Development of a high-throughput flexible quantitative suspension array assay for IgG against multiple Plasmodium falciparum antigens

Itziar Ubillos, Joseph J. Campo, Alfons Jiménez, Carlota Dobaño

Antibody responses to Plasmodium falciparum play a critical role in disease control. Finding reliable IgG biomarkers of protection is complicated by a parasite proteome of over 5000 proteins, some with polymorphisms. Studies of anti-malarial naturally acquired and vaccine immunity would benefit from a standard high-throughput immunoassay to measure multiple antibodies. A multiplex quantitative suspension assay to measure antigen-specific IgGs was developed and its precision (reproducibility and repeatability), dynamic range, limits of detection and quantification, and non-specific binding to different P. falciparum proteins tested. A set of 288 human plasma samples from a malaria-endemic region were analysed twice by two different operators. Another set of samples from 9 malaria-naïve and 10 malaria-exposed individuals were repetitively assayed during 22 consecutive days. Positive controls, negative controls, blanks and microspheres coated with bovine serum albumin were included in all assays. The multiplex quantitative suspension assay demonstrated low non-specific signal and good estimates of precision and reproducibility between operators. The overall mean of non-specific binding measured in 288 plasma samples was 32.83 to ± 44.81 median fluorescence intensity (MFI). Repeatability was 7.66% ± 15.89 between triplicates for all antigens and samples, being lower in samples from malaria-exposed than malaria-naïve individuals. No evidence of significantly different variance across days in MFI or arbitrary units (AU)/mL was found, assuming homogeneity of variance between days of analysis. Intra-class correlation coefficient between 22 days of analysis was 0.98 (0.97-0.98) for MFI units and 0.9 (0.87-0.93) for AU/mL. Reproducibility between operators for all samples and antigens had an overall adjusted correlation of 0.929 for MFI and 0.836 for AU/mL. This high-throughput multiplex immunoassay is simple and highly reproducible. This represents an asset for malaria vaccine studies involving CSP-specific antibodies and selected antigens for sero-epidemiological purposes. Measuring a multiplex antigen panel in a single reaction will help to assess not only vaccine immunogenicity but also potential malaria vaccine effects on naturally acquired immune responses. This will accelerate the identification of immune correlates of protection, down-selection of vaccine formulations, antigen discovery and guide second-generation vaccine design.