EP0766568A1 - Procede permettant d'isoler une preparation d'antigenes - Google Patents

Procede permettant d'isoler une preparation d'antigenes

Info

Publication number
EP0766568A1
EP0766568A1 EP95924903A EP95924903A EP0766568A1 EP 0766568 A1 EP0766568 A1 EP 0766568A1 EP 95924903 A EP95924903 A EP 95924903A EP 95924903 A EP95924903 A EP 95924903A EP 0766568 A1 EP0766568 A1 EP 0766568A1
Authority
EP
European Patent Office
Prior art keywords
antigen
pellet
homogenate
glycine buffer
vzv
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.)
Withdrawn
Application number
EP95924903A
Other languages
German (de)
English (en)
Inventor
Klaus-Dieter Nothacker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens Healthcare Diagnostics GmbH Germany
Original Assignee
Behringwerke AG
Behring Diagnostics GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Behringwerke AG, Behring Diagnostics GmbH filed Critical Behringwerke AG
Publication of EP0766568A1 publication Critical patent/EP0766568A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A61K39/25—Varicella-zoster virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5768—Hepatitis A
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16651—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16751—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32451—Methods of production or purification of viral material

Definitions

  • the present invention relates to a method for the preparation of antigen of viral origin from infected animal cells and their use in enzyme-linked immunosorbent assays (ELISAs).
  • ELISAs enzyme-linked immunosorbent assays
  • the determination of the antibody status against a wide variety of antigens is e.g. B. with the help of the ELISA a proven and widespread technique.
  • microtiter plates are coated with the anti-gene against which antibodies can be detected and then incubated with serum. If antigen-specific antibodies are present, they bind to the offered antigen and can then be detected by a detection system with e.g. B. peroxidase-conjugated anti-antibodies can be detected via an enzyme-mediated substrate conversion with photometrically detectable color development.
  • Animal cell cultures which form the desired product or can be stimulated to do so are preferably used for antigen production for the plate coating.
  • VZV Varicella zoster virus
  • DOCUMENTS referred to as (line) homogeneous antigens - can then be used for the coating of microtiter plates for the ELISA without further purification.
  • Antigen production - such as B. from M. Lehtinen et al., Intervirology 24, pp. 18-25 (1985) - from herpes simplex virus (HSV) -infected cells for the ELISA for the detection of anti-HSV antibodies contains comparable procedural steps.
  • HSV herpes simplex virus
  • the cell homogeneous ⁇ t (extract) antigens produced according to the prior art can be used, but they contain a very large proportion of cellular proteins which are immunologically irrelevant or even disruptive and in the coating of microtiter plates with the viral protein desired for the solid phase ⁇ nen and structures compete so that the occupancy density of the polystyrene surface with virus-specific components is limited prematurely. Because of this competition, it is no longer possible to increase the sensitivity (signal) in the ELISA even with a more concentrated homogenate antigen for the coating.
  • This task was solved by an isolation process in which cellular organelles (especially cell nuclei) and proteins are enriched and viral components by a differential centrifugation of virus-containing cell homogenate antigen, which is obtained by ultrasound treatment of VZV-infected cells in glycine buffer become.
  • the pellets with virions, capsids and precursors of the virus assembly present after ultracentrifugation are resuspended in buffer with the aid of ultrasound.
  • UZ pellet antigen With the antigen obtained in this way, hereinafter referred to as "UZ pellet" antigen, it is possible, compared to the homogenate antigen previously used, to with a significantly increased signal reserve to improve ELISA test properties such as sensitivity and P / N ratio drastically, to reduce the faulty batch rate in antigen processing, since weaker raw antigens serving as starting material can be processed to high-quality antigens with great prospects of success by strong increased coating dilution to achieve an effective saving in crude antigen consumption, as a result of which the cell culture effort is considerably reduced, keeping the work-up effort practically and economically viable on a production scale.
  • the invention therefore relates to a method for the preparation of antigens of viral origin from infected animal cells, the method including the following steps:
  • a method is preferred in which a glycine buffer, preferably a glycine buffer pH 7-10, is used in at least one of steps a) - c).
  • a glycine buffer preferably a glycine buffer pH 7-10
  • a method is also preferred in which the ultracentrifugation is carried out at 54,000 to 64,000 x g.
  • a method is also preferred, wherein a further centrifugation at 5,000-10,000 x g is inserted between step b) and c).
  • the virus used is preferably a herpes, hepatitis or mumps virus.
  • the invention further relates to the use of the antigen preparations obtained by one of the methods described above for coating solid phases, such as. B. microtitration plates or particles such as latices.
  • the starting material for the production of VZV antigens according to the method according to the invention are VZV-infected, diploid human fibroblasts, which are cultured, infected, incubated and harvested according to published instructions.
  • An embodiment of the method according to the invention is advantageous, in which 1 part of cell sediment (eg measured in graduated pointed tubes) in 1.5 to 5 parts, preferably 1.5 to 2 parts of distilled water. or glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 7-10, preferably 9-10, very preferably 9.5) resuspended and treated with ultrasound in an ice bath (ultrasonic homogenizer Labsonic U-2000 from B Braun Diessel Biotech GmbH, intermediate probe, power setting to 20-60, preferably 30-50 W, optionally needle probe with preferably about 50 W).
  • the duration and frequency of the ultrasound pulses are known per se to the person skilled in the art or, if appropriate, can be easily determined by simple experiments. It is preferred for volumes of e.g. B.
  • the material to be sonicated should not exceed a total volume of about 50 ml, depending on the ultrasound transmitter used.
  • the ultrasound treatment can e.g. B. in 50 ml pointed tubes (z. B. Greiner, PP, 50 ml) so that as comparable steric conditions as possible are guaranteed.
  • the diluted cell homogenate present is then adjusted to 0.05-0.1%, advantageously 0.06%, of ⁇ -propiolactone for inactivation, 10 min-16 h. incubated at 2-8 ° C and then 120 ⁇ 10 min at 37 ° C.
  • the cell homogenate which is no longer infectious by this treatment, is pelleted at low speed at 300-500 x g and 2-8 ° C. for 10 ⁇ 2 min in order to remove non-lysed cells, cell fragments and nuclei.
  • the removed centrifugation supernatant is subjected to high-speed centrifugation at 40,000-100,000 xg, preferably 54,000-64,000 xg, over 120 ⁇ 30 min at 2-8 ° C (e.g. in a Centricon T2050 ultracentrifuge from Kontron im Rotor 50.2 Ti or 45 Ti).
  • the pellet obtained by ultracentrifugation is resuspended with preferably glycine buffer (see above) in 0.05-0.5 parts by volume of the amount of homogenate after inactivation with 0.5-1.5 minute pulses (ultrasound homogenizer LABSONIC U-2000 by B. Braun Diessel Biotech GmbH, intermediate probe or needle probe, power setting 20 - 90 W) until an even suspension is obtained.
  • the "UZ pellet” antigen obtained in this way is enriched with viral constituents and depleted in cellular proteins.
  • the buffer used in the production of the cell homogenate during the UZ run and in the resuspending of the virus pellet is of crucial importance for the process.
  • this buffer By using this buffer, a further increase in quality in the ELISA is possible when preparing VZV or Mumps "UZ pellet" antigens.
  • an antigen is obtained which, when used for the ELISA, not only enables very significant quality improvements compared to the cell homogenate antigen produced according to the prior art, but also is characterized by considerable quantitative or economic advantages.
  • Tab. 1 shows the coating according to the invention with the VZV antigen preparation in comparison to the prior art.
  • "UZ pellet” antigen and homogenate antigen were prepared from the same cellular starting material. They were diluted 1:20, 40, 80, 160, 320, 640, 1,280, 2,560 and 5,120 with PBS and then used for direct coating of microtiter test plates (96 wells) with 100 ⁇ l / well.
  • the "homogenate” corresponds to the state of the art.
  • test procedure corresponded to that in the package insert for the ELI SA sales product from Behringwerke AG, Enzygnost®-Anti-VZV / lgG (test plate), product no. OWLU, prescribed procedure with a serum dilution of 1: 231.
  • the samples were incubated with the solid phase antigen, the unbound antibodies were washed out and an enzyme was conjugated to the formed solution Antibody / antigen complexes are bound, the excess conjugate solution is washed out, the coloring is effected via the enzyme by means of a chromogenic substrate solution and the reaction is then ended with a stop solution.
  • Tab. 1 a shows the mE extinctions of both sera achieved with the individual antigen settings.
  • Tab. 1 b shows the corresponding P / N relations (quotients between the extinctions of the positive and negative serum in each case in a dilution stage).
  • Immune diagnostics in the ELISA is significantly improved by the "UZ-Pellet" antigen by optimizing the discrimination of positive and negative sera and reducing the proportion of false negative sera (increase in sensitivity).
  • the large signal reserve in the "UZ pellet” antigen is striking. If a maximum OD value of 651 mE is achieved in the case of homogen antigen with the positive serum, the "UZ pellet” antigen enables extinctions of up to 1300 - 1600 mE.
  • the reaction of the negative serum is in the dilution range of 1:80 even below the OD value of 134 mE for the 1:20 diluted homogenate antigen. Only at 1:20 (double “UZ pellet” concentrate) or 1:40 (10 times “UZ pellet” concentrate) will the range of 130 mE be reached. With this constellation it is therefore possible to use the "UZ pellet” antigen to increase the extinction level in the case of positive sera of z. B. to double approx. 650 mE to approx. 1300 mE without losing specificity in the negative reaction.
  • the "UZ pellet" antigen is characterized by an identical absorbance for the negative serum in the positive serum by OD values of approx. 1400 mE with the 2-fold concentrate or approx. 1700 mE (interpolated) with the 10-fold Concentrate from (Tab. 1 c)). With an unchanged specificity, the extinction level of the positive serum is more than doubled compared to the homogenate antigen.
  • suitable antigens can also be produced from weaker starting materials (infected cells) - which must be discarded according to the prior art.
  • Table 2 also shows the P / N ratios derived from the calculated coating dilutions. They make it clear that with "UZ-Pellet" antigen in the case of positive reactions which are identical to the homogenate antigen, the extinction values with the Negative sera can be pressed by a factor of 2-4. A test improvement is thus also conceivable in such a way that the cut-off value is corrected downward, so that previously false-negative sera react correctly positively.
  • the mixture was then centrifuged at 500 ⁇ g for 10 min (at 4 ° C.). The centrifugation supernatant was removed and subjected to centrifugation at 54,000 x g for 120 min at a temperature of 4 ° C. The pellet obtained was taken up in 24 ml glycine buffer and resuspended with ultrasound.
  • the mixture was then centrifuged at 300 ⁇ g for 10 min (at 4 ° C.).
  • the centrifugation supernatant was removed and subjected to centrifugation at 64,000 x g for 120 min at a temperature of 4 ° C.
  • the pellet obtained was taken up in 11 ml glycine buffer and resuspended with ultrasound.
  • the centrifugation supernatant was removed and subjected to centrifugation at 54,000 x g for 120 min at a temperature of 4 ° C.
  • the pellet obtained was taken up in 10 ml glycine buffer and resuspended with ultrasound.
  • Tab. 4 summarizes the absorbances measured with 6 positive and one negative serum. If the P / N ratios are formed by summing up the extinctions of 4 positive sera and dividing by the corresponding OD value of the negative serum, the homogenate antigen then has an average P / N ratio of 20.2 ⁇ 1.4 (dilution range 1: 100 - 1: 1800), while the average ratio in the "UZ pellet" antigen is almost 1.5 times higher at 29.5 ⁇ 3.5. This means gain in sensitivity with the same specificity or gain in specificity with the same sensitivity.
  • 7.5 ml of a cell sediment from human fibroblasts which were infected with HAV were resuspended in 15 ml glycine buffer and treated with ultrasound in the ice bath for 4 x 1 min (ultrasound homogenizer LABSONIC U-2000 from B. Braun Diessel Biotech GmbH at 35 W, intermediate probe). After the homogenization, 210 ml of glycine buffer were added. Subsequently, ß-propiolactone was pipetted in to a final concentration of 0.1% and the mixture was incubated with gentle stirring first at 4 ° C. for 16 hours and then at 37 ° C. for 120 minutes. The mixture was then centrifuged at 300 ⁇ g for 10 min (at 4 ° C.).
  • the centrifugation supernatant was removed and subjected to centrifugation at 54,000 x g for 120 min at a temperature of 4 ° C.
  • the pellet obtained was taken up in 12.5 ml of glycine buffer and resuspended with ultrasound.
  • the antigens were coated on the well surface of microtiter plates using a polyclonal capture antibody for HAV in dilution levels of 1:10, 1:50 and 1: 100 in PBS and then tested in an ELISA.
  • Tab. 5 summarizes the extinctions measured with an undiluted positive serum. Only with the "UZ-PelIet" antigen is a desirable extinction level of> 1000 mE still exceeded by far with an antigen dilution of 1: 100, whereas the homogenate antigen does not quite reach this value with the 1:10 antigen dilution. This makes the "UZ pellet” material, if one excludes the concentration factor of 20, far more than 5 times more economical to use than the homogenate antigen.
  • the centrifugation supernatant was removed and subjected to centrifugation at 64,000 x g for 120 min at a temperature of 4 ° C.
  • the pellet obtained was taken up in 13 ml glycine buffer and resuspended with ultrasound.
  • alkaline glycine buffer over PBS shows a significant improvement in the antigen.
  • the calculable antigen coating dilution after using glycine buffer is about 1: 2000, after using PBS, on the other hand, only about 1: 600.
  • IgG serum antigen coating dilution 1 IgG serum antigen coating dilution 1:
  • Antigen coating dilution 1

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Abstract

L'invention concerne un procédé de préparation d'antigènes d'origine virale, à partir de cellules animales infectées, ainsi que leur utilisation dans des techniques de titrage avec immuno-absorbant lié à une enzyme (ELISA).
EP95924903A 1994-06-24 1995-06-22 Procede permettant d'isoler une preparation d'antigenes Withdrawn EP0766568A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE4422238 1994-06-24
DE4422238A DE4422238C2 (de) 1994-06-24 1994-06-24 Verfahren zur Isolierung einer Antigenpräparation
PCT/EP1995/002422 WO1996000083A2 (fr) 1994-06-24 1995-06-22 Procede permettant d'isoler une preparation d'antigenes

Publications (1)

Publication Number Publication Date
EP0766568A1 true EP0766568A1 (fr) 1997-04-09

Family

ID=6521477

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95924903A Withdrawn EP0766568A1 (fr) 1994-06-24 1995-06-22 Procede permettant d'isoler une preparation d'antigenes

Country Status (7)

Country Link
US (1) US5789232A (fr)
EP (1) EP0766568A1 (fr)
JP (1) JPH10504806A (fr)
AU (1) AU2923195A (fr)
CA (1) CA2193081A1 (fr)
DE (1) DE4422238C2 (fr)
WO (1) WO1996000083A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3514374A (en) * 1968-04-02 1970-05-26 Merck & Co Inc Method of purification of myxovirus vaccine
US4100267A (en) * 1975-10-01 1978-07-11 Ortho Diagnostics Inc. Method of detecting hepatitis B core antigen and antibody
JPS61176856A (ja) * 1985-02-01 1986-08-08 Mitsubishi Chem Ind Ltd 非ａ非ｂ型肝炎抗原
WO1993018790A1 (fr) * 1992-03-24 1993-09-30 Csatary Laszlo K Vaccin contenant un virus vivant pour la therapie de maladies virales et d'affections malignes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9600083A2 *

Also Published As

Publication number Publication date
WO1996000083A2 (fr) 1996-01-04
DE4422238C2 (de) 1996-05-30
CA2193081A1 (fr) 1996-01-04
DE4422238A1 (de) 1996-01-11
US5789232A (en) 1998-08-04
WO1996000083A3 (fr) 1996-02-15
AU2923195A (en) 1996-01-19
JPH10504806A (ja) 1998-05-12

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