AMA1 antibody cross-reactivity with multiple competitor alleles in competition ELISAs. Serum pools prepared from PNG children were tested for cross-reactivity of naturally acquired antibodies among five different PfAMA1 variants in competition ELISA. Competition with either two (A1 to D1) or three (A2 to D2) other alleles was tested in multiple antigen competition ELISA against the remaining three AMA1 variants to determine cross-reactivity. Enhancement of cross-reactivity by mixtures of two or three competitor alleles was dependent on the specific combination tested. The combination of 3D7 and 7G8 antibodies shows the highest degree of cross-reactivity towards the three other alleles, w2mef, FVO and HB3 (panel D1). Specific combinations of antibodies, that is, 3D7, w2mef and HB3, result in almost complete cross-reactivity towards the remaining AMA1 variants (panel C2). The degree of cross-reactivity observed with a particular combination of competitor alleles indicates the extent to which immunization with that combination might provide coverage against other alleles. A representative selection of double and triple antigen competition ELISAs is shown; all double and triple combination competition ELISAs are shown in Additional file 1: Figure S7. For each panel (A1-D1, and A2-D2), the coating antigens used in experiments are those listed on the Y-axis, and the antigens used for competition are at the top of the figure (for example, for panel A1, the coating antigens were W2mef, 7G8, FVO, 3D7, and HB3; the competitor antigens were 3D7 and W2mef). AMA1, apical membrane antigen 1; PNG, Papua New Guinea.