Specific immunoglobulin cDNA clones produced from hybridoma cell lines and murine spleen fragment cultures by 3SR amplification.
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.
Stillman
C A
CA
Life Sciences Research Laboratory, Baxter Diagnostics, Inc., San Diego, California 92121.
Linton
P J
PJ
Koutz
P J
PJ
Decker
D J
DJ
Klinman
N R
NR
Gingeras
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D046508:Culture Techniques; D004261:DNA Replication; D004958:Estradiol / Q000276:immunology; D006825:Hybridomas / Q000276:immunology*; D007136:Immunoglobulins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010767:Phosphorylcholine / Q000276:immunology; D016133:Polymerase Chain Reaction; D013154:Spleen / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007136:Immunoglobulins; D010767:Phosphorylcholine; D004958:Estradiol