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 comparabil- ity 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 avail- able 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 subclasses, IgM and IgE against a 1–4 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 responses were highest when the WHO reference reagent was incubated with antigen-coupled 1–4 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 immuno - globulin 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 meas- ure 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. *Correspondence: email@example.com ISGlobal, Hospital Clínic-Universitat de Barcelona, Carrer Rosselló 153 (CEK Building), 08036 Barcelona, Catalonia, Spain Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ubillos et al. Malar J (2018) 17:219 Page 2 of 15 Keywords: Plasmodium falciparum, Quantitative suspension array technology, Multiplex, IgG, IgG1, IgG2, IgG3, IgG4 subclasses, IgM, IgE, Reference reagent, Incubation conditions, Assay performance Background The quantitative suspension array technology (qSAT) The identification of immune correlates of protection and is an optimal platform for malaria biomarker discovery. risk against malaria is particularly challenging when deal- The qSAT is a mid-high throughput platform that allows ing with a complex pathogen like Plasmodium falcipa- measuring multiple antigen-specific antibodies (up to rum, which has a proteome of over 5000 proteins (http:// 500) in small sample volumes and in one single reaction. www.plasm odb.org), some of them polymorphic and/or To study the mechanisms of immunity in malaria, several variant. Consequently, malaria infection induces a very in-house qSAT assays using panels of up to 15 P. falcipa- broad and diverse antigen-specific immunoglobulin (Ig) rum antigens were previously developed to measure total subtype response [1, 2]. Although the crucial role of IgG IgG , IgG , IgM and IgE  and factors affect - 1–4 antibodies in protective malaria immunity was demon- ing IgG assay variability evaluated (Ubillos et al., pers. strated long time ago [3, 4], the antigenic targets of these comm.). However, a major challenge in the development antibodies have not yet been identified. However, it is of serological tests has been the lack of standardized presumed that such IgG responses are primarily directed positive controls  to allow comparability of data gen- to antigens on the surface of the P. falciparum asexual erated in different assays and laboratories, particularly blood stage (BS). Numerous immune-epidemiological when assessing large antigenic panels and diverse anti- surveys have reported significant associations between body isotypes/subclasses in samples of heterogeneous levels of BS-specific IgG antibodies and protection from origin. Recently, a P. falciparum-specific human serum clinical malaria [5–7]. However, most of these studies reference reagent (10/198) stable at high temperature and have only described the magnitude of IgG responses and up to 24 months of storage has been described  that little is known about their subtypes, quality and function- reduced inter-laboratory variation. This WHO standard ality. Thus, the mechanisms mediating antibody immu - has been characterized by ELISA to contain IgGs that nity are not fully elucidated. recognize the circumsporozoite surface protein (CSP) Early in vitro studies suggested that inhibitory IgG anti- and a handful of P. falciparum antigens from different bodies may control P. falciparum growth in collaboration genotypes: the merozoite surface protein (MSP)-1 (K1 with monocytes through opsonic phagocytosis [8–10] or strain), MSP-1 (3D7), MSP-2 (3D7), MSP-3 (K1), and antibody-dependent cellular inhibition . Collectively, the apical membrane antigen (AMA)-1 (3D7, FC27 and studies have pointed to cytophilic IgG subclasses (IgG1 FP3). The malaria community would benefit from having and IgG3) as the main contributors to naturally-acquired wider information on antigenic recognition of this refer- immunity, suggesting that cells bearing Fc-g receptors ence reagent. are involved in protective immune mechanisms [12–16]. In previous studies, antigen-coupled beads were incu- Recent studies have also highlighted the potential impor- bated with samples for 1 h at room temperature [22, 23, tance of IgM [17, 18] or IgE [19, 20] in malaria protection 26, 27]. Temperature of incubation influences the anti - or risk, respectively, but these isotypes have been much gen–antibody affinity [28, 29] and 1 h might not ensure less studied in the malaria field. Further studies address - the appropriate association/dissociation equilibrium. ing antibody isotypes, subclasses, and their antigenic Hence, expanded incubation times with lower (4 °C) breadth are needed to define correlates in natural and and higher (37 °C) temperatures could affect the assay in artificial immunity induced by vaccines such as the performance. RTS,S/AS01E and those based on attenuated sporozoites. In this study, a broader antibody reactivity profile of the RTS,S/AS01E is the most advanced malaria vaccine in WHO reference reagent and other customized positive development globally , however the immune surro- controls was examined with seven in-house qSAT anti- gates of protection, the mode of action, and how vaccina- body assays measuring IgG, IgG , IgM and IgE against a 1–4 tion affects or is affected by naturally-acquired immunity, panel of 40 antigens, including P. falciparum proteins that remain unclear. A better characterization of the malaria are part of the RTS,S/AS01E vaccine. This information serological profile at the Ig isotype and subclass lev - will be generalizable to other applications and large sero- els could help address these questions. However, widely epidemiological and vaccine studies of sporozoite and BS applicable standardized, miniaturized, multiplex, high- antigen targets, being useful for the malaria research com- throughput assays, able to measure all Ig isotypes and munity as a whole. In addition, different sample incubation subclasses, have been lacking. times and temperatures (4 °C overnight, 37 °C 2 h, room Ubillos et al. Malar J (2018) 17:219 Page 3 of 15 temperature 1 h) were tested to select the incubation con- Reference reagents and control samples ditions rendering the optimal quantification range and WHO Reference Reagent for anti-malaria (P. falciparum) higher sensitivity without increasing unspecific binding. human plasma (10/198) (referred as WHO reference rea- gent). A pool derived from plasma donations collected at the Blood Bank from individuals based in Kisumu, Kenya, Methods with a history of malaria. This reference reagent presents Antigens IgG reactivity to P. falciparum AMA-1, MSP-1 , MSP- A customized multiplex panel with 33 BS and 6 pre- 1 , MSP-2 and MSP-3 . The reagent has been defi - erythrocytic (PE) P. falciparum antigens was established brinated and diluted (1:5) with deionized sterile water (Table 1). The glycan α-Gal (Gala1–3GalB1–4GlcNAc-R), and filled into 1 mL/ampoules. Each ampoule has been detected in the surface of sporozoites, was also included, lyophilized comprising a freeze-dried residue of diluted as anti-α-Gal IgM antibodies have been associated with human plasma. malaria protection . In addition to P. falciparum RTS,S vaccine positive control (referred as WHO-CSP antigens, the hepatitis B surface antigen (HBsAg, a com- pool). An RTS,S pool prepared with plasmas from 10 ponent of the RTS,S vaccine) was added, as the assays Mozambican children vaccinated with RTS,S/AS02 with were intended to be used with samples from this vaccine known high IgG titres to CSP at peak response  was trial. Also, bovine serum albumin (BSA) and glutathione added to the WHO reference reagent (1:50 WHO refer- S-transferase (GST) were added to the panel to control ence reagent + 1:100 RTS,S pool), creating a CSP and for background signal coming from unspecific binding to HBsAg antibody enriched WHO reference reagent. the BSA used to block the coupled beads, and to the GST Malaria primo-infected plasma pool (referred as IgM present in some of the fusion proteins. pool). A customized pool prepared with plasmas from 20 malaria naïve European adults with known high anti- malaria IgM levels after being experimentally infected Coupling of antigens to microspheres with P. falciparum in a controlled human malaria infec- Coupling of carboxylated polystyrene microspheres was ® tion (CHMI) trial . To prepare the pool, we first carried out as described elsewhere . Briefly, MagPlex selected the time point that elicited the highest IgM microspheres (Luminex Corp., Austin, Texas) with differ - breadth of response to a panel of 20 BS and 1 PE antigens ent spectral signatures selected for each antigen, were from the CHMI trials conducted in Barcelona (day 35) washed with distilled water and activated with Sulfo- and Tübingen (day 84). Ten individuals from each trial NHS (N-hydroxysulfosuccinimide) and EDC (1-ethyl- with the highest IgM breadth of response were selected 3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and pooled. (Pierce, Thermo Fisher Scientific Inc., Rockford, IL), both Negative control. A pool of plasma samples from 20 at 50 mg/mL, in activation buffer (100 mM Monoba - Spanish malaria-naïve individuals. sic Sodium Phosphate, pH 6.2). Microspheres were RTS,S samples. Three samples from individuals partici - washed with 50 mM MES (4-morpholineethane sulfonic pating in the RTS,S malaria vaccine phase 2b trial con- acid, Sigma, Tres Cantos, Spain) pH 5.0 or dPBS (Dul- ducted in Mozambique  were randomly selected. becco’s Phosphate Buffered Saline, Lonza) pH 7.0 to a High, medium and low responders were defined by 10,000 beads/µL concentration, and coated with antigens tertiles. at a concentration previously established in MES or PBS and incubated in a rotatory shaker overnight (ON) at 4 °C and protected from light. Microspheres were blocked qSAT assay and incubation conditions tested with PBS-BN [PBS with 1% BSA and 0.05% sodium azide IgG, IgG subclasses, IgM and IgE levels were meas- 1–4 (Sigma, Tres Cantos, Spain)] and re-suspended in PBS- ured in the WHO reference reagent and other custom- BN to be quantified on a Guava PCA desktop cytom - ized pools against multiplexed P. falciparum antigens eter (Guava, Hayward, CA) to determine the percentage using the xMAP technology (Luminex Corp., Austin, recovery after the coupling procedure. Antigen-coupled Texas). Fifty microliter of multiplexed antigen-cou- beads were validated in singleplex and multiplex by pled beads were added to a 96-well μClear flat bot - measuring IgG in serial dilutions of a positive control. tom plate (Greiner Bio-One, Frickenhausen, Germany) Similar IgG MFI levels were obtained in singleplex and at 1000 beads/analyte/well. To assess the optimal tem- multiplex measurements, with a strong correlation for all perature and duration of sample incubation for IgG and antigens assessed (R > 0.98; p < 0.05) (Additional file 1). IgG assays, 50 µL of WHO reference reagent at 11 1–4 Coupled beads were stored multiplexed at a concentra- serial dilutions (1:3, starting at 1/150) and the negative tion of 1000 beads/µL/antigen at 4 °C and protected from control at 4 serial dilutions (1:2, starting at 1:50) were light. Ubillos et al. Malar J (2018) 17:219 Page 4 of 15 Table 1 Antigens included in the multiplex qSAT panel Antigens and genotype Life-cycle stage Rationale References Pre-erythrocytic (PE) CelTOS Sporozoite Exposure to sporozoite [31, 32] CSP full length* Sporozoite Exposure to sporozoite and RTS,S specific [30, 33] CSP NANP repeat* GST-fused Sporozoite Exposure to sporozoite and RTS,S specific  CSP C-terminus* GST-fused Sporozoite Exposure to sporozoite  SSP2 or TRAP Sporozoite Representative of exposure to sporozoite [34, 37] Liver stage LSA-1* Liver stage Liver stage antigen—infected hepatocytes [38, 39] Blood stage (BS) AMA-1 3D7 (FMP2.1)* Merozoite Involved in erythrocyte invasion [40, 41] AMA-1 FVO (FMP009) Merozoite Involved in erythrocyte invasion  CyRPA full length Merozoite Involved in erythrocyte invasion  EBA-140 GST-fused Merozoite Involved in erythrocyte invasion  EBA-175 R2 PfF2 Merozoite Involved in erythrocyte invasion  EBA-175 R3–5* GST-fused Merozoite Involved in erythrocyte invasion  EXP-1 Merozoite Involved in erythrocyte invasion  MSP-1 Block 2 3D7* GST-fused Merozoite Involved in erythrocyte invasion  MSP-1 Block 2 hybrid GST-fused Merozoite Involved in erythrocyte invasion  MSP-1 Block 2 MAD20 GST-fused Merozoite Involved in erythrocyte invasion  MSP-1 Block 2 PA17 GST-fused Merozoite Involved in erythrocyte invasion  MSP-1 Block 2 RO33 GST-fused Merozoite Involved in erythrocyte invasion  MSP-1 Block 2 Well GST-fused Merozoite Involved in erythrocyte invasion  MSP-1 3D7* Merozoite Involved in erythrocyte invasion [41, 48] MSP-1 FVO Merozoite Involved in erythrocyte invasion [41, 48] MSP-2 full length B* GST-fused Merozoite Representative of exposure to BS  MSP-2 full length A* GST-fused Merozoite Representative of exposure to BS  MSP-3 3C Merozoite Representative of exposure to BS  MSP-3 3D7* Merozoite Representative of exposure to BS  MSP-5 Merozoite Representative of exposure to BS [51, 52] MSP-6* GST-fused Merozoite Representative of exposure to BS  P41 Merozoite Involved in erythrocyte invasion  RH1 Merozoite Involved in erythrocyte invasion  RH2 (2030) GST-fused Merozoite Involved in erythrocyte invasion  RH2 b240 Merozoite Involved in erythrocyte invasion  RH4.2 GST-fused Merozoite Involved in erythrocyte invasion [58, 59] RH4.9* Merozoite Involved in erythrocyte invasion [58, 59] RH5 Merozoite Involved in erythrocyte invasion [42, 60] PTRAMP Merozoite Involved in erythrocyte invasion  DBL-α Trophozoite Involved in cytoadherence  Pregnancy-specific DBL1-DBL2 VAR2CSA Trophozoite Associated to placental malaria exposure and representa-  tive of maternally-transferred antibodies DBL3-DBL4 VAR2CSA* Trophozoite  Other antigens HBsAg* NA Hepatitis B surface antigen α-Gal Involved in malaria protection  Controls GST* Background Control fusion protein BSA* Background Control unspecific binding * Recombinant proteins used for the experimental assessment of the optimal temperature and time of samples incubation in the IgG assays. MSP-2 A corresponds to the CH150 strain and MSP-2 B to the Dd2 strain Ubillos et al. Malar J (2018) 17:219 Page 5 of 15 incubated against a panel of 14 P. falciparum antigen- at 1:10 for IgE) and incubated at 4 °C ON. Samples were coated beads in a 96-well plate (Table 1). Plates were assayed in 4 serial dilutions (1:10) starting at 1:500 for incubated in a rotatory shaker at 600 rpm and protected IgG, in 3 serial dilutions (1:10) starting at 1:100 for IgM from light under three conditions: (i) 37 °C for 2 h; (ii) and IgG1, and in 2 serial dilutions (1:10) starting at 1:50 4 °C ON and (iii) room temperature (RT) for 1 h. For the for IgG2 and IgG4. Samples were not assayed for IgG3 or IgM assay, 50 µL of the WHO reference reagent or the IgE. IgM pool were assayed in 15 serial dilutions (1:3, starting at 1/50) against a panel of 40 P. falciparum antigens plus Statistical analyses HBsAg (Table 1). Plates were incubated at two different To stabilize the variance, the analysis was done on conditions: 37 °C for 2 h and 4 °C ON. IgE levels in the log -transformed values of the MFI measurements. The WHO reference reagent assayed at 8 serial dilutions (1:2, correlation and reliability between the different sample starting at 1/10) were also measured under two different incubation conditions for the IgG and IgG subclasses 1–4 incubation conditions: 37 °C for 2 h and 4 °C ON. Finally, measured in the positive control, the negative control using the WHO-CSP pool, 23 standard curves for IgG, and the blanks were evaluated. After the Shapiro–Wilk IgG1, IgG3 and IgM were constructed; and 12 standard normality test was applied, differences between condi - curves for IgG2 and IgG4 all of 18 serial dilutions (1:2, tions were assessed by Kruskal–Wallis test with posthoc starting at 1:50). The standard curves were incubated at Tukey test. Reliability was assessed by the interclass cor- 4 °C ON against a total of 40 antigens (Table 1). Beads relation coefficient (ICC) . Titration curves of anti - coupled with BSA and GST were included in the panel as body concentrations vs. MFIs per antigen were fitted background controls to assess unspecific binding to BSA using a five-parameter (5PL), a 4PL or an exponential and GST. After the incubation, plates were washed with logistic equation depending on the best yield, following PBS-0.05% Tween 20 buffer using a manual magnetic the formula MFI = Emax + ((Emin − Emax)/((1 + ((Conc/ washer platform (Bio-Rad, Hercules, CA, USA). Second- EC )^Hill))^Asym)), where E C is the half maximal 50 50 ary antibodies were added as previously described . effective concentration, Emin is the minimum response, Briefly, biotinylated anti-human IgG at 1:2500 (Sigma Emax is the maximum response, Asym is the asymmetry B1140, polyclonal), anti-human IgM at 1:1000 (Sigma factor and Hill is the slope factor , using the drLumi B1265, polyclonal) anti-human IgG3 at 1:1000 (Sigma package . We calculated the coefficient of variation B3523, clone HP-6050), and anti-human IgG1 at 1:4000 (CV) of Emin, Emax and used the goodness of fit model (Abcam ab99775, clone 4E3). For the IgG2, IgG4 and IgE to assess the fitting of the curves. All analyses were done assays, the secondary antibodies were unconjugated to using R version 3.4.1. biotin: mouse anti-human IgG2 at 1:500 (Thermo Fisher MA1-34755, clone HP6014), mouse anti-human IgG4 Results at 1:8000 (Thermo Fisher MA1-80332, clone HP6025), Total IgG, IgG , IgM and IgE responses against RTS,S 1–4 and mouse anti-human IgE at 1:500 (Abcam ab99834, antigens in the WHO reference reagent compared to those clone HP6029). All secondary antibodies were incubated measured in sera from RTS,S-vaccinated children 60 min at RT and washed. In IgG2, IgG4 and IgE assays, a To assess the suitability of the WHO reference reagent as tertiary biotinylated goat anti-mouse IgG (Sigma B7401, a positive control to capture all responses in the context polyclonal) was added and incubated 60 min at RT. Plates of RTS,S vaccine studies, levels of IgG, IgG , IgM and 1–4 were washed as before and streptavidin-R-phycoerythrin IgE against the RTS,S-specific antigens (CSP full length, at 1:1000 (Sigma, Tres Cantos, Spain) was added to all CSP NANP repeat and CSP C-terminus) were measured wells and incubated 30 min at RT. Plates were washed and compared to levels in sera from RTS,S-vaccinated and beads re-suspended in 100 μL/well of PBS-BN, pro- children from a phase 2b trial with known IgG CSP titres tected from light and stored ON at 4 °C to be read the  (Fig. 1). The WHO reference reagent and RTS,S- next day. Plates were read using the Luminex xM AP vaccinees antibody responses to the whole antigenic 100/200 analyser (Luminex Corp., Austin, Texas) and at panel (Table 1) are shown in the Additional file 2. The least 50 microspheres per analyte were acquired per well. IgM pool and RTS,S-vaccinees IgM levels were also com- Results are expressed as Median Fluorescence Intensity pared (Fig. 1h and Additional file 2). The WHO reference (MFI). reagent presented lower IgG, IgG1, IgG2, IgG4, and IgM RTS,S-induced antibodies were measured in 3 sam- levels to RTS,S antigens than samples from RTS,S-vacci- ples from RTS,S-vaccinated children with known high, nated children who had high CSP responses (Fig. 1a–d, medium and low responses, together with serial dilu- g). Comparisons of IgG3 and IgE levels were not possi- tions of the WHO reference reagent or the IgM pool (1:3 ble because these data were not available for RTS,S sam- starting at 1:50 for IgG, IgG and IgM, and 1:2 starting ples. The IgM pool presented higher IgM levels to RTS,S 1–4 Ubillos et al. Malar J (2018) 17:219 Page 6 of 15 a IgG responses b IgG1 responses CSP full length CSP NANPrep CSP C−term CSP full length CSP NANPrep CSP C−term SampleType 4 4 WHO ref. reagent RTS,S high 3 3 RTS,S medium RTS,S low 2 2 blank 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 c IgG2 responses d IgG4 responses CSP full length CSP NANPrep CSP C−term CSP full length CSP NANPrep CSP C−term SampleType 4 4 WHO ref. reagent RTS,S high 3 3 RTS,S medium RTS,S low 2 2 blank 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 e IgG3 responses f IgE responses CSP full length CSP NANPrep CSP C−term CSP full length CSP NANPrep CSP C−term 4 4 SampleType 3 3 WHO ref. reagent blank 2 2 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 1234 1234 1234 g IgM responses h IgM responses CSP full length CSP NANPrep CSP C−term CSP full length CSP NANPrep CSP C−term SampleType SampleType 4 4 WHO ref. reagent IgM pool RTS,S high RTS,S high 3 3 RTS,S medium RTS,S medium RTS,S low RTS,S low 2 2 blank blank 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 log10(dilution) log10(dilution) 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 , IgM and IgE levels to RTS,S-specific antigens measured in 1–4 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 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, 1–4 1:50 for IgG ; and 1:2 starting at 1:10 for IgE). Isolated dots represent the levels measured in the technical blanks 1–4 antigens than the WHO reference reagent and the RTS,S primary vaccination had antibodies that cross-reacted samples. Consequently, we decided to prepare a custom- with GST. However, even if the samples contained equal ized positive control for the RTS,S immunological stud- or higher levels of antibodies to GST, this did not impede ies, containing 1:50 of the WHO reference reagent plus to accurately measure anti-malarial antibodies to the 1:100 of pooled plasma from RTS,S-vaccinated children GST fusion proteins, as shown in correlation analyses of with high CSP titres (WHO-CSP pool). IgG responses GST vs. GST fusion proteins in plasmas from RTS,S vac- were compared between the WHO-CSP pool and the cinees (Additional file 5). WHO reference reagent (Additional file 3). In addition, the EC ratio between positive controls (E C WHO 50 50 reference reagent/EC WHO-CSP) was calculated for Levels of total IgG, IgG and IgM against multiple P. 50 1–4 RTS,S-specific antigens as a proxy measure of relative falciparum antigens plus HBsAg measured in the WHO-CSP potency of the WHO-CSP pool to the WHO reference pool reagent (Additional file 4). The EC ratio for the 3 CSP The WHO-CSP pool was used to generate IgG, IgG 50 1–4 antigens was between 0.44 and 0.58 for IgG, IgG1 and and IgM titration curves incubating at 4 °C ON in the IgG3, and close to 1 for IgG2 against CSP full length. context of an RTS,S immunology study. The level of Fifteen proteins in the multiplex panel were GST-fused response was antigen-dependent; the most immunogenic proteins (AMA-1 3D7 and FVO, MSP-1 (Table 1). The WHO-CSP pool was reactive to GST 3D7 and FVO) because the sera from RTS,S vaccinees 1 month after log10(MFI) log10(MFI) log10(MFI) log10(MFI) log10(MFI) log10(MFI) log10(MFI) log10(MFI) Ubillos et al. Malar J (2018) 17:219 Page 7 of 15 WHO−CSP pool fitted Standard Curves alpha−Gal AMA−1 3D7AMA−1 FVO BSA CelTOS CSP C−term CSP full length CSP NANPrepCyRPA DBL−alpha DBL1−DBL2 DBL3−DBL4 EBA−140 EBA−175 R2 EBA−175 R3−5 EXP−1 GST HBsAg LSA_1 MSP−1 42 3D7MSP−1 42 FVO isotype IgG IgG1 IgG2 MSP−1 Bl2 3D7 MSP−1 Bl2 hybridMSP−1 Bl2 MAD20 MSP−1 Bl2 PA17 MSP−1 Bl2 RO33 MSP−1 Bl2 Wel MSP−2 A IgG3 IgG4 IgM MSP−2 B MSP−3 3C MSP−3 3D7 MSP−5MSP−6 p41 PTRAMP RH1 RH2 2030 RH2 b240 RH4.2 RH4.9 RH5 SSP2 or PTRAP 24 62 46 246 24 62 46 24 62 46 log10(ec) Fig. 2 IgG, IgG and IgM fitted curves using the WHO-CSP pool to the 40-antigen multiplex panel incubating at 4 °C ON. Lines and dots represent 1–4 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 assay performance of the WHO reference reagent against gave saturated signals even at the 1:6.5 × 10 dilution a panel of 14 P. falciparum antigens (Table 1) under three (Fig. 2). different incubation conditions (4 °C ON, 37 °C 2 h and To further characterize the IgG subclass composition RT 1 h) was compared. IgG and IgG assays varied of the WHO-CSP pool, the ratios of IgG subclasses 1–4 1–4 depending on the incubation procedure, with the larg- to total IgG [MFI IgG subclass at dilution (i)/MFI total est difference between 4 °C ON and RT 1 h (p < 0.001) for IgG at dilution (i) × 100] were measured (Fig. 3). The IgG. No differences were found between these two incu - predominance of IgG subclasses also varied depend- bation conditions for IgG2, IgG3 and IgG4. Differences ing on the antigen. For example, IgG1 responses were between 4 °C ON and 37 °C 2 h were only observed for dominant for HBsAg, LSA-1, MSP-5, P41, RH1, RH2, IgG (p = 0.026). IgG and IgG levels against BSA and PTRAMP, RH4.2, RH4.9 and SSP2, whereas MSP-2 full 1–4 blanks were not affected by the incubation conditions. length, MSP-1 block 2 and RH4 induced mainly IgG3. The MFI levels of IgG and IgG measured in the nega- IgG subclass responses to AMA-1 (3D7 and FVO), CSP 1–4 tive control only varied when comparing 4 °C ON vs. RT (C-terminus and NANP repeat), EXP-1, MSP-1 (3D7 1 h (p < 0.001) for some of the antigens. Figure 4 shows and FVO), MSP-3 and RH5 were dominated by IgG1 and examples of the results for IgG1, and the complete data IgG3. set is in the Additional file 6. The incubation at 4 °C ON, on average, showed the highest MFIs in the first dilution, Optimal temperature and time of incubation to measure except for IgG4 and reached blank levels at the lowest IgGs against P. falciparum antigens using the WHO dilution (Fig. 4 and Additional file 6). Negative control reference reagent MFI levels were also higher at 4 °C ON compared to To assess the optimal temperature and time of incubation other conditions, however the difference with the WHO for the measurement of IgG and IgG subclasses, the 1–4 predicted.log10_mfi Ubillos et al. Malar J (2018) 17:219 Page 8 of 15 Ratios of IgG subclasses/total IgG in the WHO−CSP pool alpha−Gal AMA−1 3D7 AMA−1 FVO BSA CelTOS CSP C−term CSP full length CSP NANPrep CyRPA DBL−alpha DBL1−DBL2 DBL3−DBL4 EBA−140 EBA−175 R2 EBA−175 R3−5 EXP−1 GST HBsAg LSA_1 MSP−1 42 3D7 MSP−1 42 FVO MSP−1 Bl2 3D7 MSP−1 Bl2 hybrid MSP−1 Bl2 MAD20 MSP−1 Bl2 PA17 MSP−1 Bl2 RO33 MSP−1 Bl2 Wel MSP−2 A MSP−2 B MSP−3 3C MSP−3 3D7 MSP−5 MSP−6 p41 PTRAMP RH1 RH2 2030 RH2 b240 RH4.2 RH4.9 RH5 SSP2 or PTRAP IgG1 IgG2 IgG3IgG4 IgG1IgG2 IgG3 IgG4 IgG1 IgG2 IgG3 IgG4 IgG1 IgG2 IgG3IgG4 IgG1IgG2 IgG3 IgG4 IgG1 IgG2 IgG3 IgG4 IgG1IgG2IgG3 IgG4 IgG subclass Fig. 3 Boxplots of ratios of IgG subclasses to total IgG measured in the WHO-CSP pool. Ratios are composed with the median of the 23 titration 1–4 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 AMA−1 3D7 BSA CSP C−term CSP full length CSP NANPrep condition 4ºC ON WHO reagent DBL3−DBL4 EBA−175 GST HBsAg LSA−1 5 37ºC 2h WHO reagent RT 1h WHO reagent 4ºC ON neg MSP−1 42 3D7 MSP−1 Bl2 3D7 MSP−2 A MSP−2 B MSP−3 3D7 37ºC 2h neg RT 1h neg 4ºC ON blank 2468 2468 2468 37ºC 2h blank MSP−6 RH4.9 RT 1h blank 246 8 2468 log10(dilution) 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 Ratio IgG subclass/total IgG IgG1 log10(MFI) Ubillos et al. Malar J (2018) 17:219 Page 9 of 15 Optimal temperature and time of incubation to measure reference reagent at same dilution was high enough to IgM and IgE against P. falciparum antigens using the WHO establish a positivity threshold (Fig. 4). reference reagent and an IgM customized pool Correlations between incubation conditions for IgG Incubation conditions to measure IgM and IgE and IgG subclasses measured against all antigens in 1–4 responses against a panel of 38 P. falciparum antigens the WHO reference reagent and negative control showed plus HBsAg, α-Gal, BSA and GST (Table 1) were tested a r > 0.93 for all IgG and IgG subclasses. The ICCs 1–4 using the WHO reference reagent and an alternative between incubation conditions for IgG and IgG meas- 1–4 IgM pool. The IgM pool gave higher IgM responses and ured in the WHO reference reagent showed overall good of higher range compared to those obtained with the reliability, being 0.91 (0.89–0.93) for IgG3, 0.88 (0.87– WHO reference reagent for most of the antigens, espe- 0.89) for IgG1, 0.83 (0.79–0.86) for total IgG, 0.79 (0.74– cially AMA-1s, MSP-1s and CSPs (Fig. 5). Incubation of 0.83) for IgG2 and 0.63 (0.53–0.72) for IgG4. However, as the IgM pool at 4 °C ON showed higher responses com- seen in Fig. 4 and Additional file 6, ICCs in the negative pared to incubation at 37 °C 2 h (Additional file 7A), control were of lower reliability, being of 0.85 (0.78–0.9) with 80% of the antigens studied (35/43) presenting a for IgG4, 0.74 (0.64–0.82) for IgG2, 0.38 (0.23–0.53) for higher EC (i.e. AMA-1 3D7 EC 4 °C ON 3.64 ± 0.66 total IgG, 0.39 (0.23–0.54) for IgG1 and 0.11 (− 0.03– 50 50 and EC 37 °C 2 h 2.62 ± 0.96). The IgM responses of 0.14) for IgG3. Blank levels were similar between incu- the negative control measured at first dilution were bation conditions (Fig. 4 and Additional file 6). Taking higher than those of IgG and IgG subclasses, but lev- together these results, we chose the incubation at 4 °C els dropped quickly after the first dilution. Overall, IgM ON as the optimal for the IgG assays. pool responses showed higher difference to the negative control than those obtained with the WHO reference Predicted IgM curves for WHO reagent and IgM pools alpha−Gal AMA−1 3D7 AMA−1 FVO BSA CelTOS CSP C−term CSP full length CSP NANPrep CyRPA full length DBL−alpha DBL1−DBL2 DBL3−DBL4 EBA−140 EBA−175 R2 EBA−175 R3−5 EXP−1 GST HBsAg LSA−1 MSP−1 42 3D7 MSP−1 42 FVO Control WHO reagent IgM pool MSP−1 Bl2 3D7 MSP−1 Bl2 hybrid MSP−1 Bl2 MAD20 MSP−1 Bl2 PA17 MSP−1 Bl2 RO33 MSP−1 Bl2 Well MSP−2 A 5 neg blank MSP−2 B MSP−3 3C MSP−3 3D7 MSP−5 MSP−6 P41 PTRAMP RH1 RH2 2030 RH2 b240 RH4.2 RH4.9 RH5 SSP2 or TRAP 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 2.55.0 7.5 log10(dilution) 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 log10(MFI) Ubillos et al. Malar J (2018) 17:219 Page 10 of 15 reagent (Fig. 5). Similar differences in IgM responses responses as well as immunogenicity evaluation of CSP- between incubation conditions were obtained with the based vaccine candidates. WHO reference reagent, measuring higher levels when The estimation of malaria antibody concentration in incubating at 4 °C ON than at 37 °C 2 h (Additional multiplex assays is increasingly difficult. There are not file 7B). IgM technical blanks were not affected by appropriate standards or reference sera available that incubation conditions (Additional file 7A, B). Correla- react strongly to complex antigen panels. Antibody tions for IgM responses between incubation conditions concentrations have been previously estimated using were r = 0.96 for both WHO reference reagent and an anti-human IgG curve [22, 23, 26, 27]. However, the IgM pool. For the IgE assay, there were no differences binding system and the affinity of the anti-human IgG between incubation conditions (Additional file 7 C). curve differ from that of antibodies in samples or posi - The ICCs between antibody responses measured in the tive controls. Thus, different assay conditions give differ - two incubation conditions with the WHO reference rea- ent slopes and curve parameters that could result in large gent were 0.92 (0.91–0.93) for IgM and 0.82 (0.79–0.85) deviations of concentration estimates. Thus, it has been for IgE; and the ICC between conditions for the IgM recently reported that MFI responses measured indepen- assay using the IgM pool was 0.91 (0.9–0.92). However, dently from a standard curve might reflect actual varia - IgM responses of negative controls showed moderate tion, while estimated concentration values are dictated reliability between incubation conditions, having an ICC by the precision of the standard curve . As an alter- of 0.66 (0.57–0.73). native, the use of long positive control curves provide When comparing antibody levels measured in the upper and lower asymptotes for most antigens, and allow WHO reference reagent vs. the IgM pool, there was establishing the linear quantification ranges, represent - moderate reliability, with ICC of 0.65 (0.61–0.769) at 4 °C ing the optimal range to capture the breadth of antibody ON, and 0.66 (0.61–0.7) at 37 °C 2 h, meaning that there response in individual samples. However, a reference was 35% of variability between reference pools. Con- human serum pool with known levels of anti-P. falcipa- sidering the strong correlation and reliability of the two rum antibody concentrations is highly desirable for the incubation conditions, but the higher IgM levels and MFI malaria community. The challenge remains in sourcing ranges obtained at 4 °C ON, this incubation was also cho- adequate serum/plasma pools that cover all antigens as sen for the IgM assay. panels become larger and more complex. To test the immuneprofile of the WHO reference rea - Discussion gent, antigen and isotype/subclass-specific curves con - A major challenge in large malaria sero-epidemiological structed with serial dilutions of the reagent were fitted and vaccine studies is to have access to consistent and in non-linear equations, establishing the linear quanti- unlimited control reagents that provide assay quality con- fication ranges. Generation of curves with optimal lin - trol and facilitate data consolidation. A universal malaria ear quantification ranges is important to allow selecting reference pool would be ideal to monitor performance the optimal dilution of test samples (lying on the linear of serological assays, improve inter-laboratory repro- range). In addition, the parameters of the curve may be ducibility, make data from different studies comparable, used for the quality control of the assay. The WHO ref - and potentially give quantitative antibody measures. In erence reagent is composed of samples from hyper- this study, information was provided on the expanded immune individuals from a malaria endemic region , antibody reactivity profile of the commercially available predominantly having anti-P. falciparum IgG1 and IgG3 WHO reference reagent for anti-malaria (P. falciparum) antibodies, rather than IgG2 and IgG4, reflecting the nat - human plasma (10/198)  and other customized posi- urally-acquired antibody patterns. Thus, for most anti - tive controls by using seven in-house qSAT multiplex gens, this pool is of restricted use to produce standard antibody assays to measure IgG, IgG , IgM and IgE curves for IgG2, IgG4 or IgE antibodies, and this remains 1–4 against a panel of 40 antigens, including P. falciparum a limitation. Similarly, the WHO reference reagent might proteins that are part of the RTS,S/AS01E vaccine. In not be optimal for IgM measurements, particularly if addition, different sample incubation times and temper - high responses are expected in test samples. For this atures (4 °C ON, 37 °C 2 h, RT 1 h) were tested for the reason, a customized IgM pool with plasmas from naïve qSAT assays to select the incubation conditions render- individuals experimentally challenged with P. falciparum ing the optimal quantification range and higher sensitiv - at a time point when IgM predominated over IgG was ity without increasing unspecific binding. Data generated prepared. This IgM pool proved to be very adequate for in this study will be useful for clinical malaria studies the generation of IgM titration curves in the study. Thus, involving assessment of naturally-acquired immune as the WHO reference reagent has been established to measure IgGs, a reference standard to measure IgM Ubillos et al. Malar J (2018) 17:219 Page 11 of 15 responses would still be lacking. Similarly, IgG2, IgG4 The WHO-CSP pool presented GST reactivity, mainly and IgE specific reference standards would improve the coming from the RTS,S samples, which poses the ques- reproducibility of the malaria-based immune assays. tion of whether the GST signal could be interfering with This study also aimed to assess the usefulness of the the responses to the GST-fused proteins. However, cor- WHO reference reagent as a positive control to gener- relation analysis showed that the antibody response to ate titration curves in the context of RTS,S immunology GST was not associated to the antibody response against studies. For this reason, samples from RTS,S vaccinated the GST-fused protein and, therefore, that responses children with diverse CSP and HBsAg IgG titres were were independent. For example, CSP-specific antibod - assayed together with the WHO reference reagent for ies detected upon vaccination were very high and not comparison. It is important to test samples at several interfered by anti-GST antibodies when using CSP GST dilutions to maximize the assay sensitivity, but keeping fusion proteins as capture antigens. Because of these to the minimum for cost-effectiveness, which is key in observations, the GST values were not subtracted during large sero-epidemiological studies. For this reason RTS,S data pre-processing, and it was concluded that GST reac- samples were assayed at 4 dilutions for IgG, 3 dilutions tivity was not a major part of the antibody signal to the for IgM and IgG1, and 2 dilutions for IgG2 and IgG4. P. falciparum portion of the fused proteins. Nevertheless, Samples from RTS,S vaccinated children had signifi - the GST reactivity with CSP pools remains an unsolved cantly higher CSP antibodies than individuals naturally- limitation that will be addressed in future studies upon exposed to P. falciparum sporozoites. Consequently, the the application of the assays to the analysis of samples WHO reference reagent could only be used to measure from RTS,S vaccinated volunteers using GST fusion pro- RTS,S-specific responses if a relative potency between teins, e.g. by testing the blocking of the reactivity with the WHO reference reagent and the vaccinees samples soluble GST. was calculated . Alternatively, data showed that the This first WHO reference reagent contains an arbitrary WHO reference reagent enriched with pooled sera from unitage of 100 Units per ampoule, however the concen- RTS,S-vaccinated children (WHO-CSP pool)  was trations of antibodies (IgG, IgG , IgM, IgE) specific 1–4 adequate to capture all antibody responses, including the to antigens such as those tested here remain unknown. very high anti-CSP IgG levels in vaccinated children. To u Th s, it has been suggested to the WHO Expert Commit - conserve the full reactivity of the WHO reference reagent tee on Biological Standardization to assess the specific to BS antigens, the WHO-CSP pool was constructed by antibody concentrations in this reagent to allow absolute adding half concentration of pooled plasmas from RTS,S quantifications in future studies. vaccinated children (1:50 WHO reference reagent and In a qSAT assay, temperature of incubation influences 1:100 plasma from RTS,S vaccinees), ensuring that RTS,S the reversible antigen–antibody kinetics by altering the specific antibodies were increased without diluting other constant association/dissociation equilibrium , which anti-P. falciparum antibodies. A proxy measure of rela- can impact assay sensitivity . Raising the incubation tive potency of the WHO-CSP pool vs. the WHO refer- temperature from 5 to 37 °C decreases the affinity of anti - ence reagent was estimated with E C . However, in 4PL gen–antibody complexes by decreasing the stability of the and 5PL analysis, the dose–response is not the same over docking complex [28, 74]. The conditions previously used the entire tested concentration range, and the response in our laboratory for incubation of samples with antigen- changes relative to the concentration only in the mid- coupled beads were 1 h and RT [22, 23, 26, 27]. For this dle part of the curves. Typically, these comparisons are study, it was hypothesized that incubating samples for made at the EC , however, these calculations are only 1 h might not ensure the appropriate association/disso- valid under limited conditions. For instance, the dose– ciation equilibrium. For this reason, expanded incuba- response curve would need to have a common slope, and tion times were tested and lower (4 °C) and higher (37 °C) the maximum achievable response should be identical temperatures were explored. Higher IgG and IgG lev- 1–4 . Unfortunately, these conditions are not met for the els were detected when the WHO reference reagent was curves of most of the tested antigens and IgG subclasses. incubated ON at 4 °C compared to 2 h at 37 °C or 1 h at Similarly to CSP, it would be desirable to increase the RT. The ON incubation at 4 °C increased the IgG levels WHO reference reagent reactivity to other P. falciparum detected at high concentrations of the WHO reference PE antigens that are also vaccine candidates like SSP2/ reagent, but also the negative control. Yet, the difference TRAP, LSA-1 or CelTOS. Additionally, a second genera- between the WHO reference reagent and the negative tion of the WHO reference reagent against other Plas- control was large enough to establish a positive thresh- modium species would be an advantage for other malaria old. Different incubation conditions showed small dif - immune studies in areas with P. vivax co-infections. ferences for the WHO reference reagent performance, but larger differences for the negative control, indicating Ubillos et al. Malar J (2018) 17:219 Page 12 of 15 more variability at very low IgG concentrations. The Additional files unspecific binding of IgGs to BSA-coupled beads or the background signal in the technical blanks was not Additional file 1. Correlations of antigen-specific IgG levels (log MFI) affected by the incubation conditions, suggesting that the between singleplex and multiplex coupled-beads measured in serial dilutions of a positive control pool. The positive pool was composed of specificity of the IgG binding was not affected by incuba - plasmas from Mozambican adults with life-long exposure to malaria. The tion duration or temperature. For all these reasons, 4 °C 2 panel contained 26 antigens. The correlation coefficients (r ) are indicated, ON was the incubation condition chosen for the anti-P. and the blue line corresponds to the linear fit. falciparum IgG and IgG profiling of the WHO refer - Additional file 2. Comparison of the WHO reference reagent, IgM pool 1–4 and RTS,S samples responses to the 40-antigen multiplex panel incubat- ence reagent and the WHO-CSP pool. ing at 4 °C ON. IgG, IgG subclasses, IgM and IgE were measured in the 1–4 The optimal incubation condition for the IgM assay respective pools and samples. The plots represent the levels of antibodies was assessed using the WHO reference reagent and the measured in serial dilutions of the positive pools (1:3 starting at 1:50 for IgG, IgG and IgM; and 1:2 starting at 1:10 for IgE), and the RTS,S samples IgM pool. IgM levels were higher when incubating at 4 °C 1–4 (1:10 starting at 1:500 for IgG, 1:100 for IgM, 1:50 for IgG ; and 1:2 starting 1–4 ON, although no significant differences were detected at 1:10 for IgE). Data on IgG3 and IgE levels measured in RTS,S vaccinees between incubating at 4 °C ON or 37 °C 2 h. Similarly to were not available. Isolated dots represent the levels measured in the technical blanks. IgG and IgG subclasses, IgM levels to BSA and blanks 1–4 Additional file 3. Comparison of the IgG and IgG predicted curves were low and not affected by the incubation condition. 1–4 between the WHO reference reagent and the WHO-CSP pool incubating Based in these observations, 4 °C ON was also the incu- at 4 °C ON. IgG and IgG predicted curves from a non-linear equation 1–4 bation condition chosen for the IgM assay. were measured against a 23-multiplex panel. Isolated dots represent the levels measured in the technical blanks. The main limitation of the IgM assay was the high reac - tivity of the negative control, also affected by the dura - Additional file 4. IgG and IgG 50% effective concentrations (EC ) to 1–4 50 RTS,S-specific antigens measured in the WHO reference reagent and the tion and temperature of incubation. IgMs are the first WHO-CSP pool, and EC ratios between pools. The functions used to fit class of antibodies produced during a primary immune the standard curves were 4PL (SSl4) or exponential (SSexp) equations. response. They are generated in the absence of apparent Additional file 5. Correlations between GST vs. antigens included in the stimulation by specific antigens , and are thought to RTS,S vaccine, and GST vs. non-RTS,S antigens in plasmas from RTS,S-vac- cinated children. Scatterplots with levels of IgG (log MFI) to GST alone in aid in the neutralization of pathogens prior to the devel- 10 the X-axis and to GST-fused proteins (orange) or proteins not fused to GST opment of high affinity, antigen-specific antibodies . (green) in the Y-axis. Linear regression lines with 95% confidence intervals Natural IgMs tend to have rather low antigen-binding (in grey) and Spearman correlation coefficients (r ) for each antigen. Cor- relations between IgGs to RTS,S proteins and GST were high but similar affinities, compensated (to some extent) by their pen - between GST-fused (CSP NANP & C-terminus) and non GST-fused proteins tameric nature. Thus, IgM is a highly polyreactive anti - (CSP full length and HBsAg). Antibody levels against the GST-fused CSPs body  and cross-reactivity of IgMs with antigens from (Y-axis value) were higher than to the GST alone (X-axis value). IgG levels to GST fusion proteins representing non-RTS,S antigens (e.g. EBA-175, other pathogens to which they have been exposed, or MSP-2) were not correlated with IgG levels to GST alone. There were low even pathogens that have not yet been “seen” by the host antibody responses to these antigens while there was a higher signal to immune system [77, 78], could account for the high reac- the GST alone. Overall, the patterns of correlations were similar between GST-fused and non-GST fused proteins. Responses to GST and to GST tivity observed in the negative control. Additional tests fusion proteins appeared to be independent. are currently being performed to improve the specificity Additional file 6. Levels of IgG and IgG to 15 antigens measured in the 2-4 of the IgM qSAT assay. WHO reference reagent compared to negative control and blanks under three different incubation conditions. Curve plots of the antigen-specific antibody levels measured in serial dilutions of the WHO reference reagent, Conclusion negative control and blanks at three different incubation conditions: 37 °C This study served to expand the characterization of 2 h (37 °C 2 h), 4 °C overnight (4 °C ON) and room temperature for 1 h (RT the immunogenicity profile of the WHO reference rea - 1 h). “neg” means negative control. gent, including multiple Ig isotypes/subclasses, and sig- Additional file 7. Levels of IgM and IgE measured to the 40-multiplex nificantly more P. falciparum antigens, including CSP. panel in the WHO reference reagent and IgM pool compared to negative control and blanks under two different incubation conditions. Incuba- The study also served to establish the optimal sam - tion conditions compared are: 4 °C (4 °C ON) vs 2 h at 37 °C (37 °C 2 h). A) ple incubation condition for seven qSAT assays (4 °C Predicted 5PL curves of IgM levels in the IgM pool. B) Predicted 5PL curves ON). Some of the limitations of the WHO reference of IgM levels in the WHO reference reagent. C) IgE levels in the WHO refer- ence reagent. reagent were circumvented by preparing in-house or adapted pools to quantify high anti-CSP IgG and IgM responses. Information generated here is applicable Authors’ contributions to other malaria sero-epidemiological studies of PE Designed the study: IU, RA, AJ, MV, JJC, CD; performed the assays: IU, AJ, MV; provided the WHO reference reagent: PB; produced the recombinant proteins: and BS vaccine candidates, and thus valuable for the DG, SD, BG, RC, VC, DL, CC, EA, JB, DC; wrote the first draft of the manuscript: malaria research community. IU, RA, CD. All authors read and approved the final manuscript. Ubillos et al. Malar J (2018) 17:219 Page 13 of 15 Author details 4. Sabchareon A, Burnouf T, Ouattara D, Attanath P, Bouharoun-Tayoun ISGlobal, Hospital Clínic-Universitat de Barcelona, Carrer Rosselló 153 (CEK H, Chantavanich P, et al. Parasitologic and clinical human response to Building), 08036 Barcelona, Catalonia, Spain. CIBER Epidemiología y Salud immunoglobulin administration in falciparum malaria. Am J Trop Med Pública (CIBERESP), Barcelona, Spain. Bacteriology Division, MHRA-NIBSC, Hyg. 1991;45:297–308. South Mimms, Potter Bars EN6 3QG, UK. Laboratory of Malaria and Vaccine 5. Richards JS, Arumugam TU, Reiling L, Healer J, Hodder AN, Fowkes FJI, Research, School of Biotechnology, Jawaharlal Nehru University, New Delhi, et al. Identification and prioritization of merozoite antigens as targets of India. Malaria Group, International Centre for Genetic Engineering and Bio- protective human immunity to Plasmodium falciparum malaria for vac- technology (ICGEB), New Delhi, India. U.S. Military Malaria Vaccine Program, cine and biomarker development. 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Cytophilic immunoglobulin responses to Plasmodium falciparum glutamate-rich Availability of data and materials protein are correlated with protection against clinical malaria in Dielmo, Data obtained in this study and more details are available from the corre- Senegal. Infect Immun. 2000;68:2617–20. sponding author on reasonable request. 13. Roussilhon C, Oeuvray C, Muller-Graf C, Tall A, Rogier C, Trape J-F, et al. Long-term clinical protection from falciparum malaria is strongly associ- Consent for publication ated with IgG3 antibodies to merozoite surface protein 3. PLoS Med. All data has consent for publication. 2007;4:e320. 14. Taylor RR, Allen SJ, Greenwood BM, Riley EM. IgG3 antibodies to Ethics approval and consent to participate Plasmodium falciparum merozoite surface protein 2 (MSP2): increasing Approval for the protocols was obtained from the Hospital Clínic of Barcelona prevalence with age and association with clinical immunity to malaria. Ethics Review Committee and the National Mozambican Ethics Review Am J Trop Med Hyg. 1998;58:406–13. Committee. 15. Stanisic DI, Richards JS, McCallum FJ, Michon P, King CL, Schoepflin S, et al. Immunoglobulin G subclass-specific responses against Plasmodium falci- Funding parum merozoite antigens are associated with control of parasitemia and This work received support from the Instituto de Salud Carlos III (Grant protection from symptomatic illness. Infect Immun. 2009;77:1165–74. Numbers PS11/00423, PI14/01422), NIH-NIAID (Grant Number R01AI095789), 16. Weaver R, Reiling L, Feng G, Drew DR, Mueller I, Siba PM, et al. The asso- PATH Malaria Vaccine Initiative, the Agency for Management of University and ciation between naturally acquired IgG subclass specific antibodies to Research Grants (AGAUR Grant Number 2014SGR991). ISGlobal is a member of the PfRH5 invasion complex and protection from Plasmodium falciparum the CERCA Programme, Generalitat de Catalunya. malaria. 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