PAM1.4 stained VSAPAM-expressing IEs, but did not yield any bands in Western blots, and did not react with any of the VAR2CSA constructs when tested in ELISA or by flow cytometry (Tables 1 and 2 ). These observations are compatible with recognition by this antibody of a conformational epitope in VAR2CSA, but also with recognition of an unidentified non-VAR2CSA PAM-specific IE surface antigen. To address this question, we tested the ability of PAM1.4 to enrich VSAPAM-expressing IEs in two parasite lines (EJ24 and EJ27) initially expressing non-PAM-type VSA and only marginally recognized by PAM1.4 ( Fig. 5A and B, and data not shown). Although both isolates were originally obtained from the peripheral blood of pregnant women, and thus expected to express VSAPAM, isolates expressing non-PAM VSA -such as EJ24 and EJ27 -are occasionally found (Ofori et al., 2003, and our unpublished data) . Remarkably, a single round of PAM1.4 antibody selection of EJ27 ( Fig. 5C and D) and EJ24 (data not shown) resulted in rapid emergence of IEs uniformly recognized by PAM1.4 and expressing VSAPAM ( Fig. 5C and D). Quantitative real-time polymerase chain reaction (PCR) analysis of the isolates showed increases in var2csa transcription in response to the selection for PAM1.4 reactivity (EJ24: twofold and EJ27: 30-fold). In addition, EJ24 acquired reactivity with the VAR2CSAspecific antibodies PAM2.8, PAM3.10, PAM6.1 and PAM7.5 following selection for PAM1.4 reactivity ( Table 1) . EJ27 did not acquire additional reactivity following PAM1.4 selection, probably because of interclonal C. Western blots of recombinant 3D7-and FCR3-specific VAR2CSA DBL3-X constructs, and of the above-mentioned chimeric construct, probed with loading control antibody V5 (left) and PAM8.1 (right). MW, molecular weight. differences in the VAR2CSA epitopes recognized by the other monoclonal antibodies. Taken together, these findings are consistent with VAR2CSA being the antigenic target of PAM1.4.