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

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 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 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 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. Electronic supplementary material The online version of this article (10.1186/s12936-018-2369-3) contains supplementary material, which is available to authorized users.

Background The identification of immune correlates of protection and risk against malaria is particularly challenging when dealing with a complex pathogen like Plasmodium falciparum, which has a proteome of over 5000 proteins (http:// www.plasm odb.org), some of them polymorphic and/or variant. Consequently, malaria infection induces a very broad and diverse antigen-specific immunoglobulin (Ig) subtype response [1,2]. Although the crucial role of IgG antibodies in protective malaria immunity was demonstrated long time ago [3,4], the antigenic targets of these antibodies have not yet been identified. However, it is presumed that such IgG responses are primarily directed to antigens on the surface of the P. falciparum asexual blood stage (BS). Numerous immune-epidemiological surveys have reported significant associations between levels of BS-specific IgG antibodies and protection from clinical malaria [5][6][7]. However, most of these studies have only described the magnitude of IgG responses and little is known about their subtypes, quality and functionality. Thus, the mechanisms mediating antibody immunity are not fully elucidated.
Early in vitro studies suggested that inhibitory IgG antibodies may control P. falciparum growth in collaboration with monocytes through opsonic phagocytosis [8][9][10] or antibody-dependent cellular inhibition [11]. Collectively, studies have pointed to cytophilic IgG subclasses (IgG1 and IgG3) as the main contributors to naturally-acquired immunity, suggesting that cells bearing Fc-g receptors are involved in protective immune mechanisms [12][13][14][15][16]. Recent studies have also highlighted the potential importance of IgM [17,18] or IgE [19,20] in malaria protection or risk, respectively, but these isotypes have been much less studied in the malaria field. Further studies addressing antibody isotypes, subclasses, and their antigenic breadth are needed to define correlates in natural and in artificial immunity induced by vaccines such as the RTS,S/AS01E and those based on attenuated sporozoites. RTS,S/AS01E is the most advanced malaria vaccine in development globally [21], however the immune surrogates of protection, the mode of action, and how vaccination affects or is affected by naturally-acquired immunity, remain unclear. A better characterization of the malaria serological profile at the Ig isotype and subclass levels could help address these questions. However, widely applicable standardized, miniaturized, multiplex, highthroughput assays, able to measure all Ig isotypes and subclasses, have been lacking.
The quantitative suspension array technology (qSAT) is an optimal platform for malaria biomarker discovery. The qSAT is a mid-high throughput platform that allows measuring multiple antigen-specific antibodies (up to 500) in small sample volumes and in one single reaction. To study the mechanisms of immunity in malaria, several in-house qSAT assays using panels of up to 15 P. falciparum antigens were previously developed to measure total IgG [22], IgG 1-4 , IgM and IgE [23] and factors affecting IgG assay variability evaluated (Ubillos et al., pers. comm.). However, a major challenge in the development of serological tests has been the lack of standardized positive controls [24] to allow comparability of data generated in different assays and laboratories, particularly when assessing large antigenic panels and diverse antibody isotypes/subclasses in samples of heterogeneous origin. Recently, a P. falciparum-specific human serum reference reagent (10/198) stable at high temperature and up to 24 months of storage has been described [25] that reduced inter-laboratory variation. This WHO standard has been characterized by ELISA to contain IgGs that recognize the circumsporozoite surface protein (CSP) and a handful of P. falciparum antigens from different genotypes: the merozoite surface protein (MSP)-1 19 (K1 strain), MSP-1 42 (3D7), MSP-2 (3D7), MSP-3 (K1), and the apical membrane antigen (AMA)-1 (3D7, FC27 and FP3). The malaria community would benefit from having wider information on antigenic recognition of this reference reagent.
In previous studies, antigen-coupled beads were incubated with samples for 1 h at room temperature [22,23,26,27]. Temperature of incubation influences the antigen-antibody affinity [28,29] and 1 h might not ensure the appropriate association/dissociation equilibrium. Hence, expanded incubation times with lower (4 °C) and higher (37 °C) temperatures could affect the assay performance.
In this study, a broader antibody reactivity profile of the WHO reference reagent and other customized positive controls was examined with seven in-house qSAT antibody assays measuring IgG, IgG temperature 1 h) were tested to select the incubation conditions rendering the optimal quantification range and higher sensitivity without increasing unspecific binding.

Antigens
A customized multiplex panel with 33 BS and 6 preerythrocytic (PE) P. falciparum antigens was established ( Table 1). The glycan α-Gal (Gala1-3GalB1-4GlcNAc-R), detected in the surface of sporozoites, was also included, as anti-α-Gal IgM antibodies have been associated with malaria protection [30]. In addition to P. falciparum antigens, the hepatitis B surface antigen (HBsAg, a component of the RTS,S vaccine) was added, as the assays were intended to be used with samples from this vaccine trial. Also, bovine serum albumin (BSA) and glutathione S-transferase (GST) were added to the panel to control for background signal coming from unspecific binding to the BSA used to block the coupled beads, and to the GST present in some of the fusion proteins.

Coupling of antigens to microspheres
Coupling of carboxylated polystyrene microspheres was carried out as described elsewhere [26].  [25]. The reagent has been defibrinated and diluted (1:5) with deionized sterile water and filled into 1 mL/ampoules. Each ampoule has been lyophilized comprising a freeze-dried residue of diluted human plasma. RTS,S vaccine positive control (referred as WHO-CSP pool). An RTS,S pool prepared with plasmas from 10 Mozambican children vaccinated with RTS,S/AS02 with known high IgG titres to CSP at peak response [65] was added to the WHO reference reagent (1:50 WHO reference reagent + 1:100 RTS,S pool), creating a CSP and HBsAg antibody enriched WHO reference reagent.
Malaria primo-infected plasma pool (referred as IgM pool). A customized pool prepared with plasmas from 20 malaria naïve European adults with known high antimalaria IgM levels after being experimentally infected with P. falciparum in a controlled human malaria infection (CHMI) trial [66]. To prepare the pool, we first selected the time point that elicited the highest IgM breadth of response to a panel of 20 BS and 1 PE antigens from the CHMI trials conducted in Barcelona (day 35) and Tübingen (day 84). Ten individuals from each trial with the highest IgM breadth of response were selected and pooled.
Negative control. A pool of plasma samples from 20 Spanish malaria-naïve individuals.
RTS,S samples. Three samples from individuals participating in the RTS,S malaria vaccine phase 2b trial conducted in Mozambique [65] were randomly selected. High, medium and low responders were defined by tertiles.

qSAT assay and incubation conditions tested
IgG, IgG 1-4 subclasses, IgM and IgE levels were measured in the WHO reference reagent and other customized pools against multiplexed P. falciparum antigens using the xMAP ™ technology (Luminex Corp., Austin, Texas). Fifty microliter of multiplexed antigen-coupled beads were added to a 96-well μClear ® flat bottom plate (Greiner Bio-One, Frickenhausen, Germany) at 1000 beads/analyte/well. To assess the optimal temperature and duration of sample incubation for IgG and IgG 1-4 assays, 50 µL of WHO reference reagent at 11 serial dilutions (1:3, starting at 1/150) and the negative control at 4 serial dilutions (1:2, starting at 1:50) were RTS,S-induced antibodies were measured in 3 samples from RTS,S-vaccinated children with known high, medium and low responses, together with serial dilutions of the WHO reference reagent or the IgM pool (1:3 starting at 1:50 for IgG, IgG 1-4 and IgM, and 1:2 starting at 1:10 for IgE) and incubated at 4 °C ON. Samples were assayed in 4 serial dilutions (1:10) starting at 1:500 for IgG, in 3 serial dilutions (1:10) starting at 1:100 for IgM and IgG1, and in 2 serial dilutions (1:10) starting at 1:50 for IgG2 and IgG4. Samples were not assayed for IgG3 or IgE.

Statistical analyses
To stabilize the variance, the analysis was done on log 10 -transformed values of the MFI measurements. The correlation and reliability between the different sample incubation conditions for the IgG and IgG 1-4 subclasses measured in the positive control, the negative control and the blanks were evaluated. After the Shapiro-Wilk normality test was applied, differences between conditions were assessed by Kruskal-Wallis test with posthoc Tukey test. Reliability was assessed by the interclass correlation coefficient (ICC) [67]. Titration curves of antibody concentrations vs. MFIs per antigen were fitted using a five-parameter (5PL), a 4PL or an exponential logistic equation depending on the best yield, following the formula MFI = Emax + ((Emin − Emax)/((1 + ((Conc/ EC 50 )^Hill))^Asym)), where EC 50 is the half maximal effective concentration, Emin is the minimum response, Emax is the maximum response, Asym is the asymmetry factor and Hill is the slope factor [68], using the drLumi package [69]. We calculated the coefficient of variation (CV) of Emin, Emax and used the goodness of fit model to assess the fitting of the curves. All analyses were done using R version 3.4.1.

Total IgG, IgG 1-4 , IgM and IgE responses against RTS,S antigens in the WHO reference reagent compared to those measured in sera from RTS,S-vaccinated children
To assess the suitability of the WHO reference reagent as a positive control to capture all responses in the context of RTS,S vaccine studies, levels of IgG, IgG 1-4 , IgM and IgE against the RTS,S-specific antigens (CSP full length, CSP NANP repeat and CSP C-terminus) were measured and compared to levels in sera from RTS,S-vaccinated children from a phase 2b trial with known IgG CSP titres [65] (Fig. 1). The WHO reference reagent and RTS,Svaccinees antibody responses to the whole antigenic panel (Table 1) are shown in the Additional file 2. The IgM pool and RTS,S-vaccinees IgM levels were also compared ( Fig. 1h and Additional file 2). The WHO reference reagent presented lower IgG, IgG1, IgG2, IgG4, and IgM levels to RTS,S antigens than samples from RTS,S-vaccinated children who had high CSP responses (Fig. 1a-d,  g). Comparisons of IgG3 and IgE levels were not possible because these data were not available for RTS,S samples. The IgM pool presented higher IgM levels to RTS,S antigens than the WHO reference reagent and the RTS,S samples. Consequently, we decided to prepare a customized positive control for the RTS,S immunological studies, containing 1:50 of the WHO reference reagent plus 1:100 of pooled plasma from RTS,S-vaccinated children with high CSP titres (WHO-CSP pool). IgG responses were compared between the WHO-CSP pool and the WHO reference reagent (Additional file 3). In addition, the EC 50 ratio between positive controls (EC 50 WHO reference reagent/EC 50 WHO-CSP) was calculated for RTS,S-specific antigens as a proxy measure of relative potency of the WHO-CSP pool to the WHO reference reagent (Additional file 4). The EC 50 ratio for the 3 CSP antigens was between 0.44 and 0.58 for IgG, IgG1 and IgG3, and close to 1 for IgG2 against CSP full length.
Fifteen proteins in the multiplex panel were GST-fused ( Table 1). The WHO-CSP pool was reactive to GST because the sera from RTS,S vaccinees 1 month after primary vaccination had antibodies that cross-reacted with GST. However, even if the samples contained equal or higher levels of antibodies to GST, this did not impede to accurately measure anti-malarial antibodies to the GST fusion proteins, as shown in correlation analyses of GST vs. GST fusion proteins in plasmas from RTS,S vaccinees (Additional file 5).

Levels of total IgG, IgG 1-4 and IgM against multiple P. falciparum antigens plus HBsAg measured in the WHO-CSP pool
The WHO-CSP pool was used to generate IgG, IgG [1][2][3][4] and IgM titration curves incubating at 4 °C ON in the context of an RTS,S immunology study. The level of response was antigen-dependent; the most immunogenic proteins (AMA-1 3D7 and FVO, MSP-1 42 3D7 and FVO)  gave saturated signals even at the 1:6.5 × 10 6 dilution (Fig. 2).

Optimal temperature and time of incubation to measure IgGs against P. falciparum antigens using the WHO reference reagent
To assess the optimal temperature and time of incubation for the measurement of IgG and IgG 1-4 subclasses, the assay performance of the WHO reference reagent against a panel of 14 P. falciparum antigens (Table 1) under three different incubation conditions (4 °C ON, 37 °C 2 h and RT 1 h) was compared. IgG and IgG 1-4 assays varied depending on the incubation procedure, with the largest difference between 4 °C ON and RT 1 h (p < 0.001) for IgG. No differences were found between these two incubation conditions for IgG2, IgG3 and IgG4. Differences between 4 °C ON and 37 °C 2 h were only observed for IgG (p = 0.026). IgG and IgG 1-4 levels against BSA and blanks were not affected by the incubation conditions. The MFI levels of IgG and IgG 1-4 measured in the negative control only varied when comparing 4 °C ON vs. RT 1 h (p < 0.001) for some of the antigens. Figure 4 shows examples of the results for IgG1, and the complete data set is in the Additional file 6. The incubation at 4 °C ON, on average, showed the highest MFIs in the first dilution, except for IgG4 and reached blank levels at the lowest dilution ( Fig. 4 IgG1  IgG2  IgG3  I gG4  IgG1  I gG2  IgG3  IgG4  IgG1  IgG2  IgG3  IgG4  IgG1  IgG2  IgG3  I gG4  IgG1  I gG2  IgG3  IgG4  IgG1  IgG2  IgG3  IgG4  IgG1  I gG2  I   reference reagent at same dilution was high enough to establish a positivity threshold (Fig. 4).

Optimal temperature and time of incubation to measure IgM and IgE against P. falciparum antigens using the WHO reference reagent and an IgM customized pool
Incubation conditions to measure IgM and IgE responses against a panel of 38 P. falciparum antigens plus HBsAg, α-Gal, BSA and GST (Table 1) were tested using the WHO reference reagent and an alternative IgM pool. The IgM pool gave higher IgM responses and of higher range compared to those obtained with the WHO reference reagent for most of the antigens, especially AMA-1s, MSP-1s and CSPs (Fig. 5) (Fig. 5). Similar differences in IgM responses between incubation conditions were obtained with the WHO reference reagent, measuring higher levels when incubating at 4 °C ON than at 37 °C 2 h (Additional file 7B). IgM technical blanks were not affected by incubation conditions (Additional file 7A, B). Correlations for IgM responses between incubation conditions were r 2 = 0.96 for both WHO reference reagent and IgM pool. For the IgE assay, there were no differences between incubation conditions (Additional file 7C). The ICCs between antibody responses measured in the two incubation conditions with the WHO reference reagent were 0.92 (0.91-0.93) for IgM and 0.82 (0.79-0.85) for IgE; and the ICC between conditions for the IgM assay using the IgM pool was 0.91 (0.9-0.92). However, IgM responses of negative controls showed moderate reliability between incubation conditions, having an ICC of 0.66 (0.57-0.73).
When comparing antibody levels measured in the WHO reference reagent vs. the IgM pool, there was moderate reliability, with ICC of 0.65 (0.61-0.769) at 4 °C ON, and 0.66 (0.61-0.7) at 37 °C 2 h, meaning that there was 35% of variability between reference pools. Considering the strong correlation and reliability of the two incubation conditions, but the higher IgM levels and MFI ranges obtained at 4 °C ON, this incubation was also chosen for the IgM assay.

Discussion
A major challenge in large malaria sero-epidemiological and vaccine studies is to have access to consistent and unlimited control reagents that provide assay quality control and facilitate data consolidation. A universal malaria reference pool would be ideal to monitor performance of serological assays, improve inter-laboratory reproducibility, make data from different studies comparable, and potentially give quantitative antibody measures. In this study, information was provided on the expanded antibody reactivity profile of the commercially available WHO reference reagent for anti-malaria (P. falciparum) human plasma (10/198) [25] and other customized positive controls by using seven in-house qSAT multiplex antibody assays to measure IgG, IgG 1-4 , IgM and IgE against a panel of 40 antigens, including P. falciparum proteins that are part of the RTS,S/AS01E vaccine. In addition, different sample incubation times and temperatures (4 °C ON, 37 °C 2 h, RT 1 h) were tested for the qSAT assays to select the incubation conditions rendering the optimal quantification range and higher sensitivity without increasing unspecific binding. Data generated in this study will be useful for clinical malaria studies involving assessment of naturally-acquired immune responses as well as immunogenicity evaluation of CSPbased vaccine candidates. The estimation of malaria antibody concentration in multiplex assays is increasingly difficult. There are not appropriate standards or reference sera available that react strongly to complex antigen panels. Antibody concentrations have been previously estimated using an anti-human IgG curve [22,23,26,27]. However, the binding system and the affinity of the anti-human IgG curve differ from that of antibodies in samples or positive controls. Thus, different assay conditions give different slopes and curve parameters that could result in large deviations of concentration estimates. Thus, it has been recently reported that MFI responses measured independently from a standard curve might reflect actual variation, while estimated concentration values are dictated by the precision of the standard curve [70]. As an alternative, the use of long positive control curves provide upper and lower asymptotes for most antigens, and allow establishing the linear quantification ranges, representing the optimal range to capture the breadth of antibody response in individual samples. However, a reference human serum pool with known levels of anti-P. falciparum antibody concentrations is highly desirable for the malaria community. The challenge remains in sourcing adequate serum/plasma pools that cover all antigens as panels become larger and more complex.
To test the immuneprofile of the WHO reference reagent, antigen and isotype/subclass-specific curves constructed with serial dilutions of the reagent were fitted in non-linear equations, establishing the linear quantification ranges. Generation of curves with optimal linear quantification ranges is important to allow selecting the optimal dilution of test samples (lying on the linear range). In addition, the parameters of the curve may be used for the quality control of the assay. The WHO reference reagent is composed of samples from hyperimmune individuals from a malaria endemic region [25], predominantly having anti-P. falciparum IgG1 and IgG3 antibodies, rather than IgG2 and IgG4, reflecting the naturally-acquired antibody patterns. Thus, for most antigens, this pool is of restricted use to produce standard curves for IgG2, IgG4 or IgE antibodies, and this remains a limitation. Similarly, the WHO reference reagent might not be optimal for IgM measurements, particularly if high responses are expected in test samples. For this reason, a customized IgM pool with plasmas from naïve individuals experimentally challenged with P. falciparum at a time point when IgM predominated over IgG was prepared. This IgM pool proved to be very adequate for the generation of IgM titration curves in the study. Thus, as the WHO reference reagent has been established to measure IgGs, a reference standard to measure IgM responses would still be lacking. Similarly, IgG2, IgG4 and IgE specific reference standards would improve the reproducibility of the malaria-based immune assays.
This study also aimed to assess the usefulness of the WHO reference reagent as a positive control to generate titration curves in the context of RTS,S immunology studies. For this reason, samples from RTS,S vaccinated children with diverse CSP and HBsAg IgG titres were assayed together with the WHO reference reagent for comparison. It is important to test samples at several dilutions to maximize the assay sensitivity, but keeping to the minimum for cost-effectiveness, which is key in large sero-epidemiological studies. For this reason RTS,S samples were assayed at 4 dilutions for IgG, 3 dilutions for IgM and IgG1, and 2 dilutions for IgG2 and IgG4. Samples from RTS,S vaccinated children had significantly higher CSP antibodies than individuals naturallyexposed to P. falciparum sporozoites. Consequently, the WHO reference reagent could only be used to measure RTS,S-specific responses if a relative potency between the WHO reference reagent and the vaccinees samples was calculated [71]. Alternatively, data showed that the WHO reference reagent enriched with pooled sera from RTS,S-vaccinated children (WHO-CSP pool) [65] was adequate to capture all antibody responses, including the very high anti-CSP IgG levels in vaccinated children. To conserve the full reactivity of the WHO reference reagent to BS antigens, the WHO-CSP pool was constructed by adding half concentration of pooled plasmas from RTS,S vaccinated children (1:50 WHO reference reagent and 1:100 plasma from RTS,S vaccinees), ensuring that RTS,S specific antibodies were increased without diluting other anti-P. falciparum antibodies. A proxy measure of relative potency of the WHO-CSP pool vs. the WHO reference reagent was estimated with EC 50 . However, in 4PL and 5PL analysis, the dose-response is not the same over the entire tested concentration range, and the response changes relative to the concentration only in the middle part of the curves. Typically, these comparisons are made at the EC 50 , however, these calculations are only valid under limited conditions. For instance, the doseresponse curve would need to have a common slope, and the maximum achievable response should be identical [72]. Unfortunately, these conditions are not met for the curves of most of the tested antigens and IgG subclasses. Similarly to CSP, it would be desirable to increase the WHO reference reagent reactivity to other P. falciparum PE antigens that are also vaccine candidates like SSP2/ TRAP, LSA-1 or CelTOS. Additionally, a second generation of the WHO reference reagent against other Plasmodium species would be an advantage for other malaria immune studies in areas with P. vivax co-infections.
The WHO-CSP pool presented GST reactivity, mainly coming from the RTS,S samples, which poses the question of whether the GST signal could be interfering with the responses to the GST-fused proteins. However, correlation analysis showed that the antibody response to GST was not associated to the antibody response against the GST-fused protein and, therefore, that responses were independent. For example, CSP-specific antibodies detected upon vaccination were very high and not interfered by anti-GST antibodies when using CSP GST fusion proteins as capture antigens. Because of these observations, the GST values were not subtracted during data pre-processing, and it was concluded that GST reactivity was not a major part of the antibody signal to the P. falciparum portion of the fused proteins. Nevertheless, the GST reactivity with CSP pools remains an unsolved limitation that will be addressed in future studies upon the application of the assays to the analysis of samples from RTS,S vaccinated volunteers using GST fusion proteins, e.g. by testing the blocking of the reactivity with soluble GST.
This first WHO reference reagent contains an arbitrary unitage of 100 Units per ampoule, however the concentrations of antibodies (IgG, IgG 1-4 , IgM, IgE) specific to antigens such as those tested here remain unknown. Thus, it has been suggested to the WHO Expert Committee on Biological Standardization to assess the specific antibody concentrations in this reagent to allow absolute quantifications in future studies.
In a qSAT assay, temperature of incubation influences the reversible antigen-antibody kinetics by altering the constant association/dissociation equilibrium [29], which can impact assay sensitivity [73]. Raising the incubation temperature from 5 to 37 °C decreases the affinity of antigen-antibody complexes by decreasing the stability of the docking complex [28,74]. The conditions previously used in our laboratory for incubation of samples with antigencoupled beads were 1 h and RT [22,23,26,27]. For this study, it was hypothesized that incubating samples for 1 h might not ensure the appropriate association/dissociation equilibrium. For this reason, expanded incubation times were tested and lower (4 °C) and higher (37 °C) temperatures were explored. Higher IgG and IgG 1-4 levels were detected when the WHO reference reagent was incubated ON at 4 °C compared to 2 h at 37 °C or 1 h at RT. The ON incubation at 4 °C increased the IgG levels detected at high concentrations of the WHO reference reagent, but also the negative control. Yet, the difference between the WHO reference reagent and the negative control was large enough to establish a positive threshold. Different incubation conditions showed small differences for the WHO reference reagent performance, but larger differences for the negative control, indicating more variability at very low IgG concentrations. The unspecific binding of IgGs to BSA-coupled beads or the background signal in the technical blanks was not affected by the incubation conditions, suggesting that the specificity of the IgG binding was not affected by incubation duration or temperature. For all these reasons, 4 °C ON was the incubation condition chosen for the anti-P. falciparum IgG and IgG 1-4 profiling of the WHO reference reagent and the WHO-CSP pool.
The optimal incubation condition for the IgM assay was assessed using the WHO reference reagent and the IgM pool. IgM levels were higher when incubating at 4 °C ON, although no significant differences were detected between incubating at 4 °C ON or 37 °C 2 h. Similarly to IgG and IgG 1-4 subclasses, IgM levels to BSA and blanks were low and not affected by the incubation condition. Based in these observations, 4 °C ON was also the incubation condition chosen for the IgM assay.
The main limitation of the IgM assay was the high reactivity of the negative control, also affected by the duration and temperature of incubation. IgMs are the first class of antibodies produced during a primary immune response. They are generated in the absence of apparent stimulation by specific antigens [75], and are thought to aid in the neutralization of pathogens prior to the development of high affinity, antigen-specific antibodies [76]. Natural IgMs tend to have rather low antigen-binding affinities, compensated (to some extent) by their pentameric nature. Thus, IgM is a highly polyreactive antibody [28] and cross-reactivity of IgMs with antigens from other pathogens to which they have been exposed, or even pathogens that have not yet been "seen" by the host immune system [77,78], could account for the high reactivity observed in the negative control. Additional tests are currently being performed to improve the specificity of the IgM qSAT assay.

Conclusion
This study served to expand the characterization of the immunogenicity profile of the WHO reference reagent, including multiple Ig isotypes/subclasses, and significantly more P. falciparum antigens, including CSP. The study also served to establish the optimal sample incubation condition for seven qSAT assays (4 °C ON). Some of the limitations of the WHO reference reagent were circumvented by preparing in-house or adapted pools to quantify high anti-CSP IgG and IgM responses. Information generated here is applicable to other malaria sero-epidemiological studies of PE and BS vaccine candidates, and thus valuable for the malaria research community.