RCA Technology

Sequencing of the variable domains of a monoclonal antibody is a long and laborious task, due to the intrinsic variability of the immune repertoire. Traditionally, multiple PCR amplification reactions are required to select a pair of nucleotide primers capable of effectively amplifying the variable domain of interest. The monoclonal antibodies obtained using mouse hybridoma technology have an additional level of complexity for sequencing, associated with the presence of an IGKV pseudogene, deriving from the myeloma line used as a partner in cell fusion. 

 

At the CIPF, we have designed and developed a novel method of sequencing the variable domains of antibodies for which the amplification steps are solely based on the knowledge of the constant domains (isotype) of the antibodies of interest. By using the principles of rolling circle amplification (RCA), specific to certain bacteriophage DNA polymerases, only three nucleotide primers are required to amplify the variable domains of an antibody. By strategically selecting the hybridization sites of the primers in the constant domains, amplification of the pseudogene is avoided. Thus the application of this new method of RCA sequencing makes it possible to reduce by 75% the number of amplification steps and by 35% the costs associated with antibody sequence determination. 

 

monoclonal antibody - CIPF

 

An additional benefit associated with this new sequencing technique is the determination of the L-PART region covering the peptide signal and untranslated sequence 5' of the antibody heavy and light chains. This information is important for later production of these antibodies by molecular engineering. This technique can also be applied to generate complete antibody libraries for presentation on the surface of eukaryotic cells. 

 

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