Improved cloning of antibody variable regions from hybridomas by an antisense-directed RNase H digestion of the P3-X63-Ag8.653 derived pseudogene mRNA

When antibody variable regions (VL or VH) are cloned from hybridoma cell lines using standard methods (1), often sequences of non-functional rearranged variable regions are obtained by PCR of the hybridoma cDNA (2,3). The origin of these sequences (GenBank accession nos X58634 for VH and X05184, K00888 and M35669 for VL) can be traced to the myeloma cell lines originally utilized for the fusion ( 4,5). Often, the non-secretor cell line P3-X63-Ag8.653 (as well as other cell lines derived from MOPC 21) is used for the establishment of hybridoma cell lines by the standard fusion technique described by G. Kohler and C. Milstein (6). In many cases these hybridoma cells not only transcribe the desired monoclonal antibody DNA, but also bear high levels of non-functionally rearranged mRNAs. These mRNAs represent pseudogenes and can greatly exceed the level of normal antibody mRNA. Due to a reading frame shift in the V‐J recombination site, these pseudogene mRNAs cannot be translated into a functional protein ( 7). Development of PCR primers which can avoid amplification of these pseudogenes was hampered by the nearly identical sequences of the N- and C-termini of the variable regions. Up to now, the discrimination between ‘wrong’ and ‘right’ clones was often achieved by DNA sequence analysis or by checking the binding properties of the expressed antibody fragments ( 2). During the process of cloning a set of monoclonal antibodies directed against cytochrome c oxidase, we ran into the pseudogene problem many times. We developed two possible ways to solve the problem. First, if the variable region of interest does not belong to the subtype γ1 for VH or κ for VL, it is possible to suppress transcription of the pseudogene mRNA (which is subtype γ1 and κ) into cDNA by using a specific primer for the first strand synthesis. This strategy worked well with a subtype IgG2a clone where the cDNA synthesis could be made subtype-specific with an IgG2a-specific primer (Fig. 1). Therefore, the VH pseudogene (which belongs to subtype IgG1) could not be transcribed into cDNA. As a result, no pseudogene sequence could be detected using this procedure for cloning of the VH domain of this clone. Secondly, we developed another strategy which works independently from the antibody subtype. We used this strategy in the case of clone 11D3 (which is an IgG1/κ monoclonal antibody