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

Abstract

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, IgG_1-4 subclasses, IgM and IgE against a panel of 40 antigens. The different positive controls were tested at different 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 primo-infected adults with adequate IgM levels proved to be more adequate as a positive control. The range and magnitude of IgG and IgG_1-4 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 difference in unspecific binding.

Conclusions: With this study, the immunogenicity profile 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.

Keywords: Assay performance; IgE; IgG, IgG1, IgG2, IgG3, IgG4 subclasses; IgM; Incubation conditions; Multiplex; Plasmodium falciparum; Quantitative suspension array technology; Reference reagent.

Fig. 1

RTS,S-specific responses measured in the WHO reference reagent, IgM pool and samples from RTS,S-vaccinated children. The 3 samples from RTS,S vaccinated children were of high, medium and low CSP IgG titres. a–g IgG, IgG_1–4, IgM and IgE levels to RTS,S-specific antigens measured in the WHO reference reagent; IgG, IgG 1, IgG2 and IgG4 also measured in RTS,S-vaccinated children; h IgM levels to RTS,S-specific antigens measured in the IgM pool vs. RTS,S-vaccinated children. The plots represent the levels of antibodies measured in serial dilutions of the positive pools (1:3 starting at 1:50 for IgG, IgG_1–4 and IgM; and 1:2 starting at 1:10 for IgE), and the RTS,S vaccinees samples (1:10 starting at 1:500 for IgG, 1:100 for IgM, 1:50 for IgG_1–4; and 1:2 starting at 1:10 for IgE). Isolated dots represent the levels measured in the technical blanks

Fig. 2

IgG, IgG_1–4 and IgM fitted curves using the WHO-CSP pool to the 40-antigen multiplex panel incubating at 4 °C ON. Lines and dots represent predicted levels from 5PL, 4PL or exponential regression equations from 23 titration curves for IgG, IgG1, IgG3 and IgM; and 12 curves for IgG2 and IgG4. Titration curves contained 18 serial dilutions (1:2) starting at 1/50 of the WHO-CSP pool to a panel of 39 P. falciparum antigens plus HBsAg, α-Gal, BSA and GST

Fig. 3

Boxplots of ratios of IgG_1–4 subclasses to total IgG measured in the WHO-CSP pool. Ratios are composed with the median of the 23 titration curves for IgG, IgG1 and IgG3 and 12 curves for IgG2 and IgG4, for each dilution point. Boxes show medians and interquartile ranges. The red star corresponds to the ratio of the median of each dilution of IgG subclass to the median of each dilution of total IgG

Fig. 4

Levels of IgG1 measured to 15 antigens in the WHO reference reagent compared to negative control and blanks under three different incubation conditions. Curve plots of the antigen-specific IgG1 levels measured in serial dilutions of the WHO reference reagent, negative control and blanks at three different incubation conditions: 37 °C 2 h, 4 °C overnight (4 °C ON) and room temperature 1 h (RT 1 h). “neg” means negative control

Fig. 5

Fitted IgM curves to the 40-multiplex panel in the WHO reference reagent and the IgM pool compared to negative control and blanks under two different incubation conditions. Curves from 4PL or 5PL logistic model equation comparing IgM levels measured in the WHO reference reagent, the IgM pool, the negative control and the blanks. Isolated dots in purple represent the IgM levels measured in the technical blanks