Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

Funaro, Ada; Gribaudo, Giorgio; Luganini, Anna; Ortolan, Erika; Lo Buono, Nicola; Vicenzi, Elisa; Cassetta, Luca; Landolfo, Santo; Buick, Richard; Falciola, Luca; Murphy, Marianne; Garotta, Gianni; Malavasi, Fabio

The variable regions of heavy chain (VH) and light chain (VL) of mAb 26A1 were sequenced according the technology established by Fusion Antibodies Ltd. Briefly, frozen cells pellets (~5 × 10 4 cells) were used for extracting total RNA with STAT-60 RNA extraction reagent. cDNA was produced by reverse transcription with an oligo(d)T primer. PCR reactions were set up to amplify VH and VL regions with a mix of IgG-and Igk/λ specific primers, respectively. The PCR products of two amplification reactions were cloned using a Eco RI restriction site in a sequencing vector (pCR2.1; Invitrogen) and used for transforming TOP10 E. coli cells, following the manufacturer's instructions. A consensus sequence was determined from at least ten clones. The resulting DNA sequences were aligned and translated into protein sequences enabling the generation of consensus DNA and protein sequence for VH 26A1 and VL 26A1. The VH 26A1 and VL 26A1 protein sequences were compared and aligned with sequences present in the GenomeQuest, GeneSeq, and EBI databases. The CDRs characterizing VH 26A1 and VL 26A1 protein sequences were predicted by the IMGT database [54] .