Note: Descriptions are shown in the official language in which they were submitted.
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The present invention is concerned with antibodies
specifically directed against Enterobacteriaceae which
have been obtained from the cell lines ECACC 89030310
and ECACC 89030311, with processes for obtaining thern
and with ~he use thereof.
In the Enterobacteriaceae family, which includes
gram negativej flagellate rods which are facultatively
aerobic, are some pathogenic micro-organisms, such as
Escherichia, Erwinia, Serratia, Proteus, Salmonella and
Shigella. The infections caused by these micro-organisms
multiply very considerably especially in the clinical
field and, furthermore, these strains develop a
resistance to antibiotics so that infections are very
difficult to combat. The Enterobacteriaceae infections
involve, on the one hand, the ascending urinary tract
and, on the other hand, the gastroenteric system. Many
deaths are brought about especially by Salmonella
typhimurium, the micro-organism which is typical for
foodstuff poisoning. Therefore, a search has been made
for an antiserum which is effective against these micro-
organisms.
A starting point common to all Enterobacteriaceae was
offered by the antigen known as the enterobacterial
common antigen (ECA). Attempts have been made to
immunise animals either with the complete micro-organism
or with this antigen and subsequently to obtain either
directly polyclonal antibodies or, after ~usion, mono-
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clonal antibodies. However, the antibodies thus obtained
showed no protection against Enterobacteriaceae
infections. Thus, it is stated in FE~S Microbiology
Letters, 27, 307-312/1985, that an anti-ECA serum
obtained from rabbits offers no protection against
infections in the case of mice. Furthermore, in
Infection and Immunity, 2, 175-182/1970, there is
described the production of a~tibodies which, however,
only react with the common antigen of quite definite
individual micro-organisms.
Therefore, it is an object of the present
invention to provide antibodies which are directed
against Enterobacteriaceae and especially against the
pathogenic species of this family. Furthermore, it is
an object of the present invention to provide a
possibility for the treatment and detection of Entero-
bacteriaceae infections.
Thus, according to the present invention, there
is provided a process for the production of antibodies
directed against Enterobacteriaceae, wherein lymphocytes
are obtained from the blood of donors in whGm an
Enterobacteriaceae infection has been detected, the
lymphocytes are immortalised, the cells which produce
antibodies with the desired ac~ivity are selec~e~,
cultured and the antibodies obtained therefrom.
Surprisingly, by the use of suitable human
lymphocytes, it is possible to produce antibodies which
are able specifically ~o bind En~erobacte iaceae.
The cell lines according to the presen~ invention7
~hlch have been obtained by the fusion of human B-
lymphocytes with myeloma cells, show no~ only a
specific binding with ECA but also, surprisingly,
bind to micro-organisms of the Enterobacteriaceae
family.
For the produc~ion of antibodies directed again~t
Enterobacteriaceae, B-lymphocytes are ob~ained in known
manner from the blood of diseased persons in whom an
Enterobacteriaceae infection has been detected. The
lymphocytes are then immortalised. Various methods are
available for t~e immor~alisation. The human lympho-
cytes can be fu.s~ lith myeloma cells of human or
murine origin a~cording to the method of Kohler and
Milstein. However, heteromyelomas can also be used for
the fusion. Furthermore, it is also possible to trans-
form the B lymphocytes with Epstein-Barr virus. In
addition, the processes described in Federal Republic
of Germany Patent Specification No. A-32 45 665 , published
on November 10, 1983, and in European Patent Specification
No. A-0,256,512, published on February 24, 1988, can also
be used. Fusioning is thereby carried out with sub-
cellular vesicles which contain a transforming DNA. The
B-lymphocytes are preferably immortalised by fusion
with cells of the human myel,oma cell line SKO-007,
ATCC CRL 8033~
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From the immortalised cells, those are selected
which produce antibodies with the deqired activit-~.
The methods of selection are sufficiently well known
to the expert and do not require a more detailed
e~planation here. For example, there can be used the
detection of the binding with lipopolysaccharide (LPS)
preparations of the Enterobacteriaceae via an ELISA
test since most commercially available LPS preparations
also contain substantial amounts of ECA.
The cells which produce the desired antibodies ar_
then cultured and the antibodies obtained in known
manner.
The present invention also provides cell lines
which have been deposited under the numbers ECACC
89030310 (L30) and 89030311 (E21) which produce mono-
clonal antibodies reacting specifically with ECA.
The present invention is also concerned with the
use of the monoclonal antibodies obtained from the
claimed cell lines for passive immunisation against
infections brought about by Enterobacteriaceae.
Since the monoclonal antibodies obtained by the
process according to the present invention can bind
specifically with ~iving Enterobacteriaceae, they can
be used for combating these micro-organisms in the human
body. It is thereby not only possible to administer the
monoclonal antibodies to patients for preventative
purposes but also to use the monoclonal antibodies
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obtained for treatment after infection has taken place.
A dosage which is suitable -~or passive immunisation
is in the range o~ from 50 to 200 mg.
Since the monoclonal antibodies obtained by the
process accordinc to the present invention are
essentiall~- re~cti~e ~ pecies of the Entero-
bacterlaceae family, the antibodies can also be used
for the qualitative and quantitative detection of
Enterobacteriaceae. The detection thereby takes place
in known manner by means of an immunological determin-
ation process. Processes of this kind are known to the
expert and do not need to be explained here in detail.
The monoclonal antibodies obtained according to the
present invention can thereby be used as labelled and/
or immobilised receptor.
Furthermore, the present invention provides a
medicament for the treatment of infections brought
about by Enterobacteriaceae, which medicaments contain
monoclonal antibodies obtained by the process according
to the present invention, preferably from the cell
lines ECACC 89030310 and/or 89030311, together with
conventional carrier and dilution agents~
Monoclonal antibodies are preferably used in a
dosage of from 50 to 200 mg.
According to the present invention, a medicament
is provided with which can be combated Enterobacteriaceae
strains which are resistant to antibiotics.
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The present invention will now be described in
more detail in the following Examples, with re~erence
to the accompanying drawings, in which:
Fig. 1 shows a diagram in which the results are shown
which have been obtained in an ELISA test after
incubation with various dilutions of the anti-
body E21 with ECA and various LPS samples, which
have been adsorbed on microtitre cups;
Fig. 2 shows a diagram in which are plotted the results
which have been obtained by preincubation of the
antibody E21 with soluble ECA or soluble LPS and
applying the mixture to microtitre plates which
have been coated with ECA from Salmonella
montevideo; antibody E21 with a concentration of
about 1 ~g./ml. was incubated for 1 hour at 25C.
with an equal volume of soluble ECA or LPS; the
mixture of antibody and inhibitor was applied to
microtitre plates coated with the ECA from
Salmonella montevideo; the curves show the results
which were obtained in comparison with the bind-
ing of antibodies to ECA without inhibitor, the
curves thereby standing for the following LPS:
x ECA (Salmonella montevideo)
0 LPS (Salmonella montevideo)
J`' LPS (Salmonella minnesota)
o LPS (Escherichia coli OlllB4)
-
a LPS (Serratia marcescens);
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Fi~. 3 shows a diagram in which are plotted the results
which have been obtained with an experiment which
was carried out as in the case of Fi2. 2 but in
which the microtitre plates had been coated with
LPS from Escherichia coli 0111B4; and
Fi~. 4 shows an immunoblot analysis; ECA and ~PS were
separated on 14% SDS/PAGE; the gel in the upper
part was stained with silver in order to show
the LPS migration pattern; ECA was not stained
by the silver reagent; an identical gel was used
for an immunoblot which had been developed with
antibody E21.
Exam~le 1.
For a selection of donors who, with great prob-
ability, show in vivo pre-activated lymphocytes against
ECA, the following process was employed: blood was taken
frorn patients with a sepsis or bacteraemia and a blood
culture set up according to known bacteriological
processes. From this blood culture, the bacteria were
identified with the help of a differentiation procedure
according to Bergey's Manual of Systematic Bacteriology
(1984, N.R. Krieg, Cr. Holt (eds.), pub. Williams and
Wilkins, Baltimore, USA). Only those persons come into
consideration as donors for lymphocytes in whorn bacteria
from the Enterobacteriaceae amily had been detected as
the cause of infection. 100 to 200 ml. of blood were
taken from these donors and the mononuclear cells
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isolated therefrom. Subsequently, these cells were
fused according to conventional methods (see Kohler
and Milstein, Nature, 256, 495-497/1975) with a human
myeloma cell line (SKO-007, ATCC CRL ~033). After 2 to
3 weeks, the culture supernatants of the hybrid cells
were tested. For this purpose 9 an ELISA test was
carried out with purified ECA, isolated from Salmonella
montevideo. For this purpose, lO0 ~1. of ECA solution
(10 ~g./ml.) in 10 mM sodium phosphate buffer (pH 7.35)
were pipetted into 96 well ELISA plates (Greiner) and
incubated for 1 hour at ambient temperature. After
washing the microtitre plates with PBS, non-specific
binding sites were blocked with bovine serum albumin.
Thereafter, 100 ~l. of culture supernatant were pipetted
into the individual wells and incubated for l hour at
ambient temperature. After washing, lO0 ~1. of
peroxidase-labelled sheep anti-human light chain anti-
bodies were added thereto and again incubated for
l hour at ambient temperature. After further washing,
the enzyme reaction was started with peroxidase
substrate (ABTS, Boehringer Mannheim GmbH, order
No. 102946). After 20 to 60 minutes at ambient temper-
ature, the extinctions were determined in a photometer
at 405 nm. The clones which produce the desired
antibodies were further cultured.
Example 2.
In order to determine the specificity of the human
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~onoclonal antibodies obtained from the cell line
ECACC 89030311 in the culture supernatant of the
hybrid cells, an ELISA test was carried out as
described in Example 1. The following results were
thereby obtained:
Table
. mE obtained with
¦ . ECA - LPS ¦ LPS
I . S. montevideo ¦ S. montevideo¦ S enteritidis
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, clone E21 1520 1 lO 100
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Example 3.
.
Monoclonal antibodies obtalned from the cell lines
ECACC 89030310 and ECACC 89030311 were tested for their
binding to intact bacterial cells. For this purpose,
bacteria killed with phenol were adsorbed on 96 well
microtitre plates by incubating overnight at 37C.
Thereafter, the plates were washed twice with phosphate-
buffered saline (PBS) and non-specific binding sites were
blocked with 1% crotein C (Boehringer Mannheim GmbH) for
1 hour at ambient temperature. After tipping off the
blocking solution, 100 ~l.of the solution containing the
monoclonal antibodies were added thereto and incubated
for 1 hour at ambient temperature. After washing three
times with PBS, 100 ~1. of peroxidase-labelled sheep
anti-human light chain antibodies were added thereto
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and again incubated for 1 hour at am~ient temperature.
After rene.wed ~ashing, the binding of the peroxidase
conjugate with ABTS was detected and the colour
reaction quantified in a photometer. The results
obtained are given in the following Table 2:
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lhese results show that the monoclonal anti~odies
accordi~g to the present invention bind ~.~ith a
plurality of members of the Enterobacteriaceae family
but not with strains which do not belong to this family,
for example Pseudo~onas.
Exam~le 4.
The immunotherapeutic effic~cy of the monoclonal
antibodies obtained from the cell line ECACC 89030311
was tested. For this purpose, monoclonal antibodies
obtained from the cell line ECACC 89030311 were
administered intravenously in various amounts to 4 to
7 week old NMRI mice and 15 minutes later a constant
amount of living bacteria was injected intraperiton-
eally. After 48 hours, the survival rate of the mice
was ~scer~ained. The following Table 3 shows the
results obtained:
Table 3
: clone ~g. H-MAB/ organism and protection
I mouse i micro-organismrate (%)
20 ' 'count
¦ E21 ' 57 1.5 x 106 CFU I80 (4/5)
14 _ rra~ia 166 (2/3)
3.5 marcescens ¦0 (0/8)
. 1 I0 (0/8)
CA13/111 63 4 x 106 CFU ¦0 (0/8)
Serratia ¦
I marcescens
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CAl3/ll: this MAB was used as control. It reacts
with the 0-antigen of Salmonella minnesota.
E~.amPle 5.
It was investigated whether antibodies obtained
from the cell line ECACC 89030311 bind specifically
with ECA or whether they display an affinity to lipo-
polysaccharides on the outer membrane of bacteria of
the Enterobacteriaceae family. For this purpose9 on
the one hand, highly purified ECA was used and, for
comparison, there was investigated the binding to lipo
polysaccharides which had been extracted from various
species. ECA and lipopolysaccharides (LPS) were
adsorbed in the same concentrations on microtitre plates
and incubated with various dilutions of the antibody.
Subsequently, the amount of bound antibody was deter-
mined by means of an EL,ISA test. Fig. 1 of the
accompanying drawings shows a graphic representation
of the results obtained.
At each antibody dilution tested, a much higher
ELISA signal was produced with immobilised ECA than
with solid phase-bound LPS. Since these differences
in reactivity could also have been brought about by the
different adsorption capacity of the antigen to the
microtitre plates, in addition, competitive binding
studies were carried out. In a first test, antibodies
were preincubated with various concentrations of ECA
from S. montevideo and LPS from various types
(S. montevideo, Escherichia coli 0111, S. minnesota
and Serratia marcescens). Figo 2 of the accompanying
drawings shows that only soluble ECA substantially
inhibits the binding of the monoclonal antibody E21 to
the solid phase-bound ECA. In a second test, there was
tested the ability of ECA and various LPS samples
competitively to inhibit the binding of the antibody
E21 to Escherichia coli LPS.
Fig. 3 of the accompanying drawings shows that
the binding of E21 to Escherichia coli LPS was inhibited
by very low ECA concentrations, whereas LPS, which had
been isolated from the same organism, showed a 50%
inhibition of the binding only when about 50 fold higher
concentrations were used. Heterologous LPS samples had
an even still smaller inhibiting capacity. These
results show that the monoclonal antibodies recognise
an epitope which is much more prominent in the ECA
molecules than in the lipopolysaccharide molecules.
Example 6.
Purified ECA from S. montevideo and LPS from
various types (including S. montevideo) was subjected
to an SDS gel electrophoresis and made visible by
Western blotting and silver stainin~. The results
obtained are to be seen from Fig. 4 of the accompany-
ing drawings. We have found that LPS gave the typical
ladder-like pattern after the staining with silver
reagent, whereas ECA could not be stained with silver
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nitrate. The detection of the b:Lotted antigens with
the antibody E21 showed a continuous ladder-like
pattern of ECA molecules, the molecular weight range
of which extended from very low to high molecular
weights. In contradistinction thereto, the monoclonal
antibody E21 reacted with LPS in the immunoblots only
with a few bands of mainly low molecular weight. The
high molecular weight bands of LPS did not react at all,
although they were present in sufficient amounts.
