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 anti-HCMV recombinant gH-specific ELISA was performed using the recombinant gH-GST antigen coated onto plastic. gH-GST corresponds to an in-frame fusion between the gH amino terminal region (amino acids 16-144) from the HCMV strain VR1814 and GST. The amino terminus of gH contains a linear antibody binding site between residues 34-43 that is recognized by neutralizing antibodies [44, 53] . The gH gene segment was PCR amplified from VR1814 DNA with the following primers: VR1814 gH F: 5'-AGTCATGGATCCCTCCTTAGTCAC-CTCA-3'; VR1814 gH R: 5'-ACTGATCTCGAGGCCT-TCAGCTGCTGC-3'. The 400 bp PCR product was then digested by XhoI and BamHI, the restricted fragment purified by agarose gel elecrophoresis and cloned into the pGEX 4T3 expression vector (Amersham) that had been restricted by XhoI and BamHI and gel purified. The recombinant gH-GST fusion protein was then expressed in E. coli BL21 and purified from the bacterial cell lysate on the basis of GST affinity.