AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas.METHODS: mRNA was isolated from the hybridoma cell lineproducing MC3 and the DNAs encoding variable domains ofheavy and light chains(VH and VL) oftthe antibody wereamplified separately byRT-PCR and assembled into ScFvDNA with a linker DNAThe ScFv DNA was iigated into thephagemid vector pCANTAB5E and the ligated sample wastransformed into E. coil TG1. The transformed cells wereinfected with M13KO7 helper phage to yield recombinantphages. After two rounds of panning with gastric carcinomacell line AGS highly expressing MC3-binding antigen, thephage clones displaying ScFv fragments of the antibodywere selected by ELISA. 4 phage clones showing strongsignal in ELISA were used to infect E. coil HB2151 toexpress soluble ScFvs. The soluble ScFve were identified byDot blot and Western blot, and their antigen-binding activitywas assayed by ELISA. The VH and VL DNAs of the ScFvDNA derived from phage clone 19 were sequenced.RESULTS: The VH, VL and ScFv DNAs were about 340 bp,320 bp and 750 bp respectively. After two rounds of panningto the recombinant phages, 18 antigen-positive phageclones were selected from 30 preselected phage clones byELISA. All the soluble ScFvs derived from the 4 out of the 18antigen-positive phage clones were about Mr 32 000 andconcentrated in periplasmatic space under the given culturecondition. The soluble ScFvs could bind the antigen, andthey shared the same binding site with MC3. The sequencesof the VH and VL DNAs of the MC3 ScFv showed that thevariable antibody genes belonged to the IgG1 subgroup,κ-type.CONCLUSION: The soluble ScFv of MC3 is successfullyproduced, which not only provides a possible novel targetingvehicle for in vivo and in vitro study on associated cancers,but also offers the anuibody a stable genetic source.
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