To accomplish these objectives, we developed a novel approach that we named multiple antigen competition enzyme-linked immunosorbent assay (ELISA) (MACE) that can also be used to define antigenic diversity of additional polymorphic antigens. Methods Study cohorts and sample collection Serum samples were collected from a cross-sectional study in Madang Province, Papua New Guinea (PNG) in 2007, and included 118 individuals: 49 adults (median age 28?years) and 69 children (median age 6?years). specific alleles for potential inclusion inside a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to variations in AMA1 sequence. Results We found that adults experienced higher cross-reactivity of antibodies than children, even though patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results display that antigenic diversity of AMA1 alleles is definitely remarkably restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be adequate to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis. Conclusions Antigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be adequate for protection against naturally-circulating strains, assisting a multi-allele approach for developing polymorphic antigens as malaria vaccines. Electronic supplementary material The online version of this article (doi:10.1186/s12916-014-0183-5) contains supplementary material, which is available to authorized users. Keywords: Malaria, data indicate that AMA1 antibodies can inhibit parasite invasion of erythrocytes [17-19]. AMA1 is definitely a encouraging blood stage vaccine candidate which is definitely presently becoming tested in medical tests. A HT-2157 recent phase II trial of a monovalent AMA 1 vaccine in one- to six-year-old children in Mali showed 65% strain specific efficacy [20]. However, AMA1 is a highly polymorphic protein with more than 60 polymorphic sites and more than 200 haplotypes per human population [21,22], probably one of the most polymorphic of all merozoite antigens. Immunization with one allele of AMA1 may not protect against parasites expressing different AMA 1 alleles, as highlighted from the Mali trial; there was HT-2157 no overall safety against medical malaria, but there was evidence of safety against malaria caused by vaccine-like alleles [20]. While sequence analysis has been used to classify AMA1 alleles into related organizations that might display cross-reactive immunity, the antigenic diversity of AMA1 and cross-reactivity of antibodies are poorly recognized, and it is unclear how sequence polymorphisms and sequence-based groupings relate to antigenic diversity and escape from acquired human being antibodies. There are only limited data on antigenic diversity in human studies and limited data to understand how sequence diversity is related to antigenic diversity, which is an impediment to vaccine development. Understanding these issues is essential for improving AMA1-centered vaccines. AMA1 also serves as an ideal model to examine antigenic diversity more broadly, the significance of polymorphism in vaccine development and the feasibility of developing multi-allele vaccines based on polymorphic ITPKB antigens. We wanted to define antigenic diversity of AMA1 and use this knowledge to understand which AMA1 alleles could be included in a multi-allele vaccine to achieve the HT-2157 broadest protection of AMA1 diversity, and establish principles that may be applied to additional polymorphic vaccine antigens. Antibody reactivity to numerous geographically-diverse AMA1 alleles was examined among children of different age groups and adults from two geographically varied malaria endemic areas (Papua New Guinea and Kenya). We examined the relationship between antigenic diversity and sequence diversity, and sought to establish whether overall antigenic diversity of AMA1 is limited and might ultimately be reduced to a small number of major serotypes. To accomplish these objectives, we developed a novel approach that we named multiple antigen competition enzyme-linked immunosorbent assay (ELISA) (MACE) that can also be used to define antigenic diversity of additional polymorphic antigens. Methods Study cohorts and sample collection Serum samples were collected from a cross-sectional study in Madang Province, Papua New Guinea (PNG) in 2007, and included 118 individuals: 49 adults (median age 28?years) and 69 children (median age 6?years). From these samples we prepared swimming pools of AMA1 antibody positive samples for screening in competition ELISAs; one pool was made from childrens samples (n?=?31; median age 7?years (range 4 to 10)) and 1 from adult samples (n?=?42; median age 28?years (range 16 to 53)). To prepare the swimming pools, all sera were first tested in standard ELISA for immunoglobulin G (IgG) reactivity against five different recombinant AMA1 alleles (3D7, W2mef, FVO, 7G8, and HB3). After testing, we.