Multiplex assay for simultaneous measurement of antibodies to multiple Plasmodium falciparum antigens.

Antibodies to Plasmodium falciparum are classically measured using the enzyme-linked immunosorbent assay (ELISA). Although highly sensitive, this technique is labor-intensive when large numbers of samples must be screened against multiple antigens. The suspension array technology (SAT) might be an alterative to ELISA, as it allows measurement of antibodies against multiple antigens simultaneously with a small volume of sample. This study sought to adapt the new SAT multiplex system for measuring antibodies against nine malarial vaccine candidate antigens, including recombinant proteins from two variants of merozoite surface protein 1, two variants of apical merozoite antigen 1, erythrocyte binding antigen 175, merozoite surface protein 3, and peptides from the circumsporozoite protein, ring erythrocyte surface antigen, and liver-stage antigen 1. Various concentrations of the antigens were coupled to microspheres with different spectral addresses, and plasma samples from Cameroonian adults were screened by SAT in mono- and multiplex formats and by ELISA. Optimal amounts of protein required to perform the SAT assay were 10- to 100-fold less than that needed for ELISA. Excellent agreement was found between the single and multiplex formats (R > or = 0.96), even when two variants of the same antigen were used. The multiplex assay was rapid, reproducible, required less than 1 mul of plasma, and had a good correlation with ELISA. Thus, SAT provides an important new tool for studying the immune response to malaria rapidly and efficiently in large populations, even when the amount of plasma available is limited, e.g., in studies of neonates or finger-prick blood.

Human leukocyte antigen class II alleles influence levels of antibodies to the Plasmodium falciparum asexual-stage apical membrane antigen 1 but not to merozoite surface antigen 2 and merozoite surface protein 1.

The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine for Plasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these antigens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5 and 70 years, were determined using recombinant AMA1, MSA2, and the N-terminal region of MSP1 (MSP1-190L). In addition, antibody responses to four variants of the C-terminal region of MSP1 (MSP1(19)) were assessed. Results showed that all individuals produced antibodies to AMA1, MSA2, and MSP1-190L; however, a proportion of individuals never produced antibodies to the MSP1(19) variants, although the percentage of nonresponders decreased with age. The influence of age and human leukocyte antigen (HLA)-DRB1/DQB1 alleles on antibody levels was evaluated using two-way analysis of variance. Age was correlated with levels of antibodies to AMA1 and MSP1(19) but not with levels of antibodies to MSA2 and MSP1-190L. No association was found between a single HLA allele and levels of antibodies to MSA2, MSP1-190L, or any of the MSP1(19) variants. However, individuals positive for DRB1*1201 had higher levels of antibodies to the variant of recombinant AMA1 tested than did individuals of all other HLA types. Since the effect was seen across all age groups, HLA influenced the level but not the rate of antibody acquisition. This association for AMA1, combined with the previously reported association between HLA class II alleles and levels of antibodies to rhoptry-associated protein 1 (RAP1) and RAP2, indicates that HLA influences the levels of antibodies to three of the five vaccine candidate antigens that we have evaluated.