VSAPAM-reactive monoclonal IgG-containing supernatants were tested in ELISA (Dodoo et al., 2000) for reactivity with the recombinant VAR2CSA proteins produced in Baculovirusinfected insect cells. In addition, the epitope specificities of monoclonal antibodies targeting DBL3-X (PAM2.8 and PAM6.1) and DBL5-e (PAM3.10, PAM5.2 and PAM7.5) were analysed by competition ELISA. PAM3.10 and PAM6.1 were purified on ÄktaXpress (GE Healthcare, Brøndby, Denmark) using a HiTrap Protein G HP 1 ml column with subsequent desalting on a HiPrep 26/10 desalting column (GE Healthcare). Purified IgG was biotinylated using EZ-link maleimide-PEO solid phase as described by the manufacturer (Pierce, Bonn, Germany). Microtitre plates (Nunc, Roskilde, Denmark) were coated with recombinant DBL3-X (8.3 mg ml -1 ) or DBL5-e (10.4 mg ml -1 ) in PBS (1 h, 37°C). After blocking of the plates, biotinylated PAM3.1-specific IgG (0.72 mg ml -1 ) or biotinylated PAM6.1-specific IgG (2.5 mg ml -1 ) and increasing concentrations of the competitor monoclonal culture supernatants were added to triplicate wells. PAM1.4 (unknown VSAPAM-specificity) and PAM2.8 (DBL3-X-specific) were added as negative controls to DBL3-X-coated and DBL5-e-coated plates respectively. Bound biotinylated IgG was detected by incubation (1 h, room temperature) of wells with horseradish peroxidase-conjugated streptavidin (1 mg ml -1 , 100 ml well -1 ; Pierce, Bonn, Germany).