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 V-L and V-H genes from cells of hybridomas and splenic fr agment cultures expressing antibodies for phosphorylcholine (PC) and e stradiol (E2), respectively. As a first step, pools of degenerate prim er pairs were identified complementary to immunoglobulin light and hea vy chain variable (V) genes and capable of amplifying immunoglobulin R NA specifically at 42 degrees C. To evaluate the functionality of the 3SR-cloned immunoglobulin genes, anti-PC V-H and V-L cDNAs were joined together to form a single chain (sc) antibody construct and were expr essed in Escherichia coli under the regulation of the alkaline phospha tase (phoA) promoter. Similarly, the combination of a murine spleen fr agment 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-driv en B-cell maturation and proliferation and RNA-specific amplification, respectively. The potential utility of these advantages for the produ ction of monoclonal antibodies and for providing the capability of stu dying memory B-cell development are discussed.