Journal of Clinical Microbiology-2020-Dobaño-JCM.01731-20.full

JCM Accepted Manuscript Posted Online 30 October 2020
J. Clin. Microbiol. doi:10.1128/JCM.01731-20

Copyright © 2020 American Society for Microbiology. All Rights Reserved.
1 Highly sensitive and specific multiplex antibody assays to quantify
2 immunoglobulins M, A and G against SARS-CoV-2 antigens
4 Carlota Dobaño1,2*, Marta Vidal1*, Rebeca Santano1, Alfons Jiménez1,2, Jordi Chi1, Diana
5 Barrios1, Gemma Ruiz-Olalla1, Natalia Rodrigo Melero3, Carlo Carolis3, Daniel Parras4, Pau
6 Serra4, Paula Martínez de Aguirre5, Francisco Carmona-Torre6, Gabriel Reina5, Pere
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7 Santamaria4,7, Alfredo Mayor1,2,8, Alberto García-Basteiro1,8,9, Luis Izquierdo1, Ruth Aguilar1,
8 Gemma Moncunill1
10 1
ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, Catalonia, Spain.
11 2
Spanish Consortium for Research in Epidemiology and Public Health (CIBERESP), Madrid,
12 Spain.
13 3
Biomolecular screening and Protein Technologies Unit, Centre for Genomic Regulation
14 (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.
15 4
Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPs), Barcelona, Spain.
16 5
Clínica Universidad de Navarra, Navarra Institute for Health Research, Pamplona, Spain.
17 6
Infectious Diseases Division and Clinical Microbiology, Clínica Universidad de Navarra,
18 Pamplona, Spain.
19 7
Julia McFarlane Diabetes Research Centre (JMDRC) and Department of Microbiology,
20 Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, Cumming School
21 of Medicine, University of Calgary, Alberta, T2N 4N1 Canada.
22 8
Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique.
23 9
International Health Department, Hospital Clinic, Universitat de Barcelona, Spain.
24
25 *Contributed equally: the order of names was determined considering that CD supervised the
26 lab work and wrote the first manuscript draft and MV performed the assay development.
27 Corresponding authors: Carlota.dobano@isglobal.org; Gemma.moncunill@isglobal.org

28 ABSTRACT
29 Reliable serological tests are required to determine the prevalence of antibodies against
30 SARS-CoV-2 and to characterise immunity to the disease in order to address key knowledge
31 gaps in the COVID-19 pandemic. Quantitative suspension array technology (qSAT) assays
32 based on the xMAP Luminex platform overcome the limitations of rapid diagnostic tests and
33 ELISA with their higher precision, dynamic range, throughput, miniaturization, cost-efficacy

34 and multiplexing capacity. We developed three qSAT assays for IgM, IgA and IgG to a panel
35 of eight SARS-CoV-2 antigens including spike (S), nucleoprotein (N) and membrane (M)
36 protein constructs. The assays were optimized to minimize processing time and maximize
37 signal to noise ratio. We evaluated their performance using 128 pre-pandemic plasmas
38 (negative controls) and 104 plasmas from individuals with SARS-CoV-2 diagnosis (positive
39 controls), of whom 5 were asymptomatic, 51 had mild symptoms and 48 were hospitalized.
40 Pre-existing IgG antibodies recognizing N, M and S proteins were detected in negative
41 controls suggestive of cross-reactive to common cold coronaviruses. The best performing
42 antibody/antigen signatures had specificities of 100% and sensitivities of 95.78% at ≥14 days
43 and 95.65% at ≥21 days since the onset of symptoms, with AUC of 0.977 and 0.999,
44 respectively. Combining multiple markers as assessed by qSAT assays has the highest
45 efficiency, breadth and versatility to accurately detect low-level antibody responses for
46 obtaining reliable data on prevalence of exposure to novel pathogens in a population. Our
47 assays will allow gaining insights into antibody correlates of immunity and their kinetics,
48 required for vaccine development to combat the COVID-19 pandemic.
49
50 Keywords
51 Antibody; immunoassay; SARS-CoV-2; COVID-19; IgM; IgA; IgG; Luminex; quantitative
52 suspension array technology; multiplex; spike; RBD; nucleocapsid; coronavirus; sensitivity;
53 specificity.
54
55 INTRODUCTION
56 In a globalized world where emerging infectious diseases of broad distribution can put at
57 stake the health and economy of millions of people, there is a need for versatile and reliable
58 serological tools that can be readily applicable (i) to determine the seroprevalence of
59 antibodies against any new pathogen and, more importantly, (ii) to characterise the immune
60 response to the disease at the individual and community levels. In the case of the COVID-19

61 pandemic caused by SARS-CoV-2, one of the main priorities since the beginning of the
62 epidemics in China by the end of 2019 (1) was to ascertain the percentage of the population
63 that had been exposed to the virus, considering that a substantial number of people could
64 have been asymptomatic (2)(3). The lack of sensitive and specific serological tests early in
65 the COVID-19 pandemic delayed the precise estimation of the burden of infection for the
66 rational implementation of public health measures to control viral spread (4). Furthermore,
67 immunological assays that can measure a high breadth of antibody types and specificities
68 are needed to dissect which are the naturally acquired protective responses and identify
69 correlates of immunity (5). Additionally, when a vaccine becomes available, such assays
70 would be valuable to evaluate immunogenicity of candidate vaccines and monitor duration of
71 immunity at the population level (6).
72 Common tools for antibody studies are (i) rapid diagnostic tests (RDT) as point of care (POC)
73 devices that usually measure either total immunoglobulins or IgG and IgM, qualitatively (7),
74 (ii) traditional enzyme-linked immunosorbent assays (ELISA) (8) that can quantify different
75 isotypes and subclasses of antibodies against single antigens at a time, and that require
76 certain previous expertise, personnel and equipment, and (iii) chemiluminescent assays
77 (CLIA), widely used in clinical practice, faster and with higher throughput than ELISA (9). The
78 performance of the best assays externally validated with >100 negative and >100 positive
79 samples (as recommended by the Foundation for Innovative New Diagnostics [FIND]),
80 mostly from symptomatic patients who are known to have higher antibody levels, ranges
81 79.7-83.1% sensitivity and 99.1-100% specificity for RDTs, 7 days post-symptoms onset
82 (10); 93.0-98.3% sensitivity and 86.3-100% specificity for ELISAs (Euroimmune, Roche); and
83 87.7-99.1% sensitivity and 96.1-100% specificity for CLIA (Siemens, DiaSorin, Abbot), 14
84 days post-symptom onset (11)(12)(13)(14).
85 A number of in-house ELISA assays have also been developed in hospital and research
86 laboratories (15), but they have the limitations that (i) a relatively large amount of sample is
87 required, (ii) the large surface area of the individual microplate wells and the hydrophobic
88 binding of capture antibody can lead to non-specific binding and increased background, and

89 (iii) most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve
90 reasonable sensitivity (16).
91 An alternative technique that offers the benefits of ELISA but also a larger dynamic range of
92 antibody quantification and higher sensitivity (16)(17) is based on the xMAP Luminex®
93 platform (www.luminexcorp.com/bibliography). Secondary antibodies are labelled with
94 fluorescent phycoerythrin (PE) directly or with biotin that mediates binding to streptavidin-R-
95 phycoerythrin (SAPE), which does not depend on an additional colorimetric reaction. The
96 technique has the added value of higher throughput (up to 384-well plate format), increased
97 flexibility, and lower cost with the same workflow as ELISA, particularly if using magnetic
98 MagPlex® microspheres. Paramagnetic beads allow for automation of workflow and better
99 reproducibility compared to the previous generation of MicroPlex® microspheres. Since the
100 beads have the capture antigen covalently-bound and immobilized on their much smaller
101 surface area compared to passive coating on a 96-well microplate well, reduced sample
102 volumes are required and non-specific binding is diminished (18). Furthermore, a chief
103 advantage over ELISA is the multiplex nature of the assay that allows measuring antibodies
104 to different antigens simultaneously. This increases the probabilities to detect a positive
105 antibody response due to the heterogeneity of the human response and therefore it has a
106 higher sensitivity relevant for identifying seropositive individuals. The Luminex technology,
107 capable of measuring simultaneously antibodies against 50 (MAGPIX®), 80 (Luminex
108 100/200®) and up to 500 different antigens (FlexMap3D®), makes it an invaluable tool for
109 antigen and epitope screening. Finally, its versatility to set up adapted antigen panels makes
110 Luminex an excellent platform to ensure better preparedness for faster response to future
111 emerging diseases and pandemics.
112 Here we report on the establishment and validation of three quantitative suspension array
113 technology (qSAT) assays to measure IgM, IgA and IgG antibodies against eight SARS-CoV-
114 2 antigens, based on the adaptation of previous in-house protocols that measured antibodies
115 to other infectious diseases, including malaria (19)(20)(21)(22). Due to the need to process a

116 large amount of samples with the minimal time and cost during a pandemic like the COVID-
117 19, we optimized several conditions to reduce the duration of the assays and report them
118 here for the three main isotypes that have proved useful for seroprevalence studies(23).
119
120 METHODS
121
122 Samples
123 Positive samples were 104 plasmas from individuals with a SARS-CoV-2 infection confirmed
124 by real time reverse-transcriptase polymerase chain reaction (rRT-PCR). Fifty-five were
125 recruited in a study of health care workers in Hospital Clínic in Barcelona, most of them with
126 mild symptoms, 1 of them hospitalized and 5 without symptoms(23). Forty-nine COVID-19
127 patients recruited at the Clínica Universidad de Navarra in Pamplona (Spain), of which 47
128 had severe symptoms and were hospitalized and 2 had mild symptoms. Time since onset of
129 symptoms ranged from 0 to 46 days. Positive samples were used as pools of up to 20
130 sample subsets for assay optimization tests, and individually for the assays evaluation.
131 Negative controls were plasmas from 128 healthy European donors collected before the
132 COVID-19 pandemic, and were used individually. Numbers of positive and negative samples
133 were in line with protocol recommendations from FIND.
134 Ethics. Samples analyzed in this study received ethical clearance for immunological
135 evaluation and/or inclusion as controls in immunoassays, and the protocols and informed
136 consent forms were approved by the Institutional Review Board (IRB) at HCB (Refs. CEIC-
137 7455 and HCB/2020/0336) or Universidad de Navarra (Ref. UN/2020/067) prior to study
138 implementation.
139
140 Antigens
141 The Receptor-Binding Domain (RBD) of the spike (S) glycoprotein of SARS-CoV-2, the
142 leading vaccine candidate target, was selected as the primary antigen to develop the initial

143 qSAT assay because (i) S is one of the most immunogenic surface proteins together with the
144 nucleocapsid protein (N) (24) (ii) RBD is the fragment of the virus that mediates binding to
145 the host receptor ACE2 in the lung cells (25) (iii) antibodies to RBD correlate with neutralizing
146 antibodies (24)(22) that could be associated with protection based on studies of other
147 coronaviruses and animal models (27–30), and (iv) an ELISA based on this same protein has
148 received FDA approval for COVID-19 serology (15). The RBD was from the Krammer lab
149 (Mount Sinai, New York, USA) (15) and the S antigen was produced in-house using Chinese
150 Hamster Ovary (CHO) cells transiently-transfected with a plasmid from the Krammer lab (15)
151 followed by purification of the recombinant protein from 4-day culture supernatants using
152 nickel affinity chromatography. The multiplex antigen panel was completed with commercial
153 S1 (GenScript Biotech, Netherlands) and S2 (Sino Biologicals, Germany) proteins, and in-
154 house produced nucleocapsid (N, full-length, N-terminal and C-terminal) and membrane (M,
155 full-length) recombinant proteins (Supplementary methods).
156
157 qSAT assays
158 The optimal concentration of protein to be coupled to beads depends on the antigen and
159 needs to be tested with each new lot. The protein antigens (10, 30 and 50 g/mL) were
160 coupled to magnetic MAGPLEX 6.5 μm COOH-microspheres from Luminex Corporation
161 (Austin, TX) in reactions of a maximum of 625,000 beads, at 10,000 beads/µl (19), and left at
162 4ºC overnight (ON) on a rotatory shaker protected from light. After blocking, washing and
163 counting, antigen-coupled beads were validated by incubating them with serial dilutions of
164 anti-histidine-Biotin antibody for antigens with a histidine-tag (Abcam, ab27025). Further
165 protocol details are provided in Supplementary Methods. Appropriate coupling
166 concentrations for maximum antibody signal were established by titrating IgG and IgM levels
167 in 3-fold 11 serial dilution curves of a pool of 20 positive samples. Batches of coupled beads
168 were stored multiplexed at 2000 beads/μL PBS-BN (phosphate buffered saline [Sigma] + 1%
169 bovine serum albumin [BSA, Biowest] + 0.05% sodium azide [Sigma, S8032]) at 4°C
170 protected from light till use.

171 We compared the performance of the assays when samples were incubated with the
172 antigen-beads for 1 h or 2 h at room temperature (RT) in relation to our previous protocol ON
173 at 4ºC. Two thousand antigen-coupled beads in 90 μL PBS-BN were added per well to a 96-
174 well μClear® flat bottom plate (Greiner Bio-One, 655096), together with individual positive
175 and individual negative plasma controls (range of dilutions 1/100 to 1/5000) at a final volume
176 of 100 μl per well. Two blank control wells with beads in PBS-BN were set up in each plate to
177 control for background signal. Plates were incubated on a microplate shaker at 600 rpm and
178 protected from light, and then washed three times with 200 μl/well of PBS-Tween20 0.05%,
179 using a magnetic manual washer (Millipore, 43-285). For more accurate IgM measurements,
180 we tested whether diluting samples 1:10 with GullSORB™ IgG Inactivation Reagent
181 (Meridian Bioscience™) prior to testing for IgM levels could reduce high responses observed
182 in some negative samples (31). Additionally, we tested the levels of RBD and S antibodies at
183 different plasma dilutions when incubated in a multiplex panel with additional antigen-beads
184 including S1, S2, M and N constructs, compared to those obtained in singleplex, to check for
185 potential interferences. Definitive plasma dilutions were established with titration experiments
186 in individual positive and negative samples once the final multiplex panel and all assay
187 conditions were selected to account for the different immunogenicities of the viral antigens.
188 For the secondary antibody reaction, we compared the performance of biotinylated
189 antibodies followed by SAPE, versus antibodies conjugated directly to PE, and at different
190 incubation times (45 versus 30 min). In all cases, each new lot of secondary antibody was
191 titrated for selecting the optimal concentration. For the first option, 100 μL of biotinylated
192 secondary antibody diluted in PBS-BN (anti-human IgG, B1140, 1/1250; anti-human IgM,
193 B1265, 1/1000; or anti-human IgA, SAB3701227, 1/500; Sigma) were added to all wells and
194 incubated for 45 min at 600 rpm at RT and protected from light. Plates were washed three
195 times, and 100 μL of SAPE (Sigma, 42250) diluted 1:1000 in PBS-BN were added and
196 incubated during 30 min at 600 rpm, RT and protected from light. For the second option, 100
197 μL of PE-secondary antibody diluted in PBS-BN (goat anti-human IgG, GTIG-001, 1/400;
198 goat anti-human IgM, GTIM-001, 1/200; or goat anti-human IgA, GTIA-001, 1/200; Moss,

199 MD, USA) were added to all wells and incubated for 45 or 30 min at 600 rpm at RT and
200 protected from light.
201 Before reading, plates were washed three times, beads resuspended in 100 μl of PBS-BN,
202 and data acquired using a Luminex® 100/200 analyzer with 70 μl of acquisition volume per
203 well, DD gating 5000-25000 settings, and high PMT option. Plates could also be kept ON at
204 4ºC, protected from light, and read the next day. At least 50 beads were acquired per antigen
205 and sample. Crude median fluorescent intensities (MFI) were exported using the xPONENT
206 software. Seropositivity threshold (cutoff) was calculated as 10 to the mean plus 3 standard
207 deviations of log10-transformed MFIs of the negative controls for each antibody isotype and
208 antigen.
209
210 Performance of the SARS-CoV-2 qSAT assays
211 The receiver operating characteristic (ROC) curves, their corresponding area under the curve
212 (AUC), and the specificity and sensitivity of the IgM, IgA and IgG assays, were established
213 testing all 104 positive samples from participants diagnosed with SARS-CoV-2 infection,
214 regardless of symptoms information and at different periods since the onset of symptoms (7,
215 14, 21 and 28 days), and 128 negative samples. For IgM, IgA and IgG assays the multiplex
216 panel including RBD, S, S1, S2, M and N antigen constructs was used, following the same
217 procedures as indicated above and after selecting the optimal assay conditions: samples at
218 1/500 or 1/3500 dilutions were incubated with antigen-coupled beads for 1 h followed by 30
219 min incubation with secondary antibodies conjugated directly to PE.
220
221 Data analysis
222 ROC curves and AUC, sensitivities and specificities were calculated using the predicted
223 values estimated by supervised machine learning Random Forest (RF) algorithm models. A
224 classification RF algorithm is a supervised machine learning method that is able to predict
225 the class of an observation based on the knowledge it has previously acquired from the
226 training data. By seeing many observations, with the value of each predictor variable and the

227 classification outcome, the algorithm is trained and is then able to classify a previously
228 unseen observation. It will do so based on the majority of votes from all the classification
229 trees that make up the forest. IgM, IgA and IgG MFIs to the different antigens or their
230 combinations were the predictors, and the outcome was SARS-CoV-2 positivity or negativity
231 determined by rRT-PCR. Antibody/antigen pairs that did not discriminate between positive
232 and negative samples were excluded from the analysis. Then the antibody/antigen variables
233 were further down-selected using an RF algorithm including all negative and positive controls
234 (N=232) or all negative controls plus positive controls corresponding to each different period
235 since onset of symptoms: ≥7 days (N=213), ≥14 days (N=199), ≥21 days (N=174), ≥28 days
236 (N=152).
237 Besides classifying, RFs also rank predictor variables according to their importance to
238 explain the outcome variable. The ranking is based on two criteria: Mean Decrease Accuracy
239 and Mean Decrease Gini. Mean Decrease Accuracy refers to the accuracy that is lost by
240 removing each predictor from the model. Mean Decrease Gini is based on the Gini impurity,
241 which measures how often a randomly chosen observation from the data set used to train
242 the model will be incorrectly classified. Using an important variable to classify the data entails
243 a high decrease in node impurity and, therefore, a high Mean Decrease in Gini.
244 Next, different RFs were built exploring all possible variable combinations at the different
245 periods since onset of symptoms based on the selected variables per period: all samples
246 (top 12 markers), ≥7 days (top 11 markers), ≥14 days (top 11 markers), ≥21 days (top 11
247 markers) and ≥28 days (top 11 markers). For each model, we calculated the AUC and
248 selected three seropositivity cutoffs aiming at specificities of i) 100%, ii) ≥ 99% and <100%,
249 and iii) ≥ 98% and <99%, and obtained the corresponding sensitivity. Models with 100%
250 specificity and the highest sensitivities were selected for ROC curve representations. The
251 analysis was carried out using the statistical software R studio version R-3.5.1 (32)
252 (packages used: randomForest (33) and pROC (34)).
253
254 RESULTS

255 The characteristics of SARS-CoV-2 infected participants whose plasma samples have been
256 used in the study, with regards to age, sex, days since rRT-PCR diagnosis and days since
257 onset of symptoms, are included in Table 1.
258
259 Optimization of assay conditions
260 Titration curves did not change substantially between 10, 30 or 50 g/mL protein, in which
261 case the lower concentration was chosen (Figure S1). We found that a 1/500-plasma dilution
262 was optimal for quantifying IgM, IgA and IgG antibodies to RBD in singleplex (Figure 1) and
263 it was used for subsequent optimizations. Our original standard operating procedures (SOP)
264 established for large sero-epidemiological and vaccine studies using Plasmodium falciparum
265 antigens were based on ON incubations of samples at 4ºC (20). For COVID-19 serology, we
266 prioritized having faster assays and thus compared the performance of ON incubations at
267 4ºC versus shorter times at RT. Reducing the incubation time to 2 h with the same plasma
268 dilutions did not change the percentage of IgG or IgA seropositive samples and it increased
269 sensitivity for IgM (Figure 2A). Although the MFI readings in positive samples generally
270 diminished, the MFI readings in the negative samples were also reduced, i.e. the signal to
271 noise ratio was the same or sometimes better, maintaining or increasing the overall
272 proportion of seropositive responses among the positive samples and thus the sensitivity.
273 We initially adopted the 2 h incubation time for a first COVID-19 seroprevalence study(23)
274 and subsequently tested shorter incubations, finding that 1 h was non-inferior to 2 h
275 incubation (Figure 2B). Treatment with GullSORB™ generally reduced the IgM reactivity in
276 negative controls and increased the signal to noise ratio, number of seropositive responses
277 and sensitivity, therefore this incubation was adopted for this isotype (Figure S2).
278 Multiplexing the antigens (8-plex panel) did not significantly decrease the MFI antibody levels
279 to RBD or S compared to singleplex testing (Figure 3A). Interestingly, there was no evidence
280 of any interference between RBD, S, S1 or S2 antigens despite sharing epitopes within the
281 same multiplex panel. A number of negative pre-pandemic samples had pre-existing

282 antibodies recognizing SARS-CoV-2 proteins for certain isotypes and dilutions: IgG to S1,
283 S2, M and N constructs, and IgA to S1 and N-term & C-term of N (Figure S2). Furthermore,
284 testing plasmas against multiple antigens increased the sensitivity of the assay since some
285 individuals who were seronegative or low responders to RBD, responded with higher
286 antibodies to S (Figure 3B).
287 One-step incubation with secondary antibodies conjugated to PE performed as well as a two-
288 step secondary antibody conjugated to biotin followed by SAPE incubation (Figure 4). A 30
289 min incubation with the PE-antibody reagent was non-inferior to a 45 min incubation (Figure
290 S3).
291
292 Sensitivity and specificity of the qSAT assays
293 We sought for the combination of immunoglobulin and antigen responses that yielded the
294 highest specificity (primarily), sensitivity and AUC to detect seropositive responses. To that
295 end, we built and assessed several RF models, each of them with all the negative controls
296 (N=128) plus one of the following: i) all the positive controls (N=104), ii) positive controls with
297 ≥ 7 days since the onset of symptoms (N=85), iii) positive controls with ≥14 days since the
298 onset of symptoms (N=71), iv) positive controls with ≥ 21 days since the onset of symptoms
299 (N=46), and v) positive controls at ≥ 28 days since the onset of symptoms (N=30).
300 Prior to fitting the RF models, exploratory dotplots were used to select antigens that could
301 discriminate well positive from negative controls and the best plasma dilution for each
302 antigen. M, S1 (IgG & IgA) and N N-term (IgM) were discarded because they had similar
303 responses in negative and positive controls. For RBD and S, samples at 1/500 dilution for
304 IgG and IgA, and samples at 1/3500 dilution for IgM, gave higher percentages of seropositive
305 responses among the positive controls and thus were selected for the calculations; for N
306 constructs, samples at 1/3500 dilution for IgG and IgA performed better except for N C-term
307 in which IgG was better for samples at 1/500 dilution.
308 The importance of the predictor variables to explain the outcome variable was ranked by the

309 RFs according to the Mean Decrease Accuracy and the Mean Decrease Gini. We down-
310 selected the top 11-12 antibody/antigen variables that entailed a bigger loss of mean
311 accuracy or Gini impurity in the RF algorithms, which were computed using positive samples
312 from different periods since onset of symptoms plus the negative controls (Figure S4). Then,
313 we ran a RF for each possible combination of the down-selected top variables per each
314 different period since onset of symptoms. The resulting RFs were ranked and selected
315 prioritizing three high specificity targets (100%, 99% and 98%). Within each specificity target,
316 the RFs were then sorted by sensitivity and AUC. When targeting a specificity of 100%, the
317 sensitivity of the qSAT assays in samples from participants with SARS-CoV-2 positive
318 diagnosis with ≥14 days since the onset of symptoms was up to 95.78%, and AUC up to
319 0.977, for the best combinations of antibody/antigen. The top 3 performing antibody/antigen
320 signatures for the three different seropositivity thresholds targeting specificities of 100%, 99%
321 and 98% are shown in Table 2, and their ROC curves with the corresponding AUC are
322 shown in Figure 5. High AUCs mean high specificity and high sensitivity and, therefore,
323 more predictive capacity of the model. For a set specificity of 100%, the sensitivity in
324 samples from participants with ≥21 days since the onset of symptoms was up to 95.65%,
325 with AUC up to 0.999 for the best combinations of antibody/antigen. Using samples from all
326 participants regardless of time since symptoms onset, the sensitivity was up to 80.77%, with
327 AUC up to 0.925 for the best combinations. The performance of the qSAT assays to predict
328 positivity was clearly superior using combinations of multiple antibody/antigens to using
329 single antibody/antigen markers (Figure 5), as higher AUCs with higher sensitivities for a
330 given specificity target were obtained in the first case, for all different periods since onset of
331 symptoms. Higher sensitivities were obtained when specificities were set to 98% or 99%
332 compared to 100% (Table 2), reaching sensitivities of 100% for samples ≥21 or ≥28 days
333 since the onset of symptoms.
334
335 DISCUSSION
336 We developed three novel multiplex immunoassays for quantifying IgM, IgA and IgG to eight

337 SARS-CoV-2 protein constructs and evaluated, by machine learning classification
338 algorithms, the performance of several antibody/antigen combinations to detect any positive
339 antibody response to infection, obtaining specificities of 100% and sensitivities of 95.78%
340 (≥14 days since symptoms onset) or 95.65% (≥21 days since symptoms onset), and very
341 high predictability (AUC ≥0.98). Our qSAT assays, based on the xMAP technology, provide
342 an excellent precision, accuracy and wide range of detection.
343 For any given test, there is usually a trade-off between sensitivity and specificity. To evaluate
344 the performance of the assays here, we prioritized specificity over sensitivity for the impact
345 that specificity has in seroprevalence studies. Particularly when prevalence of infection is
346 low, the positive predictive value of a test strongly relies on a high specificity. For example, in
347 a scenario of 5% prevalence, a decrease in specificity from 100% to 95% would cause a 5%
348 of negatives wrongly classified as positives, giving a false prevalence of 10%, the double of
349 reality (50% positive predictive value). In a scenario of 20% prevalence, a 95% specificity
350 would estimate a prevalence of 24% (83% positive predictive value). The lower the
351 prevalence, the higher the impact of a low specificity. For this reason, we highlight the
352 importance of targeting high specificities when dealing with low prevalence of infection (35).
353 However, other seropositivity thresholds can be used to have a balanced
354 specificity/sensitivity or to maximise sensitivity. For studies aiming at characterizing kinetics
355 of antibody responses, immunogenicity of vaccine candidates or correlates of protection a
356 cutoff prioritizing sensitivity over specificity would be preferable.
357 A time period after the onset of symptoms is usually established for these analyses, because
358 antibodies take an average 4-14 days since infection to be produced and detected
359 depending on the isotype and test (IgM 5-12 days, IgA 5-12 days, IgG 4-14 days)
360 (36)(37)(38)(39)(40)(41). Thus, it is not necessarily expected to detect antibodies in
361 individuals who are acutely infected and diagnosed around the time of plasma collection.
362 Accordingly, when considering all samples, which included 13 individuals with less than 6
363 days since onset of symptoms plus 1 symptomatic without information of days since onset of
364 symptoms and the 5 asymptomatic individuals, sensitivity was lower (up to 80.77%) at

365 specificities of 100%. However, we detected IgM or IgA as early as 2 days from onset of
366 symptoms. In fact, since samples were collected in the early days of the COVID-19
367 pandemic, it is expected that IgM and IgA, which are induced upon primary infection earlier
368 than IgG, could contribute to a higher sensitivity of detection. Most of the best signatures
369 identified included IgM and IgA besides IgG, regardless of the time period since onset of
370 symptoms, also beyond 28 days. However, over time, the only antibodies that would be
371 expected to remain in blood are IgG due to the decay of IgM and IgA, e.g. IgM levels may
372 become undetectable by the fifth week after symptoms onset (42). Therefore, with longer
373 days since infection, the serological assays to detect maintenance of antibodies could focus
374 on IgG detection. For diagnostic early in the disease we recommend using the combinations
375 of antibody/antigen that achieve a cutoff that results in 100% specificity with the highest
376 possible sensitivity and AUC when fitting an RF algorithm with all the negative plus positive
377 controls. Specificity would also prevail over sensitivity for the implications that a false positive
378 may have at a personal level. However, antibody assays are not able to discern current from
379 recent infection and, as mentioned, production and detection of antibodies are delayed from
380 the onset of infection.
381 The sensitivities and specificities of other SARS-CoV-2 Luminex assays evaluated with >100
382 positive and >100 negative samples, have been shown to be comparable to ours. A study
383 with sera from asymptomatic to hospitalized patients, measuring IgG and IgA to several
384 peptides derived from S and N, showed a 99% sensitivity and 98% specificity (43). Another
385 study with samples from outpatients and hospitalized cases measuring IgG to S1, RBD and
386 N reported a 90% sensitivity and 100% specificity (44). A third study with samples from
387 health care workers and hospitalized patients measuring IgG and IgM to S trimer, RBD, S1
388 and S2 estimated a 96% sensitivity and 99% specificity (45).
389 A key value of Luminex compared to ELISA is multiplexing, which allows to capture a wider
390 breadth of responses and this is needed because some individuals may not respond to one
391 antigen (e.g. RBD) but may do so to other antigens (e.g. S or N proteins) (46)(47)(48), or
392 responses may change in time. Here, we substantially increased the sensitivity of the assay

393 when combining antibodies/antigens compared to using only one. An added advantage of
394 multiplexing is the reduced usage of sample volume, resources and time, if antibodies to
395 several antigens are to be evaluated. The possibility to perform miniaturized assays in small
396 amounts of blood is very attractive in paediatric studies, in large field surveys where
397 fingerpick may be more logistically feasible, and to test special tissues of interest including
398 mucosal fluids. Those combined advantages have a direct impact on the cost-efficacy of the
399 qSAT assay, that can be less than one-fifth of the least expensive commercial ELISA and
400 less than one-sixteenth of the most expensive commercial kit. Cost is reduced because there
401 is less protein used due to the smaller surface area and less amounts of other materials and
402 reagents. Compared to the commercial xMAP® SARS-CoV-2 Multi-Antigen IgG assay, our
403 assay includes 2 additional isotypes and 5 additional antigens, allowing a better study of the
404 immune response and likely being more sensitive. We reduced the dilutions of plasma and
405 titrated the secondary antibody to use the minimal amounts of samples and reagents, without
406 compromising sensitivity. The economy of scale is improved further when the assays are
407 adapted to high throughput FlexMap3D 384-well plate format but they are also easily
408 adaptable to the bench top MagPix 96-well format that is more affordable and simpler to
409 maintain even in remote laboratory settings.
410 Positive responses to M, S1, S2 and N antigens were detected in samples collected before
411 the COVID-19 pandemic. The presence of such IgGs has been interpreted as cross-reactivity
412 with human coronaviruses causing the common cold (49)(50)(51). Indeed, higher sequence
413 homology at the protein level between SARS-CoV-2 and huCoV has been reported for N
414 (particularly N-terminal and central regions), M and S2 (49)(52)(53). The addition of a
415 commercial S1 did not have any added value and future versions of the assay will test S1
416 from different sources, as this subunit is expected to not cross-react with other -
417 coronaviruses and be specific for SARS-CoV-2 diagnostics (15)(52). Pre-existing SARS-
418 CoV-2-specific T cells have also been reported and attributed to cross-reactivity with huCoV
419 previously encountered (54)(55). The multiplex nature of the assay will allow to test this
420 hypothesis in the future with the addition of antigens to related huCoV 229E, HKU1, NL63

421 and OC43 in the same assay panel, by comparing the patterns of antibody reactivity, to
422 address the significance of this in immunity to COVID-19.
423 The assays performances were excellent but further testing needs to be performed with
424 longer periods of time since onset of symptoms, although we do expect to maintain high
425 specificity and sensitivity albeit antibody signatures would be different and based on IgG
426 only. Future studies will include additional positive samples of asymptomatic individuals, who
427 probably have lower antibody levels than mild or severe cases and are rarely included in the
428 validation of commercial kits. In addition, it will be interesting to include negative controls
429 reacting with other coronaviruses or other infections (e.g. malaria) and pathologies known to
430 induce polyclonal responses or rheumatoid factor, which may increase background
431 responses.
432 In conclusion, we developed 100% specific and fast assays with optimal diagnostic
433 characteristics to assess seroprevalence of COVID-19. Considering their high sensitivity,
434 these qSAT assays would be suited to identify individuals with low levels of antibodies, such
435 as asymptomatic children or immunosuppressed individuals, or long-term decaying
436 antibodies (56). In addition this approach would be particularly suited to identify hyper
437 immune donors with very high levels of antibodies and the largest antigenic breadth for
438 immunotherapy. The assays are highly versatile, being easily adaptable to quantify other
439 antibody IgG and IgA subclasses and avidity with the use of chaotropic agents, and even
440 functional activity like binding inhibition to the virus receptor ACE2. The multiplex capabilities
441 make them also ideal for sizeable peptide screenings to accelerate epitope mapping and
442 selection for identifying fine-specificity of immune correlates of protection for vaccine
443 development, and would also be applicable in vaccine evaluation when the first candidates
444 reach larger-scale phase 3 clinical trials.
445
446 Acknowledgements
447 We thank the volunteers who donated blood for COVID-19 studies and the clinical and
448 laboratory staff who participated in the sample collection and processing. Special thanks to

449 ISGlobal colleagues P. Cisteró, R.A. Mitchell, C. Jairoce, S. Alonso, J. Moreno, L. Puyol, C.
450 Chaccour, and those involved in data analysis and/or recruitment of volunteers at the
451 hospital, J.L. del Pozo, M. Fernández, M. Tortajada, C. Guinovart, S. Sanz, S. Méndez, A.
452 Llupià, E. Chóliz, A. Cruz, S. Folchs, M. Lamoglia, N. Ortega, N. Pey, M. Ribes, N. Rosell, P.
453 Sotomayor, S. Torres, S. Williams, S. Barroso, A. Trilla and P. Varela. We are grateful to F.
454 Krammer for donation of RBD and S plasmids, to L. Mayer for assistance with literature
455 review, and to Wilco de Jager from Luminex for technical advice.
456
457 Funding
458 The assays development and sample collection were performed with internal funds from the
459 investigators groups and institutions, and the performance analysis received support from
460 FIND. GM had the support of the Department of Health, Catalan Government
461 (SLT006/17/00109). JC is supported by SAF2016-76080-R grant from the Spanish Ministry
462 of Economy (AEI/FEDER, UE) to LI. Development of SARS-CoV-2 reagents was partially
463 supported by the NIAID Centers of Excellence for Influenza Research and Surveillance
464 (CEIRS) contract HHSN272201400008C. We acknowledge support from the Spanish
465 Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-
466 2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya
467 through the CERCA Program.
468
469 FIGURE LEGENDS
470
471 Figure 1. Levels (median fluorescence intensity, MFI) of IgM, IgA and IgG antibodies to our
472 primary SARS-CoV-2 antigen (RBD) in singleplex, using samples from positive and negative
473 individuals at different dilutions (1/100, 1/500, 1/2000, 1/5000) after overnight incubation at
474 4ºC. This served to establish the optimal range of plasma dilutions and to choose 1/500 for

475 the subsequent assay optimization experiments.
476
477 Figure 2. Levels of IgM, IgA and IgG antibodies (median fluorescence intensity, MFI) to RBD
478 antigen of SARS-CoV-2 in positive and negative plasmas (1/500 dilution) comparing
479 incubating overnight (ON) at 4ºC versus 2 h at room temperature (RT) (A), and 2 h versus 1
480 h at RT with two different secondary antibodies (B). In A, dashed lines indicate cutoff values;
481 the number and percentage of seropositive samples within rRT-PCR+ are shown at the top
482 of the dot plots. In B, the blue fitting curve was calculated using the LOESS (locally estimated
483 scatterplot smoothing) method and the black line by linear regression. Spearman test was
484 used to assess the correlations. Biotin-SAPE refers to secondary antibodies conjugated to
485 biotin and streptavidin-phycoerythrin (SAPE) and PE refers to secondary antibodies
486 conjugated with phycoerythrin. NC, negative controls; TS, test samples.
487
488 Figure 3. Levels of plasma IgM, IgA and IgG antibodies to the SARS-CoV-2 primary
489 antigens spike (S) and receptor binding domain (RBD) at different dilutions. A) Comparison
490 of antibody levels (MFI) in singleplex (orange) versus multiplex (burgundy); the first 10
491 samples from left to right are from individuals who were positive by rRT-PCR at different time
492 periods since diagnosis, and the last two samples on the right are from individuals pre-
493 COVID-19 pandemic. B) Correlation of IgG, IgM and IgA antibody levels against RBD versus
494 S at different dilutions showing the benefit of including multiple antigens in the panel to
495 maximize the detection of seropositives. Cutoff values are indicated by dashed lines.
496 Spearman test was used to assess the correlations. NC, negative controls; TS, test samples.
497
498 Figure 4. Antibody levels to RBD using different secondary antibodies and sample
499 incubations times. A) Levels of IgM, IgA and IgG antibodies (median fluorescence intensity,
500 MFI) and % seropositivity to RBD among positive controls (burgundy), comparing secondary
501 antibodies conjugated to biotin and streptavidin-phycoerythrin (SAPE) versus PE. Negative
502 controls in orange. B) Correlations between antibody levels measured using secondary

503 antibodies conjugated to biotin and SAPE versus PE, for 1 h and 2 h sample incubations.
504 Seropositivity cutoff values are indicated by dashed lines. The number and percentage of
505 seropositive samples among rRT-PCR+ is shown at the top of the dot plots. In B, the blue
506 fitting curve was calculated using the LOESS (locally estimated scatterplot smoothing)
507 method and the black line by linear regression. Spearman test was used to assess the
508 correlations.
509
510 Figure 5. Antibody Luminex assays performance. Receiver operating characteristic
511 (ROC) curve and area under the curve (AUC) using samples from pre-pandemic negative
512 controls plus either all participants with positive COVID-19 diagnosis or participants with
513 positive diagnosis at different times since onset of symptoms. ROC curves and AUC from
514 different combinations of multiple immunoglobulin isotypes to different antigens with top
515 performances are included in (A) whereas those of single isotype/antibody markers are
516 included in (B).
517
518
519 TABLES
520
521 Table 1. Characteristics of individuals from whom positive samples were tested with regards
522 to age, sex, symptoms, days since symptoms onset, and days since rRT-PCR diagnosis.
523
Continuous variable Median (IQR)

Age 50 (24.73)
Categorical variables Category N (%)
Female 61 (58.65)
Sex
Male 43 (41.35)
No 5 (4.81)
Symptoms
Yes 99 (95.19)
Hospitalized patients Yes 48 (46.15)
0-6 13 (13.13)
7-13 14 (14.14)
Days since onset of 14-20 25 (25.25)
symptoms 21-27 22 (22.22)
≥28 24 (24.24)
No information 1 (1.01)
0-6 22 (21.15)
7-13 24 (23.08)
Days since first positive 14-20 29 (27.88)
rRT-PCR ≥21 17 (16.35)
After sample collection 5 (4.81)
Not available 7 (6.73)
524
525
526 Table 2. Sensitivity and specificity of the qSAT antibody assays for negative controls
527 plus either all positive samples or positive samples at different times since onset of
528 symptoms and different thresholds targeting specificities of 100%, 99% and 98%. The top 3
529 performing signatures per each category are shown. AUC = Area under the curve.
Antibody/antigen combinations AUC Specificity Sensitivity
All samples

IgG N + IgM RBD + IgA RBD + IgM S2 0.925 100% 80.77%
IgG N + IgM RBD + IgA RBD + IgM S2 + IgG N Ct 0.922 100% 80.77%
IgG N + IgA S2 + IgM RBD + IgM S + IgM S2 + IgA N 0.919 100% 80.77%
IgG N + IgA S2 + IgM RBD 0.920 99.22% 82.69%
IgG N + IgA S2 + IgM RBD + IgG S + IgA N + IgA S 0.919 99.22% 82.69%
IgG N + IgM RBD + IgA S 0.917 99.22% 82.69%
IgG N + IgA S2 + IgM RBD 0.920 98.44% 82.69%
IgG N + IgA S2 + IgM RBD + IgG S + IgA S 0.919 98.44% 82.69%
IgG N + IgA S2 + IgM RBD + IgG S + IgA N + IgA S 0.918 98.44% 82.69%
≥7 days since onset symptoms
IgA S2 + IgM S + IgA RBD 0.975 100% 92.94%
IgA S2 + IgM S 0.975 100% 92.94%
IgA S2 + IgM S + IgM S2 + IgA RBD 0.971 100% 92.94%
IgA S2 + IgG S2 + IgM S + IgA RBD 0.981 99.22% 92.94%
IgM RBD + IgA S2 + IgG S + IgM S + IgM S2 + IgA RBD 0.980 99.22% 92.94%
IgM RBD + IgA S2 + IgG S 0.980 99.22% 92.94%
IgA S2 + IgG S + IgM S 0.983 98.44% 92.94%
IgG S2 + IgM S + IgA RBD 0.983 98.44% 92.94%
IgA S2 + IgG S2 + IgM S + IgA RBD 0.981 98.44% 92.94%
IgM RBD + IgG N + IgA S 0.977 100% 95.78%
IgA S2 + IgM S + IgG N + IgG N Ct + IgM S2 0.975 100% 95.78%
IgA S2 + IgG N + IgM S2 0.975 100% 95.78%

IgM RBD + IgG S + IgM S + IgG N 0.992 99.22% 95.78%
IgM RBD + IgG S + IgA S2 + IgM S + IgG N + IgG RBD + IgG N Ct 0.992 99.22% 95.78%
IgM RBD + IgG S + IgA S2 + IgG N + IgM S2 0.991 99.22% 95.78%
IgG S + IgG RBD + IgG N Ct + IgM S2 0.985 98.44% 97.18%
IgG S + IgG RBD 0.984 98.44% 97.18%
IgG S + IgA S2 + IgG RBD + IgM S2 + IgA RBD 0.983 98.44% 97.18%

IgG S + IgM RBD + IgG N + IgM S + IgM S2 0.999 100% 95.65%
IgM RBD + IgG N Ct + IgM S2 + IgA RBD 0.995 100% 95.65%
IgM RBD + IgM S2 + IgA S 0.992 100% 95.65%
IgG S + IgG RBD + IgM RBD 0.999 99.22% 97.83%
IgG S + IgG RBD + IgM RBD + IgG N + IgM S2 0.999 99.22% 97.83%
IgG S + IgG RBD + IgM RBD + IgG N + IgM S2 + IgA RBD 0.999 99.22% 97.83%
IgG S + IgG RBD + IgM RBD 0.999 98.44% 100%
IgG S + IgA S2 + IgG RBD + IgM RBD + IgM S2 + IgA RBD + IgA S 0.999 98.44% 100%
IgG S + IgA S2 + IgM RBD + IgM S2 + IgA RBD 0.999 98.44% 100%
IgA S2 + IgG N + IgM S2 0.9997 100% 95.83%
IgA S2 + IgM S + IgG N Ct + IgM RBD + IgM S2 0.9997 100% 95.83%
IgG N + IgG N Ct + IgM RBD 0.9997 100% 95.83%
IgA S2 + IgG N + IgM S2 0.9997 99.22% 100%
IgA S2 + IgM S + IgG N Ct + IgM RBD + IgM S2 0.9997 99.22% 100%
IgG N + IgG N Ct + IgM RBD 0.9997 99.22% 100%
IgA S2 + IgG N + IgM S2 0.9997 98.44% 100%
IgA S2 + IgM S + IgG N Ct + IgM RBD + IgM S2 0.9997 98.44% 100%
IgG N + IgG N Ct + IgM RBD 0.9997 98.44% 100%
530
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728
Highly sensitive and specific multiplex antibody assays to quantify
immunoglobulins M, A and G against SARS-CoV-2 antigens
Carlota Dobaño1,2*, Marta Vidal1*, Rebeca Santano1, Alfons Jiménez1,2, Jordi Chi1, Diana
Barrios1, Gemma Ruiz-Olalla1, Natalia Rodrigo Melero3, Carlo Carolis3, Daniel Parras4, Pau
Serra4, Paula Martínez de Aguirre5, Francisco Carmona-Torre6, Gabriel Reina5, Pere

Santamaria4,7, Alfredo Mayor1,2,8, Alberto García-Basteiro1,8,9, Luis Izquierdo1, Ruth Aguilar1,
Gemma Moncunill1
SUPPLEMENTARY MATERIAL
SUPPLEMENTARY METHODS
Production of recombinant proteins in Escherichia coli expression systems
Escherichia coli codon optimized versions of full-length N and M antigens were cloned at
ISGlobal into a pET22b expression vector, fusing an in-frame C-terminal 6xHis-tag.
Recombinant N and M proteins were expressed in E. coli BL21 DE3 by pET22b-N and

pET22b-M transformation and induction with 0.5 mM isopropyl-β-d-thiogalactopyranoside
(IPTG) when OD600 reached 0.6-0.8, followed by 5 h incubation at 37ºC or 25ºC, respectively.
Bacterial pellets were resuspended in binding buffer containing 20 mM sodium phosphate,
0.5 M NaCl, 20 mM imidazole, 0.2 mg/mL Lysozyme, 20 µg/mL DNAse, 1 mM PMSF and 1
mM MgCl2, and lysed by sonication. Lysates were centrifuged at 14,000 rpm and proteins
purified by affinity chromatography using a Ni2+ column (1 mL GE Healthcare HisTrap HP)
and imidazole gradient elution in an AKTA Start protein purification system. M and N proteins
were concentrated and buffer changed to phosphate buffered saline (PBS) using Microcon-
10 KDa centrifugal filter units (Millipore). For N-terminal (residues from 43 to 180) and C-
terminal fragments (residues from 250 to 360) of N, two constructs were designed at CRG
depending on secondary structure predictions. The encoding sequences were synthesized
and inserted into a plasmid pETM14 with the N-terminal 6xHis-tag under the control of a T7
promoter, and recombinant plasmids transformed into E. coli BL21 DE3 competent cells.
Briefly, E. coli containing the plasmid was grown and the protein expression was induced by
addition of IPTG 0.2 mM for 16 h at 18°C. Pelleted cells were resuspended in Buffer A (Tris
20 mM, 250mM NaCl, 10mM Imidazole) supplemented with 0.5% Triton-X100 Substitute
(Sigma) and complete mini EDTA-free protease (Roche), sonicated, and centrifuged (30 min,
4°C, 30000 g). The N-terminal, and the C-terminal recombinant proteins containing a N-
terminal 6xHis-tag were purified from the resulting supernatant using Hitrap Ni-NTA column
(GE Healthcare, Uppsala, Sweden) according to the manufacturer instructions. After washing
with Buffer A, the antigen was eluted using linear gradient with buffer B (Buffer A
supplemented with 500 mM Imidazol). The fractions of interest were dialyzed against PBS 1x
and concentrated by Vivaspin 5 KD (Millipore, France). The antigens produced were
quantified using a bicinchoninic acid (BCA) protein assay kit (Pierce) and their purity
controlled by Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE).
Antigen coupling to microspheres
Magnetic MAGPLEX 6.5 µm COOH-microspheres from Luminex Corporation (Austin, TX)

were washed twice with 62.5 µl of distilled water using a magnetic separator (Life
Technologies, 12321d), and resuspended in 80 µl of activation buffer, 100 mM monobasic
sodium phosphate (Sigma, S2554), pH 6.2. To activate the beads for cross-linking to
proteins, 10 µl of 50 mg/mL sulfo-N-hydroxysulfo-succinimide (Thermo Fisher Scientific,
24525) and 50 µL of 50 mg/mL 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimidehydrochloride
(Thermo Fisher Scientific, 22981) were simultaneously added to the reaction tubes, mixed
and incubated at room temperature (RT) for 20 min in a rotatory shaker and protected from
light. Next, beads were washed twice with 62.5 µl 50 mM morpholineethane sulfonic acid
(MES) (Sigma, M1317) pH 5.0, in a 10,000 beads/ µl concentration. After beads activation,
antigen were added to the reaction tubes at three different concentrations and left at 4ºC
overnight (ON) on a rotatory shaker protected from light. On the following day, coupled-
beads were brought to RT for 20 min in agitation, and blocked by incubating them with 62.5
µl PBS (Sigma) + 1% bovine serum albumin (BSA, Biowest) + 0.05% sodium azide (Sigma,
S8032) (PBS-BN) in agitation during 30 min at RT and protected from light. Beads were
washed twice with PBS-BN using the magnetic separator. To determine the percentage
recovery of beads after the coupling procedure, coupled beads were resuspended in 62.5 µL
PBS-BN and counted on a Guava PCA desktop cytometer (Guava Technologies, Automated
cell counter, PC550IG-C4C/0746-2747). In all washing and resuspension steps, beads were
softly vortexed and sonicated for 30 sec.

SUPPLEMENTARY FIGURES
Figure S1. Selection of the optimal RBD antigen coupling concentration. A) Comparison of
titration curves with an anti-histidine tag biotinylated antibody at different protein
concentrations (left panel) and with a positive plasma pool for IgG and IgM (right panel). B)
Comparison of IgG and IgM titration curves with a positive plasma pool against 5 different

batches of 30 µg/mL RBD-coupling reactions (A-E) for IgM (C) and IgG (D) showing highly
reproducible titration curves.
A)
B) IgM IgG
Figure S2. Effect of GullSORB™ treatment on IgM antibody levels to negative (NC) and
positive (PC) control samples in a multiplex antigen panel at different plasma dilutions.
GullSORB™ reduced or did not change the MFI signal, depending on the sample, antigen
and dilution (A). This additional incubation generally increased the signal to noise ratio and
thus sensitivity and number of seropositive IgM responses among the PC, particularly at the
lower dilutions, therefore the GullSORB™ incubation was adopted for this assay (B). IgM

reactivity in NC was lower against S-based antigens than against M- or N-based antigens
and thus GullSORB™ treatment benefited the most in these later proteins.
A)
B)
Figure S3. Levels of IgM, IgA and IgG antibodies (median fluorescence intensity, MFI) to
RBD in positive (TS) and negative samples (NC), and % seropositivity among TS, comparing
45 min versus 30 min incubation times of the secondary antibody conjugated to PE after 1
hour incubation of plasma samples with antigen beads.

Figure S4. Ranking of isotype/antigen markers by Random Forest models for all negative
controls plus either all positive samples or positive samples at different periods since onset of
symptoms.

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JCM Accepted Manuscript Posted Online 30 October 2020

J. Clin. Microbiol. doi:10.1128/JCM.01731-20

1 Highly sensitive and specific multiplex antibody assays to quantify

2 immunoglobulins M, A and G against SARS-CoV-2 antigens

6 Serra4, Paula Martínez de Aguirre5, Francisco Carmona-Torre6, Gabriel Reina5, Pere

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14 (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

21 of Medicine, University of Calgary, Alberta, T2N 4N1 Canada.

27 Corresponding authors: Carlota.dobano@isglobal.org; Gemma.moncunill@isglobal.org

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40 Pre-existing IgG antibodies recognizing N, M and S proteins were detected in negative

41 controls suggestive of cross-reactive to common cold coronaviruses. The best performing

46 obtaining reliable data on prevalence of exposure to novel pathogens in a population. Our

48 required for vaccine development to combat the COVID-19 pandemic.

51 Antibody; immunoassay; SARS-CoV-2; COVID-19; IgM; IgA; IgG; Luminex; quantitative

52 suspension array technology; multiplex; spike; RBD; nucleocapsid; coronavirus; sensitivity;

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70 would be valuable to evaluate immunogenicity of candidate vaccines and monitor duration of

71 immunity at the population level (6).

84 days post-symptom onset (11)(12)(13)(14).

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90 reasonable sensitivity (16).

93 platform (www.luminexcorp.com/bibliography). Secondary antibodies are labelled with

99 reproducibility compared to the previous generation of MicroPlex® microspheres. Since the

107 capable of measuring simultaneously antibodies against 50 (MAGPIX®), 80 (Luminex

111 emerging diseases and pandemics.

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133 were in line with protocol recommendations from FIND.

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155 full-length) recombinant proteins (Supplementary methods).

157 qSAT assays

160 coupled to magnetic MAGPLEX 6.5 μm COOH-microspheres from Luminex Corporation

170 protected from light till use.

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200 protected from light.

210 Performance of the SARS-CoV-2 qSAT assays

219 min incubation with secondary antibodies conjugated directly to PE.

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252 (packages used: randomForest (33) and pROC (34)).

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257 onset of symptoms, are included in Table 1.

259 Optimization of assay conditions

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286 antibodies to S (Figure 3B).

292 Sensitivity and specificity of the qSAT assays

307 in which IgG was better for samples at 1/500 dilution.

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333 since the onset of symptoms.

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342 an excellent precision, accuracy and wide range of detection.

353 However, other seropositivity thresholds can be used to have a balanced

354 specificity/sensitivity or to maximise sensitivity. For studies aiming at characterizing kinetics

355 of antibody responses, immunogenicity of vaccine candidates or correlates of protection a

356 cutoff prioritizing sensitivity over specificity would be preferable.

360 (36)(37)(38)(39)(40)(41). Thus, it is not necessarily expected to detect antibodies in

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380 the onset of infection.

388 and S2 estimated a 96% sensitivity and 99% specificity (45).

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409 maintain even in remote laboratory settings.

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422 address the significance of this in immunity to COVID-19.

433 characteristics to assess seroprevalence of COVID-19. Considering their high sensitivity,