Multiplex Assay for Simultaneous Measurement of Antibodies to Multiple Plasmodium falciparum Antigens

Abstract

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 > 0.96), even when two variants of the same antigen were used. The multiplex assay was rapid, reproducible, required less than 1 l 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.

Breadth and Magnitude of Antibody Responses to MultiplePlasmodium falciparumMerozoite Antigens Are Associated with Protection from Clinical Malaria

“…With the completion of the P. falciparum genome, numerous new (and old) antigens of the parasite have been identified and are being characterized. High-throughput assays employing suspension array technology (27) or microarrays (29,64) now allow for simultaneous analysis of antibodies to multiple antigens using minimal amounts of sera. This technology has not been matched with equivalently efficient tools for identifying protective immune responses.…”

Ex Vivo Cytokine and Memory T Cell Responses to the 42-kDa Fragment of Plasmodium falciparum Merozoite Surface Protein-1 in Vaccinated Volunteers

“…Serum samples from volunteers vaccinated with MSP1 42 -FVO/Alhydrogel (n ϭ 3 volunteers providing samples) and MSP1 42 -3D7/Alhydrogel (n ϭ 5 volunteers providing samples), which produced the highest levels of specific IgG Abs against homologous Ag (400 or more ELISA units on day 194) (21), were selected for IgE evaluation against homologous and heterologous Ags using suspension array technology (24). Serum samples from days 0 and 194 were diluted 1/50 in 1% BSA/PBS and incubated for 1 h with microspheres coupled with MSP1 42 -FVO or MSP1 42 -3D7 in Multiscreen plates (Millipore).…”

Acquired Immunity to Malaria

“…Alternatively, the protection of infants may be associated with parasite growth-inhibitory factors such as lactoferrin and secretory IgA found in breast milk and in maternal and infant sera (165). At between 4 and 10 months of age many infants are seronegative to specific malaria antigens (1,254), but the kinetics of decay of maternal antibodies show interindividual variations which depend on the antigen and the epidemiological setting (119).…”