CA2012079A1 - Monoclonal antibodies directed against eca and the use thereof - Google Patents
Monoclonal antibodies directed against eca and the use thereofInfo
- Publication number
- CA2012079A1 CA2012079A1 CA002012079A CA2012079A CA2012079A1 CA 2012079 A1 CA2012079 A1 CA 2012079A1 CA 002012079 A CA002012079 A CA 002012079A CA 2012079 A CA2012079 A CA 2012079A CA 2012079 A1 CA2012079 A1 CA 2012079A1
- Authority
- CA
- Canada
- Prior art keywords
- enterobacteriaceae
- lymphocytes
- ecacc
- eca
- antibodies
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
ABSTRACT
Monoclonal antibodies directed against ECA and the use thereof The present invention provides a process for the production or antibodies directed against Entero-bacteriaceae, wherein B-lymphocytes are obtained from the blood of donors in whom an Enterobacteriaceae infection has been detected, the lymphocytes are immortalised, the cells which produce antibodies with the desired activity are selected, cultured and the antibodies obtained therefrom.
The present invention also provides medicaments containing antibodies produced by this process. The antibodies can be used for passive immunisation against infections brought about by Enterobacteriaceae and for the qualitative and quantitative detection of Entero-bacteriaceae.
Monoclonal antibodies directed against ECA and the use thereof The present invention provides a process for the production or antibodies directed against Entero-bacteriaceae, wherein B-lymphocytes are obtained from the blood of donors in whom an Enterobacteriaceae infection has been detected, the lymphocytes are immortalised, the cells which produce antibodies with the desired activity are selected, cultured and the antibodies obtained therefrom.
The present invention also provides medicaments containing antibodies produced by this process. The antibodies can be used for passive immunisation against infections brought about by Enterobacteriaceae and for the qualitative and quantitative detection of Entero-bacteriaceae.
Description
~, r~ 3 r t t _ ~ _ 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-6- r~ r~
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~
.. ~ ,, .
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 ~ ~ ~ 2~ I ~
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.
~.~;J ~ L ~ ~ J o~
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);
~rr~ ~i n. ~
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 7 ? J
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 .. a ~
~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 . ~
, clone E21 1520 1 lO 100 , ,_____ ! . _ .
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 ~ ~ 7 ;? 3 rt ~
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:
t.1 a 3 . . . __ o _ _ __ __ ~ U o o _ _ ~ C
_~ _ ~ ..
~ ~ ' _ c~ r3 o 5 E
~ ~ - ~
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 cp ~ ~, r'~
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 ~; ,r~ ~ s~ ~3 r1 ~
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.
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-6- r~ r~
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~
.. ~ ,, .
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 ~ ~ ~ 2~ I ~
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.
~.~;J ~ L ~ ~ J o~
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);
~rr~ ~i n. ~
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 7 ? J
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 .. a ~
~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 . ~
, clone E21 1520 1 lO 100 , ,_____ ! . _ .
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 ~ ~ 7 ;? 3 rt ~
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:
t.1 a 3 . . . __ o _ _ __ __ ~ U o o _ _ ~ C
_~ _ ~ ..
~ ~ ' _ c~ r3 o 5 E
~ ~ - ~
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 cp ~ ~, r'~
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 ~; ,r~ ~ s~ ~3 r1 ~
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.
Claims (11)
1. Process for the production of antibodies directed against Enterobacteriaceae, wherein B-lymphocytes are obtained from the blood of donors in whom an Enterobacteriaceae infection has been detected, the lymphocytes are immortalized, the cells which produce antibodies with the desired activity are selected, cultured and the antibodies obtained therefrom.
2. Process according to claim 1, wherein, for the immortalization, the lymphocytes are fused with myeloma cells of human or murine origin with sub-cellular vesicles which contain a transforming DNA or are transformed with Epstein-Barr virus.
3. Process according to claim 1, wherein, for the immortalization, the lymphocytes are fused with cells of the human myeloma cell line SKO-007, ATCC CRL 8033.
4. Process according to claim 2, wherein, for the immortalization, the lymphocytes are fused with cells of the human myeloma cell line SKO-007, ATCC CRL 8033.
5. Antibodies directed against Enterobacteriaceae, whenever produced by the process according to claim 1, 2, 3 or 4.
6. The cell line ECACC 89030311 (E21).
7. The cell line ECACC 89030310 (L30).
8. The use of monoclonal antibodies obtained by the process according to claim 1, 2, 3 or 4 or from at least one of the cell lines ECACC 89030310 and ECACC
89030311 for the passive immunization against infections brought about by Enterobacteriaceae.
89030311 for the passive immunization against infections brought about by Enterobacteriaceae.
9. The use of monoclonal antibodies obtained by the process according to claim 1, 2, 3 or 4 or from at least one of the cell lines ECACC 89030310 and 89030311 for the qualitative or quantitative detection of Enterobacteriaceae.
10. Medicaments for the treatment of infections brought about by Enterobacteriaceae, containing monoclonal antibodies obtained by the process according to claim 1, 2, 3 or 4, together with conventional carrier and dilution agents.
11. Medicaments for the treatment of infections brought about by Enterobacteriaceae, containing monoclonal antibodies obtained from at least one of the cell lines ECACC 89030310 and ECACC 89030311, together with conventional carrier and dilution agents.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3908520 | 1989-03-15 | ||
| DEP3908520.1 | 1989-03-15 | ||
| DE3925161A DE3925161A1 (en) | 1989-03-15 | 1989-07-28 | MONOCLONAL ANTIBODIES AGAINST ECA AND THEIR USE |
| DEP3925161.6 | 1989-07-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2012079A1 true CA2012079A1 (en) | 1990-09-15 |
Family
ID=25878846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002012079A Abandoned CA2012079A1 (en) | 1989-03-15 | 1990-03-13 | Monoclonal antibodies directed against eca and the use thereof |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0387850B1 (en) |
| JP (1) | JPH0335799A (en) |
| AT (1) | ATE123309T1 (en) |
| CA (1) | CA2012079A1 (en) |
| DE (2) | DE3925161A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2674866A1 (en) * | 1991-04-08 | 1992-10-09 | Chemunex | ANTI-ACE ANTIBODIES AND THEIR APPLICATIONS TO SPECIFIC DETECTION AND POSSIBLELY TO THE ENTRIES OF ENTEROBACTERIES ENTIRELY BY AN IMMUNOCHEMICAL METHOD. |
| PL404229A1 (en) | 2013-06-06 | 2014-12-08 | Wrocławskie Centrum Badań Eit + Spółka Z Ograniczoną Odpowiedzialnością | Isolated immunogenic bacterial antigen and its use in the prevention and treatment of infections caused by Gram-negative bacteria |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL81370A (en) * | 1986-02-07 | 1991-06-30 | Genetic Systems Corp | Pharmaceutical and diagnostic compositions comprising a plurality of human monoclonal antibodies for the treatment of bacterial diseases,methods for the preparation of the compositions and antibodies and kits containing said compositions |
-
1989
- 1989-07-28 DE DE3925161A patent/DE3925161A1/en not_active Withdrawn
-
1990
- 1990-03-13 CA CA002012079A patent/CA2012079A1/en not_active Abandoned
- 1990-03-14 DE DE59009169T patent/DE59009169D1/en not_active Expired - Fee Related
- 1990-03-14 EP EP90104839A patent/EP0387850B1/en not_active Expired - Lifetime
- 1990-03-14 AT AT90104839T patent/ATE123309T1/en not_active IP Right Cessation
- 1990-03-15 JP JP2062857A patent/JPH0335799A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DE3925161A1 (en) | 1990-09-27 |
| ATE123309T1 (en) | 1995-06-15 |
| EP0387850A1 (en) | 1990-09-19 |
| JPH0335799A (en) | 1991-02-15 |
| EP0387850B1 (en) | 1995-05-31 |
| DE59009169D1 (en) | 1995-07-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |