WO1999019358A2 - Antibody against baculoviruses - Google Patents
Antibody against baculoviruses Download PDFInfo
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- WO1999019358A2 WO1999019358A2 PCT/DE1998/003036 DE9803036W WO9919358A2 WO 1999019358 A2 WO1999019358 A2 WO 1999019358A2 DE 9803036 W DE9803036 W DE 9803036W WO 9919358 A2 WO9919358 A2 WO 9919358A2
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- antibodies
- baculoviruses
- antibody
- antibody according
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- 241000701447 unidentified baculovirus Species 0.000 title claims abstract description 57
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 30
- 229920001184 polypeptide Polymers 0.000 claims description 18
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 18
- 241001465754 Metazoa Species 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 9
- 239000006166 lysate Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 5
- 102000003886 Glycoproteins Human genes 0.000 claims description 3
- 108090000288 Glycoproteins Proteins 0.000 claims description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 210000004989 spleen cell Anatomy 0.000 claims description 3
- 238000002965 ELISA Methods 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 230000003053 immunization Effects 0.000 claims description 2
- 238000002649 immunization Methods 0.000 claims description 2
- 238000010166 immunofluorescence Methods 0.000 claims description 2
- 238000001114 immunoprecipitation Methods 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 claims description 2
- 238000001262 western blot Methods 0.000 claims description 2
- 101710141347 Major envelope glycoprotein Proteins 0.000 claims 1
- 230000004927 fusion Effects 0.000 claims 1
- 238000000746 purification Methods 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 description 11
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 8
- 102100031013 Transgelin Human genes 0.000 description 8
- 239000013592 cell lysate Substances 0.000 description 6
- 241000238631 Hexapoda Species 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 230000002349 favourable effect Effects 0.000 description 3
- 102000005927 Cysteine Proteases Human genes 0.000 description 2
- 108010005843 Cysteine Proteases Proteins 0.000 description 2
- 108010039471 Fas Ligand Protein Proteins 0.000 description 2
- 102000015212 Fas Ligand Protein Human genes 0.000 description 2
- 101000652570 Homo sapiens Antigen peptide transporter 1 Proteins 0.000 description 2
- 101100207063 Homo sapiens FASLG gene Proteins 0.000 description 2
- 101100369993 Mus musculus Tnfsf10 gene Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 102000057131 human TAP1 Human genes 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
Definitions
- the present invention relates to antibodies against baculoviruses, in particular recombinant baculoviruses, methods for producing the antibodies and a kit containing them, and the use of the antibodies or the kit.
- Baculoviruses are suitable for the expression of polypeptides in cells.
- DNA sequences which code for the polypeptides are introduced into the genome of baculoviruses, and the recombinant baculoviruses are used to infect cells.
- the polypeptides are then obtained as expression products.
- the object of the present invention is therefore to provide an agent with which the infection of cells with recombinant baculoviruses can be controlled.
- the present invention relates to antibodies which recognize baculoviruses, in particular recombinant baculoviruses.
- the present invention is based on the knowledge of the applicant that with cells which are infected with recombinant baculoviruses, antibodies can be produced in animals which recognize baculoviruses, in particular recombinant baculoviruses. It was also found that the recognition of recombinant Baculovirus is independent of the recombinant (foreign) polypeptide expressed by it. Furthermore, it was found that some of the antibodies recognize a polypeptide derived from a baculovirus-encoded cysteine protease (Protein v-cath) or a precursor thereof, while other antibodies recognize a polypeptide that is encoded from a baculovirus glycoprotein (gp 64/67 ) comes from.
- Protein v-cath baculovirus-encoded cysteine protease
- gp 64/67 baculovirus glycoprotein
- an antibody according to the invention recognizes baculoviruses, in particular recombinant baculoviruses.
- recombinant baculoviruses includes any baculovirus that expresses one or more recombinant (foreign) polypeptides.
- foreign polypeptides encompasses any polypeptides which, as such or in their nature, are not naturally expressed by baculoviruses. These polypeptides are preferably those which can be used diagnostically and / or therapeutically.
- An antibody according to the invention is characterized in that, in addition to baculoviruses, it also recognizes recombinant baculoviruses, the recognition of the latter being independent of the recombinant polypeptide expressed by them.
- An antibody according to the invention preferably recognizes a polypeptide which is derived from a baculovirus-encoded cysteine protease, in particular protein v-cath, or a precursor thereof. It is also particularly preferred if an antibody according to the invention recognizes a polypeptide which is derived from a glycoprotein encoded by a baculovirus, in particular gp 64/67.
- An antibody according to the invention can be a polyclonal or monoclonal antibody, with a monoclonal antibody being preferred.
- the antibody can be obtained from any animal, with rabbits being preferred for a polyclonal antibody and rabbits being preferred for a monoclonal mouse.
- Particularly preferred monoclonal antibodies were obtained at the DSMZ (German Collection of Microorganisms and Cell Cultures) as SM22 and SM62 under the designation DSM ACC2324 and ACC2325 on the 21st Aug. 1 997 deposited.
- the above antibodies can be produced by conventional methods. If polyclonal or monoclonal antibodies are to be produced, it is favorable to immunize animals, in particular rabbits for the former and mice for the latter antibodies, with cells infected with recombinant baculoviruses, in particular SF9 insect cells, or lysates thereof. The animals can be further boosted with the same or the same antigens. The polyclonal antibodies can then be obtained from the serum of the animals. For the monoclonal antibodies, spleen cells from the animals are fused with myeloma cells.
- an antibody according to the invention can be synthetic, parts or parts which are not necessary for the above recognition being missing in whole or in part, or these parts being replaced by others which give the antibody further favorable properties.
- Antibodies according to the invention are distinguished by the fact that they recognize baculoviruses, in particular recombinant baculoviruses. The recognition of the latter is independent of the recombinant polypeptide expressed by this. Antibodies according to the invention are therefore suitable for controlling an infection of cells which has occurred with baculoviruses, in particular with recombinant baculoviruses. Such control is particularly important when recombinant baculoviruses are used for diagnostic and / or therapeutic measures, especially in vivo or ex vivo experiments.
- Recombinant baculoviruses can be detected by means of antibodies according to the invention in any detection method, in particular in a Western blot, an ELISA, an immunoprecipitation or an immunofluorescence.
- the antibodies according to the invention if appropriate, can be labeled or in
- Antibodies according to the invention are also suitable for baculoviruses, in particular recombinant baculoviruses, e.g. to be cleaned from cell extracts. This can e.g. in that the antibodies are coupled to column material and the (recombinant) baculoviruses present in the cell extracts bind to the antibodies. The (recombinant) baculoviruses can then be isolated by conventional methods.
- kits are also provided for the use of antibodies according to the invention, which is also a subject of the present invention.
- a kit comprises the following: an above antibody and conventional auxiliaries such as buffers, carriers, detection reagents and controls.
- Lanes 1 and C are lysates of cells not infected with baculoviruses. Lanes 2- 5 and 7,
- FIGS 7, 48, 72 and 96 are lysates of cells infected with baculoviruses.
- Figures 7, 48, 72 and 96 represent the time at which the lysates were obtained after infection.
- Lanes 1-5 show lysates from infected with different recombinant baculoviruses Cells. Lanes 1-3 result from infections with an MOI of 10, while lanes 4 and 5 originate from infections with an MOI of 2.5.
- Sf9 insect cells (Gibco BRL, Gaithesburg, MD, USA) were treated with recombinant baculoviruses required for human CD95L, mouse CD95L
- mice used. The mice were "boosted” with the same antigen at half the dose at a two-week interval. After killing the mice, these spleen cells were removed and fused with myeloma cells, Ag8. Monoclonal antibodies were obtained. Two of these were used at the DSM as SM22 and SM62 under ACC2324 and ACC2325 on the 21st Aug. 1 9957 deposited.
- Sf9 insect cells were infected with recombinant baculoviruses encoding mouse trail.
- the "multiplicity of infection” (MOI) was 1.0. Lysates were obtained from the cells at different times, namely 7, 48, 72 and 96 hours after infection. These lysates were combined with a lysate from non-infected Sf9 insect cells on an SDS-PAGE and subsequently an electroblot Procedure. The nitrocellulose membranes obtained were incubated with the antibody SM22 according to the invention or with an anti-trail rabbit anti-trail antibody. In the first case, an HRPO-labeled goat anti-mouse igG Fc-
- Antibody and in the latter case an HRPO-labeled goat anti-rabbit IgG antibody is used.
- Cells i.e. recognizes recombinant baculoviruses, but not cell lysates from uninfected cells (see FIG. 1 (A).
- the cell lysates from infected cells were also detected by binding the anti-trail antibody (see FIG. 1 (B)).
- Sf9 insect cells were infected with recombinant baculoviruses coding for mouse CD95L, human CD95L and human TAP1, respectively.
- the "multiplicity of infection” (MOI) was 10 and 2.5, respectively.
- cell lysates were obtained from the cells which were subjected to SDS-PAGE and a subsequent electroblot process.
- the nitrocellulose membrane obtained was incubated with the antibody SM22 according to the invention.
- An HRPO-labeled goat anti-mouse IgG Fc antibody was used as the detection reagent.
- Non-infi- Decorated cell lysates were not recognized by the antibody according to the invention.
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Abstract
The invention relates to antibodies against baculoviruses, in particular recombinant baculoviruses. The invention also relates to a method for producing said antibodies and a kit containing them, as well as the use of the antibodies and of the kit.
Description
Antikörper gegen Baculoviren Antibodies against baculoviruses
Die vorliegende Erfindung betrifft Antikörper gegen Baculoviren, insbesondere rekombinante Baculoviren, Verfahren zur Herstellung der Antikörper und einen sie enthaltenden Kit sowie die Verwendung der Antikörper bzw. des Kits.The present invention relates to antibodies against baculoviruses, in particular recombinant baculoviruses, methods for producing the antibodies and a kit containing them, and the use of the antibodies or the kit.
Baculoviren eignen sich zur Expression von Polypeptiden in Zellen. Hierzu werden in das Genom von Baculoviren DNA-Sequenzen eingeführt, die für die Polypeptide kodieren, und die rekombinanten Baculoviren werden zur Infektion von Zellen verwendet. Die Polypeptide werden dann als Expressionsprodukte erhalten.Baculoviruses are suitable for the expression of polypeptides in cells. For this purpose, DNA sequences which code for the polypeptides are introduced into the genome of baculoviruses, and the recombinant baculoviruses are used to infect cells. The polypeptides are then obtained as expression products.
Vielfach ist es notwendig, die Infektion von Zellen mit rekombinanten Baculoviren zu kontrollieren. Hierzu werden Mittel verwendet, die für die einzelnen Polypeptide spezifisch sind. Solche Mittel sind aber teuer und vielfach nur schwer verfügbar.It is often necessary to control the infection of cells with recombinant baculoviruses. For this purpose, means are used which are specific for the individual polypeptides. However, such funds are expensive and often difficult to obtain.
Der vorliegenden Erfindung liegt somit die Aufgabe zugrunde, ein Mittel bereitzustellen, mit dem die Infektion von Zellen mit rekombinanten Baculoviren kontrolliert werden kann.The object of the present invention is therefore to provide an agent with which the infection of cells with recombinant baculoviruses can be controlled.
Erfindungsgemäß wird dies durch die Gegenstände in den Patentansprüchen erreicht.According to the invention, this is achieved by the subject matter in the claims.
Gegenstand der vorliegenden Erfindung sind Antikörper, die Baculoviren, insbesondere rekombinante Baculoviren, erkennen.The present invention relates to antibodies which recognize baculoviruses, in particular recombinant baculoviruses.
Die vorliegende Erfindung beruht auf der Erkenntnis des Anmelders, daß mit Zellen, die mit rekombinanten Baculoviren infiziert sind, in Tieren Antikörper produziert werden können, die Baculoviren, insbesondere rekombinante Baculoviren, erkennen. Ferner wurde gefunden, daß die Erkennung von rekombinanten
Baculoviren unabhängig von dem durch diese exprimierten rekombinanten (Fremd)-Polypeptid ist. Desweiteren wurde gefunden, daß einige der Antikörper ein Polypeptid erkennen, das von einer Baculovirus kodierten Cystein-Protease (Protein v-cath) oder einem Vorläufer davon stammt, während andere Antikörper ein Polypeptid erkennen, das von einem Baculovirus kodierten Glycoprotein (gp 64/67) stammt.The present invention is based on the knowledge of the applicant that with cells which are infected with recombinant baculoviruses, antibodies can be produced in animals which recognize baculoviruses, in particular recombinant baculoviruses. It was also found that the recognition of recombinant Baculovirus is independent of the recombinant (foreign) polypeptide expressed by it. Furthermore, it was found that some of the antibodies recognize a polypeptide derived from a baculovirus-encoded cysteine protease (Protein v-cath) or a precursor thereof, while other antibodies recognize a polypeptide that is encoded from a baculovirus glycoprotein (gp 64/67 ) comes from.
Ein erfindungsgemäßer Antikörper erkennt Baculoviren, insbesondere rekombinante Baculoviren. Der Ausdruck "rekombinante Baculoviren" umfaßt jegliche Baculoviren, die ein oder mehrere rekombinante (Fremd)-Polypeptide exprimieren. Der Ausdruck "Fremd-Polypeptide" umfaßt jegliche Polypeptide, die als solche oder in ihrer Art nicht natürlicherweise durch Baculoviren exprimiert werden. Vorzugsweise sind diese Polypeptide solche, die diagnostisch und/odertherapeutisch verwendbar sind. Ein erfindungsgemäßer Antikörper zeichnet sich dadurch aus, daß er neben Baculoviren auch rekombinante Baculoviren erkennt, wobei die Erkennung letzterer unabhängig von dem durch diese exprimierten rekombinanten Polypeptid ist. Vorzugsweise erkennt ein erfindungsgemäßer Antikörper ein Polypeptid, das von einer Baculovirus kodierten Cystein-Protease, insbesondere Protein v-cath, oder einem Vorläufer davon stammt. Ebenso ist es besonders bevorzugt, wenn ein erfindungsgemäßer Antikörper ein Polypeptid erkennt, das von einem Baculovirus kodierten Glycoprotein, insbesondere gp 64/67, stammt.An antibody according to the invention recognizes baculoviruses, in particular recombinant baculoviruses. The term "recombinant baculoviruses" includes any baculovirus that expresses one or more recombinant (foreign) polypeptides. The term "foreign polypeptides" encompasses any polypeptides which, as such or in their nature, are not naturally expressed by baculoviruses. These polypeptides are preferably those which can be used diagnostically and / or therapeutically. An antibody according to the invention is characterized in that, in addition to baculoviruses, it also recognizes recombinant baculoviruses, the recognition of the latter being independent of the recombinant polypeptide expressed by them. An antibody according to the invention preferably recognizes a polypeptide which is derived from a baculovirus-encoded cysteine protease, in particular protein v-cath, or a precursor thereof. It is also particularly preferred if an antibody according to the invention recognizes a polypeptide which is derived from a glycoprotein encoded by a baculovirus, in particular gp 64/67.
Ein erfindungsgemäßer Antikörper kann ein polyklonaler oder monoklonaler Antikörper sein, wobei ein monoklonaler Antikörper bevorzugt ist. Der Antikörper kann aus jeglichem Tier erhalten sein, wobei für einen polyklonalen Antikörper Kaninchen und für einen monoklonalen Mäuse bevorzugt sind. Besonders bevorzugte monoklonale Antikörper wurden bei der DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) als SM22 bzw. SM62 unter der Bezeichnung DSM ACC2324 bzw. ACC2325 am 21 . Aug. 1 997 hinterlegt.An antibody according to the invention can be a polyclonal or monoclonal antibody, with a monoclonal antibody being preferred. The antibody can be obtained from any animal, with rabbits being preferred for a polyclonal antibody and rabbits being preferred for a monoclonal mouse. Particularly preferred monoclonal antibodies were obtained at the DSMZ (German Collection of Microorganisms and Cell Cultures) as SM22 and SM62 under the designation DSM ACC2324 and ACC2325 on the 21st Aug. 1 997 deposited.
Vorstehende Antikörper können nach üblichen Verfahren hergestellt werden.
Sollen polyklonale bzw. monoklonale Antikörper hergestellt werden, ist es günstig, Tiere, insbesondere Kaninchen für erstere und Mäuse für letztere Antikörper, mit mit rekombinanten Baculoviren infizierten Zellen, insbesondere SF9-Insektenzellen, oder Lysaten davon zu immunisieren. Weiteres Boostern der Tiere kann mit dem oder den gleichen Antigenen erfolgen. Die polyklonalen Antikörper können dann aus dem Serum der Tiere erhalten werden. Für die monoklonaien Antikörper werden Milzzellen der Tiere mit Myelomzellen fusioniert.The above antibodies can be produced by conventional methods. If polyclonal or monoclonal antibodies are to be produced, it is favorable to immunize animals, in particular rabbits for the former and mice for the latter antibodies, with cells infected with recombinant baculoviruses, in particular SF9 insect cells, or lysates thereof. The animals can be further boosted with the same or the same antigens. The polyclonal antibodies can then be obtained from the serum of the animals. For the monoclonal antibodies, spleen cells from the animals are fused with myeloma cells.
Ferner kann ein erfindungsgemäßer Antikörper synthetisch sein, wobei ihm ggfs. Teile, die für vorstehende Erkennung nicht notwendig sind, ganz oder teilweise fehlen bzw. diese Teile durch andere ersetzt sind, die dem Antikörper weitere günstige Eigenschaften verleihen.Furthermore, an antibody according to the invention can be synthetic, parts or parts which are not necessary for the above recognition being missing in whole or in part, or these parts being replaced by others which give the antibody further favorable properties.
Zur Herstellung von synthetischen Antikörpern kann z.B. von vorstehend erhaltenen, monoklonaien Antikörpern ausgegangen werden. Hierzu bietet sich an, die Antigen-Bindungsregionen der monoklonaien Antikörper zu analysieren und die für vorstehende Erkennung notwendigen und nicht notwendigen Teile zu identifizieren. Die notwendigen Teile können dann modifiziert und die nicht notwendigen ganz oder teilweise eliminiert bzw. durch Teile ersetzt werden, die den Antikörpern weitere günstige Eigenschaften verleihen. Auch können Teile außerhalb der Bindungsregionen der Antikörper modifiziert, eliminiert oder ersetzt werden. Der Fachmann weiß, daß sich für vorstehende Maßnahmen insbesondere die DNA-Rekombinationstechnologie eignet. Diese ist ihm bestens vertraut.For the production of synthetic antibodies e.g. from monoclonal antibodies obtained above. For this purpose it is advisable to analyze the antigen binding regions of the monoclonal antibodies and to identify the parts that are necessary and not necessary for the above recognition. The necessary parts can then be modified and the unnecessary parts can be completely or partially eliminated or replaced with parts that give the antibodies further favorable properties. Parts outside the binding regions of the antibodies can also be modified, eliminated or replaced. The person skilled in the art knows that DNA recombination technology is particularly suitable for the above measures. He is very familiar with this.
Erfindungsgemäße Antikörper zeichnen sich dadurch aus, daß sie Baculoviren, insbesondere rekombinante Baculoviren, erkennen. Die Erkennung letzterer ist dabei unabhängig von dem durch diese exprimierten rekombinanten Polypeptid. Erfindungsgemäße Antikörper eignen sich somit zur Kontrolle einer mit Baculoviren, insbesondere mit rekombinanten Baculoviren, erfolgten Infektion von Zellen. Eine solche Kontrolle ist insbesondere wichtig, wenn rekombinante Baculoviren für diagnostische und/oder therapeutische Maßnahmen, insbesondere bei in vivo
oder ex vivo Versuchen, eingesetzt werden. Der Nachweis von rekombinanten Baculoviren kann mittels erfindungsgemäßer Antikörper in einem beliebigen Nachweisverfahren, insbesondere in einem Western Blot, einem ELISA, einer Immunpräzipitation oder einer Immunfluoreszenz, erfolgen. Hierzu können die erfindungsgemäßen Antikörper, wenn es angebracht ist, markiert sein oder inAntibodies according to the invention are distinguished by the fact that they recognize baculoviruses, in particular recombinant baculoviruses. The recognition of the latter is independent of the recombinant polypeptide expressed by this. Antibodies according to the invention are therefore suitable for controlling an infection of cells which has occurred with baculoviruses, in particular with recombinant baculoviruses. Such control is particularly important when recombinant baculoviruses are used for diagnostic and / or therapeutic measures, especially in vivo or ex vivo experiments. Recombinant baculoviruses can be detected by means of antibodies according to the invention in any detection method, in particular in a Western blot, an ELISA, an immunoprecipitation or an immunofluorescence. For this purpose, the antibodies according to the invention, if appropriate, can be labeled or in
Kombination mit markierten gegen sie gerichteten Antikörpern eingesetzt werden. Auch eignen sich erfindungsgemäße Antikörper, um Baculoviren, insbesondere rekombinante Baculoviren, z.B. aus Zellextrakten zu reinigen. Dies kann z.B. dadurch erfolgen, daß die Antikörper an Säulenmaterial gekoppelt sind und die in den Zellextrakten vorliegenden (rekombinanten) Baculoviren an die Antikörper binden. Die (rekombinanten) Baculoviren können dann durch übliche Verfahren isoliert werden.Combination with labeled antibodies directed against them can be used. Antibodies according to the invention are also suitable for baculoviruses, in particular recombinant baculoviruses, e.g. to be cleaned from cell extracts. This can e.g. in that the antibodies are coupled to column material and the (recombinant) baculoviruses present in the cell extracts bind to the antibodies. The (recombinant) baculoviruses can then be isolated by conventional methods.
Zur Verwendung erfindungsgemäßer Antikörper wird auch eine Kit bereitgestellt, der ebenfalls ein Gegenstand der vorliegenden Erfindung ist. Ein solcher Kit umfaßt folgendes: einen vorstehenden Antikörper und übliche Hilfsmittel, wie Puffer, Träger, Nachweisreagentien und Kontrollen.A kit is also provided for the use of antibodies according to the invention, which is also a subject of the present invention. Such a kit comprises the following: an above antibody and conventional auxiliaries such as buffers, carriers, detection reagents and controls.
Kurze Beschreibung der ZeichnungBrief description of the drawing
Fig. 1 zeigt den Nachweis von rekombinanten Baculoviren durch den erfindungsgemäßen Antikörper SM22. Spuren 1 und C sind Lysate von nicht mit Baculoviren infizierten Zellen. Spuren 2- 5 bzw. 7,1 shows the detection of recombinant baculoviruses by the antibody SM22 according to the invention. Lanes 1 and C are lysates of cells not infected with baculoviruses. Lanes 2- 5 and 7,
48, 72 und 96 sind Lysate von mit Baculoviren infizierten Zellen. Die Angaben 7, 48, 72 und 96 geben die Zeit wieder, zu der die Lysate nach Infektion erhalten wurden.48, 72 and 96 are lysates of cells infected with baculoviruses. Figures 7, 48, 72 and 96 represent the time at which the lysates were obtained after infection.
Fig. 2 zeigt den Nachweis von rekombinanten Baculoviren durch den erfindungsgemäßen Antikörper SM22. Spuren 1 - 5 zeigen Lysate von mit unterschiedlichen, rekombinanten Baculoviren infizierten
Zellen. Die Spuren 1 - 3 gehen aus Infektionen mit einem MOI von 1 0 hervor, während die Spuren 4 und 5 von Infektionen mit einem MOI von 2,5 stammen.2 shows the detection of recombinant baculoviruses by the antibody SM22 according to the invention. Lanes 1-5 show lysates from infected with different recombinant baculoviruses Cells. Lanes 1-3 result from infections with an MOI of 10, while lanes 4 and 5 originate from infections with an MOI of 2.5.
Die Erfindung wird durch die nachstehenden Beispiele erläutert.The invention is illustrated by the examples below.
Beispiel 1 : Herstellung von erfindungsgemäßen AntikörpernExample 1: Production of antibodies according to the invention
Sf9-Insektenzellen (Gibco BRL, Gaithesburg, MD, USA) wurden mit rekombinanten Baculoviren, die für humanes CD95L, Maus-CD95LSf9 insect cells (Gibco BRL, Gaithesburg, MD, USA) were treated with recombinant baculoviruses required for human CD95L, mouse CD95L
Maus-Trail bzw. humanes TAP1 kodieren, infiziert (vgl. Mariani, S.M. et al., J. Immunol. Methods 1 93 ( 1 996), 63; Armandola, E.A. et al. Eur. J. Immunol. 26 ( 1 996), 1 748) . Die "multiplicity of infec- tion) (MOI) betrug 0, 1 . Vier Tage nach Infektion wurden die Zellen gesammelt und jeweils 1 07 Zellen zur Immunisierung einer BALB/cEncode mouse trail or human TAP1, infected (see Mariani, SM et al., J. Immunol. Methods 1 93 (1 996), 63; Armandola, EA et al. Eur. J. Immunol. 26 (1 996 ), 1 748). The multiplicity of infection (MOI) was 0.1. Four days after infection, the cells were collected and 10 7 cells in each case for the immunization of a BALB / c
Maus verwendet. Die Mäuse wurden in einem zweiwöchigen Abstand mit dem gleichen Antigen in halber Dosierung "geboostert" . Nach Abtötung der Mäuse wurden diesen Milzzellen entnommen und mit Myelomzellen, Ag8, fusioniert. Es wurden monoklonale Antikörper erhalten. Zwei dieser wurden bei der DSM als SM22 bzw. SM62 unter ACC2324 bzw. ACC2325 am 21 . Aug. 1 9957 hinterlegt.Mouse used. The mice were "boosted" with the same antigen at half the dose at a two-week interval. After killing the mice, these spleen cells were removed and fused with myeloma cells, Ag8. Monoclonal antibodies were obtained. Two of these were used at the DSM as SM22 and SM62 under ACC2324 and ACC2325 on the 21st Aug. 1 9957 deposited.
Beispiel 2: Nachweis von rekombinanten BaculovirenExample 2: Detection of recombinant baculoviruses
(a) Sf9-Insektenzellen wurden mit rekombinanten Baculoviren, die für Maus-Trail kodieren, infiziert. Die "multiplicity of infection" (MOI) betrug 1 0. Von den Zellen wurden zu verschiedenen Zeitpunkten, nämlich 7, 48, 72 bzw. 96 Stunden nach Infektion Lysate gewonnen. Diese Lysate wurden zusammen mit einem Lysat von nicht-infizierten Sf9-Insekten- zellen einer SDS-PAGE und nachfolgend einem Elektroblot-
Verfahren unterzogen. Die erhaltenen Nitrozellulosemembranen wurden mit dem erfindungsgemäßen Antikörper SM22 bzw. mit einem gegen Trail gerichteten Kaninchen anti-Trail Antikörper inkubiert. Als Nachweisreagens wurden in ersterem Fall ein HRPO-markierter Ziege anti-Maus igG Fc-(a) Sf9 insect cells were infected with recombinant baculoviruses encoding mouse trail. The "multiplicity of infection" (MOI) was 1.0. Lysates were obtained from the cells at different times, namely 7, 48, 72 and 96 hours after infection. These lysates were combined with a lysate from non-infected Sf9 insect cells on an SDS-PAGE and subsequently an electroblot Procedure. The nitrocellulose membranes obtained were incubated with the antibody SM22 according to the invention or with an anti-trail rabbit anti-trail antibody. In the first case, an HRPO-labeled goat anti-mouse igG Fc-
Antikörper und in letzterem Fall ein HRPO-markierter Ziege anti-Kaninchen IgG-Antikörper verwendet.Antibody and in the latter case an HRPO-labeled goat anti-rabbit IgG antibody is used.
Es zeigte sich, daß der erfindungsgemäße Antikörper SM22 Zelllysate von mit rekombinanten Baculoviren infiziertenIt was found that the antibody SM22 according to the invention infected cell lysates from with recombinant baculoviruses
Zellen, d.h. rekombinante Baculoviren, nicht aber Zellysate von nicht-infizierten Zellen erkennt (vgl. Fig. 1 (A) . Die Zellysate von infizierten Zellen wurden auch durch die Bindung des gegen Trail gerichteten Antikörpers nachgewiesen (vgl. Fig. 1 (B) .Cells, i.e. recognizes recombinant baculoviruses, but not cell lysates from uninfected cells (see FIG. 1 (A). The cell lysates from infected cells were also detected by binding the anti-trail antibody (see FIG. 1 (B)).
(b) Sf9-Insektenzellen wurden mit rekombinanten Baculoviren, die für Maus-CD95L, humanes CD95L bzw. humanes TAP1 kodieren, infiziert. Die "multiplicity of infection" (MOI) betrug 1 0 bzw. 2,5. Von den Zellen wurden, wie unter (a) beschrieben, Zellysate erhalten, die einer SDS-PAGE und einem nachfolgenden Elektroblot-Verfahren unterzogen wurden. Die erhaltene Nitrozellulosemembran wurde mit dem erfindungsgemäßen Antikörper SM22 inkubiert. Als Nachweisreagens wurde ein HRPO-markierter Ziege anti-Maus IgG Fc-Antikör- per verwendet.(b) Sf9 insect cells were infected with recombinant baculoviruses coding for mouse CD95L, human CD95L and human TAP1, respectively. The "multiplicity of infection" (MOI) was 10 and 2.5, respectively. As described under (a), cell lysates were obtained from the cells which were subjected to SDS-PAGE and a subsequent electroblot process. The nitrocellulose membrane obtained was incubated with the antibody SM22 according to the invention. An HRPO-labeled goat anti-mouse IgG Fc antibody was used as the detection reagent.
Es zeigte sich, daß der erfindungsgemäße Antikörper SM22It was found that the antibody SM22 according to the invention
Zellysate von mit rekombinanten Baculoviren infizierten Zellen, d.h. rekombinante Baculoviren, erkennt, wobei seineCell lysates from cells infected with recombinant baculoviruses, i.e. recombinant baculoviruses, recognizing its
Erkennung unabhängig von den Fremd-Polypeptiden ist, welche die rekombinanten Baculoviren exprimieren. Nicht-infi-
zierte Zellysate wurden nicht durch den erfindungsgemäßen Antikörper erkannt.
Recognition is independent of the foreign polypeptides that express the recombinant baculoviruses. Non-infi- Decorated cell lysates were not recognized by the antibody according to the invention.
Claims
1 . Antikörperl gegen Baculoviren.1 . Antibody against baculoviruses.
2. Antikörper nach Anspruch 1 , wobei die Antikörper rekombinante Baculoviren erkennen.2. Antibody according to claim 1, wherein the antibodies recognize recombinant baculoviruses.
3. Antikörper nach Anspruch 1 oder 2, wobei die Antikörper polyklonal sind.3. Antibody according to claim 1 or 2, wherein the antibodies are polyclonal.
4. Antikörper nach Anspruch 1 oder 2, wobei die Antikörper monoklonal sind.4. Antibody according to claim 1 or 2, wherein the antibodies are monoclonal.
5. Antikörper nach Anspruch 4, wobei die Antikörper ein Polypeptid erken- nen, das von einem Baculovirus-kodierten Protein v-cath stammt.5. The antibody of claim 4, wherein the antibodies recognize a polypeptide derived from a baculovirus-encoded protein v-cath.
6. Antikörper nach Anspruch 5, wobei die Antikörper bei der DSM unter ACC2324 hinterlegt worden sind.6. Antibody according to claim 5, wherein the antibodies have been deposited with the DSM under ACC2324.
7. Antikörper nach Anspruch 4, wobei die Antikörper ein Polypeptid erkennen, das von einem Baculovirus-kodierten Glycoprotein gp64/67 stammt.7. The antibody of claim 4, wherein the antibodies recognize a polypeptide derived from a baculovirus-encoded glycoprotein gp64 / 67.
8. Antikörper nach Anspruch 7, wobei die Antikörper bei der DSM unter ACC 2325 hinterlegt worden sind.8. Antibody according to claim 7, wherein the antibodies have been deposited with the DSM under ACC 2325.
9. Verfahren zur Herstellung eines Antikörpers nach einem der Ansprüche 1 -9. A method for producing an antibody according to any one of claims 1 -
8, wobei ein Tier mit rekombinanten Baculoviren-infizierten Zellen immunisiert wird, und8, an animal being immunized with recombinant baculovirus-infected cells, and
(a) polyklonale Antikörper aus dem Serum des Tieres erhalten werden, oder(a) polyclonal antibodies are obtained from the animal's serum, or
(b) monoklonale Antikörper nach Fusion von Milzzellen des Tieres mit Myelomzellen erhalten werden.
(b) monoclonal antibodies are obtained after fusion of animal spleen cells with myeloma cells.
10. Verfahren nach Anspruch 9, wobei ein Lysat der Zellen zur Immunisierung verwendet wird.10. The method of claim 9, wherein a lysate of the cells is used for immunization.
1 1 . Verwendung eines Antikörpers nach einem der Ansprüche 1 - 8 in einem Nachweisverfahren für Baculoviren, insbesondere rekombinante Baculoviren.1 1. Use of an antibody according to one of claims 1-8 in a detection method for baculoviruses, in particular recombinant baculoviruses.
1 2. Verwendung nach Anspruch 1 1 , wobei das Nachweisverfahren ein We- stern-Blot, ein ELISA, eine Immunfluoreszenz oder eine Immunpräzipitation ist.1 2. Use according to claim 1 1, wherein the detection method is a Western blot, an ELISA, an immunofluorescence or an immunoprecipitation.
1 3. Verwendung eines Antikörpers nach einem der Ansprüche 1 - 8 zur Reinigung von Baculoviren, insbesondere rekombinanten Baculoviren.1 3. Use of an antibody according to any one of claims 1-8 for the purification of baculoviruses, in particular recombinant baculoviruses.
14. Kit, umfassend: einen Antikörper nach einem der Ansprüche 1 - 8, und übliche Hilfsmittel, wie Puffer, Träger, Nachweisreagentien und14. A kit comprising: an antibody according to any one of claims 1-8, and conventional auxiliaries such as buffers, carriers, detection reagents and
Kontrollen.
Controls.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1997144655 DE19744655C2 (en) | 1997-10-09 | 1997-10-09 | Antibodies against baculoviruses |
| DE19744655.8 | 1997-10-09 |
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| Publication Number | Publication Date |
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| WO1999019358A2 true WO1999019358A2 (en) | 1999-04-22 |
| WO1999019358A3 WO1999019358A3 (en) | 1999-06-17 |
| WO1999019358B1 WO1999019358B1 (en) | 1999-07-22 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DE1998/003036 WO1999019358A2 (en) | 1997-10-09 | 1998-10-09 | Antibody against baculoviruses |
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| DE (1) | DE19744655C2 (en) |
| WO (1) | WO1999019358A2 (en) |
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1998
- 1998-10-09 WO PCT/DE1998/003036 patent/WO1999019358A2/en active Application Filing
Non-Patent Citations (5)
| Title |
|---|
| MARIANI, SARA M. ET AL: "Detection of active infection of Sf9 insect cells by recombinant baculoviruses" J. VIROL. METHODS (DECEMBER 1997), 69(1,2), 19-28 CODEN: JVMEDH;ISSN: 0166-0934, XP002095592 * |
| MCCARTHY, W. J. ET AL: "Current developments in baculovirus serology" BIOL. BACULOVIRUSES (1986), VOLUME 1, 147-58. EDITOR(S): GRANADOS, ROBER R.;FEDERICI, BRIAN A. PUBLISHER: CRC, BOCA RATON, FLA. CODEN: 55ZFA2, XP002095588 * |
| OOMENS, A. G. P. ET AL: "The baculovirus GP64 envelope fusion protein: synthesis, oligomerization, and processing" VIROLOGY (1995), 209(2), 592-603 CODEN: VIRLAX;ISSN: 0042-6822, XP002095591 * |
| SLACK, JEFFREY M. ET AL: "Characterization of v - cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus" J. GEN. VIROL. (1995), 76(5), 1091-8 CODEN: JGVIAY;ISSN: 0022-1317, XP002095590 * |
| WHITFORD, MARC ET AL: "Identification and sequence analysis of a gene encoding gp67, an abundant envelope glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus" J. VIROL. (1989), 63(3), 1393-9 CODEN: JOVIAM;ISSN: 0022-538X, XP002095589 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999019358A3 (en) | 1999-06-17 |
| DE19744655C2 (en) | 2000-03-16 |
| WO1999019358B1 (en) | 1999-07-22 |
| DE19744655A1 (en) | 1999-04-15 |
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