WO1986005400A1 - Antigens, antibodies and their uses - Google Patents
Antigens, antibodies and their uses Download PDFInfo
- Publication number
- WO1986005400A1 WO1986005400A1 PCT/GB1986/000142 GB8600142W WO8605400A1 WO 1986005400 A1 WO1986005400 A1 WO 1986005400A1 GB 8600142 W GB8600142 W GB 8600142W WO 8605400 A1 WO8605400 A1 WO 8605400A1
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- WO
- WIPO (PCT)
- Prior art keywords
- antigen
- candidosis
- serum
- human
- antigens
- Prior art date
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 44
- 102000036639 antigens Human genes 0.000 title claims abstract description 44
- 108091007433 antigens Proteins 0.000 title claims abstract description 44
- 201000003984 candidiasis Diseases 0.000 claims abstract description 19
- 229960005486 vaccine Drugs 0.000 claims abstract description 4
- 230000001225 therapeutic effect Effects 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 claims description 2
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 2
- 229960003942 amphotericin b Drugs 0.000 claims description 2
- XRECTZIEBJDKEO-UHFFFAOYSA-N flucytosine Chemical compound NC1=NC(=O)NC=C1F XRECTZIEBJDKEO-UHFFFAOYSA-N 0.000 claims description 2
- 229960004413 flucytosine Drugs 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 238000002560 therapeutic procedure Methods 0.000 abstract description 2
- 229940121375 antifungal agent Drugs 0.000 abstract 1
- 239000003429 antifungal agent Substances 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 11
- 239000000499 gel Substances 0.000 description 8
- 241000283973 Oryctolagus cuniculus Species 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 229940027941 immunoglobulin g Drugs 0.000 description 6
- 239000000020 Nitrocellulose Substances 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 229920001220 nitrocellulos Polymers 0.000 description 5
- 230000009885 systemic effect Effects 0.000 description 5
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000007975 buffered saline Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 description 2
- VKYZDCTWJGBFDW-UHFFFAOYSA-N 4-chloro-2-methylaniline;hydron;chloride Chemical compound Cl.CC1=CC(Cl)=CC=C1N VKYZDCTWJGBFDW-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241000222122 Candida albicans Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000007973 glycine-HCl buffer Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 208000030289 Lymphoproliferative disease Diseases 0.000 description 1
- 238000011887 Necropsy Methods 0.000 description 1
- 239000006159 Sabouraud's agar Substances 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000000521 hyperimmunizing effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000001268 lymphoproliferative syndrome Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
Definitions
- This invention relates to antigens, their isolation from subjects suffering from the associated malady, to antibodies against the antigens, and to their respective uses in therapy and diagnosis.
- Candida is a fungus which is pathogenic in humans who are i muno-suppressed.
- Systemic candidosis is a well-known complaint. It is associated with dissemination of Candida species throughout the blood stream and invading deep tissues. This occurs in patients with an underlying lympho-proliferative disorder, or after surgery.
- the diagnostic criteria are (1) cultural and histological evidence from a deep organ biopsy at necropsy or during life, and (2) positive blood cultures.
- EP-A-0141616 and O-A- disclose monoclonal antibodies against antigens of the genus Candida. A description of the various species within the genus is given in the latter, the relevant contents of which are incorporated herein by reference.
- a first aspect of the present invention lies in candidosis antigens, for therapeutic or diagnostic use.
- a second aspect of the present invention lies in antibodies, more preferably human antibodies and most preferably human monoclonal antibodies, against a candidosis antigen, e.g. an antigen as defined above, for therapeutic or diagnostic use, and in such human monoclonal antibodies per se.
- An antigen of the invention may be obtained by purification of a blood sample obtained from a subject suffering from systemic candidosis, followed by purification.
- An antigen of the invention may be provided in suitable form for therapeutic or diagnostic use, e.g. in substantially pure form and/or as part of the kit containing one or more other reagents.
- the antigen is preferably provided for use in the form of a vaccine, e.g. for intravenous or percutaneous administration to a human subject.
- An antigen of the present invention may be characterised by a molecular weight o ⁇ from 45 to 55 kD, and more specifically about 47 kD.
- An antigen of the invention may be used to raise serum in an animal, e.g. in rabbits.
- the rabbit anti-serum may then be used to detect candidal antigens in human sera.
- An antigen of the invention may be of general use as a diagnostic reagent, as the basis of antigen tests for systemic candidosis.
- Antigens of the invention, refractionated if necessary, may be used in the invention .described in O-A-8600927.
- Antibodies of the invention may be provided for use in the form of a pharmaceutical composition. They may be used in the treatment of systemic candidosis. They may be given directly or coupled to certain drugs, e.g. anti-candidosis drugs such as amphotericin B or 5-fluorocytosine.
- This Example illustrates how an antigen of the invention may be obtained from yeast.
- Yeasts (Candida albicans species) were grown overnight in Sabouraud's agar or in a defined minimal accommodation supplemented with amino-acids. They were harvested in distilled water, and crushed using a press; a "pressate” was prepared by fragmentation in an "X press” (LKB, Bromnia, Sweden ⁇ at a pressure of 200 mPa. Any suitable form of mechanical destruction may be used instead of a press, e.g. glass balls may be used.
- the pressate was frozen at -70 C overnight and then loaded with yeast. This product was then placed within plastic bags in a 70% methanol bath containing cardice, for 30 minutes. The plastic bags prevented methanol from penetrating into the pressate containing the yeast. The pressate was taken from the bath, the plastic bags were removed, and a pressure of 200 mPa was exerted.
- This Example illustrates the preparation of an antigen from human serum.
- Serum was obtained from patients with systemic candidosis, and desalted. This desalted serum was used as an antigen preparation in an immunoblotting system.
- the nitrocellulose was then incubated at 25 C for 2 hours with the anti-serum raised against the yeast "pressate", diluted 1:50 in 3% BSA - 0.05% Tween 20 in buffered saline. After washing five times over 30 minutes in 0.9% saline - 0.05% Tween 20, it was incubated for 1 hour at 25 C with alkaline phosphatase-conjugated goat anti-rabbit IgG. The conjugate was diluted 1:1000, immediately before use in 3% BSA in buffered saline. After washing, the nitrocellulose was incubated for 15 minutes at 25 C in a freshly prepared and filtered mixture of equal volumes of naphthol ASMX phosphate and Fast Red TR salt.
- This Example illustrates the further purification of an antigen obtained, as in Example 2, from human serum.
- Serum was obtained from a patient with candidosis, and was shown, by enzyme immunoassay against antigen prepared as in Examples 1 and 2, to contain antibody against antigens of Candida albicans, including the antigen of 45 to 47 kD described above.
- Immunoglobulin G (IgG) from the serum was purified by adsorption on to protein A (which has a specific affinity for IgG) and then elution. This yielded 110 mg IgG which was shown, by polyacrylamide gel electrophoresis, to be pure.
- the IgG was coupled to 20 ml Sepharose CL 6B using cyanogen bromide (a standard method) .
- the IgG-Sepharose complex was packed into a column and washed, firstly with 3 column volumes of glycine-HCl buffer, pH 2.8, and secondly with 5 column volumes of PBS.
- the column was then washed through with glycine-HCl buffer, pH 2.8, to elute the Candida antigen specifically bound to the IgG-Sepharose.
- the eluate was collected in
- the column was then washed again with PBS in readiness for the next sample of desalted serum.
- a sodium dodecyl polyacrylamide gel was prepared by:
- Transblot cell A current of 350 mA was applied for 1.5 hours, so that antigen was transferred from the gel to the paper. The paper was then removed, and soaked in
- 35 paper was then washed five times in normal saline with 0.05% Tween 20, and then incubated at 25 C for 1 hour with a goat antibody against rabbit or human (as appropriate) immunoglobulin conjugated with alkaline phosphatase, diluted 1:1000 in BSA-TRIS.
- the paper was washed as before, and then incubated for 15 min at 25 C in a freshly prepared and filtered mixture of equal volumes of naphthol ASMX phosphate and Fast Red TR salt.
- the paper was then washed again as before, and dried in air, when characteristic bands of stained immune complexes were visible on the paper.
- monoclonal antibody may be used, prepared by the procedure described in either WO-A-8600927 or O-A-8600928 , the contents of which are incorporated herein by reference.
- the antibody may be diluted 1:7 in the buffered BSA.
- the antigenic supernatant obtained by the procedure of Example 1 is used to raise hyperimmune serum in New ' Zealand white rabbits.
- the rabbit anti-serum is then used to detect candidal antigens in human sera as described in Example 4.
- the rabbits are immunised either by direct intravenous injection or else by combining antigen with Full Freund's adjuvant and immunising subcutaneously.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A purified Candidosis antigen, having a molecular weight of 45 to 55 kD, can be obtained and used in a vaccine. Antibodies, e.g. a human monoclonal antibody, can be obtained and used in therapy, e.g. together with an anti-fungal agent.
Description
AXON
-1-
ANTIGENS, ANTIBODIES AND THEIR USES FIELD OF THE INVENTION
This invention relates to antigens, their isolation from subjects suffering from the associated malady, to antibodies against the antigens, and to their respective uses in therapy and diagnosis. BACKGROUND OF THE INVENTION
Candida is a fungus which is pathogenic in humans who are i muno-suppressed. Systemic candidosis is a well-known complaint. It is associated with dissemination of Candida species throughout the blood stream and invading deep tissues. This occurs in patients with an underlying lympho-proliferative disorder, or after surgery. The diagnostic criteria are (1) cultural and histological evidence from a deep organ biopsy at necropsy or during life, and (2) positive blood cultures.
EP-A-0141616 and O-A- (PCT/GB85/00476) disclose monoclonal antibodies against antigens of the genus Candida. A description of the various species within the genus is given in the latter, the relevant contents of which are incorporated herein by reference. SUMMARY OF THE INVENTION
A first aspect of the present invention lies in candidosis antigens, for therapeutic or diagnostic use. A second aspect of the present invention lies in antibodies, more preferably human antibodies and most preferably human monoclonal antibodies, against a candidosis antigen, e.g. an antigen as defined above, for therapeutic or diagnostic use, and in such human monoclonal antibodies per se. DETAILED DESCRIPTION OF THE INVENTION
An antigen of the invention may be obtained by purification of a blood sample obtained from a subject suffering from systemic candidosis, followed by purification. An antigen of the invention may be
provided in suitable form for therapeutic or diagnostic use, e.g. in substantially pure form and/or as part of the kit containing one or more other reagents. The antigen is preferably provided for use in the form of a vaccine, e.g. for intravenous or percutaneous administration to a human subject. An antigen of the present invention may be characterised by a molecular weight o± from 45 to 55 kD, and more specifically about 47 kD. An antigen of the invention may be used to raise serum in an animal, e.g. in rabbits. The rabbit anti-serum may then be used to detect candidal antigens in human sera. An antigen of the invention may be of general use as a diagnostic reagent, as the basis of antigen tests for systemic candidosis. Antigens of the invention, refractionated if necessary, may be used in the invention .described in O-A-8600927.
Antibodies of the invention may be provided for use in the form of a pharmaceutical composition. They may be used in the treatment of systemic candidosis. They may be given directly or coupled to certain drugs, e.g. anti-candidosis drugs such as amphotericin B or 5-fluorocytosine.
The following Examples 1, 2, 3 and 5 illustrate the invention. The following abbreviations are used: PBS = phosphate-buffered saline; BSA = bovine serum albumin; BSA-TRIS = 3% BSA in TRIS-buffered saline, pH 7.4. Percentages are by weight, or by volume with respect to liquids. ("Millipore" , "Millex" , "Sepharose" and "Tween" may be registered Trade Marks) . Example 1
This Example illustrates how an antigen of the invention may be obtained from yeast.
Yeasts (Candida albicans species) were grown overnight in Sabouraud's agar or in a defined minimal
mediu supplemented with amino-acids. They were harvested in distilled water, and crushed using a press; a "pressate" was prepared by fragmentation in an "X press" (LKB, Bromnia, Sweden^ at a pressure of 200 mPa. Any suitable form of mechanical destruction may be used instead of a press, e.g. glass balls may be used.
The pressate was frozen at -70 C overnight and then loaded with yeast. This product was then placed within plastic bags in a 70% methanol bath containing cardice, for 30 minutes. The plastic bags prevented methanol from penetrating into the pressate containing the yeast. The pressate was taken from the bath, the plastic bags were removed, and a pressure of 200 mPa was exerted.
The process was repeated three times, to obtain 90% or more disruption of cells. The distintegrated cells were removed from the press and centrifuged at 38,000 g for 1.5 hours at 4 C. The supernatant was filtered through a 0.22 μm Millipore-membrane (Millex) , to ensure sterility. Example 2
This Example illustrates the preparation of an antigen from human serum.
Serum was obtained from patients with systemic candidosis, and desalted. This desalted serum was used as an antigen preparation in an immunoblotting system.
It was run on a SDS Polyacrylamide electrophoresis gel to separate it from the other components of serum. A Transblotting set was used to transfer it from the gel to nitrocellulose paper. It was detected by a modified enzyme-linked immunosorbent assay, using a rabbit anti-serum raised against a yeast "pressate" (see above) . Transfer was performed at room temperature, using a current of 350 mA for 1.5 hours, in a Bio-Rad Trans-Blot cell. Free protein sites were saturated by incubation in
3% BSA in buffered saline (0.9% NaCl - 10 mM TRIS, pH 7.4) at 4 C overnight.
The nitrocellulose was then incubated at 25 C for 2 hours with the anti-serum raised against the yeast "pressate", diluted 1:50 in 3% BSA - 0.05% Tween 20 in buffered saline. After washing five times over 30 minutes in 0.9% saline - 0.05% Tween 20, it was incubated for 1 hour at 25 C with alkaline phosphatase-conjugated goat anti-rabbit IgG. The conjugate was diluted 1:1000, immediately before use in 3% BSA in buffered saline. After washing, the nitrocellulose was incubated for 15 minutes at 25 C in a freshly prepared and filtered mixture of equal volumes of naphthol ASMX phosphate and Fast Red TR salt. Example 3
This Example illustrates the further purification of an antigen obtained, as in Example 2, from human serum. Serum was obtained from a patient with candidosis, and was shown, by enzyme immunoassay against antigen prepared as in Examples 1 and 2, to contain antibody against antigens of Candida albicans, including the antigen of 45 to 47 kD described above. Immunoglobulin G (IgG) from the serum was purified by adsorption on to protein A (which has a specific affinity for IgG) and then elution. This yielded 110 mg IgG which was shown, by polyacrylamide gel electrophoresis, to be pure.
The IgG was coupled to 20 ml Sepharose CL 6B using cyanogen bromide (a standard method) . The IgG-Sepharose complex was packed into a column and washed, firstly with 3 column volumes of glycine-HCl buffer, pH 2.8, and secondly with 5 column volumes of PBS.
2-3 ml desalted serum (as from Example 2) were passed through the column at a rate of 0.5 l/min, and the column was then washed with PBS until the optical
densi'ty of the filtrate, measured at 280 nm, was zero.
The column was then washed through with glycine-HCl buffer, pH 2.8, to elute the Candida antigen specifically bound to the IgG-Sepharose. The eluate was collected in
5 vessels containing solid TRIS , to raise the pH to 8.0.
The column was then washed again with PBS in readiness for the next sample of desalted serum.
Each sample of eluted antigen was placed in a dialysis sac, and covered with solid sucrose to
10 concentrate the antigen. The antigen was then dialysed against distilled water.
Example 4
This Example illustrates an assay for antigen obtained by any of the methods described in Examples 1, 2
15 and 3.
A sodium dodecyl polyacrylamide gel was prepared by
. a standard method. Aliquots of the antigen (0.025 ml) in distilled water were applied into sample slots of the gel, and a current of 50 mA at 150 volts -was applied
20 across the gel for 6 hours. During this time, the components of the sample migrated in the gel at a rate roughly proportional to their molecular size.
The current was switched off. The gel was removed from its glass supporting plates and mounted, together
25 with a sheet of nitrocellulose paper, in a Bio-Rad
Transblot cell. A current of 350 mA was applied for 1.5 hours, so that antigen was transferred from the gel to the paper. The paper was then removed, and soaked in
BSA-TRIS at 4 C overnight, to saturate free binding sites
30 on the paper. _% • The nitrocellulose paper was then incubated with anti-serum prepared as in Example 3 (or from patients with, or convalescing from, candidosis) diluted 1:50 in
BSA-TRIS with 0.05% Tween 20 for 2 hours at 25 C. The
35 paper was then washed five times in normal saline with
0.05% Tween 20, and then incubated at 25 C for 1 hour with a goat antibody against rabbit or human (as appropriate) immunoglobulin conjugated with alkaline phosphatase, diluted 1:1000 in BSA-TRIS. The paper was washed as before, and then incubated for 15 min at 25 C in a freshly prepared and filtered mixture of equal volumes of naphthol ASMX phosphate and Fast Red TR salt. The paper was then washed again as before, and dried in air, when characteristic bands of stained immune complexes were visible on the paper.
Instead of anti-serum, monoclonal antibody may be used, prepared by the procedure described in either WO-A-8600927 or O-A-8600928 , the contents of which are incorporated herein by reference. The antibody may be diluted 1:7 in the buffered BSA. Example 5
The antigenic supernatant obtained by the procedure of Example 1 is used to raise hyperimmune serum in New ' Zealand white rabbits. The rabbit anti-serum is then used to detect candidal antigens in human sera as described in Example 4. The rabbits are immunised either by direct intravenous injection or else by combining antigen with Full Freund's adjuvant and immunising subcutaneously.
Claims
1. A candidosis antigen, for therapeutic or diagnostic use.
2. An essentially pure candidosis antigen.
3. An antigen according to claim 1 or claim 2, which has a molecular weight of from 45 to 55 kD.
4. An antigen according to claim 1 or claim 2, which has a molecular weight of about 47 kD.
5. A human antibody against a candidosis antigen, for therapeutic or diagnostic use.
6. A human monoclonal antibody against a candidosis antigen.
7. A human monoclonal antibody against a candidosis antigen, for therapeutic or diagnostic use.
8. An antibody according to any of claims 5 to 7, in which the antigen is as defined in claim 3 or claim 4.
9. A vaccine which comprises an antigen according to any of claims 1 to 4.
10. A -pharmaceutical composition which comprises an antibody according to any of claims 5 to 8.
11. A composition according to claim 10, which additionally comprises an anti-candidosis drug.
12. A composition according to claim 11, in which the drug is amphotericin B or 5-fluorocytosine.
13. A method of treating a human or other animal subject prone to or suffering from candidosis, which comprises administering to the subject an anti-candidosis effective amount of an antigen according to any of claims 1 to 4, an antibody according to any of claims 5 to 8, a vaccine according to claim 9 or a composition according to any of claims 10 to 12.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8506373 | 1985-03-12 | ||
GB858506373A GB8506373D0 (en) | 1985-03-12 | 1985-03-12 | Antigens antibodies |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1986005400A1 true WO1986005400A1 (en) | 1986-09-25 |
Family
ID=10575866
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1986/000142 WO1986005400A1 (en) | 1985-03-12 | 1986-03-12 | Antigens, antibodies and their uses |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0214265A1 (en) |
JP (1) | JPS62502193A (en) |
GB (1) | GB8506373D0 (en) |
WO (1) | WO1986005400A1 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0406029A1 (en) * | 1989-06-30 | 1991-01-02 | The Victoria University Of Manchester | Fungal stress proteins |
WO1996007426A1 (en) * | 1994-09-06 | 1996-03-14 | Auspharm International Limited | Enteral vaccine for vaginal infections |
GB2304347A (en) * | 1995-08-11 | 1997-03-19 | Boeringer Ingelheim Vetmedica | Antigenic preparations |
US6136794A (en) * | 1998-02-02 | 2000-10-24 | Merck & Co., Inc. | Platelet aggregation inhibition using low molecular weight heparin in combination with a GP IIb/IIIa antagonist |
US7722869B2 (en) | 2007-04-27 | 2010-05-25 | Novartis Ag | Antibody molecules and nucleic acids |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU4281001A (en) * | 2000-03-27 | 2001-10-08 | Mochida Pharmaceutical Co., Ltd. | Kit for detecting fungal infection of specimens in oral cavity |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2108384A (en) * | 1981-11-03 | 1983-05-18 | Pal Sutka | Immunobiological preparations and processes for their production |
FR2543969A1 (en) * | 1983-04-08 | 1984-10-12 | Kureha Chemical Ind Co Ltd | ANTIBODY AGAINST MUSHROOMS OF THE GENUS CANDIDA, HYBRIDOMA AND PROCESS FOR THE PREPARATION OF SAID ANTIBODY, METHOD OF IDENTIFYING AND / OR CLASSIFYING FUNGI OF THE GENUS CANDIDA USING THE ANTIBODY OR ONE OF ITS DERIVATIVES OR RESTRICTION PRODUCTS AND MEDICAMENT AGAINST CANDIDOSES CONTAINING THIS ANTIBODY OR ITS DERIVATIVES |
EP0145333A2 (en) * | 1983-12-02 | 1985-06-19 | Temple University - Of The Commonwealth System Of Higher Education | Candida albicans cytoplasmic antigen, monoclonal antibodies against it and methods for the preparation |
WO1986002365A1 (en) * | 1984-10-19 | 1986-04-24 | Technology Licence Company Limited | Monoclonal antibodies and their use |
-
1985
- 1985-03-12 GB GB858506373A patent/GB8506373D0/en active Pending
-
1986
- 1986-03-12 EP EP86901937A patent/EP0214265A1/en not_active Withdrawn
- 1986-03-12 JP JP61501599A patent/JPS62502193A/en active Pending
- 1986-03-12 WO PCT/GB1986/000142 patent/WO1986005400A1/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2108384A (en) * | 1981-11-03 | 1983-05-18 | Pal Sutka | Immunobiological preparations and processes for their production |
FR2543969A1 (en) * | 1983-04-08 | 1984-10-12 | Kureha Chemical Ind Co Ltd | ANTIBODY AGAINST MUSHROOMS OF THE GENUS CANDIDA, HYBRIDOMA AND PROCESS FOR THE PREPARATION OF SAID ANTIBODY, METHOD OF IDENTIFYING AND / OR CLASSIFYING FUNGI OF THE GENUS CANDIDA USING THE ANTIBODY OR ONE OF ITS DERIVATIVES OR RESTRICTION PRODUCTS AND MEDICAMENT AGAINST CANDIDOSES CONTAINING THIS ANTIBODY OR ITS DERIVATIVES |
EP0145333A2 (en) * | 1983-12-02 | 1985-06-19 | Temple University - Of The Commonwealth System Of Higher Education | Candida albicans cytoplasmic antigen, monoclonal antibodies against it and methods for the preparation |
WO1986002365A1 (en) * | 1984-10-19 | 1986-04-24 | Technology Licence Company Limited | Monoclonal antibodies and their use |
Non-Patent Citations (1)
Title |
---|
CHEMICAL ABSTRACTS, Volume 100, No. 17, 23 April 1984, Columbus, Ohio, (US) N.A. STOCKBINE et al.: "Production and Characterization of Three Monoclonal Antibodies to Candida Albicans Proteins", see page 484, Abstract No. 137106s & Infect. Immun. 1984, 43 (3), 1012-18 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0406029A1 (en) * | 1989-06-30 | 1991-01-02 | The Victoria University Of Manchester | Fungal stress proteins |
WO1991000351A1 (en) * | 1989-06-30 | 1991-01-10 | Burnie James P | Fungal stress proteins |
GB2240979A (en) * | 1989-06-30 | 1991-08-21 | Burnie James P | Fungal stress proteins |
GB2240979B (en) * | 1989-06-30 | 1993-03-17 | Burnie James P | Fungal stress proteins |
US5288639A (en) * | 1989-06-30 | 1994-02-22 | The Victoria University Of Manchester | Fungal stress proteins |
US5541077A (en) * | 1989-06-30 | 1996-07-30 | The Victoria University Of Manchester | Fungal stress proteins |
US5686248A (en) * | 1989-06-30 | 1997-11-11 | The Victoria University Of Manchester | Fungal stress proteins |
WO1996007426A1 (en) * | 1994-09-06 | 1996-03-14 | Auspharm International Limited | Enteral vaccine for vaginal infections |
GB2304347A (en) * | 1995-08-11 | 1997-03-19 | Boeringer Ingelheim Vetmedica | Antigenic preparations |
US6136794A (en) * | 1998-02-02 | 2000-10-24 | Merck & Co., Inc. | Platelet aggregation inhibition using low molecular weight heparin in combination with a GP IIb/IIIa antagonist |
US7722869B2 (en) | 2007-04-27 | 2010-05-25 | Novartis Ag | Antibody molecules and nucleic acids |
Also Published As
Publication number | Publication date |
---|---|
JPS62502193A (en) | 1987-08-27 |
GB8506373D0 (en) | 1985-04-11 |
EP0214265A1 (en) | 1987-03-18 |
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