A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex technology (Bio-Plex 200 or MAGPIX).

Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.

Introduction

Over the past 5–10 years there has been a rapid advancement of Luminex bead-based technologies to measure antibody responses to multiple proteins simultaneously. These assays have numerous advantages over traditional enzyme-linked immuosorbent assays (ELISA), such as a reduction in sample volume required and reduced laboratory time if choosing multiple targets to assay for, as well as the main advantage of allowing multiplexed detection of antibody responses. This is particularly relevant for the detection of antibodies against complex pathogens that express many hundreds to thousands of proteins, such as the Plasmodium parasites (the causative agent of malaria). Access to standardized control reagents [1] will also allow results from these assays to be reliably compared between different laboratories, which may result in more consistent findings among different studies [2].

Multiplexed bead-based assays use Luminex xMAP® technology [3] (https://www.luminexcorp.com/xmap-technology/), which centers on use of beads (microspheres) with different fluorescent colours that can be detected in unique regions on a compatible instrument such as a Luminex® 200™ (also known as a Bio-Plex® 200, sold by Bio-Rad), MAGPIX® or FLEXMAP 3D (https://www.luminexcorp.com/xmap-instruments/). The beads are internally labeled with different ratios of two fluorophores, one in a red wavelength and the other infrared. The compatible instruments and related software have pre-gated channels that detect the internal fluorophores in discrete regions. Proteins of interest can be coupled to a unique set of beads, facilitating multiplexed detection of antibody responses to multiple proteins. Coupling is the process of attaching a specific protein to the bead, through carboxyl groups on the bead surface (covalent bonding). Several studies have been conducted with a focus on optimizing various steps of the coupling process or assay work-flow, in the context of detection of antibodies against Plasmodium proteins, such as bead coupling [4], sample pre-dilution [4], assay temperature [4], plate washing [4], operator expertise [4], incubation times [1], and bead numbers [5]. Two different types of bead compositions are available for coupling proteins: non-magnetic and magnetic. Non-magnetic beads can only be run on flow-based instruments such as the Luminex® 200™/Bio-Plex® 200 or FLEXMAP 3D®, whilst magnetic beads can be run on both flow-based instruments and the MAGPIX®. The MAGPIX® is based on CCD imaging technology, and offers some advantages over the flow-based systems such as reduced use of reagents such as sheath fluid and the reduced cost of the MAGPIX® instrument compared to the Luminex® 200™/Bio-Plex® 200 instruments.

The primary aim of this study was to perform a series of comparisons of both non-magnetic and magnetic beads and assaying those beads on the Bio-Plex® 200 or the MAGPIX®. A secondary aim was to determine whether this assay is reproducible in an independent laboratory through an external comparison. This study used a panel of 19 different P. vivax proteins and plasma samples from P. vivax-endemic areas to detect P. vivax-specific IgG responses.

Materials and methods

Plasma samples

For all assays described here, a pool of samples from individuals from Papua New Guinea (PNG) with high levels of anti-Plasmodium antibodies was used as a positive control for the standard curve dilution to adjust for plate to plate variation, as previously described [6]. The standard curve was run on every plate.

Two sets of plasma samples from malaria-endemic areas were used for comparisons of non-magnetic and magnetic beads, and the different acquisition instruments. These were 80 individuals from a longitudinal observational cohort study in Thailand, conducted in the Kanchanaburi and Ratchaburi provinces in 2013–2014. This cohort has previously been described in detail [7, 8], and the 80 plasma samples used were collected at the last visit of the cohort. The second set of samples came from a longitudinal observational cohort study in the Solomon Islands, conducted on the island Ngella in 2013–2014. This cohort has previously been described in detail [7, 9], and 83 plasma samples were used from individuals at the last visit of this cohort.

An additional set of plasma samples from a cohort study in PNG was used for external comparison of the assay. Samples were selected from the Mugil II paediatric cohort study. The study enrolled 450 children aged 5–12 years old in 2012 from the Mugil area on the North Coast of Madang province. All children were given antimalarial drugs to eliminate blood-stage Plasmodium spp. and blood samples were collected for parasitological and immunological studies. For the external comparison, a set of 425 samples was used from the baseline timepoint (collected 2 weeks after drug treatment).

Ethics statement

All samples were collected after approval from local ethics committees, with volunteers/participants providing written informed consent and/or assent (and parents or guardians providing informed consent for children). The Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand approved the Thai cohort study (MUTM 2013-027-01). The National Health Research and Ethics Committee of the Solomon Islands Ministry of Health and Medical Services (HRC12/022) approved the Solomon Islands study. The Mugil II paediatric cohort was approved by the PNG Institute of Medical Research Institutional Review Board (IMR IRB) (1116/1204), the PNG Medical Research Advisory Committee (MRAC) (11.21/1206), the Walter and Eliza Hall Institute Human Research Ethics Committee (WEHI HREC) (12/09), and the Case Western Reserve University Hospitals of Cleveland Medical Center (CWRU UHCMC) (05-11-11). The HREC at WEHI approved samples for use in Melbourne (#14/02).

Coupling P. vivax proteins to non-magnetic and magnetic beads

The carboxylated beads were sourced from Bio-Rad (Bio-Plex COOH Beads, 1ml, 1.25x107 beads/ml and Bio-Plex Pro Magnetic COOH Beads, 1ml, 1.25x107 bead/ml) and stored at 2–4°C. Optimisation of coupling procedures for non-magnetic and magnetic beads were done separately, due to the larger size of the magnetic beads generally requiring more protein (see Results). To be able to measure all plasma samples at the same dilution, we optimized all protein amounts by generating a log-linear standard curve with a positive control plasma pool from immune PNG donors (high responders to Plasmodium antigens). The positive control pool was used to generate a standard curve running from a 1/50 dilution to a 1/25,600 dilution (10 point standard curve, 2-fold serial dilution). One set amount of protein was selected that resulted in a log-linear standard curve over this dilution series; the amounts optimized are not saturating but enable one dilution of plasma (1/100) to be run for all samples. As different amounts of protein are coupled for each protein construct, the MFI cannot be directly compared between proteins. A representative standard curve for both non-magnetic and magnetic beads is shown in S1 Fig.

Coupling of P. vivax proteins to non-magnetic beads was performed as previously described [7]. Briefly, the optimised antigen concentration (Table 1) was coupled to 2.5x106 pre-activated microspheres, in 100 mM monobasic sodium phosphate buffer pH 6.0, using 50mg/ml sulfo-NHS and 50 mg/ml of EDC to cross-link the proteins to the beads. The activated beads were washed and stored in PBS, 0.1% BSA, 0.02% Tween-20, 0.05% Na-azide, pH 7.4 at 4°C until use. For the coupling to magnetic beads, a magnet rack was used for pelleting the beads, instead of the centrifugation step for non-magnetic beads. The coupling is random, not directional, which is optimal when the epitopes within the proteins are unknown. We qualitatively assessed the stability of the coupled beads by visual comparison of the MFI of the standard curve over a nine-month period. A reduction in the MFI or loss of log-linearity were considered markers of instability.

Table 1

P. vivax proteins used in the comparison experiments, with the concentration of protein coupled per non-magnetic and magnetic beads indicated (note amount is listed per 1x106 beads, 2.5x106 beads were used for a bulk coupling).

Gene Annotation	Protein ID	Expression System	Protein Concentration (μg/ul)	Construct, amino acids (size)	Protein amount (μg/1x10^6) non-magnetic beads	Protein amount (μg/1x10^6) magnetic beads
RBP2b (P25)	PVX_094255	E. coli	4.15	161–1454 (1294)	0.21	0.24
MSP1-19	PVX_099980	WGCF	1.55	1622–1729 (108)	0.30	1.60
RBP2b	PVX_094255	WGCF	2.06	1986–2653 (667)	0.28	3.20
RAMA	PVX_087885	WGCF	0.78	462–730 (269)	0.06	0.48
PvEBPII	KMZ83376.1	E. coli	10	109–432 (324)	0.08	0.20
SSA-s16	PVX_000930	WGCF	0.41	31-end (110)	0.40	0.80
PvRIPR	PVX_095055	E. coli	1	552–1075 (524)	0.40	0.80
MSP3.10	PVX_097720	WGCF	0.64	25-end (828)	0.40	0.80
Hyp. Protein	PVX_097715	WGCF	0.7	20-end (431)	0.14	1.20
PvDBPII (AH)	AAY34130.1	E. coli	0.6	1–237 (237)	0.43	0.56
MSP8	PVX_097625	WGCF	0.39	24–463 (440)	0.28	0.56
Unspecified/	PVX_112670	WGCF	1.13	34-end (302)	0.45	0.90
Pv-fam-a
Pv-fam-a	PVX_096995	WGCF	1.7	61-end (420)	0.34	1.20
MSP3.3	PVX_097680	WGCF	0.55	21-end (996)	0.48	0.32
MSP7.1	PVX_082700	WGCF	0.33	23-end (397)	0.40	0.60
MSP5	PVX_003770	WGCF	0.58	23–365 (343)	0.01	0.016
MSP7	PVX_082670	WGCF	0.61	24-end (388)	0.40	0.40
PvTRAP/	PVX_082735	WGCF	0.9	26–493 (468)	0.40	0.80
SSP2
PvDBPII (sal1)	PVX_110810	E. coli	1.2	193–521 (329)	0.29	0.24

Gene annotations and protein IDs were sourced from PlasmoDB (release 36, http://plasmodb.org/plasmo/), or GenBank when necessary. All proteins have previously been used and described in our past work [7].

Plasmodium vivax recombinant antigens were expressed and purified in three countries: Japan (Takafumi Tsuboi, Ehime University & Matthias Harbers, CellFree Sciences), Australia (Wai-Hong Tham and Julie Healer, Walter & Eliza Hall Institute of Medical Research) and France (Chetan Chitnis, Institut Pasteur). Proteins were expressed either in the wheat-germ cell-free expression system (WGCF) or E. coli. See Table 1 for a complete list of proteins and the optimised amount coupled to non-magnetic and magnetic beads.

Multiplexed assay for measurement of P. vivax-specific antibody responses

To measure the IgG levels, a multiplexed bead based assay was used, as previously described [7]. Briefly, antigen-specific IgG was detected by incubating 500 beads of each antigen per well with plasma diluted at 1:100, in a final volume of 100μl. Non-magnetic beads were washed using a vacuum manifold, whereas magnetic beads were washed using a magnetic plate washer. After the washings, 100μl of a 1:100 dilution of 0.5mg/ml PE-conjugated Donkey F(ab)2 anti-human IgG (JIR 709-116-098) was added. At least 15 beads of each region/antigen were then acquired and analysed on a Bio-Plex® 200 instrument and/or a MAGPIX® instrument as per the manufacturer’s instructions. Note that for comparing data between Bio-Plex® 200 and MAGPIX® instruments it is important that the “high RP1” target is not selected on the Bio-Plex® 200, as this option is not available on the MAGPIX®. On each plate, a twofold serial dilution from 1/50 to 1/25,600 of a seropositive control plasma pool (generated from PNG adults) was included. Note that for the external comparison both labs used the same PNG control pool to generate the standard curve. Each instrument was maintained as instructed by the manufacturer, with the relevant calibration, validation and/or verification beads run daily or as indicated by the manufacturer. Note that the Bio-Plex® calibration beads used were different between the two laboratories for the external comparison.

The results were expressed as mean fluorescence intensity (MFI) of at least 15 beads for each antigen. We have previously determined that data from at least 15 beads is required per antigen for consistent and repeatable results.

Instruments

Antibody measurements were acquired using a Bio-Plex® 200 Multiplexing Analyzer System from Bio-Rad for all non-magnetic coupled beads (Bio-Plex® 200System, Bio-Plex® high-throughput fluidics system, microplate platform and a computer with the Bio-Plex® manager software v.5.0). Washing steps were carried out on a Bio-Rad Aurum vacuum manifold.

For all magnetic coupled beads a MAGPIX® Multiplexing System from Millipore was used (MAGPIX® System and the Xponent software V.4.2). Washing steps were carried out using a magnetic plate washer from BioTek Instruments (BioTek ELx50). A Bio-Rad Sure Beads magnetic rack was used during the coupling process.

Plates were incubated on a Ratek Platform shaker (Microtiter/PCR Plate Shaker). A Vortex Sonicator (Branson 2200), a BioSan Vortex V-1 plus and a Table centrifuge (Eppendorf Centrifuge 5424) were also used during the coupling process.

Statistical analysis

The raw MFI results were converted to relative antibody units (RAU) using protein-specific standard curve data (see S1 Fig for examples of standard curves). A log–log model was used to obtain a more linear relationship, and a five-parameter logistic function was used to obtain an equivalent dilution value compared to the PNG control plasma. We extrapolated one step further beyond the lowest dilution (i.e. from 1/25,600 to 1/51,200), resulting in converted data units ranging from 1.95×10−5 to 0.02, as previously described [7]. This was performed in R. Pearson’s r correlations were performed to determine the strength of correlation and the statistical significance for all comparisons. To enable these parametric correlations, data were log-transformed prior to the analysis to better fit the normal distribution. Pearson r values <0.3 were considered weak, 0.3–0.7 moderate, and >0.7 strong correlations.

Results and discussion

Comparison of total IgG antibodies detected against P. vivax antigens coupled to either non-magnetic or magnetic beads and assayed by a Bio-Plex® 200 instrument

Total IgG antibody levels against a panel of 19 P. vivax proteins, measured in plasma samples from 163 individuals living in malaria-endemic areas of Thailand and the Solomon Islands, were assayed using either non-magnetic or magnetic beads and run on a Bio-Plex® 200 instrument. IgG levels to 18 of 19 proteins were moderate-strongly correlated between non-magnetic and magnetic assays, with Pearson r-values ranging from 0.53–0.98 (all p<0.0001) (Fig 1), supporting previous findings based on P. falciparum proteins [10]. This is despite different amounts of each protein being coupled to non-magnetic versus magnetic beads (Table 1). The exception was for the protein PVX_003770 (MSP5), with the lowest correlation coefficient at r = 0.27 (p<0.001). A sub-set of the samples that had relatively high antibody levels for PVX_003770 when assayed with non-magnetic beads had relatively low antibody levels when assayed with magnetic beads, likely accounting for the low correlation coefficient observed. Interestingly, the amount of protein coupled for PVX_003770 (for both non-magnetic and magnetic beads) was substantially lower than for the other proteins. Future experiments are planned to determine whether increasing the protein amount for PVX_003770 could result in a higher correlation between the two platforms.

Fig 1

IgG antibody levels (RAU) measured against 19 P. vivax proteins in samples from malaria-endemic areas, using either non-magnetic or magnetic beads and run on a Bio-Plex® 200 instrument.

*** p<0.001, **** p<0.0001.

Comparison of total IgG antibodies detected against P. vivax antigens coupled to magnetic beads and assayed using either a Bio-Plex® 200 instrument or a MAGPIX® instrument

For this comparison, all 19 P. vivax antigens were coupled to magnetic beads only, at the optimised antigen concentrations. Total IgG antibody levels were measured in the same set of 163 plasma samples, with the assay run on both a Bio-Plex® 200 and a MAGPIX® instrument. To our knowledge, this is the first published report of this comparison. Here, the Pearson r correlation coefficients indicated a strong correlation between samples run on both instruments (r = 0.985–0.999, p<0.0001, Fig 2). These results indicate that results obtained on either platform, when antigens are coupled at the same optimised concentrations to magnetic beads, are highly comparable. The strength of the correlations in this comparison is stronger than the previous analysis (which compared non-magnetic versus magnetic beads on the same instrument), presumably because the same sets of coupled beads were run on both instruments. The strength of the correlations suggests that results obtained on the Bio-Plex® 200 and MAGPIX® are interchangeable.

Fig 2

IgG antibody levels (RAU) measured against 19 P. vivax proteins in samples from malaria-endemic areas, using magnetic beads and run on either a Bio-Plex® 200 instrument or MAGPIX® instrument.

**** p<0.0001.

Comparison of total IgG antibodies against P. vivax antigens coupled to non-magnetic beads and analyzed on a Bio-Plex® 200 instrument and antigens coupled to magnetic beads and analyzed on a MAGPIX® instrument

The final comparison we wanted to conduct was of antigens coupled to non-magnetic beads and assayed on a Bio-Plex® 200 instrument with antigens coupled to magnetic beads and assayed on a MAGPIX® instrument. As non-magnetic beads are cheaper to purchase, users that have only a Bio-Plex® 200 instrument would potentially favour this configuration (even though the instrument can run both non-magnetic and magnetic beads). Conversely, for users that only have a MAGPIX® instrument, they are only able to run magnetic beads as the instrument cannot detect non-magnetic beads. To our knowledge, this is the first published report of this comparison for a non-commercial assay.

It was again observed that there was a moderate-strong correlation between results obtained using the non-magnetic beads/Bio-Plex® 200 and magnetic beads/MAGPIX® platforms, with Pearson r correlation coefficients ranging from 0.42–98 (p<0.0001, Fig 3). These correlation coefficients are similar to those obtained in the first comparison (non-magnetic versus magnetic beads both run on the Bio-Plex® 200 instrument), and provide further support for our finding that antigens coupled to either type of beads and run on either instrument generally give very comparable total IgG measurements. As we observed in the first comparison, the weakest correlation was again for the protein PVX_003770 (with a moderate r correlation coefficient of 0.42).

Fig 3

IgG antibody levels (RAU) measured against 19 P. vivax proteins in samples from malaria-endemic areas, using non-magnetic beads and run on a Bio-Plex® 200 compared to use of magnetic beads run on a MAGPIX® instrument.

**** p<0.0001.

External comparison of a multiplexed assay using P. vivax antigens coupled to non-magnetic beads and analyzed on a Bio-Plex® 200 instrument

The results thus far indicate that IgG levels measured using either non-magnetic or magnetic beads and assayed on either a Bio-Plex® 200 or MAGPIX® instrument are highly comparable. A group of 3 staff members, but all at the same Institute (Walter & Eliza Hall Institute, WEHI) using the same instruments, performed these measurements. Therefore an additional comparison was performed: external comparison of the assay at an independent research Institute located overseas (Case Western Reserve University, CWRU). It is important to note that each Institute used their own (commercial) Bio-Plex® calibration and validation beads to set-up and maintain their respective instruments, which may contribute to some variation in results between laboratories.

A set of 425 plasma samples were aliquoted at CWRU and shared with WEHI. At the same time, a set of 12 P. vivax proteins (Table 2) were coupled to non-magnetic beads at WEHI and shared with CWRU. During the same week assays were performed to measure total IgG antibodies against these P. vivax antigens in the 425 plasma samples on Bio-Plex® 200 instruments independently at each Institute (total of 6 plates run at each Institute). After exclusion of plates or samples following quality control checks (positive control–non log-linear standard curve; bead counts < 15), data from 318 samples was directly compared between sites. The drop from 425 to 318 samples was largely due to one plate with failed standard curves that could not be repeated due to sample availability. IgG levels were compared first using raw data (MFI values). The Pearson r correlation coefficients indicated a strong correlation for all proteins with r-values ≥ 0.76 (p<0.0001), with the exception of PVX_094255 (RBP2b) (r = 0.57, p<0.0001) (Table 3, scatter plots in S2 Fig). The same correlation analysis was then performed on data converted in R using the standard curves (to account for any plate-plate variation). Strong correlation coefficients were observed for all 12 proteins, including PVX_094255 (r values ≥ 0.72, p<0.0001) (Table 3, scatter plots in S3 Fig). For the majority of proteins, the correlation was stronger after conversion (Table 3). This is expected given the conversion, based on the standard curve generated with a plasma pool from immune PNG donors that is run on every plate, is used to account for any plate-plate variation and potentially to overcome differences that might be attributed to different machines maintained and set-up with different calibration and validation bead sets.

Table 2

P. vivax proteins used for the external comparison.

Gene Annotation	Protein ID	Expression System
MSP1-19	PVX_099980	WGCF
Pv-fam-a	PVX_096995	WGCF
hypothetical protein, conserved	PVX_094830	WGCF
Pv-fam-a	PVX_112670	WGCF
MSP7	PVX_082650	WGCF
RBP2b	PVX_094255	WGCF
hypothetical protein, conserved	PVX_001000	WGCF
merozoite surface protein 8	PVX_097625	WGCF
PvTRAP/SSP2	PVX_082735	WGCF
MSP7	PVX_082645	WGCF
PvRBP-2, putative	PVX_090330	WGCF
sexual stage antigen s16	PVX_000930	WGCF

Proteins were coupled to non-magnetic beads at WEHI and half of each batch of bead-conjugated protein was shipped to CWRU. All proteins have previously been used and described in our past work [7].

Table 3

External comparison of the non-magnetic bead assay run on the Bio-Plex® 200.

Protein ID	Correlation MFI (n = 318)	Correlation RAU (n = 318)
PVX_099980	0.87 ****	0.92 ****
PVX_096995	0.83 ****	0.87 ****
PVX_094830	0.76 ****	0.74 ****
PVX_112670	0.79 ****	0.81 ****
PVX_082650	0.85 ****	0.84 ****
PVX_094255	0.57 ****	0.72 ****
PVX_001000	0.83 ****	0.83 ****
PVX_097625	0.86 ****	0.85 ****
PVX_082735	0.90 ****	0.92 ****
PVX_082645	0.89 ****	0.87 ****
PVX_090330	0.84 ****	0.81 ****
PVX_000930	0.89 ****	0.91 ****

Pearson r correlation coefficients are shown for both the raw data (MFI) and the standard curve converted data (RAU).

**** p<0.0001.

These results indicate that data generated using this multiplexed assay are highly reproducible in a different laboratory setting when the same coupled-beads are used, particularly if both laboratories have access to the same positive control for standardization. Unfortunately, whilst there is a WHO reference reagent for P. falciparum serology studies [11], there is not yet a similar product available for P. vivax. Importantly, we also assessed the stability of the coupled beads by running the standard curve 10 times over a period of 9 months (intensely for 2 months) (S4 Fig). For most proteins the coupled beads were highly stable (11/16 tested over 9-months), with the MFI dropping for three proteins and increasing for two proteins. This is supported by previous research that has indicated the stability of protein-coupled beads [10], noting that the stability may vary by antigen [12].

Conclusions

The aim of this study was to determine whether multiplexing assays performed using magnetic beads or non-magnetic beads are highly comparable, independent of the beads and platform used to analyze the assays. We compared here a total of 19 P. vivax proteins that were coupled to both magnetic beads and non-magnetic beads. The protein concentration used for the couplings was individually determined by optimisation for each protein for the chosen bead type (Table 1). For this, a dilution series from the positive control plasma pool, prepared from immune PNG donors, was used to generate a log-linear standard curve for each protein. The non-magnetic beads are 5.5μm in size, whilst the magnetic beads are 6.5μm in size, likely accounting for the need to couple on average 0.3μg of protein to non-magnetic versus 0.8 μg of protein to magnetic beads (per 1x106 beads). One coupling reaction using these amounts of protein is enough to assay > 3000 samples in singlicate, thus the slightly higher amount of protein required for magnetic beads is unlikely to be a limitation to using this format. We did not assess the efficiency of antigen coupling, which could potentially be an important variable impacting the amount of protein required for coupling.

We have demonstrated that results are moderately-strongly correlated whether using proteins coupled to magnetic beads or non-magnetic beads and analysed using either a Bio-Plex® 200 (non-magnetic and magnetic beads) or MAGPIX® (magnetic beads only). Our external comparison has also demonstrated that results generated in different laboratories are strongly correlated, if a reference standard curve is included for standardization. Therefore researchers can, in principle, compare data generated with a different type of bead or assayed using a different instrument platform, if the amount of protein coupled is optimised for the correct type of bead. Overall, the choice of assay platform and instrument used is up to the user. However, we do suggest that selecting one bead composition for running experiments is preferred, given the variation in strength of correlation between proteins (Fig 1). Running magnetic beads on a Bio-Plex® 200 or a MAGPIX® generates data that is so highly correlated they could be considered interchangeable (Fig 2). An important consideration is that up to 100 different proteins can be assayed simultaneously using non-magnetic beads and a Bio-Plex® 200 instrument, whereas the maximum is 50 proteins using a MAGPIX®. If less than 50 proteins will be used, the MAGPIX® instrument is cheaper and enables washing steps to be conducted with magnets, which improves both bead retention [10, 13] and speed of the assay.

For future use and development of the assay, we recommended that a reference laboratory provide both protein-coupled beads and a positive control, along with a Standard Operating Procedure for the assay. All protein-coupled beads should be tested for stability and researchers provided with an expiry date for their use, in addition to checking the performance of the standard curve before each use. This should ensure repeatable and comparable measurements are generated between different research groups. A key focus of P. vivax serology efforts should be to develop a standard WHO reference reagent for P. vivax that is available to any research group worldwide.

Whilst these results were obtained in the context of P. vivax-specific IgG responses in individuals from malaria-endemic areas, the large panel of proteins used and consistent results obtained for all proteins suggest these results can be applied to guide studies in other fields. Luminex xMAP® technology has been used to measure antibody responses against other infectious pathogens, such as HIV and influenza [14, 15], to a variety of vaccine antigens such as tetanus toxoid [16], and more recently to SARS-CoV-2 [17-19].

Representative example of standard curves generated for each P. vivax protein on non-magnetic and magnetic beads.

MFI = median fluorescent intensity. S1 –S10 = standard 1 to standard 12 (2 fold serial dilution of positive plasma pool, starting at 1/50 dilution). The data are converted from MFI to relative antibody units (RAU) using a five-parameter logistic function to obtain an equivalent dilution value compared to the PNG control plasma. For example, an MFI of similar value to that of the 1/50 dilution of the standard curve would result in an RAU of around 0.02 (or 1/50). The RAU values therefore range from 1.95×10−5 (equivalent to 1/51,200 or S11, as the curve is extrapolated one step further) to 0.02.

(TIF)

Click here for additional data file.

Comparison of IgG antibody levels against 12 P. vivax proteins when run at WEHI compared to CWRU: Raw MFI values.

(TIF)

Click here for additional data file.

Comparison of IgG antibody levels against 12 P. vivax proteins when run at WEHI compared to CWRU: Converted RAU values.

(TIF)

Click here for additional data file.

Stability of protein-coupled magnetic beads over 9-months.

The original coupled beads were tested at every week for 2 months after coupling, then again at 9 months post-coupling. The MFI of the standard curves are presented (S1 = 1/50, then 2-fold serial dilution). New vials of secondary antibodies were opened on 19/02/19, 26/02/19 and 08/03/19. Protein PVX_094255 (WGCF construct) was not tested in this experiment.

(TIF)

Click here for additional data file.

25 Aug 2020

PONE-D-20-24214

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against P. vivax antigens in a multiplexed bead-based assay using Luminex® technology (Bio-Plex®200 or MAGPIX®)

PLOS ONE

Dear Dr. Longley,

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**********

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Reviewer #3: Yes

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Reviewer #1: The authors describe a systematic comparison of IgG antibody data as collected against a panel of 19 P. vivax antigens and human plasma samples from three endemic settings. This is an important study with the increase in use of the bead-based multiplex platform as performed by multiple groups worldwide, and the need to assess comparability among bead and platform variations. The study does well to directly compare variables of interest, but statistical presentation of results is only limited to correlation coefficients and p values inappropriately presented. The comparison should also include slope estimates for the regression models and p values corresponding to those.

Major concerns:

In the Introduction, the authors could do a better job of explaining the xMAP technology to the lay person unfamiliar with bead-based multiplex serology. Some suggestions are below, but review and imagine you’ve never heard of the multiplexing technology before when revising.

Table 1 and Table 2. For the benefit of the reader, for antigens that had been published on before, please include appropriate reference(s).

Throughout the paper, the authors state what appear to be p values for the r^2 correlation coefficient, which is inappropriate since r simply indicates goodness of fit for the regression model. For each comparison in the study, the authors should report both the r^2 as well as the slope, and can report on the p value of the slope. Additionally, if the slope deviates from 1.0, this is important information for the reader for generalizing if one bead type or platform would give consistently higher/lower MFI values. Instead of presenting all this information in the Figure panels, perhaps Tables (in a similar manner to Table 3) would make it easiest to digest. The authors should also state in the Methods what is criteria for ‘strong’ correlation.

In Conclusions, the authors accentuate the good correlation of responses between the various two comparators, but do not address potential reasons for when correlation (and slope) is not good. For example, why is MSP5 appear to have more issues than other Pv antigens? Many of the scatterplots in Figs 1 and 3 have very poor correlation and some points with differences >2 orders of magnitude, and this should be addressed.

Minor concerns:

- In title, spell out entire parasite name

- Throughout the document, if choosing to use the (R) symbol for company information, use consistently each time company is mentioned

- Abstract: instead of just mentioning responses were ‘strongly correlated’ also provide quantitative measures of correlation/difference (r^2 and slope estimates)

- Line 51-52: “such as a reduction in sample volume required and reduced laboratory time” if choosing multiple targets to assay for

- Line 57: “in more consistent findings among different studies”

- Line 59-62: if wanting to discuss the xMAP technology, need to be specific here about the IR dyes and pre-gating bead regions to allow multiplex data collection. Also need to mention the FlexMAP platform here. Provide a link to manufacturer’s website that explains this technology so readers can investigate for themselves.

- Line 62: explain what ‘coupling’ is

- Line 67: “Two different types of bead compositions are available…”

- Line 69-72: need to mention FlexMAP 3D system here

- Line 70-71: “offers advantages over the flow-based systems such as faster acquisition time” not true, and this is dependent on how many beads present in each assay well. Flow-based machines can actually read a plate faster than a MAGPIX

- Line 75-77: “A secondary aim was to demonstrate that this assay is highly reproducible in an independent laboratory through an external validation.” As currently worded, this isn’t an aim, but a pre-determined conclusion.

- Line 78-80: “however the large number of proteins assessed and consistent results obtained, suggest these findings should be generalizable for optimization of the multiplexed bead-based assay for other pathogens.” Even though there’s 19 antigens on your panel, they’re only to one pathogen. Not appropriate to extrapolate to the other myriad of human pathogens from this data alone.

- Line 80-82: “This is important in the context of the ongoing SARS-CoV-2 pandemic, as multiple laboratory assays based on Luminex technology are under development [6-8].” I get it that it’s really cool to talk about nCoV right now, but this sentence has nothing to do with your Pv malaria study and should be removed.

- Line 133-134: need to state here what would indicate that the beads were not stable

- Line 153-155, 162: Luminex recommends at least 35 beads acquired per region. Need to state here why 15 was chosen for this study.

- Line 266: “bead counts < 15”

- For Table 3 data, this is more of an “external comparison” rather than validation work

- Table 4: the majority of these factors (cost, time, etc.) are relative for an institution and the SOP being used, and this table should be removed since many of the statements are subjective

Reviewer #2: R. Mazhari and colleagues report the results from a straight-forward study that compared results using the original non-magnetic bead Luminex (100/200 – BioPlex) method with the newer magnetic bead-MAGPIX approach, by measuring antibody (Ab) levels to a series of P. vivax antigens. Although many researchers have conducted a few comparative assays when switching from the non-magnetic to magnetic format, to this reviewer’s knowledge, this is the first study to conduct an in-depth comparison between the two methods. Overall, the study is well described, the results clearly presented, and the results confirm what most investigators have assumed, namely, the two methods give very similar (but not identical) results. The study will provide reassurance to the research community that the two methods provide similar answers.

The following comments are meant to be helpful for improving the manuscript.

1. Methods: Line 153. “a 1:100 dilution of PE-conjugated Donkey …. was added.” More information is needed: What was the concentration used? How many ul were added? (e.g., the reagent (1 mg/ml) was diluted 1:1000 and 50uL was added to each well.”

2. Line 123-111124: “…, we optimized all protein concentrations …… .” This reviewer is not exactly sure what the statement means. Does it mean you coupled various concentrations of each antigen to beads, created a log-linear curve, and then selected a concentration of antigen that would give you a specific MFI after coupling using a 1:100 dilution of the plasma standard? This point is important for understanding Table 1, as the amounts listed do not appear to be “saturating” concentrations. It would also be useful the authors commented if it is possible to compare MFI between antigens, i.e., if 10,000 MF1 for antigen X means there are more Ab to X than 5,000 MFI to Ab Y? Some researchers report that if the beads are coupled with equal amounts of antigen, then comparison can be made across antigens (although I’m not sure I agree), but the question could be addressed.

3. It is surprising that the authors did not mention calibration or verification beads (standard), which I believe differ between BioPlex and MAGIX. Some mention of this would be useful. Were the same calibration beads (standards) used in the external validation study? If so, one might have expected even better agreement in SFig. 1 for MFI. Some mention of the influence (or lack thereof) of instrument calibration should be included.

4. In sFig. 1, it is somewhat difficult to interpret/compare the results, especially between antigens, because different Y- and X- axes were used. For example, for MSP1 the Y-Axis is from 100 to 10,000, but the X-axis is from 10 to 10,000. If similar results were obtained between the two assays, one should be able to draw a diagonal (45o) line, and half the data points would be above and half below. So, having the same X- and Y- axes is beneficial. It appears that higher MFI were obtained at CASE for antigens 112670, MSP7, and MSP8 (majority data points above the diagonal line), but lower values for 096995, PVx-001000, 094830. Do the authors have an explanation for this?

5. Converting raw data to arbitrary units using a standard curve is always difficult for readers to truly envision. It would be beneficial if a standard curve was included in the Supplemental Information section and some discussion provided on how MFI were converted to relative antibody units (RAU). That is, in sFig. 1 the MFI values range from 10 to >30,000 MFI (i.e., numbers normally found in the literature; readers will be able to identify with the numbers). In SFig. 2, data were transformed using the standard curve and range from 0.00001 to 0.1 arbitrary units. How did you get these numbers? Based on the patterns in paired figures, it appears 10,000 MFI equals ~0.01 RAU, right? This area of the manuscript would benefit from clarification. Based on the data provided and the reproducibility of the results across the various formats, it seems that reporting data as MFI would be beneficial, since Luminex data produced in all laboratories around the world are generated as MFI. Since the data using the standard curve is reported to be only slightly better, the authors might consider including a discussion/comment about the process of data transformation using R compared to simply using MFI.

6. SFig. 1 Y-axis is labeled Case; whereas, in sFig. 2 it is labeled CWRU. The labels should have the same nomenclature.

Minor comments:

1. Abstract: Technically, the word data is a plural term (datum singular). So, Line 34 should read “Data are lacking …..”. However, the world may be changing in how this term is perceived.

2. Line 49: “…. Has been a rapid uptake of Luminex ……” The word “uptake” is colloquial, so suggest changing to another term, such as, increased, development, advancement, etc.

3. Line 54: Do Plasmodial parasites really express hundreds of thousands of proteins?

Just comments for the authors (FYI: no response needed)

1. Line 162: Our biostatisticians also calculated that 15 beads was the lowest number of beads needed to provide representative data. Alas, most investigators think that bead counts need to be ≥100. So, your manuscript will help set the record straight.

2. We have also found that antigen-coupled beads are quite stable. We have used the same batch of coupled beads, without loss of MFI, for over 10 years. We have also found that antigens tend to be more stable after they are coupled than if they are re-frozen and recoupled.

Reviewer #3: The authors presented a technical- descriptive paper which aimed to compared two different tools (beads) applied to serological multiplex methods. This bring interesting results that could help cutting steps on laboratory work and to choose the better technique for serological studies. The authors cited previews studies where beads-based multiplex assays have been used, suggesting that kind a technical is going to be the future for serological studies, as screening to evaluation of the antibody responses. But the standard immuno-assay (ELISA) is still a standard protocol to this kind of evaluation. Also, the use of a coupling beads needs more validations regarding the integrity of the protein conformation, once the coupled protein could hide some important epitope to antigen recognition by antibodies. Taken this and to make the present study afford to publication, I’d like to make some suggestions:

1. Show the standard curve for both type beads in the main manuscript. This is a big step to select the optimized antigen concentrations of the protein and beads necessary the assay. Also, will be interesting show the detail how MFI is converted to relative antibody units (RAU) using protein-specific standard curve data.

2. As the author are presenting a technical paper, showing a technique that could replace the ELISA, I suggest show the antibody titles do not change for coupled and non-coupled antigen for both type beads, using for example, the standard ELISA for at least an antigen with weak correlation (PVX_003770), and with an antigen with a high correlation, and decenter controls. Those technique aim to show the antibodies titles, which cannot be significant different compare with a standard immunological assay (ELISA).

3. I’m concern about the protein conformation preservation when it is coupled. Maybe a assay using monoclonal antibodies for which recognize the specific epitopes should be interesting to show that protein conformation are being preserved.

**********

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15 Oct 2020

Reviewer #1: The authors describe a systematic comparison of IgG antibody data as collected against a panel of 19 P. vivax antigens and human plasma samples from three endemic settings. This is an important study with the increase in use of the bead-based multiplex platform as performed by multiple groups worldwide, and the need to assess comparability among bead and platform variations. The study does well to directly compare variables of interest, but statistical presentation of results is only limited to correlation coefficients and p values inappropriately presented. The comparison should also include slope estimates for the regression models and p values corresponding to those.

Major concerns:

In the Introduction, the authors could do a better job of explaining the xMAP technology to the lay person unfamiliar with bead-based multiplex serology. Some suggestions are below, but review and imagine you’ve never heard of the multiplexing technology before when revising.

We thank the reviewer for the suggestions below and have updated our introduction section on the xMAP technology.

Table 1 and Table 2. For the benefit of the reader, for antigens that had been published on before, please include appropriate reference(s).

As all proteins have been used and published by our group before, we have included this sentence in the Table headings: All proteins have previously been used and described in our past work [7].

Throughout the paper, the authors state what appear to be p values for the r^2 correlation coefficient, which is inappropriate since r simply indicates goodness of fit for the regression model. For each comparison in the study, the authors should report both the r^2 as well as the slope, and can report on the p value of the slope. Additionally, if the slope deviates from 1.0, this is important information for the reader for generalizing if one bead type or platform would give consistently higher/lower MFI values. Instead of presenting all this information in the Figure panels, perhaps Tables (in a similar manner to Table 3) would make it easiest to digest. The authors should also state in the Methods what is criteria for ‘strong’ correlation.

We thank the reviewer for this feedback. We think the confusion has come from our use of reporting r^2. This was an error and we should have reported the pearson r value (the r^2 indicates the goodness of fit of the model as the reviewer indicates). We ran a pearson parametric correlation on log-transformed data, and not a regression model, so will report the r. We have updated all Figures, table and text to include the pearson r value and not the r^2. We believe that the Figures are useful for the reader to interpret the data, and prefer to keep those rather than tables.

We have now added into the statistics section that we considered r values of <0.3 to be weak, 0.3 – 0.7 to be moderate, and > 0.7 to be indicative of a strong correlation.

In Conclusions, the authors accentuate the good correlation of responses between the various two comparators, but do not address potential reasons for when correlation (and slope) is not good. For example, why is MSP5 appear to have more issues than other Pv antigens? Many of the scatterplots in Figs 1 and 3 have very poor correlation and some points with differences >2 orders of magnitude, and this should be addressed.

We note that in the Results/Discussion we suggested that the potential reason for the lower correlation for MSP5 was the much smaller amount of antigen coupled to beads for this protein. Whilst this amount was optimised, perhaps the lower total amount of protein coupled leaves more space available on the beads for non-specific binding, resulting in differing results between the two bead compositions. We plan to investigate this further and potentially re-optimise coupling for this particular protein. We have changed our language in the discussion to more simply state that the correlations were moderate-strong, and suggest users stick to one type of bead to run their experiments if possible: “However, we do suggest that selecting one bead composition for running experiments is preferred, given the variation in strength of correlation between proteins (Fig 1). Running magnetic beads on a Bio-Plex® 200 or a MAGPIX® generates data that is so highly correlated they could be considered interchangeable (Fig 2).”

All minor concerns have been addressed as indicated below.

Minor concerns:

- In title, spell out entire parasite name

This has been done.

- Throughout the document, if choosing to use the (R) symbol for company information, use consistently each time company is mentioned

This has been done.

- Abstract: instead of just mentioning responses were ‘strongly correlated’ also provide quantitative measures of correlation/difference (r^2 and slope estimates)

This has been done.

- Line 51-52: “such as a reduction in sample volume required and reduced laboratory time” if choosing multiple targets to assay for

This has been added.

- Line 57: “in more consistent findings among different studies”

Between has been changed to “among”.

- Line 59-62: if wanting to discuss the xMAP technology, need to be specific here about the IR dyes and pre-gating bead regions to allow multiplex data collection. Also need to mention the FlexMAP platform here. Provide a link to manufacturer’s website that explains this technology so readers can investigate for themselves.

We have included the FLEXMAP 3D platform and added in a link to the Luminex Corp website, along with more information on the bead dyes and pre-gating on the software.

- Line 62: explain what ‘coupling’ is

We have added: “Coupling is the process of attaching a specific protein to the bead, through carboxyl groups on the bead surface (covalent bonding).”

- Line 67: “Two different types of bead compositions are available…”

We have added the requested change to the sentence.

- Line 69-72: need to mention FlexMAP 3D system here

This has been added.

- Line 70-71: “offers advantages over the flow-based systems such as faster acquisition time” not true, and this is dependent on how many beads present in each assay well. Flow-based machines can actually read a plate faster than a MAGPIX

We thank the reviewer for this comment. Whilst our assay is run faster on a MAGPIX, it is true that this is highly dependent on the beads present in each assay well. We have removed this part of the sentence.

- Line 75-77: “A secondary aim was to demonstrate that this assay is highly reproducible in an independent laboratory through an external validation.” As currently worded, this isn’t an aim, but a pre-determined conclusion.

We have re-worded the aim.

- Line 78-80: “however the large number of proteins assessed and consistent results obtained, suggest these findings should be generalizable for optimization of the multiplexed bead-based assay for other pathogens.” Even though there’s 19 antigens on your panel, they’re only to one pathogen. Not appropriate to extrapolate to the other myriad of human pathogens from this data alone.

We appreciate the reviewers point and have removed this text.

- Line 80-82: “This is important in the context of the ongoing SARS-CoV-2 pandemic, as multiple laboratory assays based on Luminex technology are under development [6-8].” I get it that it’s really cool to talk about nCoV right now, but this sentence has nothing to do with your Pv malaria study and should be removed.

This has been removed.

- Line 133-134: need to state here what would indicate that the beads were not stable

This has been added: “A reduction in the MFI or loss of log-linearity were considered markers of instability.”

- Line 153-155, 162: Luminex recommends at least 35 beads acquired per region. Need to state here why 15 was chosen for this study.

In our hands, with at least 15 beads our data is consistent and highly repeatable. We have added the sentence: “We have previously determined that data from at least 15 beads is required per antigen for consistent and repeatable results.”

- Line 266: “bead counts < 15”

Updated.

- For Table 3 data, this is more of an “external comparison” rather than validation work

We have changed reference to this work to “external comparison” throughout the paper.

- Table 4: the majority of these factors (cost, time, etc.) are relative for an institution and the SOP being used, and this table should be removed since many of the statements are subjective

We appreciate this point and have removed the Table.

---------------------------------

Reviewer #2: R. Mazhari and colleagues report the results from a straight-forward study that compared results using the original non-magnetic bead Luminex (100/200 – BioPlex) method with the newer magnetic bead-MAGPIX approach, by measuring antibody (Ab) levels to a series of P. vivax antigens. Although many researchers have conducted a few comparative assays when switching from the non-magnetic to magnetic format, to this reviewer’s knowledge, this is the first study to conduct an in-depth comparison between the two methods. Overall, the study is well described, the results clearly presented, and the results confirm what most investigators have assumed, namely, the two methods give very similar (but not identical) results. The study will provide reassurance to the research community that the two methods provide similar answers.

We thank the reviewer for the positive feedback on our paper and constructive comments below.

The following comments are meant to be helpful for improving the manuscript.

1. Methods: Line 153. “a 1:100 dilution of PE-conjugated Donkey …. was added.” More information is needed: What was the concentration used? How many ul were added? (e.g., the reagent (1 mg/ml) was diluted 1:1000 and 50uL was added to each well.”

This has been added.

2. Line 123-111124: “…, we optimized all protein concentrations …… .” This reviewer is not exactly sure what the statement means. Does it mean you coupled various concentrations of each antigen to beads, created a log-linear curve, and then selected a concentration of antigen that would give you a specific MFI after coupling using a 1:100 dilution of the plasma standard? This point is important for understanding Table 1, as the amounts listed do not appear to be “saturating” concentrations. It would also be useful the authors commented if it is possible to compare MFI between antigens, i.e., if 10,000 MF1 for antigen X means there are more Ab to X than 5,000 MFI to Ab Y? Some researchers report that if the beads are coupled with equal amounts of antigen, then comparison can be made across antigens (although I’m not sure I agree), but the question could be addressed.

We have added in further details as follows: “The positive control pool was used to generate a standard curve running from a 1/50 dilution to a 1/25,600 dilution (10 point standard curve, 2-fold serial dilution). One set amount of protein was selected that resulted in a log-linear standard curve over this dilution series; the amounts optimized are not saturating but enable one dilution of plasma (1/100) to be run for all samples.” In essence, we use a process of trial and error to determine an optimal amount of protein to couple to the beads, based on the standard curve generated from a 2-fold serial dilution of positive pool plasma. We also added the following statement: “As different amounts of protein are coupled for each protein construct, the MFI cannot be directly compared between proteins.”

3. It is surprising that the authors did not mention calibration or verification beads (standard), which I believe differ between BioPlex and MAGIX. Some mention of this would be useful. Were the same calibration beads (standards) used in the external validation study? If so, one might have expected even better agreement in SFig. 1 for MFI. Some mention of the influence (or lack thereof) of instrument calibration should be included.

We thank the reviewer for this comment; it was not something we had considered. We have added in more information in the methods on this point: “Each instrument was maintained as instructed by the manufacturer, with the relevant calibration, validation and/or verification beads run daily or as indicated by the manufacturer. Note that the Bio-Plex® calibration beads used were different between the two laboratories for the external comparison.” We have added this sentence in the results: “It is important to note that each Institute used their own (commercial) Bio-Plex® calibration and validation beads to set-up and maintain their respective instruments, which may contribute to some variation in results between laboratories.” We also updated the following sentence to include the part in italics: “This is expected given the conversion, based on the standard curve generated with a plasma pool from immune PNG donors, is used to account for any plate-plate variation and potentially to overcome differences that might be attributed to different machines maintained and set-up with different calibration and validation bead sets.”

4. In sFig. 1, it is somewhat difficult to interpret/compare the results, especially between antigens, because different Y- and X- axes were used. For example, for MSP1 the Y-Axis is from 100 to 10,000, but the X-axis is from 10 to 10,000. If similar results were obtained between the two assays, one should be able to draw a diagonal (45o) line, and half the data points would be above and half below. So, having the same X- and Y- axes is beneficial. It appears that higher MFI were obtained at CASE for antigens 112670, MSP7, and MSP8 (majority data points above the diagonal line), but lower values for 096995, PVx-001000, 094830. Do the authors have an explanation for this?

We appreciate the point made by the reviewer and have updated all Figures to ensure the Y and X-axes have the same scale. However, we believe that there should be some degree of variation that is random – so having some proteins have higher values and Case whilst some have higher values at WEHI is a good thing (if they were all higher at one Institute this would be a systematic bias).

5. Converting raw data to arbitrary units using a standard curve is always difficult for readers to truly envision. It would be beneficial if a standard curve was included in the Supplemental Information section and some discussion provided on how MFI were converted to relative antibody units (RAU). That is, in sFig. 1 the MFI values range from 10 to >30,000 MFI (i.e., numbers normally found in the literature; readers will be able to identify with the numbers). In SFig. 2, data were transformed using the standard curve and range from 0.00001 to 0.1 arbitrary units. How did you get these numbers? Based on the patterns in paired figures, it appears 10,000 MFI equals ~0.01 RAU, right? This area of the manuscript would benefit from clarification. Based on the data provided and the reproducibility of the results across the various formats, it seems that reporting data as MFI would be beneficial, since Luminex data produced in all laboratories around the world are generated as MFI. Since the data using the standard curve is reported to be only slightly better, the authors might consider including a discussion/comment about the process of data transformation using R compared to simply using MFI.

We thank the reviewer for this comment and agree that some further detail would be beneficial in this section. We have updated this section to read: “The raw MFI results were converted to relative antibody units (RAU) using protein-specific standard curve data (see Figure S3 for examples of standard curves from non-magnetic bead couplings). A log–log model was used to obtain a more linear relationship, and a five-parameter logistic function was used to obtain an equivalent dilution value compared to the PNG control plasma. We extrapolated one step further beyond the lowest dilution (i.e. from 1/25,600 to 1/51,200), resulting in converted data units ranging from 1.95×10−5 to 0.02, as previously described [7]. This was performed in R.”

Whilst the correlations of data between WEHI and CWRU were quite good just when using the MFI, we believe that having a standard curve and converting the data to relative antibody units is beneficial for accounting for inter-operator variability and inter-assay variability.

6. SFig. 1 Y-axis is labeled Case; whereas, in sFig. 2 it is labeled CWRU. The labels should have the same nomenclature.

We have updated all to CWRU.

Minor comments:

1. Abstract: Technically, the word data is a plural term (datum singular). So, Line 34 should read “Data are lacking …..”. However, the world may be changing in how this term is perceived.

We have made this change.

2. Line 49: “…. Has been a rapid uptake of Luminex ……” The word “uptake” is colloquial, so suggest changing to another term, such as, increased, development, advancement, etc.

We changed to advancement.

3. Line 54: Do Plasmodial parasites really express hundreds of thousands of proteins?

It reads “many hundreds TO thousands” of proteins, which is true.

Reviewer #3: The authors presented a technical- descriptive paper which aimed to compared two different tools (beads) applied to serological multiplex methods. This bring interesting results that could help cutting steps on laboratory work and to choose the better technique for serological studies. The authors cited previews studies where beads-based multiplex assays have been used, suggesting that kind a technical is going to be the future for serological studies, as screening to evaluation of the antibody responses. But the standard immuno-assay (ELISA) is still a standard protocol to this kind of evaluation. Also, the use of a coupling beads needs more validations regarding the integrity of the protein conformation, once the coupled protein could hide some important epitope to antigen recognition by antibodies. Taken this and to make the present study afford to publication, I’d like to make some suggestions:

1. Show the standard curve for both type beads in the main manuscript. This is a big step to select the optimized antigen concentrations of the protein and beads necessary the assay. Also, will be interesting show the detail how MFI is converted to relative antibody units (RAU) using protein-specific standard curve data.

We have provided an additional supplementary figure (Figure S1 in the revised manuscript) that shows a representative example of the standard curve for each protein for each bead type. Following a similar comment from reviewer 2 we have provided more details of the standard curve conversion in the methods.

2. As the author are presenting a technical paper, showing a technique that could replace the ELISA, I suggest show the antibody titles do not change for coupled and non-coupled antigen for both type beads, using for example, the standard ELISA for at least an antigen with weak correlation (PVX_003770), and with an antigen with a high correlation, and decenter controls. Those technique aim to show the antibodies titles, which cannot be significant different compare with a standard immunological assay (ELISA).

We acknowledge the reviewers comment, however, we believe there is sufficient prior evidence that ELISA and Luminex assay results are correlated (i.e. https://jcm.asm.org/content/55/1/165#sec-2; https://malariajournal.biomedcentral.com/articles/10.1186/s12936-018-2465-4#Sec16; https://www.sciencedirect.com/science/article/pii/S0022175914000465?via=ihub), also noting that Luminex assays for some targets are more sensitive (https://malariajournal.biomedcentral.com/articles/10.1186/s12936-019-3027-0). Our aim for this manuscript was not to establish Luminex as a new assay, but rather to compare different versions of the assay that are available.

3. I’m concern about the protein conformation preservation when it is coupled. Maybe a assay using monoclonal antibodies for which recognize the specific epitopes should be interesting to show that protein conformation are being preserved.

Again we appreciate the reviewers comment but this is beyond the aims and scope of our manuscript. For most of the proteins we have tested, the epitopes are unknown (and mAbs have not been produced), so this would not be feasible to undertake. What we have done is to include further text that explains the binding to the beads is random, not directional, and that this is optimal when we don’t know the epitopes. “The coupling is random, not directional, which is optimal when the epitopes within the proteins are unknown.”

Submitted filename: response_to_reviewers.docx

Click here for additional data file.

12 Nov 2020

PONE-D-20-24214R1

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex® technology (Bio-Plex®200 or MAGPIX®)

PLOS ONE

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

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Reviewer #3: All comments have been addressed

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: (No Response)

Reviewer #2: The authors have made changes in the manuscript that enhance its clarity and addressed the issues raised by the reviewers. The study clearly makes the point that a correlation exists among the data generated using difference beads-based systems. The authors might consider the following suggestions:

1. The authors added the sentence: “This is expected given the conversion, based on the standard curve generated with a plasma pool from immune PNG donors…..” Was the standard curve run on each plate? If so, please add this information to the text.

2. Supporting Fig. 1 shows the linearity of the dilution curve. However, it is not totally clear how one obtains relative arbitrary unit (RAU) from the curve (MFI vs S1, S2, etc.). Could the authors please add information to the figure legend on how to convert the data from MFI to RAU?

3. Supportive Fig. 2 – YEAH! This figure shows the raw MFI and that subsequent statistical refinements not really needed. All readers will appreciate this figure.

4. Supportive Fig. 3 – shows the relationship of the adjusted values and documents the association after statistical adjustments. Interestingly, adjustments changed the correlations insignificantly. Nice to see the comparison.

5. Supplemental Figures 3 and 4 appear to be the same – comparison of RAU at CWRU and WHEI. Maybe the wrong figure was uploaded into the copy I received. Supportive Figure 4 doesn’t show changes in MFI over time. The authors need to check sFig. 4.

Reviewer #3: (No Response)

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Reviewer #1: Yes: Eric Rogier

Reviewer #2: Yes: Diane Wallace Taylor

Reviewer #3: No

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15 Nov 2020

Reviewer #2: The authors have made changes in the manuscript that enhance its clarity and addressed the issues raised by the reviewers. The study clearly makes the point that a correlation exists among the data generated using difference beads-based systems. The authors might consider the following suggestions:

1. The authors added the sentence: “This is expected given the conversion, based on the standard curve generated with a plasma pool from immune PNG donors…..” Was the standard curve run on each plate? If so, please add this information to the text.

We have updated the sentence to read “This is expected given the conversion, based on the standard curve generated with a plasma pool from immune PNG donors that is run on every plate, is used to account for any plate-plate variation and potentially to overcome differences that might be attributed to different machines maintained and set-up with different calibration and validation bead sets.

We also updated the methods text to make it clear that the standard curve is run on every plate.

2. Supporting Fig. 1 shows the linearity of the dilution curve. However, it is not totally clear how one obtains relative arbitrary unit (RAU) from the curve (MFI vs S1, S2, etc.). Could the authors please add information to the figure legend on how to convert the data from MFI to RAU?

We have added the following text supporting Fig. 1 legend:

The data are converted from MFI to relative antibody units (RAU) using a five-parameter logistic function to obtain an equivalent dilution value compared to the PNG control plasma. For example, an MFI of similar value to that of the 1/50 dilution of the standard curve would result in an RAU of around 0.02 (or 1/50). The RAU values therefore range from 1.95×10−5 (equivalent to 1/51,200 or S11, as the curve is extrapolated one step further) to 0.02.

3. Supportive Fig. 2 – YEAH! This figure shows the raw MFI and that subsequent statistical refinements not really needed. All readers will appreciate this figure.

Thank you for your positive response!

4. Supportive Fig. 3 – shows the relationship of the adjusted values and documents the association after statistical adjustments. Interestingly, adjustments changed the correlations insignificantly. Nice to see the comparison.

Thank you for your positive response!

5. Supplemental Figures 3 and 4 appear to be the same – comparison of RAU at CWRU and WHEI. Maybe the wrong figure was uploaded into the copy I received. Supportive Figure 4 doesn’t show changes in MFI over time. The authors need to check sFig. 4.

Thank you for noticing this, we have replaced sFig. 4. with the correct figure.

Submitted filename: ResponsetoReviewers_16Nov2020.docx

Click here for additional data file.

18 Nov 2020

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex® technology (Bio-Plex®200 or MAGPIX®)

PONE-D-20-24214R2

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23 Nov 2020

PONE-D-20-24214R2

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against Plasmodium vivax antigens in a multiplexed bead-based assay using Luminex technology (Bio-Plex 200 or MAGPIX).

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