Background: Antibody responses to Plasmodium falciparum play a critical role in disease control. Finding reliable IgG biomarkers of protection is complicated by a parasite proteome of over 5000 proteins, some with polymorphisms. Studies of anti-malarial naturally acquired and vaccine immunity would benefit from a standard high-throughput immunoassay to measure multiple antibodies. A multiplex quantitative suspension assay to measure antigen-specific IgGs was developed and its precision (reproducibility and repeatability), dynamic range, limits of detection and quan- tification, and non-specific binding to different P. falciparum proteins tested. A set of 288 human plasma samples from a malaria-endemic region were analysed twice by two different operators. Another set of samples from 9 malaria- naïve and 10 malaria-exposed individuals were repetitively assayed during 22 consecutive days. Positive controls, negative controls, blanks and microspheres coated with bovine serum albumin were included in all assays. Results: The multiplex quantitative suspension assay demonstrated low non-specific signal and good estimates of precision and reproducibility between operators. The overall mean of non-specific binding measured in 288 plasma samples was 32.83 to ± 44.81 median fluorescence intensity (MFI). Repeatability was 7.66% ± 15.89 between trip- licates for all antigens and samples, being lower in samples from malaria-exposed than malaria-naïve individuals. No evidence of significantly different variance across days in MFI or arbitrary units (AU)/mL was found, assuming homogeneity of variance between days of analysis. Intra-class correlation coefficient between 22 days of analysis was 0.98 (0.97–0.98) for MFI units and 0.9 (0.87–0.93) for AU/mL. Reproducibility between operators for all samples and antigens had an overall adjusted correlation of 0.929 for MFI and 0.836 for AU/mL. Conclusions: This high-throughput multiplex immunoassay is simple and highly reproducible. This represents an asset for malaria vaccine studies involving CSP-specific antibodies and selected antigens for sero-epidemiological purposes. Measuring a multiplex antigen panel in a single reaction will help to assess not only vaccine immunogenic- ity but also potential malaria vaccine effects on naturally acquired immune responses. This will accelerate the iden- tification of immune correlates of protection, down-selection of vaccine formulations, antigen discovery and guide second-generation vaccine design. Keywords: Quantitative suspension array technology, Multiplex, IgG, Plasmodium falciparum, Assay conditions, Assay performance, Precision, Reproducibility *Correspondence: firstname.lastname@example.org Itziar Ubillos and Joseph J. Campo contributed equally to this work ISGlobal, Hospital Clínic–Universitat de Barcelona, 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) 17:216 Page 2 of 15 Precision and accuracy are common metrics for evalu- Background ation of new methodologies, as well as establishing the Antibody responses to Plasmodium falciparum parasites assay’s limits of detection and quantification. The pre - play a critical role in disease control . Finding reliable cision of an analytical procedure expresses the close- biomarkers associated with malaria protection or risk is ness of agreement (degree of scatter) between a series complicated by a parasite with a proteome of over 5000 of measurements obtained from multiple sampling of a proteins (http://www.plasm odb.org), many of them dif- homogeneous sample under prescribed conditions. The ferently expressed during distinct life-cycle stages and precision of an analytical procedure is usually expressed presenting genetic polymorphisms . Antibodies can as the variance, standard deviation (SD) or coefficient persist in blood for months after infection  and there- of variation (CV) of a series of measurements. Precision fore have been used as indicators of malaria transmission may be considered at three levels: repeatability, interme- [4–7]. In a low endemic setting, the use of direct para- diate precision and reproducibility. Repeatability refers sitological methods such as microscopy, rapid diagnostic to a precision estimate obtained from replicate meas- tests (RDTs) or quantitative polymerase chain reaction urements made in one laboratory by a single operator (qPCR) lack sensitivity because of low numbers of posi- using the same equipment over a short time scale. It is tive samples . Also, direct parasitological detection typically used to estimate the likely difference between methods only provide a snapshot of the present malaria replicate measurements obtained in a single batch of infection, whereas serological markers can better cap- analysis. Therefore, repeatability is also referred to as ture past (as well as present) malaria exposure and reflect intra-assay precision . Intermediate precision refers malaria transmission over a prolonged period of time . to a precision estimate obtained from replicate measure- A high-throughput assay for the measurement of multi- ments made in a single laboratory under more variable ple antigen-specific immunoglobulins (Ig) would be very conditions than repeatability conditions. Intermediate useful for studies of malaria naturally acquired or vac- precision is also known as ‘between-laboratory repro- cine-induced immunity, as well as other sero-epidemio- ducibility’ if tested in different laboratories or ‘between- logical biomarker studies. day reproducibility’ if tested in different days [14, 15]. Quantitative suspension array technology (qSAT) is a Reproducibility commonly refers to a precision estimate high-throughput immunoassay that has advanced bio- obtained from replicate measurements carried out with marker research compared to traditional methods, such different laboratories, operators and equipment. It is as ELISA, and has been employed for measurement also known as ‘inter-laboratory reproducibility’ . of DNA, cytokines and antibodies . When used in Accuracy refers to the closeness of agreement between a multiplex format, qSAT dramatically reduces labour measurement and the true value, and therefore includes input, reagents and sample volume requirements . the effect of both precision and bias [14, 16]. It encom- Quantitative measurements are as important in immu- passes both systematic and random error components. nological investigations as qualitative determinations of Accuracy of a method can be estimated by using certified seropositivity or seronegativity, where definitive thresh - reference materials by the new method, or by comparing olds may be identified. Inclusion of calibration curves the results of a new method with the results of a refer- using standard reference samples allows for quantitative ence method. or semi-quantitative estimates, either in physiological The high-throughput qSAT IgG assay described in concentration (e.g., μg/mL) or arbitrary units (e.g., AU/ this study could be used as a standard immune-assay mL). The problem with many standardized assays is the for malaria immunology and vaccine studies of multiple use of arbitrary measurements, such as ELISA units/mL antibody targets, and to characterize antibody reactivi- (EU/mL), which often make results incomparable with ties for sero-epidemiological purposes. Here, a qSAT IgG other studies or across laboratories. A standardized assay assay with a set of malaria antigens was developed and would be more useful if it had easily interpretable meas- the dynamic range and the limits of detection and quanti- urements that could be replicated in other laboratories fication assessed. The analysis of the assay for nonspecific or with different protocols. One challenge of multiplex binding and precision (reproducibility and repeatability) technology is the increasing difficulty of procuring spe - is reported. cies-specific reference standards that can be employed in a multiplex panel to produce calibration curves for all Methods targets in the panel. Another intrinsic problem of ELISA Study samples or qSAT is that human sera may contain up to 40% heter- Plasma samples used in the establishment of the qSAT ophilic antibodies that non-specifically bind to different assay were obtained from children and adult volunteers antigen-coupled beads, resulting in some levels of back- naturally exposed to malaria, participating in two studies ground [12, 13] that need to be evaluated. Ubillos et al. Malar J (2018) 17:216 Page 3 of 15 conducted at the Centro de Investigação em Saúde da xMAP technology (Luminex Corp., Austin, TX, USA) Manhiça (CISM), southern Mozambique. One set of and the Bio-Plex 100 platform (Bio-Rad, Hercules, CA, samples were from a clinical trial of intermittent pre- USA). MagPlex polystyrene 6.5 μm COOH-microspheres ventive treatment during pregnancy with sulfadoxine- (Luminex Corp, Austin, TX, USA) of different ID regions pyrimethamine (IPTp-SP) (Clinicaltrials.gov identifier were selected for each antigen, including one for bovine NCT00209781) conducted in 2003–2005 . A unique serum albumin (BSA). For the standard curve, micro- operator in 22 consecutive days and in triplicates assayed spheres were coupled to anti-human IgG F’ab antibody plasmas from this study by qSAT to assess precision of (Sigma-Aldrich, Madrid, Spain). For comparison of the the assay. Another set of samples were from a clinical anti-IgG standard curve with a curve generated from trial of different chemoprophylaxis schedules to selec - the anti-CSP Sp3C6 mAb, microspheres were coupled to tively control first exposure to P. falciparum in infancy anti-mouse IgG F’ab antibody (Jackson ImmunoResearch (Clinicaltrials.gov identifier NCT00231452) conducted Inc. PA, USA). Briefly, microspheres were washed, soni - in 2005–2009 . Two operators assayed samples from cated and activated with Sulfo-NHS (N-hydroxysulfosuc- this second study in 5 different days. A pool of hyperim - cinimide) and EDC (1-Ethyl-3-[3-dimethylaminopropyl] mune plasmas (HIP) from Mozambican adults life-long carbodiimide hydrochloride) (Pierce, Thermo Fisher exposed to malaria was used as a polyclonal positive Scientific Inc., Rockford, IL, USA). Microspheres were control and antigen-specific reference standard . washed and resuspended in cold Dulbecco’s PBS (dPBS) Negative controls from malaria-naïve volunteers were pH 7.2 (Invitrogen, Carlsbad, CA, USA) or 50 mM MES obtained from ISGlobal repository. The analysis of all the pH 5.0 (Sigma, Tres Cantos, Spain), depending on the samples was covered under protocols approved by the optimal buffering system for each individual antigen. The National Mozambican Ethics Review Committee and the recombinant proteins were added to the tubes in concen- Hospital Clínic of Barcelona Ethics Review Committee, trations ranging from 30 to 50 μg/mL and left at 4°C on and written informed consent was obtained from all par- a shaker overnight. Coupled microspheres were resus- ticipants or their parents/guardians before collection of pended in PBS with 1% BSA and 0.05% sodium azide specimens. (PBS-BN) to block. Microspheres recovery was quanti- fied on a Guava PCA desktop cytometer (Guava, Hay - Antigens and antibodies ward, CA, USA). Equal amounts of each antigen-coupled A combination of 11 antigens expressed during the pre- microspheres were combined in multiplex tubes and erythrocytic and erythrocytic stages of P. falciparum stored at 1000 microspheres/μL at 4°C, protected from life cycle was selected for the qSAT multiplex panel. The light. Anti-IgG-coupled microspheres were stored at apical membrane antigen (AMA)-1 of the 3D7 parasite 2000 microspheres/μL at 4°C, protected from light in sin- strain [20–22], the F-2 region of the erythrocyte bind- gleplex. Bead blocking agent BSA in the coupling buffers ing antigen (EBA)-175 [22, 23], the Duffy binding-like to covalently ‘block’ the free carboxylic group (-COOH) (DBL)3x domains and DBL-α of the erythrocyte mem- from the microspheres was included, absorbing most of brane protein (PfEMP)-1 were produced at ICGEB the non-specific binding to secondary or tertiary anti - (Delhi, India) [24, 25]. The AMA-1 and the 42 kDa frag - bodies during assay steps  and heterophilic antibody ment of the merozoite surface protein 1 (MSP-1 ) from binding seen in previous systems . Also a BSA-coated the FVO strain were provided by WRAIR (Walter Reed microsphere in the multiplex panel was included, to Army Institute of Research, MD, USA) [26–28]. The determine non-specific ‘bead binders’ of serum IgG to liver stage antigen (LSA)-1 [29, 30], the sporozoite sur- the BSA. face protein 2 (SSP2 or TRAP) , the circumsporozoite protein (CSP)  and the cell traversal-ookinete sur- qSAT assay face antigen (CelTOS)  were purchased from Protein Antigen-coupled microspheres were added to a 96-well Potential, LLC (Rockville, MD, USA). μClear flat bottom plate (Greiner Bio-One, Fricken - Sp3C6 monoclonal antibody (mAb) produced in mice hausen, Germany) in multiplex (1000 microspheres per was gifted to this study by the Pluschke lab at the Swiss analyte per well) in a volume of 50 μL of Luminex Buffer TPH Institute (Basel, Switzerland). Sp3C6 mAb gives (PBS-BN). Anti-IgG-coupled microspheres were added specific responses to CSP of the 3D7 P. falciparum strain to the plate in singleplex (2000 microspheres per analyte . per well) in 50 μL of Luminex Buffer. 50 µL of test plasma samples diluted 1:250 and 1:10,000 in Luminex Buffer were Microsphere coupling added to the plates in duplicates (final dilutions of 1:500 A qSAT multiplex panel was constructed to quantify and 1:20,000, respectively). The HIP pool was used as a IgG responses to P. falciparum antigens using Luminex positive control and included on each assay plate diluted Ubillos et al. Malar J (2018) 17:216 Page 4 of 15 1:150,000. Technical blanks consisting of Luminex Buffer regression model . If the 5-PL regression model did and microspheres without samples were added in dupli- not converge, then, a 4-PL method without asymmetry cate wells to detect and adjust for non-specific microsphere factor G was fitted instead. To obtain AU/mL, corre - signal. Plates were incubated for 1 h at room temperature sponding MFI values were adjusted by their correspond- in agitation and protected from light. Then, washed three ing blank values before curve fitting. times with 100 μL PBS-T (0.05% Tween 20 in PBS) on a An r cut-off value of 0.994 was used for each standard Bio-Plex Pro wash station with magnetic platform (Bio- curve as acceptability criteria. Additionally, blank values Rad, Hercules, CA, USA). 100 μL of biotinylated anti- of all antigen-coated microspheres had to be below 200 human IgG (Sigma-Aldrich, Tres Cantos, Spain) diluted MFI, and for the anti-IgG-coated microspheres had to be 1:2500 in Luminex buffer was applied to all wells and below 300 MFI. The parameters of the anti-IgG standard incubated for 45 min as before. For the assay of anti-CSP curve were used in a Microsoft Excel template to cal- Sp3C6 mAb and mouse IgG standard curves, biotinylated culate antigen-specific AU/mL, respectively. Using the anti-mouse IgG (Sigma-Aldrich, Madrid, Spain) was used. derived parameters of the standard curve, the estimates After washing plates, 100 μL of streptavidin-conjugated of concentration were multiplied by corresponding dilu- R-phycoerythrin (Invitrogen, Carlsbad, CA, USA) diluted tion factors to calculate antigen-specific AU/mL. 1:1000 (1 μg/mL) in Luminex Buffer was applied to all wells and incubated for 25 min as before. Plates were washed and Determinations of limit of blank (LOB) and limit microspheres resuspended with 100 μL of Luminex Buffer, of quantification (LOQ) and covered with an adhesive film and stored at 4°C over - LOBs were estimated by measuring replicates of a techni- night to be read the next morning. Data were acquired on cal blank (well without sample) and calculating the lo g a Bio-Plex 100 reader using Bio-Plex Manager version 4.0 MFI mean and the SD (LOB = mean blank + 1.645 × (SD (Bio-Rad, Hercules, CA, USA). At least 50 microspheres blank)). To calculate concentration in AU/mL for each per analyte were acquired, and median fluorescence inten - sample and antigen tested, we first adjusted each MFI sity (MFI) was reported for each analyte. value by its corresponding blank values. Then, the param - eters from a non-linear 5-PL regression model obtained Standard curve from the singleplex IgG standard curve were fitted in the A heterologous reference standard [35–37] for estimat- inverse 5-PL equation for each sample and antigen. We ing concentration of antibodies in plasma was constructed established a ‘good range’ of quantification where percent using microspheres coupled to anti-human IgG F’ab region change in AU/mL does not exceed 5% for a 1% change and dilution series of IgG purified from human serum in MFI, and these were considered the assay LOQ. Only (Sigma-Aldrich, Tres Cantos, Spain). The commercially AU/mL measurements were adjusted by their corre- available purified human IgG was incubated with the anti- sponding blanks values. human IgG F’ab microspheres in a 10-step dilution series (twofold) starting at 250 ng/mL and producing an 11-point Assessment of precision curve. For comparison of a heterologous standard with a For the determination of repeatability and intermediate homologous standard, a curve was generated with purified precision, antibody levels (in log MFI or AU/mL) were mouse IgG (ThermoFisher, Spain) and microspheres cou - measured against BSA, AMA-1 3D7, AMA-1 FVO, MSP- pled with anti-mouse IgG F’ab region (Jackson ImmunoRe- 1 3D7, MSP-1 3D7, EBA-175, LSA-1 and CSP in 10 42 19 search Inc. PA, USA) plus biotinylated anti-mouse IgG malaria-exposed and 9 non-exposed individuals span- (Sigma-Aldrich, Madrid, Spain). This curve was contrasted ning a large range of immunogenicities. Samples were with a curve generated with a dilution series of anti-CSP measured in triplicates on 22 different days, equivalent to Sp3C6 mAb with CSP-coupled microspheres in singleplex. 1254 measurements for the 8 antigens included. Repeat- The 5-PL regression was the selected method for fitting ability between replicates for each antigen and day was curves due to its superior fit to antibody data: assessed by the Intra-class Correlation Coefficient (ICC) SD , one way ANOVA  and CV ( × 100 ) [3, 15, Mean 41]. Bland–Altman plots were also used to assess ‘within- y = A + day reproducibility’ . 1 + To assess reproducibility, two operators performed the assay on 5 different days in the same laboratory and where A is the lower asymptote (Emin), B is the slope using the same apparatus. Operator and day effects at the inflection point (Hill), C is the concentration at were assessed, but inter-laboratory variation could not the inflection point (EC50), D is the upper asymptote be assessed. IgG (in lo g MFI levels and AU/mL) to (Emax), and G is a factor of asymmetry added in the 5-PL 11 P. falciparum antigens (AMA-1 3D7, AMA-1 FVO, Ubillos et al. Malar J (2018) 17:216 Page 5 of 15 Assessment of non‑specific background reactivity MSP-1 3D7, MSP-1 FVO, EBA-175, CelTOS, LSA-1, 42 42 To assess non-specific background reactivity, MFI levels SSP2, DBL-α, DBL3x and CSP) and BSA were measured to BSA-coupled beads were measured in 288 samples and in 282 samples from malaria-exposed individuals, and 52 blanks. Signal to blanks had values below 150 MFI for malaria-naïve individuals. Positive controls and blanks both operators (Fig. 2a). The overall mean of non-specific were included in all plates, and all samples were meas- binding to BSA in samples was 32.83 ± 44.81 MFI, with ured in duplicates. a range 0–654.5 MFI, and was different depending on the operator (Fig. 2b). In 2 of 288 samples for operator 2 (0.69%) and one for operator 1 (0.34%), the non-specific Assessment of accuracy binding to BSA was above 250 MFI, one sample being the Since samples in this study had unknown antibody con- same in both operators. centrations, standard curves with known concentra- tions of total IgG measured on 22 different days were used to assess accuracy and the observed and expected concentrations for each day of analysis compared. Also, Assay precision as performance of total IgG measurement might not be Precision was assessed for two measurements: antibody representative of the performance of antigen-specific level estimates from heterologous interpolation of an IgG measurements, parameters from the curve fitting of anti-human IgG standard curve (AU/mL) and lo g of standard curves with mouse IgG were compared with MFI (henceforth, “MFI”). The repeatability of the assay, anti-CSP mouse mAb Sp3C6 IgG standard curves. measured using the ICC between replicates for all anti- gens and days of analysis, gave a value of 1 for samples from malaria-exposed individuals (n = 1540 obser va- tions) and 0.99 for the non-exposed donors (n = 1386 Statistical methods observations). No evidence of significantly different vari - All MFI or AU/mL measurements were lo g -transformed ance across replicates was seen, therefore homogene- for statistical analysis. Means and 95% confidence inter - ity of variance in triplicate measurements was assumed vals (CI) were calculated for repeated measures. T-tests (p-value = 0.73) (Fig. 3a). Repeatability or intra-assay were used to assess differences between means. One-way variability was also assessed by analysing the CV of ANOVA and Levene’s test  were used to assess dif- MFI for triplicate measures [3, 16] with an overall CV ferences between replicates. Agreement was assessed by of 7.66% ± 15.89 between triplicates for all antigens and performing Bland–Altman plots [40, 43], and reliabil- samples. CV was lower in samples from malaria-exposed ity by the ICC from psych R package [39, 44]. SD and (2.81% ± 7.85 n = 10) than malaria-naïve (13.07% ± 20.25, CVs were calculated for precision measurements. A p n = 9) individuals (p-value < 0.001), suggesting higher var- value < 0.05 was considered statistically significant. Data iability between replicates of low immunoreactive sam- were analysed using R software version 3.4.1. ples. Intermediate precision or inter-assay variability was assessed by the ICC between the 22 days of analysis: 0.98 (0.97–0.98) for MFI and 0.9 (0.87–0.93) for AU/mL. Simi- Results larly, no evidence of significantly different variance across Limits of blanks and limits of quantification days in MFI (p-value = 0.613) or AU/mL (p-value = 0.446) The LOB ranged from 95.18 MFI for the DBL-α to 150.33 measurements was shown, assuming homogeneity of for MSP-1 FVO. LOB for the other antigens were: variance between days of analysis. Overall, between- 98.43 MFI for AMA-1 3D7, 103.31 for MSP-1 3D7, day or inter-assay CV was 9.24% ± 8.98 for MFI data 103.68 for LSA-1, 106.05 for DBL3x, 107.46 for CSP, [3, 16] and was lower in samples from malaria-exposed 121.42 for CelTOS, 122.45 for AMA-1 FVO, 124.78 for (5.25% ± 6.16, n = 10) than non-exposed (14.16% ± 9.44, SSP2, 148.44 for EBA-175 and 127.62 for BSA. The lower n = 9) individuals (p-value < 0.001) (Fig. 3b). Simi- LOQ (LLOQ), based on an anti-IgG standard curve, was larly, between-day CV in AU/mL was lower in malaria- estimated at 237.88 ± 70.86 MFI, and the upper LOQ exposed individuals (31.28% ± 25.56) than malaria-naïve (ULOQ) at 23,355.48 ± 489.99 MFI (Fig. 1a). The analyti - individuals (38.2% ± 14.78) (p-value < 0.001). To assess cal range was found to be 0.007–9.95 AU/mL, resulting if variability depended on the antigen immunogenic- in 2.61–233,979.78 AU/mL after correcting for the sam- ity, mean CV was measured between replicates and days ple dilution factor (1:500 and 1:20,000) (Fig. 1b). The for each antigen (Table 1). BSA-coated microspheres LLOQ (237.88 ± 70.86 MFI) was above the LOB, and used to control for non-specific binding gave the high - measurements below the LLOQ were considered to not est between-day variability. Between-day variability in be quantifiable. AU/mL increased compared to that obtained with MFI, Ubillos et al. Malar J (2018) 17:216 Page 6 of 15 Fig. 1 Serial dilutions of human IgG fitted into a non-linear 4-parameter logistic (4-PL) regression model. Median fluorescence intensity (MFI) values versus log IgG concentration (ng/mL). Blue line represents Loess fitted line for all days of analysis. Error bars represent the standard deviation of the mean between days of analysis. a Standard curves performed in 5 different days. Solid horizontal lines represent the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ). b Standard curves performed on 22 different days of analysis suggesting that the curve fitting did not correct the and the inter-assay variability for each operator dif- day-to-day variability. Correlations (r ) between days of fered depending on the dilution factor, with higher var- analysis in MFI and AU/mL, all antigens together, were iability at lower concentrations (Fig. 4). For correlation greater than 0.9 (p < 0.001) (Additional file 1). The differ - between operators for all samples and antigens (Fig. 5), ence in the daily MFI mean of triplicates and mean of all all antigens together had an overall adjusted r of 0.929 days was plotted against the daily mean of triplicates for for MFI and r of 0.836 for AU/mL (log scale). Corre- each antigen and for test samples and negatives controls lation of AU/mL between operators tended to increase (Fig. 3c), which showed that variation was higher at lower with higher MFI, such as AMA-1, MSP-1 and EBA- MFI values. 175 (Fig. 5a, b). Compared to MFI, the measurements in AU/mL had very different ranges (Fig. 5b, c), with Assay reproducibility higher estimates of AU/mL by operator 1 for the anti- To assess the operator effect, the sample size was gens with higher MFI; this was due to the different expanded to 288 samples against a panel of 11 P. fal- estimates of the parameters from the 5-PL or 4-PL ciparum antigens. The inter-assay CV of the standard regression model by operator. The between-day opera- curves differed between operators (p-value = 0.023), tor CV for blank and positive controls based on MFI, Ubillos et al. Malar J (2018) 17:216 Page 7 of 15 a Blank non−specific binding to BSA bead operator operator1 operator2 1 23456789 10 11 12 13 14 15 16 Blanks on BSA (n=16 plates) b Human samples non−specific binding to BSA bead operator operator1 operator2 0100 200 300 Sample id (n=288) Fig. 2 Non-specific binding in blanks and plasma samples to bovine serum albumin (BSA)-coated microspheres by operator. a MFI values to BSA-coated microspheres in blanks from 16 plates assayed by 2 operators; b MFI values to BSA-coated microspheres in 288 plasma samples, from a chemoprophylaxis clinical trial in Mozambique, assayed by 2 operators. The horizontal red line corresponds to 250 MFI. Red columns represent operator 1 and green columns operator 2 plate-to-plate signals and trend lines for each operator Discussion were not aligned for blanks (p < 0.001), although posi- A high-throughput immunoassay such as the qSAT tive controls were aligned (Fig. 6). described herein is highly useful for biomarker research using custom antigen panels for complex pathogens, such as P. falciparum. It can be used as a standard immune- Assay accuracy assay for malaria vaccine studies involving multiple Measurements of the observed versus the expected antibody specificities, and to select antigens for sero-epi - concentrations from the IgG standard curves assayed demiological purposes. The qSAT assay developed here over 22 different days had a minimum r value of 0.854 demonstrated low non-specific signal, good estimates on day 22 and all remaining days with r > 0.95 (Fig . 7). of precision and correlation between operators, albeit When a standard curve based on mouse IgG was com- with operator-dependent differences in range of AU/mL pared to a curve based on a monoclonal mouse anti- estimates. CSP IgG, the curves showed very different estimates For calibration purposes, the use of a standard curve of the 5-PL or 4-PL regression model parameters fitting an inverse 4-PL or 5-PL regression model to (Table 2), particularly EC50 and minimum estimates. estimate concentration is a valid method to normal- Measurements and concentrations of monoclonal anti- ize MFI and provide quantifiable antibody measures CSP IgG and purified mouse IgG were not comparable , accounting for day-to-day variability. Typically, (Fig. 8). low multiplexity assays would utilize a reference sam- ple or pool of mAb to produce standard curves for MFI levels MFI levels Ubillos et al. Malar J (2018) 17:216 Page 8 of 15 a Repeatability between replicates (intra−assay) b Coefficient of Variation between replicates AMA−1 3D7 AMA−1 FVO BSA 4 30 CSP EBA−175 LSA−1 sample.cat Malaria exposed 30 2 20 Malaria non−exposed Malaria exposed Malaria non−exposed MSP−1 19 MSP−1 42 r1 r2 r3 Malaria exposed Malaria non−exposed Malaria exposed Malaria non−exposed Repeats Sample Type c Intermediate precision between days (inter−assay) d Coefficient of Variation between days AMA−1 3D7 AMA−1 FVO BSA 4 30 CSP EBA−175 LSA−1 sample.cat Malaria exposed Malaria non−exposed Malaria exposed Malaria non−exposed MSP−1 19 MSP−1 42 123456789 10 11 12 13 14 15 16 17 18 19 20 21 22 Malaria exposed Malaria non−exposed Malaria exposed Malaria non−exposed Days of analysis Sample Type e Between day reproducibility AMA−1 3D7 AMA−1 FVO BSA −1 −2 CSP EBA−175 LSA−1 sample.cat 0 Malaria exposed −1 Malaria non−exposed −2 1 234 MSP−1 19 MSP−1 42 −1 −2 123 4 1234 mean log10(MFI) day Fig. 3 Repeatability and intermediate precision of sample measurements. IgG in samples from malaria-naïve (non-exposed) and malaria-exposed individuals from an IPTp clinical trial in Mozambique, were measured in triplicates on 22 different days against 7 P. falciparum antigens. a Repeatability represented by boxplots of log MFI distributions for all antigens together at each repetition (r1, r2, r3); b boxplots of coefficient of variation between replicates by antigen and sample type. c Intermediate precision represented by boxplots of log MFI distribution for all antigens together at each day of analysis, and d boxplots of coefficient of variation between days of analysis by antigen and sample type. Boxplot horizontal lines represent median and interquartile range. e Bland–Altman plots showing the difference of each day versus the mean of all days of analysis for each antigen and both sample types Table 1 Mean coefficient of variation (CV) and standard deviation (SD) Analyte CV between repeats (%) CV between days (%) CV between days (%) log MFI (3 repeats) log MFI (n = 22) AU/mL (n = 22) 10 10 Intra‑assay Inter‑assay Inter‑assay CV SD CV SD CV SD AMA-1 3D7 2.31 ± 4.47 4.35 ± 4.07 25.79 ± 12.16 AMA-1 FVO 4.96 ± 16.59 7.57 ± 9.76 28.65 ± 14.7 EBA-175 1.47 ± 2.79 4.59 ± 3.16 35.25 ± 33.16 CSP 2.29 ± 7.92 7.97 ± 9.61 27.73 ± 11.46 LSA-1 11.05 ± 18.43 13.21 ± 8.12 43.27 ± 17.81 Control BSA 22.54 ± 23.74 21.38 ± 7.08 64.12 ± 31.18 Overall 7.66 ± 15.89 9.24 ± 8.98 31.28 ± 22.74 Between triplicates and days of analysis in log MFI or AU/mL for each antigen of the multiplex panel Difference day(i)− mean days log10(MFI) log10(MFI) %Coeffcient of Variation (inter−assay) %Coeffcient of Variation (intra−assay) Ubillos et al. Malar J (2018) 17:216 Page 9 of 15 a IgG Standard Curve Operator1 Operator2 day −3 −2 −1 01 2−3−2−10 12 log10(ng/mL) b Variation by day on Standard Curve Operator Operator1 Operator2 0.001 0.004 0.013 0.038 0.114 0.343 1.029 3.086 9.259 27.778 83.333 250 ng/mL Fig. 4 Reproducibility between days of the IgG standard curve by operator. a MFI versus log ng/mL by operator. Blue line represents Loess fit, and the error bars represent the standard deviation of the mean between days of analysis. b Between-day coefficient of variation at each dilution point and by operator each analyte, known as ‘homologous interpolation’ the secondary antibody and streptavidin-conjugated . Higher levels of multiplexing require standards detection molecule (R-PE) are the same for all beads. that react strongly to all antigens in the panel or larger Thus, the read-out of anti-hIgG and malaria antigen pools of mAb, both resources that are likely limited beads are the same: fluorescence emitted from IgG and not widely available. The hypothesis of the current bound (or captured) to the bead. Indeed, this approach, system tested was that a single curve generated from known as ‘heterologous interpolation’ has been used anti-human IgG coated beads and commercially avail- previously in development of ELISA and similar assays able purified IgG could close the gap for increasingly [19–21] and is useful when standard reference reagents complex multiplex assays. The validity of this approach, for homologous interpolation are unavailable. The despite the differences in binding mechanism between possibility exists that systematic effects may alter the IgG capture and antigen-specific IgG binding, is that MFI Coefficient of Variation Ubillos et al. Malar J (2018) 17:216 Page 10 of 15 a log10(MFI) between operators c AU/mL between operator AMA−1 3D7 AMA−1 FVO BSA CelTOS 0123 40 1234 0123 40 1234 log10(operator1) Operator b log10(AU/mL) between operators 2.5 0.0 −2.5 −5.0 2.5 0.0 −2.5 −5.0 2.5 0.0 −2.5 −5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 log10(operator1) Fig. 5 Correlation of samples measurements between operators. Plasma samples (n = 288) from a chemoprophylaxis clinical trial in Mozambique, with a correlation of log MFI data; b correlation of log AU/mL data; and c AU/mL at each dilution and antigen by operator. r is Pearson’s 10 10 correlation coefficient relationship of IgG captured to beads by the Fc region should be identical . Unfortunately, these conditions with that of IgG binding specifically to antigen. were not met for the curves generated with anti-mouse For most antigens, between-day variability differed IgG and specific mouse anti-CSP mAb. Also, polyclonal from estimated concentration in AU/mL and fluores - biological samples like plasma or serum contain antibod- cence-based crude MFI measurements. This variability ies with a range of affinities, suggesting that calibration was attributed to operator-dependent differences in the curves from either antigen-specific mAbs (homologous) slopes and curve parameters, resulting in large deviations or purified human IgG (heterologous) may provide a bet - in AU/mL estimates. Furthermore, the comparison of ter relative estimate of antibody concentration (AU/mL) the anti-IgG curve with a curve generated with a specific than physiological concentration (ng/mL). The stand - mAb against the CSP protein (Fig. 8) showed differing ard curve with quantifiable human IgG was expected to slopes and limits of the curves. These differences are pos - reflect levels of IgG antibodies in serum/plasma and to sibly due to higher affinity of the mAb to the target anti - be a widely applicable resource for estimating concentra- gen compared to the affinity of anti-IgG-coated beads. To tion. However, the poor reproducibility of AU/mL meas- estimate relative potency, both curves would need to have urements using a single IgG standard curve suggests that a common slope and the maximum achievable response a pool of known antibody concentrations may perform operator operator operator operator operator1operator operator operator 1 2 1 2 1 2 log10(operator2) log10(operator2) AU/mL Ubillos et al. Malar J (2018) 17:216 Page 11 of 15 a Blanks in 16 plates b Coeff.Variation Blanks between day by operator AMA−1 3D7 AMA−1 FVOBSA CelTOS 2.1 1.9 1.7 1.5 CSPDBL3x DBLalpha EBA−175 Operator Operator 2.1 Operator1 1.9 Operator1 Operator2 1.7 Operator2 1.5 LSA−1 MSP−1 42 3D7 MSP−1 42 FVOSSP2 2.1 1.9 1.7 1.5 123456789 10 11 12 13 14 15 16 123456789 10 11 12 13 14 15 16 123456789 10 11 12 13 14 15 16 123456789 10 11 12 13 14 15 16 Operator Plate c Positive Controls in 16 plates d Coeff.Variation Positive control between day by operator AMA−1 3D7 AMA−1 FVOBSA CelTOS CSPDBL3x DBLalpha EBA−175 Operator Operator Operator1 Operator1 Operator2 Operator2 LSA−1 MSP−1 42 3D7 MSP−1 42 FVOSSP2 48 12 16 48 12 16 48 12 16 48 12 16 Operator Plate Fig. 6 Reproducibility of blanks and positive controls. a log MFI levels of blanks assayed in 16 plates, and b boxplots of coefficient of variation of blanks in each operator between days. c log MFI levels of positive controls assayed in 16 plates, and d boxplots of coefficient of variation of positive controls in each operator between days. Boxplot horizontal lines represent the median and interquartile range better in measuring antibodies against P. falciparum. In studies have assessed non-specific binding of IgG directly the absence of a homologous reference standard, a stand- to the microsphere surface by using unconjugated blank ardized assay with heterologous interpolation may be beads . While useful in identifying ‘bead binding’ used. activity, the approach does not adequately quantify the The multiplex assays were performed with 1000 contribution of bead-binding signal to the overall back- microspheres/analyte/well and the singleplex stand- ground signal of the current assay using antigen-coupled/ ard curve with 2000 microspheres/analyte/well. Other BSA-blocked microspheres. BSA-coated microspheres studies found no differences between both bead con - better account for the signal attributable to non-specific centrations , although they did not measure differ - IgG binding to (1) the microsphere surface, (2) free – ences when assays were performed in singleplex at 1000 COOH and (3) BSA. An added benefit is identifying microspheres/analyte/well. Here, a higher concentration outliers that represent IgG binding through specific rec - of beads in singleplex assays was included for improved ognition of BSA as an antigen (e.g., through various types acquisition of the minimum number of events. of bovine exposure), allowing for sample censorship or Findings from other laboratories indicate that some adjustment of signals. Maintaining aseptic technique serum/plasma samples contain antibodies that appear is critical throughout the procedure, and all batches of to bind directly to the carboxylated surface of the micro- coupling reactions were tested with ‘blanks’ before com- spheres even in the absence of coupled antigen [12, 13]. bining into a single batch for a series of experiments. Here, an overall low level of non-specific binding of the Therefore, when preparing the assay, coupling reactions samples assayed was observed by measuring response to of P. falciparum antigens with blanks above 250 MFI BSA protein, with MFI generally below 150 when using should be excluded from the multiplex panel. blanks and with only 2 of 288 (0.69%) samples with crude Repeatability has traditionally been assessed by esti- MFI above 250 to BSA-coupled microspheres. Other mating the CV or relative SD [3, 16, 47, 48]. Similar to op1 op2 op1 op2 log10(MFI) log10(MFI) %Coefficient of Variation %Coefficient of Variation Ubillos et al. Malar J (2018) 17:216 Page 12 of 15 a b Observed vs Expected concentration by day of analysis MFI Standard Curve 1 2 3 4 5 2 2 2 2 2 r = 0.954 r = 0.971 r = 0.969 r = 0.974 r = 0.947 6 7 8 9 10 2 2 2 2 r = 0.975 r = 0.976 r = 0.963 r = 0.978 r = 0.976 11 12 13 14 15 2 2 2 2 r = 0.98 r = 0.956 r = 0.974 r = 0.967 r = 0.972 16 17 18 19 20 2 2 2 r = 0.982 2 r = 0.97 r = 0.972 r = 0.972 r = 0.956 −2 −1 01 2 −2 −1 01 2 −2 −1 01 2 21 22 2 2 r = 0.972 r = 0.854 1 −2 −1 01 2 −2 −1 01 2 −2 −1 01 2 Expected log10(ng/mL) IgG concentration (ng/mL) Fig. 7 Accuracy of standard curve with human IgG assayed in 22 plates. a Observed versus expected concentrations after inverse non-linear 4-parameter logistic (4-PL) regression model fitting and r from Pearson’s correlation coefficient b log MFI to IgG concentration (ng/mL) median and error bar at each dilution point in the 22 plates other studies, higher variability at lower MFI values The qSAT assay gave highly reproducible MFI meas -  and comparable values of inter-assay and intra- urements, but the variation in the performance of the assay variation [3, 41] were found, indicating accept- standard curves between operators may limit the use of able precision of the assay. However, those measures AU/mL to a normalization method for single-operator do not take into account the multi-dimensionality of a studies until future assay development improves the multiplexed assay where antibodies against more than reproducibility of IgG standard curves between opera- one antigen are measured at a time. ICC and ANOVA tors and, presumably, laboratories. This suggests that a analyses can account for this dimensionality. reference pool of known specific antibody concentra - tions may perform better across studies in measuring antibodies against P. falciparum . The challenge remains in sourcing adequate serum/plasma/mAb pools that cover all antigens as panels become larger and more Table 2 Parameters from the non-linear 4-parameter complex. logistic (4-PL) regression function for singleplex curves Analyte Parameters Coefficient Standard 95% CI error Conclusion IgG Emax 30,929.13 286.52 30,280.97 31,577.28 The qSAT assay developed here demonstrated low non- Emin 423.37 420.67 − 528.26 1375.00 specific signal, gave reproducible MFI measurements EC50 3.53 0.15 3.18 3.88 and good estimates of precision and correlation between Hill 1.41 0.08 1.23 1.60 operators. The use of a singleplex standard curve to mCSP Emax 32,000.25 3650.55 23,742.15 40,258.36 measure antibody concentration through a non-linear Emin 4000.20 1547.16 500.27 7500.13 parameter function added more variability to the assay. EC50 20.44 7.90 2.58 38.30 The assay with heterologous IgG standard may be used Hill 0.87 0.27 0.26 1.48 when homologous antigen-specific standards are unavail - Anti-mouse IgG-coupled beads incubated with purified mouse IgG and CSP- able, but should be further standardized for broad appli- coupled beads incubated with mouse anti-CSP monoclonal antibody (mCSP) at cation. RTS,S and sporozoite malaria vaccine immune known IgG concentrations studies could benefit from the use of a multiplex antigen CI confidence interval Observed log10(ng/ml) log10(MFI) Ubillos et al. Malar J (2018) 17:216 Page 13 of 15 anti−mouse IgG and CSP Standard Curves analyte IgG mCSP −1 012 log10 Concentration (ng/mL) Fig. 8 Standard curve with mouse IgG (red) and monoclonal anti-CSP IgG (blue). Lines represent Loess fitting and grey area the 95% confidence interval Author details panel to measure not only vaccine-specific but also nat - ISGlobal, Hospital Clínic–Universitat de Barcelona, Barcelona, Catalonia, Spain. urally-acquired immune responses and accelerate the 2 3 Antigen Discovery, Inc, Irvine, CA, USA. CIBER Epidemiología y Salud Pública identification of immune correlates of protection. (CIBERESP), Barcelona, Spain. Acknowledgements Additional file We thank volunteers from whom samples were derived, and the clinical and laboratory teams conducting the trials, particularly Azucena Bardají, Clara Additional file 1. Correlation matrix of MFI or AU/mL between days of Menéndez, Caterina Guinovart, Augusto Nhabomba, and Pedro Alonso. We analysis. All samples and controls are included. Upper panel report cor- are grateful to Evelina Angov’s and Sheetij Dutta’s Labs ( Walter Reed Army relation coefficient (r) and p-value (p) and between days of analysis. Lower Institute of Research, USA) and Chetan Chitnis’s Lab (International Centre for panels plot variables against each other. Genetic Engineering and Biotechnology, India) for protein supply, and Claudia Daubenberger and Gerd Pluschke’s Labs (SwissTPH) for anti-CSP mAb supply. We thank Ruth Aguilar for critical review of the manuscript. Authors’ contributions Competing interests Conceived the study: IU, JJC, CD. Carried out experimental design and The authors declare that have no competing interests. Luminex experiments: AJ and JJC. Performed database management and sta- tistical analysis: IU. Supervised the study: CD. Wrote the first manuscript draft: Availability of data and materials IU, JJC. All authors read and approved the final manuscript. The data can be made available upon request from the corresponding author. MFI Ubillos et al. Malar J (2018) 17:216 Page 14 of 15 Consent for publication 15. 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