The animals were subcutaneously immunized with either single VAR2CSA-domain constructs (DBL1x-6e) or with FV2. Control groups were immunized with the DBL3c-VAR1-FCR3 in the same concentration as the tested VAR2CSA proteins [15] . Groups of BALB/c mice (n = 8) (inbred) (Taconic, Denmark) were subcutaneously immunized with 15 mg per animal of recombinant protein in Freund's complete adjuvant, followed by three followup immunizations of 10 mg of protein in Freund's incomplete adjuvant at two-week intervals. However, due to insufficient amounts of protein only four mice received DBL2x, and six mice received DBL6x. For immunizations with FV2 (done on a later time point) the mouse strain used was CB6F1 mice (F1 hybrid between BALB/c females and C57BL/6 males) (Harlan, USA) following the same protocol as previously described for the BALBc mice. Seven groups of Wistar rats (outbred) (Taconic, Denmark) were immunized with 40 mg per animal of recombinant protein in Freund's complete adjuvant, followed by three booster injections of 20 mg of protein in Freund's incomplete adjuvant at 2K-week intervals. The rat groups consisted of three animals except the group receiving the DBL4e (n = 8) and DBL3c (n = 2). New Zealand White rabbits (outbred)(HB Lidköping Kaninfarm, Sweden) were immunized with 40 mg of recombinant protein in Freund's complete adjuvant, followed by three booster immunizations of 20 mg of protein in Freund's incomplete adjuvant at 3 week intervals. For each recombinant protein one rabbit was immunized with the exception of rabbits receiving DBL2x & FV2 (n = 2) and DBL4e & DBL5e (n = 3). For all animals sera were collected 8 days after the final immunization. Sera were stored at 220uC until use.