TEST KIT AND METHOD OF DIAGNOSING SEVERE ACUTE PANCREATITIS

ENSEMBLE DE TEST ET PROCEDE PERMETTANT DE DIAGNOSTIQUER UNE PANCREATITE AIGUE SEVERE

English Abstract

A method of in vitro diagnosing severe acute pancreatitis, monoclonal
antibodies binding to carboxypeptidase B activation peptide (CAPAP) having SEQ
ID NO:1 (aa 1 - 81) or fragments thereof, and a test kit, preferably a
dipstick, are described. The method comprises A) bringing a sample of body
fluid into contact with a) a first antibody binding to the CAPAP of SEQ ID
NO:1, and b) a second antibody binding to the CAPAP of SEQ ID NO:1, other than
the side in a), or c) a solid-phase bound CAPAP of SEQ ID NO:1 or fragments
thereof, wherein one of the antibodies of a) and b) or the peptide of c) is
labeled with a non-radioactive label, and wherein at least one of the
antibodies of a) and b) is monospecific for CAPAP, and B) determining the
level of antibody-antigen-antibody complex formed, or the level of an excess
component in the competitive antigen-antibody reaction, and C) using the
determined level for estimation of the amount of CAPAP in the sample for the
diagnosis of severe acute pancreatitis in the patient.

French Abstract

L'invention concerne un procédé permettant de diagnostiquer in vitro une pancréatite aiguë sévère, des anticorps monoclonaux se fixant sur les peptides d'activation de la carboxypeptidase B (CAPAP) présentant la séquence SEQ ID NO:1 (aa 1 81) ou des fragments de ces derniers, et un ensemble de test, se présentant de préférence sous forme d'une bandelette réactive. Ce procédé comprend les étapes suivantes: A) on met en contact un échantillon de fluide corporel avec a) un premier anticorps se fixant sur les peptides CAPAP présentant la SEQ ID NO:1, et b) un second anticorps se fixant sur les CAPAP présentant la SEQ ID NO:1, en un site différent de a), ou c) un CAPAP présentant la SEQ ID NO:1 ou des fragments de celui-ci, fixés sur un support solide, un des anticorps a) et b) ou le peptide c) étant marqué au moyen d'un marqueur non radioactif, et au moins un des anticorps a) et b) étant monospécifique de CAPAP; et B) on mesure la concentration de complexe anticorps-antigène-anticorps formé ou la concentration d'un composant excédentaire dans la réaction compétitive antigène-anticorps et C) on utilise les valeurs correspondant à la concentration de CAPAP dans l'échantillon pour diagnostiquer une pancréatite aiguë sévère chez le patient.

Claims

1
Claims
1. A method in in vitro diagnosing severe acute pancreatitis in a patient
comprising
the steps of
A) bringing a sample of body fluid from the patient into contact with a
sufficient amount of
a) at least one first antibody binding to an antigenic site on the
carboxypeptidase B
activation peptide (CAPAP) having the amino acid sequence SEQ ID NO:1, and
b) at least one second antibody binding to an antigenic site on the CAPAP
having the
amino acid sequence SEQ ID NO: 1, other than the site in a), or
c) a solid-phase bound CAPAP having the amino acid sequence SEQ ID NO:1 or a
peptide fragment thereof containing an epitope,
wherein at least one antibody of a) and b) or the peptide of c) is labeled
with a non-
radioactive label, and
wherein at least one of the first and second antibodies is monospecific for
CAPAP,
and
B) determining the level of the antibody a) - CAPAP - antibody b) complex
formed, or the
level of an excess component in the competitive reaction of antibody a) -
CAPAP and
antibody a) - peptide c) with the aid of the label used, and
C) using the determined level for estimation of the amount of CAPAP in the
sample in the
diagnosis of severe acute pancreatits in the patient.
2. The method according to claim 1, wherein the determination B) is performed
with
a double sandwich enzyme-linked immunosorbent assay (ELISA) or a competitive
immunosorbent assay.
3. The method according to claim 1 or 2, wherein the label in b) is selected
from the
group consisting of gold, carbon, and latex particles, and enzymes.
4. The method according to any one of claims 1 - 3, wherein the sample of body
fluid
in A) is a blood sample, and when the estimated amount of CAPAP in C) is > 6 -
10 nmol/L
of urine, the patient is diagnosed positive for severe acute pancreatitis.
5. The method according to any one of claims 1 - 3, wherein the sample of body
fluid
in A) is a urine sample, and when the estimated amount of CAPAP in C) is > 60 -
100 nmol/L
of blood, the patient is diagnosed positive for severe acute pancreatitis.
6. A monoclonal antibody binding to an antigenic site on CAPAP having the
amino
acid sequence SEQ ID NO:1 or a peptide fragment thereof containing an epitope.

2
7. Test kit for severe acute pancreatitis comprising
a) at least one first antibody binding to an antigenic site on the
carboxypeptidase B activation
peptide (CAPAP) having the amino acid sequence SEQ ID NO:1, and
b) at least one second antibody binding to an antigenic site on the CAPAP
having the amino
acid sequence SEQ ID NO:1, other than the site in a), or
c) a solid-phase bound CAPAP having the amino acid sequence SEQ ID NO:1 or a
peptide
fragment thereof containing an epitope,
wherein at least one antibody of a) and b) or the peptide of c) is labeled
with a non-radioactive
label, and
wherein at least one of the first and second antibodies is monospecific for
CAPAP.
8. Test kit according to claim 7, wherein the label is selected from the group
consisting of gold, carbon and latex particles, and enzymes.
9. Test kit according to claim 7 or 8, wherein the kit is a dipstick.

Description

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Test kit and method of diagnosing severe acute ~ancreatitis
The present invention relates to a test kit and a method of in vitro
diagnosing severe
acute pancreatitis with the aid of at least two antibodies each binding to a
different site on the
carboxypeptidase B activation peptide, CAPAP, or with one antibody
specifically binding to
CAPAP and a solid-phase bound epitope-containing peptide of CAPAP. Further,
the
invention relates to a monoclonal antibody binding to an antigenic site on
CAPAP.
Background
The clinical rationale for assessment of severity of an attack of acute
pancreatitis has
not become obvious until recently when more specific treatments have emerged.
Anticytokines, antibiotics and endoscopic sphincterotomy have shown promising
results but
only when the treatment has been instituted within 24-48 hours of onset of
pain. Furthermore,
four out of five cases of acute pancreatitis are mild and do not benefit from
any expensive
specific treatment.
During recent years many new laboratory variables have been proposed as early
single tests for severity prediction in acute pancreatitis.
Measurement of substances that mirror the activation of trypsinogen like the
trypsinogen activation peptide, TAP, the carboxypeptidase B activation
peptide, CAPAP, and
trypsin-a,-antitrypsin complexes seem to be able to predict severity already
within 24 hours
of onset of acute pancreatitis. Inflammatory mediators like interleukin-6 and
PNM-elastase
are also useful.
The carboxypeptidase B activation peptide (CAPAP) is the largest activation
peptide
(MW 9400) released from any pancreatic proenzyme. The activation occurs
normally in the
intestine during the digestion of food. Serum and urine normally contain no or
very low levels
of immunoreactive CAPAP. During the initial phase of acute pancreatitis this
activation
occurs prematurely within and around the pancreatic gland. CAPAP then leaks to
the
circulation from where it will be eliminated trough glomerular filtration and
excreted in the
urine. Only one small clinical study has been published so far [Appelros S,
Thim L,
Borgstrom A. The activation peptide of carboxypeptidase B in serum and urine.
Gut 1998;
42:97-102J.
CAPAP is very stable in serum and urine, and the determination of the protein
is
enabled by a CAPAP RIA kit, marketed by EuroDiagnostica AB, Malmo, Sweden.
However,
handling of reagents comprising a radioactive label require specialized
laboratories and

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2
trained personnel. Once the sample to be tested reaches the laboratory, the
test may be
performed in approximately two hours.
The US patent 5,356,781 discloses an immunological method for detecting the
activation of pancreatic zymogens for diagnosing or monitoring the progress of
pancreatic
disease. The method comprises detection of the absence or presence in a sample
of peptides
having the same carboxy-terminal pentapeptide sequence as the activation
peptides of
pancreatic zymogens. Among these procarboxypeptidases A and B are mentioned.
However,
no antibodies binding to procarboxypeptidases are disclosed.
There are good reasons for an early assessment of severity in acute
pancreatitis
since this could discount the majority of the attacks which are mild and have
a self limiting
course and do not require any treatment other than general support and
parenteral fluid. Thus,
there is a need for a rapid method for identification of patients with severe
attack of acute
pancreatitis already at the time of admission to the Emergency room. The
perfect method for
determination of severity in regular clinical practice does not yet exist, and
a simple and
reliable test that can be used to predict the severity in acute pancreatitis
would be desirable.
Description of the invention
The present invention provides a method of diagnosing severe acute
pancreatitis, a
test kit and monoclonal antibodies binding to CAPAP.
The CAPAP used in the present invention is the human carboxypeptidase B
activation peptide of 81 amino acids, which results from the N-terminal 95-
amino acid
activation peptide after further degradation of the C-terminal end by the dual
action of active
carboxypeptidase B and trypsin. The amino acid sequence of this 81 amino-acid
CAPAP is
disclosed in the Sequence listing part of this description and it is nominated
SEQ ID NO:1.
More precisely, the present invention provides a method of in vitro diagnosing
severe acute pancreatitis in a patient comprising the steps of
A) bringing a sample of body fluid from the patient into contact with a
sufficient amount of
a) at least one first antibody binding to an antigenic site on the
carboxypeptidase B
activation peptide (CAPAP) having the amino acid sequence SEQ ID NO:1, and
b) at least one second antibody binding to an antigenic site on the CAPAP
having the
amino acid sequence SEQ ID NO:l,other than the site in a), or
c) a solid-phase bound CAPAP having the amino acid sequence SEQ ID NO: l or a
peptide fragment thereof containing an epitope,
wherein at least one antibody of a) and b) or the peptide of c) is labeled
with a non-
radioactive label, and

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3
wherein at least one of the first and second antibodies is monospecific for
CAPAP,
and
B) determining the level of the antibody a) - CAPAP - antibody b) complex
formed, or the
level of an excess component in the competitive reaction of antibody a) -
CAPAP and
antibody a) - peptide c) with the aid of the label used, and
C) using the determined level for estimation of the amount of CAPAP in the
sample for the
diagnosis of severe acute pancreatits in the patient.
The amount of CAPAP in the sample of body fluid which will be regarded as cut-
off
level for severe acute pancreatitis will be determined emprically for each
specific assay set-up,
but will normally be in the range of 60 to 100 nmol/L , such as 80 nmol/L, for
a urine sample,
and 6 to 10 nmol/L , such as 8 nmol/L, for a blood or serum sample.
The phrase "at least one first antibody" is used to define that there may be
several
different antibodies which, however, do not bind to the same sites on the
protein as the second
antibody. The phrase " at least one second antibody" is used to define that
there may be
several different antibodies which, however, do not bind to the same sites on
the protein as the
first antibody.
The phrase " antibody monospecific for CAPAP" is used to define that the
antibody
binds to only one specific site on the CAPAP, and examples of monospecific
antibodies are
monoclonal antibodies, monospecific polyclonal antibodies and recombinant
modified
antibodies having specific binding to the protein.
In a preferred embodiment of the invention the first and the second antibodies
are
monoclonal antibodies.
The solid phase used for the solid-phase bound CAPAP having the amino acid
sequence SEQ ID NO:1 or a peptide fragment thereof containing an epitope may
be any solid
phase used in the art of immunoassays, such as cellulose particles, glass
surfaces, plastics
surfaces, magnetic beads, etc.
In an embodiment of the invention the determination B) is performed with a
double
sandwich enzyme-linked immunosorbent assay (ELISA) or a competitive
immunosorbent
assay.
In another embodiment the label in b) is selected from the group consisting of
gold,
carbon, and latex particles, and enzymes.

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In yet another embodiment the sample of body fluid in A) is a urine sample,
and
when the estimated amount of CAPAP in C) is > 60 -I00 nmoUL of urine, the
patient is
diagnosed positive for severe acute pancreatitis.
In still another embodiment the sample of body fluid in A) is a blood sample,
and
when the estimated amount of CAPAP in C) is > 6 - 10 nmol/L of blood, the
patient is
diagnosed positive for severe acute pancreatitis.
The present invention further provides a monoclonal antibody binding to an
antigenic
site on CAPAP having the amino acid sequence SEQ ID NO:1 or a peptide fragment
thereof
containing an epitope.
To our knowledge, no monoclonal antibodies against CAPAP have been previously
reported. However, now several different antibodies against CAPAP having the
amino acid
sequence SEQ ID NO:1 are produced for selection of at least two antibodies
binding to
different sites on CAPAP. These may then be used in the method and test kit of
the invention.
The present invention also provides a test kit for acute pancreatitis
comprising
a) at least one first antibody binding to an antigenic site on the
carboxypeptidase B activation
peptide (CAPAP) having the amino acid sequence SEQ ID NO:1, and
b) at least one second antibody binding to an antigenic site on the CAPAP
having the amino
acid sequence SEQ ID NO:1, other than the site in a), or
c) a solid-phase bound CAPAP having the amino acid sequence SEQ ID NO:1 or a
peptide
fragment thereof containing an epitope,
wherein at least one antibody of a) and b) or the peptide of c) is labeled
with a non-radioactive
label, and
wherein at least one of the first and second antibodies is monospecific for
CAPAP.
In an embodiment the label is selected from the group consisting of gold,
carbon and
latex particles, and enzymes.
In a preferred embodiment the test kit is a dipstick.
Preparation of monoclonal antibodies
Immunization
Balb/c mice were inoculated with CAPAP purified protein having the amino acid
sequence SEQ ID NO:1, covalent linked to BSA. All injections were performed
with the
antigen mixed with RIBI adjuvant (MPL + TDM). Each mouse was injected with 10
pg
conjugate, followed one and two weeks later by injections of 1 pg per mouse
and then a
resting period of seven weeks. The mice now received a booster dose of 1 p,g
per mouse and

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10 days later blood samples were collected and analyzed for antibodies against
CAPAP in
ELISA. Pure CAPAP was coated at a concentration of 1 p,g/ml in carbonate
buffer pH 9.0, 60
p,l per well in Maxisorp ELISA plates at room temperature over night.
S Fusion
The mouse with the highest concentration of antibodies in the serum was
injected
with 1 pg of CAPAP having the amino acid sequence SEQ ID NO:1 conjugated to
BSA in
RIBI adjuvant intraperitoneally. Three days later the mouse was killed by
cervical dislocation
and the spleen was removed. The spleen cells were harvested as a single cell
suspension and
washed three times with serum free medium and counted. The spleen cells were
mixed with
half their number of SP 2/0 mouse myeloma cells similarly washed in serum free
medium and
pelleted together by centrifugation at 250 x g and after careful removal of
all liquid placed in a
37°C water-bath. The fusion procedure is performed at this temperature
and with added
reagents pre-warmed to 37°C. The cell pellet is gently broken with the
tip of a pipette and 1
ml of SO% poyethylene glycol 1500 solution (PEG 1500, Boehringer Mannheim) is
slowly
added during the period of one minute and gentle mixing. The reaction mixture
is gently
mixed for one minute. During continued gentle mixing 1 ml of serum free medium
is added
during one minute, 2 ml during the second minute, 4 ml during the third and 8
ml during the
fourth minute. The cells are centrifuged at 200 x g for seven minutes, the
supernatant removed
and the cell pellet with minimal disturbance transferred to 100 ml of pre-
warmed medium
supplemented with 17% FCS and HAT for selection of hybridomas. The cells were
distributed
to ten 96-well plates, the previous day seeded with intraperitoneal lavage
cells, 3000 per well,
as feeder cells. Supernatants from wells with growing hybridomas were tested
in ELISA as
above when the cells covered approximately SO% of the bottom area.
Selection of monoclonal antibodies
Positive and negative screening procedures are performed on the 20 possible
hybridoma cell lines to select monoclonal antibodies binding to different
sites on the CAPAP
protein.
One-step rapid tests
At least two of the produced monoclonal antibodies binding to different sites
on the
CAPAP having the amino acid sequence SEQ ID NO:1 are used for dipsticks. In
this regard,
use may be made of a commercially available test set-up, e.g. Clear View from
Johnson &

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6
Johnson or Test Pac from ABBOTT in accordance with the recommendations of the
manufacturer. Both these test systems give a visual reading within seconds to
minutes.

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SEQUENCE LISTING
<110> EURODIAGNOSTICA AB
<120> Test kit and method of diagnosing acute severe pancreatitis.
<130> 192962200BN
<140>
<141>
<150> SE 9804069-4
<151> 1998-11-26
<160> 1
<170> PatentIn Ver. 2.1
<210> 1
<211> 81
<212> PRT
<213> Homo sapiens
<400> 1
His His Gly Gly Glu His Phe Glu Gly Glu Lys Val Phe Arg Val Asn
1 5 10 IS
Val Glu Asp Glu Asn His Ile Asn Ile Ile Arg Glu Leu Ala Ser Thr
20 25 30
Thr Gln Ile Asp Phe Trp Lys Pro Asp Ser Val Thr Gln Ile Lys Pro
35 40 45
His Ser Thr Val Asp Phe Arg Val Lys Ala Glu Asp Thr Val Thr Val
SO 55 60
Glu Asn Val Leu Lys Gln Asn Glu Leu Gln Tyr Lys Val Leu Ile Ser
65 70 75 80
Asn