This report was funded by Luminex Corporation (Austin, TX). The authors thank Matthew Silverman PhD (Biomedical Publishing Solutions, Delray Beach, FL) for scientific editing assistance.

<ol>
	<li>Cameron, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+microsphere+IgG+assay+for+SARS-CoV-2+using+ACE2-mediated+inhibition+as+a+surrogate+for+neutralization.">A multiplex microsphere IgG assay for SARS-CoV-2 using ACE2-mediated inhibition as a surrogate for neutralization.</a> Journal of Clinical Microbiology. 59 (2), 02489 (2020).</li><li>Waterboer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+human+papillomavirus+serology+based+on+in+situ-purified+glutathione+s-transferase+fusion+proteins.">Multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins.</a> Clinical Chemistry. 51 (10), 1845-1853 (2005).</li><li>Pickering, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplexed+fluorescent+microsphere+immunoassay+for+antibodies+to+pneumococcal+capsular+polysaccharides.">A multiplexed fluorescent microsphere immunoassay for antibodies to pneumococcal capsular polysaccharides.</a> American Journal of Clinical Pathology. 117 (4), 589-596 (2002).</li><li>Ayouba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+sensitive+and+specific+serological+assay+based+on+Luminex+technology+for+detection+of+antibodies+to+Zaire+Ebola+virus.">Development of a sensitive and specific serological assay based on Luminex technology for detection of antibodies to Zaire Ebola virus.</a> Journal of Clinical Microbiology. 55 (1), 165-176 (2017).</li><li>Hornbeck, P., Fleisher, T. A., Papadopoulos, N. M., Vastl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotype+determination+of+antibodies.">Isotype determination of antibodies.</a> Current Protocols in Immunology. 116, 1-7 (2017).</li><li>. Multiplex Bead Mixes Made Easy with an Excel-Based Bead Calculator Available from: <a target="_blank" href="https://www.luminexcorp.com/blog/mmultiplex-bead-mixes-made-easy-with-an-excel-based-bead-calculator/">https://www.luminexcorp.com/blog/mmultiplex-bead-mixes-made-easy-with-an-excel-based-bead-calculator/</a> (2020)</li><li>EUA Authorized Serology Test Performance. FDA Available from: <a target="_blank" href="https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/eus-authorized-serology-test-performance">https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/eus-authorized-serology-test-performance</a> (2020)</li><li>Indirect ELISA protocol. Abcam Available from: <a target="_blank" href="https://www.abcam.com/ps/pdf/protocols/Indirect%20ELISA%20protocol.pdf">https://www.abcam.com/ps/pdf/protocols/Indirect%20ELISA%20protocol.pdf</a> (2021)</li><li>Baker, H. N., Murphy, R., Lopez, E., Garcia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+a+capture+ELISA+to+a+Luminex+xMAP+assay+using+a+multiplex+antibody+screening+method.">Conversion of a capture ELISA to a Luminex xMAP assay using a multiplex antibody screening method.</a> Journal of Visualized Experiments. (65), e4084 (2012).</li><li>Pickering, J. W., Martins, T. B., Schroder, M. C., Hill, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+multiplex+flow+cytometric+assay+with+enzyme-linked+immunosorbent+assay+for+auantitation+of+antibodies+to+tetanus,+diphtheria,+and+Haemophilus+influenzae+Type+b.">Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.</a> Clinical and Diagnostic Laboratory Immunology. 9 (4), 872-876 (2002).</li></ol>

Stephen Angeloni and Sherry Dunbar are employees of Luminex Corporation that produces some of the reagents and instruments that were used in this study.&#160;Open Access publication of this article was sponsored by Luminex Corporation

This study expanded the functionality of a multiplex fluorescent microsphere assay, 3Flex, that qualitatively assesses the IgG response to infection by SARS-CoV-21. While many serological assays for COVID-19 detect a single antigen, this assay simultaneously evaluates S, RBD, and Nc antigens, and includes an internal antibody isotype control7. In addition, ACE2 is used as an indicator to detect neutralizing antibody titers to SARS-CoV-2.
Multi-antigen assays may be particularly useful in the future for discriminating immune responses between infected and vaccinated persons. With SARS-CoV-2, if only S and RBD antibodies are evaluated, it would be impossible to discern whether this was due to a past infection or to a vaccination antibody response. None of the vaccines that are currently in use target the Nc antigen, so by evaluating S/RBD and Nc antigens, it is possible to distinguish past viral infection (Nc- and S/RBD-positive) from a vaccination response (S/RBD-positive only; Nc-negative).
In the current study, the 3Flex serological and neutralization assays (sections 5 and 6) were transferred onto a new dual reporter flow analysis instrument (sections 7 and 8). Typical immunoassay protocols for bead-based multiplex assays are similar to standard ELISA protocols, following comparable steps for sample incubation, detection antibody/reporter incubation, and analysis of results8. Previous studies have also demonstrated how standard ELISA assays can be transferred to a bead-based multiplexing platform9.
The assay protocols could be seamlessly transferred from the predecessor single reporter instrument to the new dual reporter system, as shown by the results obtained for the test samples and controls (Figure 4 and Figure 5). In the serological assay, all positive serum samples demonstrated a characteristic dose-responsive decrease in signal corresponding to both a higher sample dilution and a lower detection antibody concentration, whereas the signals for the negative samples remained at background levels. Similarly, for the neutralization assay, decreasing signals for S and RBD, but not Nc, corresponded to increasing concentrations of the ACE2 competitor, whereas the IgG stripped serum was much less affected by the addition of ACE2. Pre-incubating the beads with ACE2 for 2 min prior to addition of the serum sample (as described in sections 6 and 8), was also compared to combining the beads with ACE2 and serum at the same time and produced little difference in the results (data not shown). However, because the results obtained with the beads plus ACE2 pre-incubation step were more consistent, it was the final procedure adopted for the neutralization assay protocol (sections 6 and 8).
Because the dual reporter system incorporates a second fluorescent reporter channel, both assays were converted into isotyping assays to simultaneously measure both IgG and IgM responses. The dual reporter functionality of the flow analyzer allows collection of fluorescent signals from two reporter detection reagents for each analyte in the multiplex for each sample, effectively doubling the data generated per sample in an assay. For development and optimization of the dual reporter assay protocols, several commercially available reporter dyes and detection antibodies were evaluated for the new RP2 channel (Figure 7 and Figure 8) to determine the best option(s) available. The anti-IgM antibody conjugated with the DL 405 reporter dye did not perform well in the RP2 channel (Figure 7), so biotin anti-IgG with SASB was subsequently evaluated for detection in the RP2 channel (Figure 8). Three combinations of RP1 and RP2 detection reagents were compared in the dual reporter neutralization assay (Table 1 and Figure 9) to determine the best performing combinations for simultaneous detection of IgG and IgM neutralizing antibodies. While there were significant differences in the signal intensity between the RP1 channel using PE-anti IgM and the RP2 channel using DL 549 anti-IgM, there was no significant difference in the measurement of the percent binding inhibition by ACE2.
Several limitations to the current method and this study are acknowledged. First, the samples tested and used in the method development and optimization were limited to sets of 8-11 samples. More samples will be tested in expanded studies to verify the method is fully optimized. Second, a limited number of reporter fluorophores and reporter pairs were tested. Further testing of all available reporters in all possible combinations will determine which reagents can be used successfully in this method. The anti-IgG antibody conjugated to biotin was different than the anti-IgG antibody conjugated to the BV 421 reporter. So, although variability in the signal intensity for the BV 421 anti-IgG existed, it could be due to the specificity of the antibody and not related to the reporter dye. Testing other available BV 421-conjugated antibodies may identify another antibody that would perform well in the RP2 channel. Future studies will also evaluate the combination of PE anti-IgM with BV 421 anti-human IgG for detection in RP1 and RP2, respectively. Last, even with the dual reporter capability of the instrument, assays are limited to two isotypes (two parameters) per reaction. If additional isotypes are to be measured or full isotyping is desired, then additional reactions using the same two reporter channels would need to be run in separate wells.
Bead-based multiplexing technology allows for rapid and flexible assay design with easy implementation into routine laboratory workflows3,4,10. This study demonstrated the ease in transferring the previously described 3Flex serological and neutralization assays for SARS-CoV-2 onto a flow analysis system for measuring IgG with a single reporter, or with slight modification, simultaneously measuring both IgG and IgM responses using two reporters. The dual reporter option has clear advantages over single reporter platforms because it can provide twice as much data per sample, using half the sample volume, and in half the number of reaction wells. With the appropriate reporter dyes and detection antibodies, isotyping assays can be easily developed and performed on the dual reporter flow analysis instrument.

The COVID-19 pandemic has emphasized the importance of being able to rapidly develop and implement fast, high-throughput, high-performing serological tests that can be used for diagnosis and surveillance of novel pathogens such as SARS-CoV-2. Multiplex serological testing can be extremely useful in a pandemic, as it can simultaneously analyze antibodies to multiple antigens, which provides for broad pathogen coverage and controls for variability in the organism and the host response to infection. The development of a multiplex fluorescent bead-based assay, the SARS-CoV-2 IgG 3Flex (3Flex), that can detect antibodies to the spike (S) protein, the spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc) of SARS-CoV-2 was recently reported1. 3Flex was shown to have comparable performance to a reference SARS-CoV-2 IgG assay for serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Additionally, inhibition of antibody binding was demonstrated for S and RBD antigens using soluble ACE2 in a neutralization assay format. Multiplex bead-based technology has been widely adopted as a proven technology for multiplex serological testing and has distinct advantages for large-scale screening, including short time-to-results, small sample requirements, and reduced labor2,3,4.
Recently, a new flow analysis instrument has become available that provides the same high-plex, high-throughput capability of the system demonstrated in previous work1, but with the added benefit of two reporter channels. The ability to measure two fluorescent reporter signals in a single reaction allows the assessment of two results per analyte for each sample and is ideal for isotyping applications, which typically must be performed in separate reaction wells5. The dual reporter capability provides twice the data for each sample in half as many reaction wells with half the sample volume requirements, and thus has a clear advantage over single reporter systems. This study details the standard serological assay protocol and describes the adaptation to a neutralization assay. Additionally, this report describes the conversion of the single reporter assay to a dual reporter serological assay, and subsequently, a dual reporter neutralization assay, to simultaneously measure neutralizing antibodies of both IgG and IgM isotypes against the SARS-CoV-2 S, RBD, and Nc antigens. A visual overview of the assay protocol is provided in Figure 1.

<table><tbody><tr><td>ACE2 (human) (rec.)</td><td>AdipoGen Life Sciences</td><td>AG-40B-0192-3050</td><td></td></tr><tr><td>Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, Fc&amp;gamma; fragment specific</td><td>Jackson ImmunoResearch Laboratories</td><td>109-065-098</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>MilliporeSigma</td><td>A7888</td><td>Sigma-Aldrich</td></tr><tr><td>Branson 1800 Cleaner/water bath sonicator</td><td>Various</td><td></td><td></td></tr><tr><td>Brilliant Violet 421 AffiniPure Donkey Anti-Human IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>709-675-149</td><td></td></tr><tr><td>Corning 96-well Black Flat Bottom Polystyrene NBS Microplate</td><td>Corning</td><td>3650</td><td></td></tr><tr><td>Corning 96-well Microplate Aluminum Sealing Tape, Nonsterile</td><td>Corning</td><td>6570</td><td></td></tr><tr><td>DyLight 405 AffiniPure Goat Anti-Human IgM, Fc5&amp;mu;&amp;nbsp;fragment specific</td><td>Jackson ImmunoResearch Laboratories</td><td>109-475-043</td><td></td></tr><tr><td>DyLight 549 AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Goat Anti-Human IgM Fc5&amp;mu; specific</td><td>Jackson ImmunoResearch Laboratories</td><td>109-506-129</td><td>Discontinued but available from multiple vendors</td></tr><tr><td>eBioscience Streptavidin Super Bright 436 Conjugate</td><td>ThermoFisher Scientific</td><td>62-4317-82</td><td></td></tr><tr><td>Fisherbrand Incubating Microplate Shaker</td><td>ThermoFisher Scientific</td><td>02-217-757</td><td>Fisher Scientific</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, PE</td><td>ThermoFisher Scientific</td><td>P-2771MP</td><td></td></tr><tr><td>Luminex Magnetic Plate Separator</td><td>Luminex Corporation</td><td>CN-0269-01</td><td></td></tr><tr><td>MagPlex beads</td><td>Luminex Corporation</td><td>MC100XX-ID</td><td></td></tr><tr><td>PE AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Goat Anti-Human IgM Fc5&amp;mu; specific</td><td>Jackson ImmunoResearch Laboratories</td><td>109-116-129</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>MilliporeSigma</td><td>P3813</td><td>Sigma-Aldrich</td></tr><tr><td>SARS Nucleocapsid Protein Antibody</td><td>Novos Biologicals</td><td>NB100-56683</td><td>Unconjugated, rabbit, polyclonal, Protein G purified</td></tr><tr><td>SARS Nucleocapsid Protein Antibody</td><td>Novos Biologicals</td><td>NB100-56049</td><td>Unconjugated, rabbit, polyclonal, Protein A purified</td></tr><tr><td>SARS Spike Protein Antibody</td><td>Novos Biologicals</td><td>NB100-56047</td><td>Unconjugated, rabbit, polyclonal, Protein G purified</td></tr><tr><td>SARS Spike Protein Antibody</td><td>Novos Biologicals</td><td>NB100-56048</td><td>Unconjugated, rabbit, polyclonal, Protein G purified</td></tr><tr><td>SARS-CoV-2 (2019-nCoV) Spike S1 Antibody, Rabbit MAb</td><td>SinoBiological</td><td>40150-R007</td><td></td></tr><tr><td>Sodium Azide</td><td>MilliporeSigma</td><td>S2002</td><td>Sigma-Aldrich</td></tr><tr><td>Streptavidin, R-Phycoerythrin Conjugate (SAPE)</td><td>ThermoFisher Scientific</td><td>S21388</td><td>premium grade</td></tr><tr><td>Tube Revolver/ Rotator</td><td>ThermoFisher Scientific</td><td>88881001</td><td></td></tr><tr><td>TWEEN 20</td><td>MilliporeSigma</td><td>P9416</td><td>Sigma-Aldrich</td></tr><tr><td>xMAP Antibody Couplng Kit</td><td>Luminex Corporation</td><td>40-50016</td><td></td></tr><tr><td>xMAP INTELLIFLEX DR-SE</td><td>Luminex Corporation</td><td>INTELLIFLEX-DRSE-RUO</td><td></td></tr></tbody></table>

a rapid, multiplex dual reporter igg and igm sars-cov-2 neutralization assay for a multiplexed bead-based flow analysis system

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

Optimization of the Single Reporter Serological Assay. 
The coupling strategy for all SARS-CoV-2 antigens is described in Cameron, et al., 20201 and in step 2.1 above. The strategy for coupling confirmation is described in step 2.3 above. For an efficient coupling, the median fluorescence intensity (MFI) values should be significantly higher than the no serum/no antibody negative control reaction(s) and should demonstrate a dose response of decreasing MFI signal with decreasing amounts of detection reagent. MFIs of approximately 1,000 MFI units or greater over background MFI generally indicate efficient protein coupling and are dependent on the concentration and specificity of the detection reagent used. Using this method, confirmation of S, RBD, and Nc antigen coupling were tested for two lots of beads (Figure 2). The MFI signals for all antigens were significantly higher than the background MFI and showed a dose response with titration of the detection reagents. The data also showed good reproducibility of MFI levels between the two different lots of coupled beads.
Optimization of the single reporter assay was done as described in Cameron, et al., 20201. Each antigen was tested at different coupling concentrations with different dilutions of several serum samples representing high, medium, low, and negative samples. In addition, because the assay measures IgG titers, an IgG-stripped serum was used as an additional negative sample. Results of a representative sample dilution series with different antigen coupling levels are shown in Figure 3. This initial dilution series was tested with a fixed concentration of detection reagents to get a potential range of representative antigen-specific and background MFI levels. From these data, additional sample dilution ranges and antigen coupling levels were determined and tested further with additional samples (data not shown).
Next, the concentration of the detection reagent was optimized for use with the different serum dilutions and antigen coupling levels. Representative data using 6 positive and 2 negative samples are shown in Figure 4. Following additional testing with several hundred negative pre-COVID-19 serum samples, the final optimal antigen coupling levels and detection regent concentrations were selected for the final assay protocol, as described in section 5.
Conversion to a single reporter neutralization assay 
Conversion of the single reporter serological assay to a neutralization assay was achieved by adding an incubation step using ACE2 (as a competing reagent) with the beads prior to adding the serum sample. By titrating the amount of ACE2 added, inhibition of IgG binding to the S and RBD antigens was observed, as shown in Figure 5. While the 11 patient sera had different titers of IgG, each showed a significant decrease in MFI signal with increasing ACE2 concentration. As expected, ACE2 only impacts the MFI signals of S and RBD, but not the Nc antigen.
The percent residual MFI signal relative to 0 &#181;g/mL ACE2 for all samples is shown in Figure 6. For most samples, a signal loss &#62;30% was observed for S and RBD with increasing ACE2 concentration. As expected, there was no effect of ACE2 on the Nc signal.
Conversion to a dual reporter IgG and IgM serological assay 
For the dual reporter IgG and IgM assay, the standard single reporter assay conditions were used to test different antibody and reporter combinations for the two reporter channels. The RP1 channel is similar to previous flow analysis instruments with detection of &#34;orange&#34; fluorescence for reporter dyes such as PE. The second channel, RP2, employs a violet excitation laser that can be used for detecting the &#34;blue&#34; fluorescence of dyes excited at 402 nm with emission spectra peaks in the 421-441 nm range.
Several different anti-human IgG and IgM antibody combinations were tested at various concentrations with the standard 2,000-fold sample dilution and incubation conditions detailed in section 6. An initial test of anti-IgM conjugated to the DyLight 405 reporter dye (DL 405; excitation at 400 nm and emission at 420 nm) for the RP2 channel was performed using a set of 11 PCR-positive samples with DFSO ranging from 5 to &#62;60 days, and an IgG-stripped serum sample. This detection reagent did not produce a high signal in the RP2 channel as shown in Figure 7A,B,C. For samples with the high IgM RP2 MFI, the highest signals were seen for RBD and Nc (Figure 7B,C). While IgM titers should be elevated at this time in some samples, the observed MFI levels did not exceed 140 MFI units with the IgM detection reagent. Furthermore, the control bead for IgM lacked a significant dynamic range for MFI when using DL 405 conjugated to anti-IgM compared to a PE-labeled anti-IgM RP1 reporter used at the same concentrations (Figure 7D).
Because a suitable RP2 reporter-labeled detection reagent for IgM was not available, the original biotin anti-IgG with SASB was tested for use in the RP2 channel. The signal for IgG in the RP2 channel with SASB did not have the same dynamic range as with SAPE, but it still had a suitable range for measuring IgG titers for S, RBD, and Nc (Figure 8). These data led to testing different RP1 IgM and RP2 IgG detection reagent combinations, as described below.
Conversion to dual reporter neutralization assay 
The dual reporter serological assay was converted to a neutralization assay by adding an incubation step with ACE2 and beads, prior to adding the serum sample. A 2-fold dilution series of ACE2 starting at 16 &#181;g/ml was used, and then tested with sets of 11 samples and stripped sera as described above. In addition, 3 different combinations of RP1 IgM and RP2 IgG detection antibody mixes were tested on a set of 11 samples and stripped serum controls. The final combinations and reagent concentrations determined to be optimal are detailed in Table 1.
For IgG detection, the biotin anti-human IgG/SASB used in the original single reporter assay was tested along with another anti-IgG conjugated to the BV reporter dye. While the BV conjugate had high RP2 MFI signals, the MFI signal intensity for IgG titer varied across the ACE2 titrations (Figure 9A,C,E). The biotin anti-human IgG/SASB combination had more consistent MFI signal decrease across the ACE2 titration compared to the BV conjugate, demonstrating a dose response, although the MFI levels were lower. The signal fluctuation by the BV conjugate across ACE2 concentrations also interfered with determining the percent inhibition by ACE2 across the range of IgG titers compared to the biotin anti-human IgG/SASB combination (Figure 9B,D,F). Samples with low MFI signals, such as the DFSO 3 sample, do not have high enough titers to evaluate the performance of these reagents or measure IgG neutralizing capacity.
For determining IgM titers in the RP1 channel, a PE-conjugated anti-IgM was compared to an anti-human IgM conjugated to the DyLight 549 reporter dye (DL 549; excitation at 562 nm and emission at 576 nm) at the concentrations listed in Table 1. Of these two reagents, the PE anti-IgM displayed higher MFIs than those generated by DL 549 anti-human IgM for the samples tested (Figure 9A,C,E). The IgM control bead measuring the addition of these reagents had different average MFIs of &#8776;17,000 for the PE anti-IgM and 2,723 for the DL 549 conjugate. Even with these differences, the impact of the various ACE2 concentrations on MFI was measurable with the DL 549 conjugate. For determining ACE2 percent inhibition of IgM binding, there was a slight but insignificant difference between the two IgM detection reagents (Figure 9B,D,F).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Combination</td>
			<td rowspan="2">Reporter</td>
			<td colspan="2">Final Concentration In Well (&#181;g/mL)</td>
		</tr>
		<tr>
			<td>RP1</td>
			<td>RP2</td>
		</tr>
		<tr>
			<td rowspan="3">1</td>
			<td>PE anti-IgM</td>
			<td>2</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biotin-Ig</td>
			<td>-</td>
			<td>0.62</td>
		</tr>
		<tr>
			<td>SASB</td>
			<td>-</td>
			<td>4</td>
		</tr>
		<tr>
			<td rowspan="2">2</td>
			<td>DyLight 549 Anti-Human IgM</td>
			<td>1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 Anti-Human IgG</td>
			<td>-</td>
			<td>1</td>
		</tr>
		<tr>
			<td rowspan="3">3</td>
			<td>DyLight 549 Anti-Human IgM</td>
			<td>1</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biotin-Ig</td>
			<td>-</td>
			<td>0.62</td>
		</tr>
		<tr>
			<td>SASB</td>
			<td>-</td>
			<td>4</td>
		</tr>
	</tbody>
</table>
Table 1: List of RP1 and RP2 reporter dye combinations tested. Several different RP1 and RP2 reporter dyes were evaluated for measuring IgG and IgM titers. The three combinations shown above were tested at different RP1 detection modes and for optimization of the neutralization assay. For RP2 combinations using SASB, concentrations for the biotinylated detection antibody and SASB are shown. *Final Concentration represents the final concentration in the reaction well.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62487/62487fig01.jpg" /> 
Figure 1: Flowchart highlighting main assay protocol steps. <a href="https://www.jove.com/files/ftp_upload/62487/62487fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62487/62487fig02.jpg" /> 
Figure 2: Confirmation of antigen coupling. Antigens were coupled at 100, 10 and 1 pmol/106 beads. For S and RBD antigens, rabbit anti-S sera was used and detected with PE anti-rabbit IgG. For Nc, a Protein G-purified rabbit polyclonal anti-Nc was used. The titration curves for beads coupled with 10 pmol S (A), 100 pmol RBD&#160;(B), and 100 pmol Nc (C) are shown. <a href="https://www.jove.com/files/ftp_upload/62487/62487fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62487/62487fig03.jpg" /> 
Figure 3: Optimization of serum dilution with different antigen coupling levels. Serum samples representing high, medium, low, and negative samples were tested with the different pmol antigen/106 bead couplings. A 4-fold serum titration was tested with a multiplex mix of antigen-coupled beads using the assay protocol described in section 5. The MFI values of the titration curves for S (A), RBD (B), and Nc (C) are shown. <a href="https://www.jove.com/files/ftp_upload/62487/62487fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62487/62487fig04.jpg" /> 
Figure 4: Determination of optimal serum dilution and detection reagent concentration. Eight serum samples, including negative patients (2 samples) and low- to high-positive (6 samples), were tested at additional serum dilutions with three different detection reagent concentrations. The MFI results are shown for S (A), RBD (B), and Nc (C). Based on these and other data (not shown), a sample dilution of 1:2,000 and a biotin detection antibody concentration of 0.62 &#181;g/mL was selected. <a href="https://www.jove.com/files/ftp_upload/62487/62487fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62487/62487fig05.jpg" /> 
Figure 5: Optimization of single reporter neutralization assay. Modification of the single reporter serological assay to a neutralizing antibody detection assay was performed by adding an incubation step with ACE2 as a competitor, as described in Section 6. A set of 11 samples, ranging from low- to high-positive titer sera, and an IgG-striped serum sample were tested with a two-fold dilution series of ACE2 starting at 4 &#181;g/mL, using standard assay conditions. Across this range of ACE2 concentrations, a decrease in MFI can be seen with increased ACE2 for S (A) and RBD (B), but not for Nc (C). <a href="https://www.jove.com/files/ftp_upload/62487/62487fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62487/62487fig06.jpg" /> 
Figure 6: Percent inhibition of signal with ACE2. The percent residual signals for the set of 11 samples (from Figure 4) are shown. For each sample, regardless of its IgG titer, a maximum decrease of &#8805;30% MFI signal was achieved for S (A) and RBD (B) at the highest ACE2 concentration. For the Nc antigen (C) there was no significant inhibition of signal with any amount of ACE2 tested. <a href="https://www.jove.com/files/ftp_upload/62487/62487fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62487/62487fig07.jpg" /> 
Figure 7: Test of DyLight 405 anti-IgM as RP2 IgM detection reagent. A set of 11 samples and IgG-stripped sera were used to test DL 405-conjugated anti-IgM as an RP2 detection reagent. The RP2 IgM MFI signals for S (A), RBD (B), and Nc (C) are shown. The highest MFI signal for any antigen with any sample was approximately 140 MFI units for RBD with sample H (B). Even the signal on the IgM control bead set was less than 1,000 MFI in the RP2 channel across the whole antibody titer range and did not show the increased dynamic range observed with the PE anti-IgM detection antibody in the RP1 channel (D). <a href="https://www.jove.com/files/ftp_upload/62487/62487fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62487/62487fig08.jpg" /> 
Figure 8: Optimization of SASB as RP2 reporter with Biotin anti-IgG. A set of 11 samples and IgG-stripped sera were used to test a dilution series of SASB with the standard 0.62 &#181;g/mL biotin anti-IgG. The resulting IgG MFIs in the RP2 channel for S (A), RBD (B), and Nc (C) are shown. <a href="https://www.jove.com/files/ftp_upload/62487/62487fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62487/62487fig09.jpg" /> 
Figure 9: Comparison of three different combinations of RP1 IgM and RP1 IgG detection mixes. Three different detection antibody mixes were tested on 11 samples and IgG stripped sera. The combinations and final concentrations used are described in Table 1. All samples were tested with a 2-fold ACE2 dilutions, starting at 16 &#181;g/mL. Data for samples at DFSO of 3, 12, and 30 days are shown. The MFI signals for RP1 IgM (orange) and RP2 IgG (blue) are shown in A (DFSO 3), C (DFSO 12), and E (DFSO 30). The ACE2 percent residual MFIs are shown in B (DFSO 3), D (DFSO 12), and F (DFSO 30). Signals representing a &#62;30% decrease as compared to No ACE2 controls are highlighted light green.&#160;<a href="https://www.jove.com/files/ftp_upload/62487/62487fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System at JoVE.com

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

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Research

JoVE Journal

Immunology and Infection

4.9K Views.  Luminex Corporation. A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

Video: A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

Flow Analysis of PDL1-Vaxx Antibody Blockade Using Magnetic Fluorescent Beads

Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer patients and has become a mainstay approach in modern cancer immunotherapy. However, only a subset of patients respond to checkpoint monoclonal antibody immunotherapy. Therefore, it is urgent to develop new therapeutic strategies against cancer. A novel B-cell peptide epitope PDL1 (programmed death ligand 1) cancer vaccine has been developed, with amino acids 130-147 linked to the MVF peptide (&#34;promiscuous&#34; T-cell measles virus fusion protein) via a GPSL linker. Preclinical testing has indicated that this PDL1 vaccine (PDL1-Vaxx) effectively stimulates highly immunogenic antibodies in animals. Animals immunized with PDL1-Vaxx show reduced tumor burden and extended survival rates in various animal cancer models. The mechanisms of action indicate that vaccine-elicited antibodies inhibit tumor cell proliferation, induce apoptosis, and block the PD-1/PD-L1 interaction. This manuscript introduces a magnetic bead-based assay that uses a dual-reporter flow analysis system to evaluate the PD-1/PD-L1 interaction and its blockade by the anti-PDL1 antibodies raised against the PDL1-Vaxx.

Author Spotlight: Magnetic Fluorescent Bead-Based Dual-Reporter Flow Analysis of PDL1-Vaxx Peptide Vaccine-Induced Antibody Blockade of the PD-1/PD-L1 Interaction 

Checkpoint inhibitors are important targets in developing therapies for the battle against cancer. This report introduces a novel PDL1 peptide-based cancer vaccine, PDL1-Vaxx, which induces neutralizing polyclonal antibody production that blocks PD-1/PDL1 complex formation. This work also details the development and testing of a fluorescent bead-based assay for analyzing this activity.

The inhibition of checkpoint receptors (PD-1, PD-L1, and CTLA-4) with monoclonal antibodies has shown great benefit in clinical trials for treating cancer ...

Cancer Research

Multiplexed Fluorometric ImmunoAssay Testing Methodology and Troubleshooting

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (&alpha;Ig) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The &alpha;Ig control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Using Luminex Corporation&#x2019;s xMAP microsphere technology, we have developed the Multiplexed Fluorometric ImmunoAssay (MFIA) for serosurveillance of various laboratory animal species. The MFIA is a suspension microarray where antigen, tissue control or immunoglobulins are covalently linked to color-coded polystyrene microspheres. The MFIA testing method as well as various troubleshooting topics is addressed.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if ...

Biology

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

Multiplex Immunoassay for Borrelia-Specific IgG and IgM in Serum

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

Author Spotlight: Expanding the Scope of Multiplex Immunoassays for Lyme Borreliosis Diagnostics and Pathogen Research 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

Medicine

Multianalyte Immunobead Assay: A Flow-Based Immunoassay Technique for Concurrent Detection of Multiple Antibody Subtypes in Human Serum Sample

Source: Fhied, C. L. et al., <a href="https://www.jove.com/v/63352">Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19.</a> J. Vis. Exp. (2022).
This video describes an immunoassay technique using fluorescent magnetic beads or microspheres coupled with different antigens to simultaneously detect various antibody isotypes in a serum sample. The assay is helpful in understanding seroconversion during an infection or post-vaccination.

Source: Fhied, C. L. et al., Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19. J. Vis. Exp. (2022).
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Immunoassays

Bioreporter Assay: A Sensitive Technique using a Bioluminescent Reporter System to Detect SARS-CoV-2 Antibodies

Source: Rezaei, R. et al., <a href="https://app.jove.com/v/62488">Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter.</a> J. Vis. Exp. (2021)
In this video, we show the HiBit-RBD bioreporter system-based assay for detecting SARS-CoV-2 neutralizing antibodies. This system targets the viral RBD domain by using a bioluminescent reporter system containing HiBiT fused to the RBD domain.

Source: Rezaei, R. et al., Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter. J. Vis. Exp. (2021)
In this video, we ...

A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

Source: Lehmann, J. S., et al. <a href="https://app.jove.com/v/56440">Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.</a> J. Vis. Exp. (2017).
This video demonstrates a multiplex bead-based immunoassay for quantifying multiple cytokine targets in a sample. A mixture of distinct microspheres conjugated to cytokine-specific antibodies is used to bind the cytokines present in the sample. Streptavidin-tagged reporters are immobilized on the bound-cytokines using biotinylated detection antibodies. Flow cytometry identifies the bound cytokines based on the unique sizes and internal fluorescence intensities of the microspheres, while the fluorescence intensity of the reporters indicates the number of cytokines.

1. Biological Sample Preparation
NOTE: The chosen anatomical site and volume of blood collection are left to the discretion of each individual ...

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres

This video demonstrates multiplexed quantitative bacterial detection employing carboxylated magnetic polystyrene microspheres dyed into distinct sets. The procedure encompasses the formation of bead-bacteria complexes, magnetic separation, and flow-based analysis, enabling precise and simultaneous quantification of multiple bacteria in the same reaction well.

1. Covalent Coupling of Capture Antibodies to MagPlex Microspheres
Each capture antibody is conjugated to a specific carboxylated magnetic microsphere set ...