# name
Optimization of incubation conditions of Plasmodium falciparum
                antibody multiplex assays to measure IgG, IgG1-4, IgM and IgE
                using standard and customized reference pools for
                sero-epidemiological and vaccine studies

# metadata.dc.description.abstract[].value
Background: The quantitative suspension array technology (qSAT) is a useful platform for malaria immune marker
discovery. However, a major challenge for large sero-epidemiological and malaria vaccine studies is the comparability across laboratories, which requires the access to standardized control reagents for assay optimization, to monitor
performance and improve reproducibility. Here, the Plasmodium falciparum antibody reactivities of the newly available WHO reference reagent for anti-malaria human plasma (10/198) and of additional customized positive controls
were examined with seven in-house qSAT multiplex assays measuring IgG, IgG1–4 subclasses, IgM and IgE against a
panel of 40 antigens. The diferent positive controls were tested at diferent incubation times and temperatures (4 °C
overnight, 37 °C 2 h, room temperature 1 h) to select the optimal conditions.
Results: Overall, the WHO reference reagent had low IgG2, IgG4, IgM and IgE, and also low anti-CSP antibody levels,
thus this reagent was enriched with plasmas from RTS,S-vaccinated volunteers to be used as standard for CSP-based
vaccine studies. For the IgM assay, another customized plasma pool prepared with samples from malaria primoinfected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude
of IgG and IgG1–4 responses were highest when the WHO reference reagent was incubated with antigen-coupled
beads at 4 °C overnight. IgG levels measured in the negative control did not vary between incubations at 37 °C 2 h
and 4 °C overnight, indicating no diference in unspecifc binding.
Conclusions: With this study, the immunogenicity profle of the WHO reference reagent, including seven immunoglobulin isotypes and subclasses, and more P. falciparum antigens, also those included in the leading RTS,S malaria
vaccine, was better characterized. Overall, incubation of samples at 4 °C overnight rendered the best performance for
antibody measurements against the antigens tested. Although the WHO reference reagent performed well to measure IgG to the majority of the common P. falciparum blood stage antigens tested, customized pools may need to be
used as positive controls depending on the antigens (e.g. pre-erythrocytic proteins of low natural immunogenicity)
and isotypes/subclasses (e.g. IgM) under study.

# metadata.dc.source[].value
Articles publicats en revistes (ISGlobal)

# metadata.dc.title[].value
Optimization of incubation conditions of Plasmodium falciparum
                antibody multiplex assays to measure IgG, IgG1-4, IgM and IgE
                using standard and customized reference pools for
                sero-epidemiological and vaccine studies