US4670382A
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Monoclonal antibody to Candida albicans cytoplasmic antigens and methods of preparing same
Assignee: Temple University
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Filing: 1984-01-16
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Granted: 1987-06-02
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Expires: 2004-01-16
Abstract
Abstract Two novel hybridoma cell lines, ATCC #HB-8397 and ATCC #HB-8398 produce monoclonal antibody monospecific to a single determinant shared by a set of three closely related cytoplasmic antigens of Candida albicans. The antigens have molecular weights of 120-135 Kd, 48-52 Kd, and 35-38 Kd. The hybridomas are formed by fusing splenocytes from immunized BALB/c mice with SP2/O-Ag 14 myeloma cells. Monoclonal and monospecific, polyclonal antibodies to these cytoplasmic antigens find application in the immunodiagnosis of Candida infections. A procedure is provided for preparing partially purified cytoplasmic antigen of pathogenic Candida species for administration to splenocyte-donating mice. Also provided is a method for the biochemical purification of cytoplasmic antigen of a pathogenic Candida species used for the preparation of monoclonal and monospecific, polyclonal antisera thereto.
Inventors
- 👤 Helen R. Buckley
- 👤 Michael T. Largen
- 👤 Nancy A. Strockbine
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 21:13
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/37; A; A61; A61P; A61P31/00; A61P31/04; C07K16/00; C07K16/14; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/948
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
28 words
Mouse monoclonal antibody which reacts with 3 non-cell-wall cytoplasmic antigens of C. albicans having apparent molecular weights 120-135 Kd, 48-52 Kd, and 35-38 Kd as determined by SDS-PAGE.
Claim 2
Dependent Claim
28 words
Mouse monoclonal antibody which reacts with 3 non-cell-wall cytoplasmic antigens of C. albicans having apparent molecular weights 120-135 Kd, 48-52 Kd, and 35-38 Kd as determined by SDS-PAGE.
Claim 3
Dependent Claim
147 words
A mouse monoclonal antibody which binds a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections, said antibody prepared by the method which comprises the steps of: a. Immunizing mice with cytoplasmic extract of C. albicans; b. Removing the spleens from said mice and making a suspension; c. Fusing said spleen cells with mouse myeloma cells in the presence of a fusion promoter; d. Diluting and culturing the fused cells in separate wells in a medium which will not support the unfused myeloma or spleen cells; e. Evaluating the supernatant in each well for the presence of antibody to said non-cell-wall cytoplasmic antigen of C. albicans; f. Selecting and cloning an antibody-producing hybridoma which binds said antigen; g. Recovering the antibody from the supernatant above said clones.
Claim 4
Dependent Claim
147 words
A mouse monoclonal antibody which binds a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections, said antibody prepared by the method which comprises the steps of: a. Immunizing mice with cytoplasmic extract of C. albicans; b. Removing the spleens from said mice and making a suspension; c. Fusing said spleen cells with mouse myeloma cells in the presence of a fusion promoter; d. Diluting and culturing the fused cells in separate wells in a medium which will not support the unfused myeloma or spleen cells; e. Evaluating the supernatant in each well for the presence of antibody to said non-cell-wall cytoplasmic antigen of C. albicans; f. Selecting and cloning an antibody-producing hybridoma which binds said antigen; g. Recovering the antibody from the supernatant above said clones.
Claim 5
Dependent Claim
27 words
A monoclonal antibody according to claim 2 which binds to three cytoplasmic antigens of C. albicans of apparent molecular weights 120-135 Kd, 48-52 Kd and 35-38 Kd.
Claim 6
Dependent Claim
168 words
A mouse monoclonal antibody which binds a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections, said antibody prepared by the method which comprises the steps of: a. Immunizing mice with cytoplasmic extract of C. albicans; b. Removing the spleens from said mice and making a suspension of the spleen cells; c. Fusing said spleen cells with mouse myeloma cells in the presence of a fusion promoter; d. Diluting and culturing the fused cells in separate wells in a medium which will not support the unfused myeloma or spleen cells; e. Evaluating the supernatant in each well for the presence of antibody to said non-cell-wall cytoplasmic antigen of C. albicans; f. Selecting and cloning a hybridoma producing antibody which binds said antigen; g. Transferring said clones intraperitoneally into mice and; h. Harvesting the malignant ascites or serum from said mice, which ascites or serum contains the desired antibody.
Claim 7
Dependent Claim
168 words
A mouse monoclonal antibody which binds a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections, said antibody prepared by the method which comprises the steps of: a. Immunizing mice with cytoplasmic extract of C. albicans; b. Removing the spleens from said mice and making a suspension of the spleen cells; c. Fusing said spleen cells with mouse myeloma cells in the presence of a fusion promoter; d. Diluting and culturing the fused cells in separate wells in a medium which will not support the unfused myeloma or spleen cells; e. Evaluating the supernatant in each well for the presence of antibody to said non-cell-wall cytoplasmic antigen of C. albicans; f. Selecting and cloning a hybridoma producing antibody which binds said antigen; g. Transferring said clones intraperitoneally into mice and; h. Harvesting the malignant ascites or serum from said mice, which ascites or serum contains the desired antibody.
Claim 8
Dependent Claim
27 words
A monoclonal antibody according to claim 4 which binds to three cytoplasmic antigens of C. albicans of apparent molecular weights 120-135 Kd, 48-52 Kd and 35-38 Kd.
Claim 9
Independent Claim
48 words
A method of preparing monoclonal antibodies against cytoplasmic antigens of C. albicans with molecular weights of 35-38 Kd, 48-52 Kd, and 120-135 Kd, which comprises culturing the hybridomas ATCC #HB-8397 or ATCC #HB-8398 in a suitable medium and recovering the antibody from the supernatant of above said hybridomas.
Claim 10
Independent Claim
48 words
A method of preparing monoclonal antibodies against cytoplasmic antigens of C. albicans with molecular weights of 35-38 Kd, 48-52 Kd, and 120-135 Kd, which comprises culturing the hybridomas ATCC #HB-8397 or ATCC #HB-8398 in a suitable medium and recovering the antibody from the supernatant of above said hybridomas.
Claim 11
Dependent Claim
10 words
The monoclonal antibody prepared by the method of claim 7.
Claim 12
Independent Claim
49 words
A method of preparing monoclonal antibodies against cytoplasmic antigens of C. albicans with molecular weights of 35-38 Kd, 48-52 Kd, and 120-135 Kd, which comprises injecting into a mouns the hybridomas ATCC #HB-8397 or ATCC #HB-8398 and recovering the antibody from the malignant ascites or serum of said mice.
Claim 13
Independent Claim
49 words
A method of preparing monoclonal antibodies against cytoplasmic antigens of C. albicans with molecular weights of 35-38 Kd, 48-52 Kd, and 120-135 Kd, which comprises injecting into a mouns the hybridomas ATCC #HB-8397 or ATCC #HB-8398 and recovering the antibody from the malignant ascites or serum of said mice.
Claim 14
Dependent Claim
10 words
The monoclonal antibody prepared by the method of claim 1.
Claim 15
Dependent Claim
77 words
A composition comprising a continuous cell line which produces monoclonal antibodies of class lgG to a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections, comprising a fused cell hybrid of a BALB/c mouse spleen cell previously immunized with a cytoplasmic antigen of C. albicans fused to a mouse myeloma and a culture medium for said hybrid.
Claim 16
Dependent Claim
77 words
A composition comprising a continuous cell line which produces monoclonal antibodies of class lgG to a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections, comprising a fused cell hybrid of a BALB/c mouse spleen cell previously immunized with a cytoplasmic antigen of C. albicans fused to a mouse myeloma and a culture medium for said hybrid.
Claim 17
Dependent Claim
12 words
The composition of claim 10 wherein said mouse myeloma is SP2/O-Ag 14.
Claim 18
Dependent Claim
21 words
The composition of claim 11 wherein the cell line is selected from the group consisting of ATCC #HB-8397 and ATCC #HB-8398.
Claim 19
Dependent Claim
26 words
The composition of claim 10 wherein the monoclonal antibodies react with three cytoplasmic antigens of apparent molecular weights of 120-135 Kd, 48-52 Kd, and 35-38 Kd.
Claim 20
Dependent Claim
17 words
The composition of claim 10 wherein the monoclonal antibodies are of the IgG 1 , kappa subclass.
Claim 21
Dependent Claim
36 words
A monoclonal antibody which binds to a cytoplasmic non-cell-wall antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections.
Claim 22
Dependent Claim
36 words
A monoclonal antibody which binds to a cytoplasmic non-cell-wall antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in humans during disseminated candidiasis but not during non-invasive C. albicans infections.
Claim 23
Dependent Claim
34 words
A monoclonal antibody according to claim 15 produced by a hybridoma formed by fusion of cells from a myeloma line with spleen cells from a donor previously immunized by cytoplasmic antigen from C. albicans.
Claim 24
Dependent Claim
16 words
A monoclonal antibody according to claim 16 wherein the myeloma line and spleen cells are murine.
Claim 25
Dependent Claim
10 words
A monoclonal antibody according to claim 17 of class IgG.
Claim 26
Dependent Claim
10 words
A monoclonal antibody according to claim 18 of kappa subclass.
Claim 27
Dependent Claim
64 words
A monoclonal antibody according to claim 18 which: reacts with three cytoplasmic antigens of C. albicans with apparent molecular weights in SDS-PAGE of 120-135 Kd, 48-52 Kd, and 35-38 Kd; is monospecific for a single antigenic determinant shared by the cytoplasmic antigens of C. albicans with the above apparent molecular weights; and is not contaminated with other immunoglobulins directed against any other Candida antigens.
Claim 28
Dependent Claim
37 words
A monoclonal antiboyd according to claim 17 which is produced from hybridomas formed by the fusion of SP2/O-Ag 14 mouse myeloma cells and spleen cells from a BALB/c mouse previously immunized with cytoplasmic antigen of C. albicans.
Claim 29
Dependent Claim
33 words
Polyclonal, monospecific antisera against a cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detected in humans during disseminated candidiasis but not during non-invasive C. albicans infection.
Claim 30
Dependent Claim
33 words
Polyclonal, monospecific antisera against a cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detected in humans during disseminated candidiasis but not during non-invasive C. albicans infection.
Claim 31
Dependent Claim
28 words
Antisera according to claim 22 which reacts with three cytoplasmic antigens of C. albicans with apparent molecular weights in SDS-PAGE of 120-135 Kd, 48-52 Kd and 35-38 Kd.
Claim 32
Dependent Claim
56 words
A diagnostic method for disseminated or invasive candidiasis comprising contacting body fluids with a monoclonal antibody which binds to a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in said fluids during disseminated candidiasis but not during non-invasive infection, and detecting the material bound by said antibody.
Claim 33
Dependent Claim
56 words
A diagnostic method for disseminated or invasive candidiasis comprising contacting body fluids with a monoclonal antibody which binds to a non-cell-wall cytoplasmic antigen of C. albicans of 48-52 Kd apparent molecular weight in SDS-PAGE which is detectable in said fluids during disseminated candidiasis but not during non-invasive infection, and detecting the material bound by said antibody.
Claim 34
Dependent Claim
33 words
A diagnostic method according to claim 24 wherein the monoclonal antibody reacts with three cytoplasmic antigens of C. albicans with apparent molecular weights in SDS-PAGE of 120-135 Kd, 48-52 Kd and 35-38 Kd.
Claim 35
Dependent Claim
13 words
A diagnostic method according to claim 24 wherein the fluid is blood serum.
Claim 36
Dependent Claim
29 words
A diagnostic method according to claim 24 wherein the monoclonal antibody is produced by a hybridoma selected from the group of hybridomas consisting of ATCC #HB-8397 and ATCC #HB-8398.
Claim 37
Dependent Claim
25 words
A diagnostic method according to claim 25 wherein the detecting means is selected from the group consisting of latex agglutination, radioimmunoassay and enzyme-linked immunosorbent assay.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-06-01 | 1990-12-01 | 1991-06-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-06-01 | 1994-12-01 | 1995-06-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-06-01 | 1998-12-01 | 1999-06-02 | $7700.00 | Window Closed / Lapsed |