Weissenhorn, Winfried; Weiss, Elisabeth H.; Schwirzke, Marina; Kaluzab, Brigitte; Weidle, Ulrich H.
Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction.
In: Gene, Vol. 106: pp. 273-277
We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the
variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the
chimerization ofAbs. A fundamental prerequisite for this is the knowledge ofthe exact sequences in the 5’-untranslated region
of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated
oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments
into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig)
promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte
antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into
non-Ig-producing myeloma cells.