In contrast to the cases of HCV, HIV or MSP1, the non-AMA1 IgGs tested in this study showed negligible level of binding to the AMA1 protein as judged by ELISA (Table 1 ). In addition, the non-AMA1 IgGs did not compete with US-total IgG in a competition ELISA ( Figure 5 ). We tested up to ,10 times greater ratio of non-AMA1 IgG to US-total IgG compared with the ratio tested in GIA ( Figure 3) where non-AMA1 IgG showed this interference effect, but still there was no competition observed. Since the non-AMA1 IgGs had some residual anti-AMA1 antibodies in the final preparation, one may consider the possibility that the residual antibody in the non-AMA1 IgGs masked the competition effect. However, we think this is not the case. When non-AMA1 IgGs were tested at 11-14 mg/ml (which gave the same ratio of non-AMA1 IgG and US-total IgG as tested in Figure 3 ), the amount of anti-AMA1 antibody coming from the non-AMA1 IgG was less than 5% compared to the one from UStotal IgG. Even at the highest concentration of non-AMA1 IgG tested (i.e., 133 mg/ml), anti-AMA1 antibody from non-AMA1 IgG was less than 30% compared to the one from US-total IgG. Therefore, we concluded the competition effect of non-AMA1 was not obvious if any. These results suggest that either binding between AMA1 protein and non-AMA1 IgGs was too weak to detect by conventional ELISA and/or the mechanism of interference by non-AMA1 IgG is an indirect effect. Not only the function of AMA1 during the invasion process, but also the mechanism of invasion inhibition by anti-AMA1 antibodies is still controversial. Some studies show that a rabbit polyclonal growthinhibitory anti-AMA1 antibody disrupts proteolytic processing of AMA1 [40, 41] and other studies with inhibitory mAb or peptide show that they block complex formation of AMA1 and rhoptry neck proteins RON2, RON4 and RON5 [42, 43] . In our previous study [27] , we have shown that the interfering activity was due to the malaria-specific IgGs in the non-AMA1 IgGs population. However, because the growth-inhibitory activity of non-AMA1 IgGs in the previous study was high (the IgGs were collected mainly from Malian adults), we couldn't measure the strength of the interference effect. In this study with IgGs from Malian children, we could not perform malaria-extract-specific IgG purification from the non-AMA1 IgGs because of the limited quantity of blood samples available. On the other hand, as the intrinsic growth-inhibitory activity of non-AMA1 IgGs from Figure 4 . The correlation between anti-AMA1 antibody levels in the original total IgG pool and the interference effect of the corresponding non-AMA1 IgG. Anti-AMA1(3D7) (A) or anti-AMA1(FVO) (B) antibody levels (mg/ml) in the original total IgG pools (x-axis) are plotted against delta % inhibition of non-AMA1 IgGs (y-axis) tested with P. falciparum 3D7 (A) or FVO (B) parasites. Delta % inhibition of each non-AMA1 IgG was calculated using the data presented in Figure 3 as follows: delta % inhibition = (% inhibition of the US-total IgG alone (black bar in Figure 3 )) -(% inhibition of a mixture of the non-AMA1 IgG and US-total IgG). doi:10.1371/journal.pone.0020947.g004 Malian children is relatively low, we can measure the strength of the interference effect. We found that the interference effect of non-AMA1 IgG from total IgG pools with higher AMA1 titers was greater than that from total IgG pools with lower AMA1 titers. We believe the higher AMA1 titer reflects more malaria exposure, because the Malian children with higher AMA1 titer also display higher titers against other malaria antigens such as MSP1 (our unpublished observation). Therefore, the correlation between anti-AMA1 antibody levels in the original total IgG pool and the strength of interference effect in non-AMA1 IgG suggests that the interference effect is due to antibody against a malaria antigen other than AMA1. If the interference mechanism of the non-AMA1 IgGs is indirect, as the ELISA and competition ELISA results suggest, it is possible that the non-AMA1 IgGs may bind to RON2, RON4 and/or RON5 first and then may block the ability of the anti-AMA1 growth-inhibitory antibody to bind to the critical site of complex formation. Further studies are required to reveal the mechanism of the interference IgGs, and such studies may enhance our understanding of the invasion-inhibition mechanism by human anti-AMA1 polyclonal antibodies.