BIOMAT Database Logo BIOMAT Database Logo

Specific immunoglobulin cDNA clones produced from hybridoma cell lines and murine spleen fragment cultures by 3SR amplification. 1994

C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Life Sciences Research Laboratory, Baxter Diagnostics, Inc., San Diego, California 92121.

The isothermal 3SR amplification method has been employed to assist in cloning the VL and VH genes from cells of hybridomas and splenic fragment cultures expressing antibodies for phosphorylcholine (PC) and estradiol (E2), respectively. As a first step, pools of degenerate primer pairs were identified complementary to immunoglobulin light and heavy chain variable (V) genes and capable of amplifying immunoglobulin RNA specifically at 42 degrees C. To evaluate the functionality of the 3SR-cloned immunoglobulin genes, anti-PC VH and VL cDNAs were joined together to form a single chain (sc) antibody construct and were expressed in Escherichia coli under the regulation of the alkaline phosphatase (phoA) promoter. Similarly, the combination of a murine spleen fragment and 3SR methodologies were employed to clone a selected pool of cDNAs for cultures producing anti-estradiol antibodies. This approach of using the murine spleen fragment and 3SR isothermal amplification offers the advantages of B-cell follicle architecture for antigen-driven B-cell maturation and proliferation and RNA-specific amplification, respectively. The potential utility of these advantages for the production of monoclonal antibodies and for providing the capability of studying memory B-cell development are discussed.

UI MeSH Term Description Entries
D007136 Immunoglobulins Multi-subunit proteins which function in IMMUNITY. They are produced by B LYMPHOCYTES from the IMMUNOGLOBULIN GENES. They are comprised of two heavy (IMMUNOGLOBULIN HEAVY CHAINS) and two light chains (IMMUNOGLOBULIN LIGHT CHAINS) with additional ancillary polypeptide chains depending on their isoforms. The variety of isoforms include monomeric or polymeric forms, and transmembrane forms (B-CELL ANTIGEN RECEPTORS) or secreted forms (ANTIBODIES). They are divided by the amino acid sequence of their heavy chains into five classes (IMMUNOGLOBULIN A; IMMUNOGLOBULIN D; IMMUNOGLOBULIN E; IMMUNOGLOBULIN G; IMMUNOGLOBULIN M) and various subclasses. Globulins, Immune,Immune Globulin,Immune Globulins,Immunoglobulin,Globulin, Immune
D008807 Mice, Inbred BALB C An inbred strain of mouse that is widely used in IMMUNOLOGY studies and cancer research. BALB C Mice, Inbred,BALB C Mouse, Inbred,Inbred BALB C Mice,Inbred BALB C Mouse,Mice, BALB C,Mouse, BALB C,Mouse, Inbred BALB C,BALB C Mice,BALB C Mouse
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010767 Phosphorylcholine Calcium and magnesium salts used therapeutically in hepatobiliary dysfunction. Choline Chloride Dihydrogen Phosphate,Choline Phosphate Chloride,Phosphorylcholine Chloride,Choline Phosphate,Phosphocholine,Chloride, Choline Phosphate,Chloride, Phosphorylcholine,Phosphate Chloride, Choline,Phosphate, Choline
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004261 DNA Replication The process by which a DNA molecule is duplicated. Autonomous Replication,Replication, Autonomous,Autonomous Replications,DNA Replications,Replication, DNA,Replications, Autonomous,Replications, DNA
D004958 Estradiol The 17-beta-isomer of estradiol, an aromatized C18 steroid with hydroxyl group at 3-beta- and 17-beta-position. Estradiol-17-beta is the most potent form of mammalian estrogenic steroids. 17 beta-Estradiol,Estradiol-17 beta,Oestradiol,17 beta-Oestradiol,Aerodiol,Delestrogen,Estrace,Estraderm TTS,Estradiol Anhydrous,Estradiol Hemihydrate,Estradiol Hemihydrate, (17 alpha)-Isomer,Estradiol Monohydrate,Estradiol Valerate,Estradiol Valeriante,Estradiol, (+-)-Isomer,Estradiol, (-)-Isomer,Estradiol, (16 alpha,17 alpha)-Isomer,Estradiol, (16 alpha,17 beta)-Isomer,Estradiol, (17-alpha)-Isomer,Estradiol, (8 alpha,17 beta)-(+-)-Isomer,Estradiol, (8 alpha,17 beta)-Isomer,Estradiol, (9 beta,17 alpha)-Isomer,Estradiol, (9 beta,17 beta)-Isomer,Estradiol, Monosodium Salt,Estradiol, Sodium Salt,Estradiol-17 alpha,Estradiol-17beta,Ovocyclin,Progynon-Depot,Progynova,Vivelle,17 beta Estradiol,17 beta Oestradiol,Estradiol 17 alpha,Estradiol 17 beta,Estradiol 17beta,Progynon Depot
D006825 Hybridomas Cells artificially created by fusion of activated lymphocytes with neoplastic cells. The resulting hybrid cells are cloned and produce pure MONOCLONAL ANTIBODIES or T-cell products, identical to those produced by the immunologically competent parent cell. Hybridoma
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein

Related Publications

C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Autoagglutinin produced by organ fragment cultures of mouse spleen.
November 1977, European journal of immunology,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Antigen-specific suppressor factors from hybridoma cell lines.
January 1985, Methods in enzymology,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Isolation of cDNA clones encoding a T-cell receptor beta-chain from a beef insulin-specific hybridoma.
December 1985, Proceedings of the National Academy of Sciences of the United States of America,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells.
October 1990, Hybridoma,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
cDNA clones encoding IgE-binding factors from a rat-mouse T-cell hybridoma.
April 1985, Proceedings of the National Academy of Sciences of the United States of America,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.
April 1995, PCR methods and applications,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Construction of a partial rabbit spleen cDNA library and identification of immunoglobulin clones.
October 1982, European journal of immunology,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Establishment of antigen-specific and MHC-restricted B cell hybridoma clones: a model for studying antigen presentation to T cell lines and clones.
January 1988, Archivum immunologiae et therapiae experimentalis,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.
October 1997, Leukemia,
C A Stillman, and P J Linton, and P J Koutz, and D J Decker, and N R Klinman, and T R Gingeras
Nucleotide sequences of the gene/cDNA coding for anti-murine erythrocyte autoantibody produced by a hybridoma from NZB mouse.
April 1990, Nucleic acids research,
Copied contents to your clipboard!