have been examined. The best
and more reproducible results have been obtained when
incubation time is at least seven days at 4°C, probably
because higher incubation times increase the number of
molecules of IPG that are bound to the solid phase.
Step 3: Blocking reagent for ELISA is a registered
product of Boehringer Mannheim. In order to prepare the
solution we must dissolve the content (27g) in 100 ml
redist. Water at room temperature while stirring for
approx 30 minutes. The concentrate is stable when stored
in aliquots at -20°C. For use, dilute one aliquot in a
ratio of 1:10 with redistilled water to yield a working
solution containing 1~ protein mixture (w/v) that was
obtained by proteolytic degradation of purified gelatin,
in 50mM Tris-HC1, 150mM NaCl, pH ca.7.4.
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Step 5: A purified monoclonal and/or polyclonal antibody
are used in an indirect ELISA (initial concentration
approx 2mg/ml). Working dilutions are normally between
1:100 and 1:200, getting final concentrations of 20 ug/ml
5 and 10 pg/ml respectively).
Incubation time for rapid assays can be two hours at room
temperature.
10 Step 7: The anti-rabbit and the anti-mouse antibodies are
used at 1:1000 dilution straight from the stock bottle.
The stock bottle is stored at 2-8°C. Under these
conditions the product is stable for at least six months,
after which any remaining solution is discarded.
iml of biotinylated reagent is supplied in 0.1 M
phosphate-buffered saline of pH7.5 containing 1% (w/v)
bovine serum albumin and 0.05% (w/v) sodium azide.
Step 9: Streptavidin-biotinylated/HRP is used at 1:500
dilution straight from the stock bottle. The stock
bottle is stored at 2-8°C. Under rhAQO ~~ra;~;..~,..
product is stable for at least three months, after which
any remaining solution is discarded.
The complex is supplied in 2m1 of phosphate-buffered
saline pH7.5 containing 1% (w/v) bovine serum albumin and
an antimicrobial agent.
Y Step 11: ARTS buffer is a registered product of
Boehringer Mannheim. The solution consists of sodium
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perborate, citric acid and disodium hydrogen phosphate.
The solution is stored in aliquots of 1ml at -20°C. For
use, dilute one aliquot in a ratio of 1:10 with
redistilled water.
ExamDl~
Sandwich ELISA Aeeay
The following ELISA protocol is provided as an example of
an assay for IPGs employing the deposited monoclonal
antibodies.
Add 100 ul/well in all the steps.
(1) Anti-IPG monoclonal antibody was diluted 1:100 in
PBS in added to a F96 Maxisorp Nunc-Immuno plate. The
plate was incubated for at least 2 days at 4°C.
(2) The plate was washed with PBS three times.
(3) Blocking reagent for Elisa (Boehringer Mannheim) in
distilled water (1.5:10 v/v) was added for 2 hours at
room temperature.
(4) The plate was washed with PBS-Tween 20 (0.1~) three
times.
(5) Human serum diluted 1:4 in PBS was added to the
plate overnight at 4°C.
(6) The plate was wash with PHS-Tween 20 (0.1~) three
times.
(7) A purified polyclonal antibody (diluted 1:100 in
PBS) was added overnight at 4°C.
(8) The plate was washed with PBS-Tween 20 (0.1~) three
times.
(9) An anti-rabbit Ig, biotinylated species-specific
whole antibody (from donkey )(Amersham) was diluted
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1:1000 in PHS. and added to the plate for 1 h 30 min at
room temperature.
(10) Wash with PBS-Tween 20 (0.1%) three times.
(11) Add a streptavidin-biotinylated horseradish
peroxidase complex (Amersham) diluted 1:500 in PBS, 1 h
30 mires at room temperature.
(12) The plate was washed with PBS three times.
(13) 2,2~-Azino-di-(3-ethylbenzthiazoline sulfonate (6))
diammonium salt crystals (ARTS) was made up in buffer for
ARTS (Boehringer Mannheim). The buffer for ARTS was
added to distilled water (1:10 v/v). lmg of ABTS was
added to 1ml of diluted buffer for ABTS.
(14) The absorbance was read in a Multiskan Pius P 2.01
using a 405 nm filter in 15-30 mires.
CommentB:
The solvents used were phosphate-buffered saline (PBS)
pH7.2 or PeS-Tween 20 (0.1%).
In step (1): The examples employed purified monoclonal
antibody 2D1 in the Sandwich ELISA assays (initial
concentration 2.5mg/ml). Working dilutions varied
normally between 1:100 and 1:200, with final
concentrations of 25pg/ml and 12.5ug/ml, respectively.
The incubation time used in the assay can be varied. For
rapid assays, incubation time can vary between two hours
at room temperature or overnight at 4°C. However.
improved results are obtained when incubation time is at
least two days at 4°C. This improvement may be due to
the higher incubation times increasing the number of
molecules of monoclonal antibody that are bound to the
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solid phase.
Step (3): Blocking reagent for ELISA is a registered
product of Boehringer Mannheim. In order to prepare the
solution, the content (27g) was dissolved in 100m1
redistilled water at room temperature while stirring for
approximately 30mins. The concentrate is stable when
stored in aliquots at -20°C. For use, aliquots were
diluted in a ratio of 1:10 with redistilled water to
yield a working solution containing 1~ protein mixture
(w/v) that was obtained by proteolytic degradation of
purified gelatin, in 50 mM Tris-HC1, 150 mm NaCl, pH ca.
7~.4.
Step (5): Human serum was probed in several dilutions
(1:1, 1:2, 1:4...). Working dilutions are normally
between 1:2 and 1:4. IPG in several dilutions was used
as a standard (positive control). Wells without serum
are used as negative control.
The incubation time for rapid assays was two hours at
room temperature.
Step (7): A purified polyclonal antibody was used in~
Sandwich ELISA (initial concentration 2 mg/ml). Working
dilutions were between 1:100 and 1:200, with final
concentrations of 20 ug/ml and 10 ug/ml respectively.
Step (9): The anti-rabbit was used at 1:1000 dilution
straight from the stock bottle, stored at 28°C. Under
these conditions the product is stable for at least six
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34
months.
lml of biotinylated reagent is supplied in 0.1 M
phosphate-buffered saline pH 7.5 containing 1~ (w/v)
bovine serum albumin and 0.05 (w/v) sodium azide.
Step (11): Streptavidin-biotinylated/HRP was used at
1:500 dilution straight from the stock bottle, stored at
2-8°C. Under these conditions the product is stable for
at least three months.
The complex is supplied in 2m1 of phosphate-buffered
saline pH 7.5 containing 1~ (w/v) bovine serum albumin
and an antimicrobial agent.
Step (13): ARTS buffer is a registered product of
Hoehringer Mannheim. The solution consists of sodium
perborate, citric acid and disodium hydrogen phosphate.
The solution is stored in aliquots of lml at -20°C. For
use, one aliquot is diluted in a ratio of 1:10 with
redistilled water.
Figure 1 shows a dose-response curve of assay response
(absorbance) plotted against IPG concentration,
demonstrating that the antibodies can be used to assay
for IPGs.
Example 4
Assay for Type I Diabetes and Controls
The protocol set out in example 3 was used to measure IPG
levels in a different patients in response to a glucose
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load (75g). Figures 2 to 4 plot the changes in IPG
levels in the patients over time after the glucose load.
The profiles shown in the figures and/or the levels of
the IPGs can be used in the diagnosis of the risk of
5 developing type I diabetes by comparing them with the
profile or level obtained from a sample from a patient
suspected of being at risk of developing type I diabetes.
In particular, examining the change in IPG levels has the
advantage that it isolates the change in IPG level caused
10 by the glucose load from the effect of IPGs acting as
second messengers for other growth factors in the
patient.
Figure 2 shows the response obtained in a control patient
15 with no familial history of diabetes. -Thus, after
administration of the glucose, the IPG level in the
patient rose to a maximum around 2 hours after the load,
and then fell again to the background level.
20 Figure 4 shows a second control profile obtained from a
patient with carbohydrate intolerance, i.e. who was
unable to metabolise the glucose via insulin and
subsequent IPG production. Thus, the IPG profile
following the glucose load is approximately flat.
Figure 3 shows a profile obtained from a patient with a
familial history of type I diabetes. No elevation in IPG
is observed indicating that there has been an inadequate
release of insulin in response to the glucose challenge.
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Example 5
Properties of the Anti-IPG Antibody 2D1
The properties of monoclonal antibody 2D1 were further
investigated in CVG culture, measuring cellular
proliferation in presence of 2D1 anti-IPG antibody, with
and without IGF-I.
CVGs were isolated from E3.5 chicken embryos as described
in Varela-Nieto et al, 1991, and cultured for 24 hours in
4-well multidishes (NUNC) in 0.25 ml of M199 culture
medium with Earle~s.salts, 2mM L-glutamine, 25mM HEPES
and antibiotics. Incubations were carried out at 37°C in
a humidified atmosphere with 5~ CO2.
The photomicrographs of figure 5 show CVG proliferation
under the following conditions: control medium with no
additions (A), 25ug/ml 2D1 antibody (B), 1nM IGF-I alone
(C), 1 nM IGF-1 plus 25pg/ml 2D1 (D). The exogenous
addition of 100 uM of synthetic compound (3-methyl-3,4-
bis(disodium phosphate) galactose (GP2) that alone do not
stimulate CVG growth (E), was able to rescue the 2D1
inhibitory effect on CVG growth induced by IGF-I (F).
IPG type-A (1/100), that alone has a slight effect on
CVG-growth (G), completely recovered the growth
inhibitory effect caused by the 2D1 antibody (H).
Figure 6A shows percent of inhibition of 2D1 antibody on
CVG cultured in presence of IGF-I, in these experiments,
CVGs were isolated as described above. The DNA synthesis
was determined by culturing the CVG explants in M199
medium containing 10 uCi/ml of ['H]thymidine of 24 hours.
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Radioactivity incorporated by the explants was extracted
with 10~ trichloroacetic acid and quantified by
scintillation counting. (A) Percent of inhibition in
CVGs cultured with 1 nM IGF-I in presence of increasing
concentrations of 2D1 antibody from 25 to 100 ug/ml. (B)
Acid-precipitable ['HJ thymidine uptake into CVGs cultured
in parallel experiments with the additions indicated in
Figure 5 (A-H). The exogenous addition of 100 uM of
synthetic analogue C3 was unable to rescue the 2D1
inhibitory effect on CVG growth induced by IGF-I (I).
Data are representative of at least 3 different
experiment with an average of 4 CVGs per condition.
Examvle 6
Properties of Anti-IPG Antibody 5H6
As discussed above, a prior art polyclonal antibody
raised against the GPI-anchor of VSG was found to cross-
react with IPG containing fractions. Figure 7 shows that
a polyclonal antibody raised against IPGs cross reacted
with GPI-anchors. Thus, these results are consistent and
demonstrate that there is some cross reactivity between
the IPG and GPI anchors. In contrast, when the
reactivity of monoclonal antibody 5H6 towards soluble and
membrane bound VSG was tested, figure 7 (left hand
column) shows that it does not cross react in the same
way as the polyclonal antibody raised against IPGs
(middle column). In these experiments, a polyclonal
antibody raised against the common reactive determinant
(CRD) common to GPI anchored proteins was used as a
positive control (right hand column).
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Thus, figure 7 shows that antibody 5H6 does not cross
react with either soluble or membrane bound VSG, whereas
both polyclonal antibodies react strongly. This property
distinguishes the antibodies of the invention from cross
reacting polyclonal antibodies. This in turn suggests
that polyclonal antibodies will not specifically detect
IPGs, but are a valuable reagent in ELISA sandwich assays
which require two anti-IPG antibodies, one of which is
very specific (monoclonal) and the other of general
reactivity (polyclonal).
Figure 8 shows that monoclonal antibody 5H6 is able to
inhibit the action of P-type IPG from rat liver in a P-
type phosphatase assay. Thus, this and similar
antibodies could be used as P-type IPG antagonists, e.g.
in the treatment of conditions associated with elevated
levels of P-type IPGs such as pre-eclampsia.
Example 8
Assay for Urine IPG Levels (e. g, for use in the diagnosis
of pre-eclampsia)
The following assay was carried out using anti-IPG
monoclonal 2D1-the capture antibody and polyclonal rabbit
IgG anti-IpG as the secondary antibody. The assay was
carried out using samples from pre-eclamptic and control
pregnant patient urine samples.
A Maxisorb plate was coated with 50m1 of 2D1 antibody at
2.5mg/ml in PHS and incubated overnight at 4°C. The
plate was then blocked with 200m1 of blocking reagent and
incubated for lh at 37°C in a sealed container floating
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on a waterbath.
50m1/well of the urine sample diluted 20 fold in blocking
reagent was added and incubated for 2h at 37°C. The
wells were then emptied and washed 5 times with
approximately 100m1 of 0.05 Tween20/PHS. 50m1/well of
2mg/ml polyclonal rabbit IgG anti-IPG diluted 1/500 in
blocking reagent was then added and incubated for 1.5h
37°C. The wells were then washed 5 times with 100m1 of
0.05 Tween20/PBS and 50m1/well of goat anti-rabbit IgG-
HRP diluted 1/6000 in blocking reagent was added. This
was left to incubate for lh at 37° C and wells washed
again before adding 50m1 of room temperature pre-warmed
TMB solution/well. This was allowed to incubate~for 20
minutes at room temperature, after which the colour
reaction was topped with the addition of 50m1 of iM HC1.
The result of the assay was obtained by reading
absorbance at 450nm.
Figure 9 shows results from the assay comparing two
samples from pre-eclamptic patients with non-pre-
eclamptic controls. Figure 10 shows the results obtained
from a blinded study of 24 samples, 12 from pre-eclamptic
patients. This shows that the assay successfully
identified 10/12 of the pre-eclamptic samples with no
false positives.
Figure 11 shows the correlation between platelet counts
and the reaction of 2D1 monclonal antibody with urine
from pre-eclamptic women. The inverse correlation is
significant p < 0.05. In pre-eclamptic women, there is a
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decrease in platelet counts. The figure demonstrates
that the higher the level of 2D1 reactive material, the
lower the platelet counts, suggesting that the 2D1
antibody is detecting a factor which correlates with
5 pathology in pre-eclamptic women.
Figure 12 shows the correlation between plasma aspartic
transaminase (AST) and the binding reactivity of 2D1 to
urine from pre-eclamptic women. AST (liver derived)
10 levels are elevated in women with pre-eclampsia and the
correlation suggests that the IPG levels may correlate
with pathology in the disease.
Deposit
15 The deposit of hybridomas 2F7, 2D1 and 5H6 in support of
this application was made at the European Collection of
Cell Cultures (ECACC) under the Budapest Treaty by
Rademacher Group Limited (RGL), The Windeyer Building, 46
Cleveland Street, London WiP 6D8, UK. The deposits have
20 been accorded accession numbers accession numbers
98051201, 98031212 and 98030901. RGL give their
unreserved and irrevocable consent to the materials being
made available to the public in accordance with
appropriate national laws governing the deposit of these
25 materials, such as Rules 28 and 28a EPC. The expert
solution under Rule 28(4) EPC is also hereby requested.
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41
Ref erences :
The reference mentioned herein are all incorporated by
reference in their entirety.
Caro et al, Biochem. Molec. Med., 61:214-228, 1997.
Kunjara et al, In: Hiopolymers and Bioproducts:
Structure, Function and Applications, Ed Svati et al,
301-305, 1995.
Rademacher et al, Brazilian J. Med. Biol. Res., 27:327-
341, 1994.
Nestler et al, Endocrinology, 129:2951-2956, 1991.
Romero et al, P.N:A.S., 87:1476-1480, 1990.
Huang et al, Endocrinology, 132:652-657, 1993.
Represa et al, P.N.A.S., 88:8016-8019, 1991.
Varela-Nieto et al, Dev. Biol., 143:432-435, 1991.
Mato et al, J. Biol. Chem., 262:2131-2137, 1987.
Clemente et aI, Cell Signalling, 7:411-421, 1991.
Vareia-Nieto et al, In Handbook of Endocrine Research
Techniques: Intracellular Mediators of Peptide Hormone
Action: Glycosylphospatidylinositol/Inositol
Phosphoglycan System, ed de Pablo et al, San Diego:
CA 02321113 2000-08-18
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42
Academic Press, 391-406, 1993.
Avila et al, Hiochem. J., 282:681-686, 1992.
Gaulton, Diabetes, 40:1297-1304, 1991.
W098/011116 and W098/011117 (Rademacher Group Limited)
