The LMO has optimized the oligonucleotide primer design for efficient mouse scFv cloning. With the newly-designed primers, it has been possible to successfully clone the variable domains of both the light chain and heavy chain of anti-ETS1 monoclonal antibody E44 and anti-ETS1 mouse spleen antibodies. The Laboratory is now making an effort to solve the problem in light chain and heavy chain assembly by replacing the problematic PCR assembly step by more predictable enzymatic ligation. After functional anti-ETS1 scFv's have been made, progress toward the goal of ETS1 intracellular function perturbation can be realized. Expression of the cloned anti-ETS1 scFv in eukaryotic cells, and a colon carcinoma cell line, which has been reverted back to normal after ETS1 overexpres- sion, will be attempted. Those designed scFv's will also be applied to various ETS1 projects ongoing within the LMO. To further explore the usage of the mouse scFv primers, a mouse naive scFv library has been developed in order to be expressed and test-selected against various pure antigens and oncoproteins of interest. A """"""""Universal Immuno-PCR"""""""" method has been developed, based on the recently described procedure, """"""""Immuno-PCR."""""""" This is, thus far, the most sensitive method available for antigen detection and can be applied to various laboratory and/or clinical studies or diagnosis.
