Note: Descriptions are shown in the official language in which they were submitted.
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PANCREAS ELASTASE 1-SPECIFIC ANTIBODY, A PROCESS FOR
OBTAINING IT, AND A TEST RIT CONTAINING SUCH ANTIBODY
This invention relates to a highly sensitive and selective
antielastase 1 antibody, a process for its manufacture, and
a highly sensitive diagnostic test kit containing said
antibody.
Instances of inflammatory diseases of the pancreas are
constantly increasing in industrial countries (W. Rosch,
Deutsches Arzteblatt 84: C-397-398, 1987). These diseases
usually have an intermittent course and can finally lead to
complete loss of the gland. Acute episodes are recognizable
by severe abdominal pain and nausea, but intermediate phases
are usually experienced by the patient as free from pain.
They evolve only with uncharacteristic digestive complaints,
so that they are hard to recognize. Consideration is
therefore to be given to a chronic pancreatic disease in all
digestive disorders.
Determination of the serum amylase level has hitherto
usually been made in laboratory diagnoses of pancreatitis.
However, an elevation in serum amylase also occurs in other
intra-abdominal inflammations, e.g., in intestinal
perforation, mumps or renal failure. Moreover, an elevation
in the serum amylase level may also be observed following
the administration of morphines. Another laboratory
diagnostic possibility consists of determining the ratio of
amylase to creatinine clearance, which ratio increases in
acute pancreatitis. Unfortunately, amylase values elevated
in acute pancreatitis normalize very rapidly, so that normal
values are already found 48 hours after the onset of the
disease in one-third of the patients (J. A. Eckfeldt et al.,
Arch. Pathol. Lab. Med. 109:316-319, 1985).
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Lipase determination represents another diagnostic
possibility. However, determination of either lipase or
amylase is not suitable for detecting chronic pancreatitis.
This disease has hitherto only been insufficiently
demonstrated by determining the activity of the pancreatic
enzyme chymotrypsin in stools. The disadvantage of this
method of determination is based on the fact that only a
small part of the chymotrypsin excreted by the pancreas is
detectable in the stool, which part, moreover, is also
subject to very considerable fluctuations (Goldberg et al.,
Gut 10:477-483, 1969). This makes the determination of
normal values extremely difficult.
It is known from A. Sziegoleit, Biochem. J. 219:735-742,
1984, that pancreatic elastase 1 (E1), also called protease
E, is exclusively formed in the pancreas and is separated
out in the duodenum with digestive juice. Attempts have
already been made to determine the level of elastase 1 in
the stool to avoid the above-mentioned disadvantages, since
the level of this enzyme in the stool represents the
exocrine function of the pancreas substantially better than
does the chymotrypsin activity (A. Sziegoleit et al., Clin.
Biochem. 22:85-89, 1989).
It was also found that acute pancreatitis can be detected by
determining E1 in serum (A. Sziegoleit et al., Clin.
Biochem. 22:79-83, 1989).
It has hitherto been assumed that, unlike other enzymes, E1
is not degraded, or only unsubstantially degraded, during
intestinal passage. Its level in the stool accordingly
indicates the degree of pancreatic exocrine function.
Moreover, the enzyme also enters the blood stream in acute
pancreatic disease phases.
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A radioimmunologic test is already available for measuring
serum elastase 1 (A. Murata et al., Enzyme 30:29-37, 1983;
Elastase-1-RIA-Kit, Abbott Diagnostic).
However, such a radiologic (RIA) determination presents a
disadvantage, in that the radioactive reagents have only
limited stability and therefore must continuously be
resynthesized. Moreover, the radioactive material must be
disposed of carefully, and the measurement of radioactive
materials requires specially trained personnel and special
laboratory equipment. In addition, it is not possible, or
is only insufficiently possible, to determine the E1 level
in the stool using this test.
The object of the invention is consequently that of
developing a test process with which human elastase 1 can be
determined for the diagnosis of both acute and chronic
pancreatitis, and which is sufficiently sensitive for
determining elastase 1 in serum and stools.
According to this invention, this object is achieved by
means of an antibody directed against the epitope having the
amino acid sequence Thr-Met-Val-Ala-Gly-Gly-Asp-Ile-Arg.
Surprisingly, it was found that antibodies directed against
this epitope of human elastase 1 selectively recognize the
marker enzyme and thereby discriminate against other
antigens. .
Accordingly, this invention also relates to a process for
preparing anti-elastase antibodies in a known way,
characterized by the fact that the previously defined
epitope is used as an antigen. It is also possible,
according to the invention, to use parts and fragments
of this epitope for immunization or preparation of
antibodies, provided the parts and fragments engender an
immune response. Such fragments are obtainable either
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synthetically or by chemical and/or biological degradation
of elastase 1. In the process of the invention, it was
found suitable to bind the peptide or peptide part to a
carrier, by means of a spacer if necessary. Suitable
carriers are known to those skilled in the art and are, for
example, synthetic and natural membrane parts,
polysaccharides, peptides or proteins. Albumins and
hemocyanins are especially preferable. The spacers to be
used are also known to those skilled in the art. With the
epitope or its fragments, it is possible, according to the
invention, to obtain both selective monoclonal and
polyclonal antibodies. Antibodies preferred according to
the invention are able to recognize paraffin-embedded thin
sections.
Antisera containing antibodies according to the invention
are obtained by immunizing experimental animals with highly
purified human elastase 1 or fragments of this enzyme.
Experimental animals such as mice, rats, rabbits, goats or
horses are thereby immunized in a known way, and antisera
with polyclonal antibodies are thus obtained from which
antibodies according to the invention are also obtainable in
a known way. Antibodies preferred according to the
invention are able to recognize paraffin-embedded thin
sections.
In a preferred embodiment, monoclonal antibodies are
suitably obtained by means of the epitope according to the
invention using the method of G. Kohler and C. Milstein
(Nature 256:495-497, 1975).
A further object of the invention is consequently a
monoclonal antibody specifically capable of binding with E1.
Such an antibody is obtainable by immunizing mice or rats
with highly purified E1 or the epitope to be used according
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to the invention, fusing a-lymphocytes from the spleens of
immunized animals with myeloma cells, cloning the hybridoma
cells formed, cloning and culturing hybridoma cells which
secrete antibodies capable of binding E1, and then obtaining
5 the monoclonal antibodies formed by them.
It is especially preferable to use a cell line which does
not itself produce any immunoglobulin.
The monoclonal antibodies obtainable according to the
invention do not react with other substances, but are
specific for E1. The monoclonal antibodies according to the
invention are preferably able to recognize paraffin-embedded
thin sections.
Antibodies preferred according to the invention are
obtainable from hybridoma cell lines filed with the European
Collection of Animal Cell Cultures (ECACC), Vaccine Research
and Production Laboratory, Public Health and Laboratory
Service, Center for Applied Microbiology and Research,
Porton Down, GB Salisbury, Wiltshire SP4 OJG, on Dec. 21,
1990, which were given application numbers 90 121 90 6 and
90 121 90 7. Both antibodies obtainable from these cell
lines are able to recognize paraffin-embedded thin sections.
Another object of the invention is the use of the El-
specific antibodies according to the invention for the
qualitative and/or quantitative determination of E1. It is
accordingly possible to specifically detect elastase 1 in
bodily fluids and stools with the use of the antibody. The
invention therefore also relates to a test kit containing
antibodies according to the invention, especially
immunologic test kits for the diagnosis and course
monitoring of chronic pancreatitis, acute pancreatitis and
mucoviscidosis in bodily fluids and/or stools. Suitable
bodily fluids are blood, plasma and serum.
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Indirect, competitive and sandwich ELISAs were introduced in
experiments for detecting E1 in blood, plasma, serum or
stools. However, it was found that a sandwich ELISA is most
suitable for rapid diagnosis on a large specimen scale,
since it is independent of other serum factors not capable
of being calculated. At least two different monoclonal
antibodies directed against different epitopes of the enzyme
are necessary for this purpose.
Enzyme value changes in serum or stools, for example, can be
demonstrated with such tests, especially the appearance of
these displacements in the event of changes in pancreas
status.
Determination processes based on the immunoassay principle
have been widely developed. Advantages of these
determination methods include precision and rapidity (great
reliability and sample processing) as well as the
possibility of being able to detect very small quantities of
substance (in the nanogram range). Various process variants
are possible for conducting the determination, with both
homogeneous and heterogeneous phases. In the embodiment
with the heterogeneous phase, one of the receptors is bound
to a carrier. In the sandwich process, for example, a fist
antibody is bound to the carrier as a receptor, or a so-
called catcher, and the test solution is added, whereby the
antigen to be determined in the test solution is fished out
and bound. A second tagged antibody is then added, which
reacts specifically with the antigen or antigen-receptor
complex. With the aid of a calibrated solution (isolated,
purified human elastase 1), it is then possible to determine
the quantity of antigen by tagging the second antibody.
In another preferred embodiment of the invention, a first
antibody is bound as a receptor to a carrier matrix of
membrane, tissue, or flowing structure, so that it does not
CA 02088354 1999-06-16
represent the usual floor of the depression of an ELISA
immunoplate, but is instead present as bound to the matrix.
Preferred matrices are microporous flat membranes or hollow
fiber membranes provided in a special embodiment with ion
exchange groups. Microporous flat membranes such as those
marketed by Pall Corp., New Jersey, USA, for example, are
preferably used for this purpose. Hollow fiber membranes to
be used according to the invention are also available on the
market, and sold, for example, by Sepracor Inc. Mass., USA.
It is possible to develop particularly rapid and
uncomplicated detection processes by means of such carrier
materials.
Many variation possibilities exist for this general
principle. For example, it is possible to make a
determination with three receptors, whereby one of the three
receptors is present in the heterogeneous phase, and the
other two receptors are soluble. One of the two soluble
receptors is tagged, whereas the other is untagged. The
soluble receptor is then directed against the untagged
receptor.
The use of the E1-specific antibody according to the
invention for the selective quantitative determination of E1
based on the immunoassay principle is done by incubation
with at least two different receptors. Both receptors,
e.g., monoclonal antibodies, must be specific for E1, which
must be bound to different epitopes (binding sites) in all
cases.
One of the two receptors is bound to a solid phase. The
binding to the solid phase is done in the usual way, as
known to those skilled in the art. In addition, at least
one other receptor is used, present in soluble form.
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This other receptor bears a label. If several receptors are
used, only one of them carries a label. Receptor tagging is
done in a usual way, known to those skilled in the art.
S Tagging in a test kit according to the invention is done in
a known way, especially by radioactive tagging, binding of
biotin (biotin/avidin), by an enzyme releasing a measurable
reaction, or by a chemiluminescent or fluorescent compound.
Tagging with an enzyme is especially preferred, particularly
with peroxidase or phosphatase. In a special embodiment,
tagging with an enzyme also permits the introduction of this
antibody into a second enzyme amplification system (C. J.
Stanley; F. Paris; A. Plumb; A. Webb; A Johansson, American
Biotechnology Laboratory: May-June 1985; C.H. Self, J.
Immunol. Meth. 1985).
In an especially preferred embodiment of this process,
either a receptor capable of binding unspecifically to E1 or
preferably a receptor capable of binding specifically to E1
is bound to a solid phase. This receptor bound to the solid
phase is then incubated with the solution containing the E1
to be determined and an antibody which is specifically
capable of binding with E1, present in soluble form, and
bears a label.
If the receptor bound to the solid phase is capable of
unspecifically binding with E1, not only E1 but other
antigens also form a complex with the solid phase. The
second antibody, which is capable of specifically binding to
E1, nevertheless forms a complex only with E1, so that only
E1 molecules specifically bear a tagged antibody; whereas
other antigens are not labeled. After separation of the
solid from the liquid phase, it is possible to determine the
E1 content in this way by measuring the labeling.
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If a first receptor capable of specifically binding to E1 is
fixed to the solid phase, only E1 is specifically bound to
the solid phase. During incubation with the soluble E1-
specific second receptor or antibody, the latter also reacts
exclusively with E1. Accordingly, since almost no binding
of other antigens to the solid phase takes place, this
process is highly specific and therefore makes very precise
determinations possible. E1 is thereby selectively bound to
the solid phase; other antigens remain in solution. In
addition, the soluble labeled antibody capable of binding
with E1 forms a complex with E1. After separation of the
solid from the liquid phase, the E1 content can again be
determined very precisely by the labeling. In a
particularly preferred embodiment, a third antibody is added
to further increase the selectivity. The third antibody is
directed against the second antibody, which bears the label.
Other process variants with three receptors known to those
skilled in the art are also possible using antibodies
capable of specifically binding with E1. They do not
require any further comments here.
Preferably at least one of the antibodies used for carrying
out the process of the invention is a monoclonal antibody.
In a preferred embodiment, only monoclonal antibodies are
used as receptors.
The antibody specifically capable of binding to E1 can be
present either bound to the solid phase or as a soluble
tagged or untagged receptor. This receptor is preferably a
monoclonal antibody. It is especially preferred for all
receptors used to be monoclonal antibodies.
The process according to the invention, as well as the test
kit, are especially suitable for automated analysis systems,
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especially for systems which are based on a biosensor and
which make use of chip technology.
The invention is illustrated in greater detail by the
5 following examples.
Example 1
a) Preparation of monoclonal antibodies by means of
highly purified El:
Highly purified human E1, the production of which from
the human pancreas has been described (S. Sziegoleit,
Purification and characterization of a cholesterol-binding
protein from human pancreas. Biochem. J. 207:573-582,
1982), is dissolved in PBS and mixed in equal parts with
Freund's adjuvant. In all cases, 100 ~.cg of this mixture are
injected IP and SC into Balb/c mice 6 to 8 weeks old. These
injections are repeated twice at an interval of 3 to 4
weeks. In this design, mice immunized with highly purified
E1 receive an IV injection of 100 ,ug of highly-purified E1
dissolved in PBS 3 days before removal of the spleen.
About 100 million cells from the spleen of an immunized
mouse are fused with 50 million myeloma cells (x 63-Sp8-653,
a cell line which does not synthesize any immunoglobulin;
obtainable from The Salk Institute, Cell Distribution
Center, San Diego CA 92112, USA) in the presence of
polyethyleneglycol (MG 3400). Fused cells are disseminated
on 8 plates, each containing 24 depressions. Each of these
depressions contains 50 million spleen cells, from
unimmunized isologous mice, in nutrient medium containing
hypoxanthine, aminopterin and thymidine.
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The antibody-containing supernatant liquids of these fused
cells (hybridomas) are tested 10 to 14 days later for their
specificity to highly purified human E1 by means of ELISA,
Western blot, frozen sections and paraffin sections.
To obtain monoclonal antibodies directed only against E1,
hybridoma cells whose supernatant liquid does not contain
any antibodies directed against other antigens are cloned
two times.
b) Preparation of monoclonal antibodies by means of an
immunogenic peptide
The peptide having the amino acid sequence Thr-Met-val-Ala-
Gly-Gly-Asp-Ile-Arg is prepared by solid phase synthesis
after Merrifield and, as described above, is injected into 6
to 8 week-old Balb/c mice. By the procedure described
above, monoclonal antibodies which are highly specific
against human elastase 1 from both stools and serum are
obtained.
Determination of El in plasma with monoclonal
E1-specific antibodies
Example 2
E1 in plasma is determined by ELISA. For this purpose,
monoclonal E1-specific antibodies obtained according to
Example 1, which are dissolved in PBS at pH 7.2, are
immobilized on polystyrene as a carrier. After a washing
step, dilute E1-containing plasma or serum is added. The
plasma or serum is diluted in a buffer of PBS, 5 mmol EDTA
and 0.2°s Tween 20*.
* Trademark
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After a washing step in PBS and 0.2% Tween 20, the E1 bound
to the antibodies is incubated for one hour at room
temperature with a polyclonal antibody which also binds E1
and is coupled to phosphatase, which is dissolved in PBS
containing 0.2% Tween 20 at pH 7.2. After another washing
step, p-nitrophenylphosphate disodium hexahydrate is added,
and the change in optical density is measured in the
reaction vessels in which the monoclonal antibodies have
reacted with E1.
Determination of E1 in plasma with two different
El-specific antibodies
Example 3
A monoclonal antibody directed against E1 is fixed to a
carrier as described in Example 2. After a washing step,
E1-containing serum or plasma is incubated with the
monoclonal antibodies under the same conditions as in
Example 2. After another washing step, the binding of E1 is
detected by a second E1-specific monoclonal antibody
according to the invention. This second E1-specific
antibody bears covalently bound peroxidase. After a washing
step and addition of ABTS as a substrate for peroxidase, the
change in optical density is measured.
Example 4
The procedure is as described in Example 3, but biotin is
coupled to the second E1-specific antibody instead of an
enzyme. Peroxidase-conjugated avidin or peroxidase-
conjugated streptavidin is added before the addition of
substrate. The selected E1 determination thus made possible
permits quite specific determination of only E1. Other
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antigens contained in the solution do not disturb the E1
determination according to the invention.
