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 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 ≥21 days from symptom onset but had higher sensitivity with samples collected at ≤5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.
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 ≥21 days from symptom onset but had higher sensitivity with samples collected at ≤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.
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 authors have nothing to disclose.
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.
ACE2 (human) (rec.) | AdipoGen Life Sciences | AG-40B-0192-3050 | |
Biotin-SP (long spacer) AffiniPure Goat Anti-Human IgG, Fcγ fragment specific | Jackson ImmunoResearch Laboratories | 109-065-098 | |
Bovine Serum Albumin | MilliporeSigma | A7888 | Sigma-Aldrich |
Branson 1800 Cleaner/water bath sonicator | Various | ||
Brilliant Violet 421 AffiniPure Donkey Anti-Human IgG (H+L) | Jackson ImmunoResearch Laboratories | 709-675-149 | |
Corning 96-well Black Flat Bottom Polystyrene NBS Microplate | Corning | 3650 | |
Corning 96-well Microplate Aluminum Sealing Tape, Nonsterile | Corning | 6570 | |
DyLight 405 AffiniPure Goat Anti-Human IgM, Fc5μ fragment specific | Jackson ImmunoResearch Laboratories | 109-475-043 | |
DyLight 549 AffiniPure F(ab')2 Goat Anti-Human IgM Fc5μ specific | Jackson ImmunoResearch Laboratories | 109-506-129 | Discontinued but available from multiple vendors |
eBioscience Streptavidin Super Bright 436 Conjugate | ThermoFisher Scientific | 62-4317-82 | |
Fisherbrand Incubating Microplate Shaker | ThermoFisher Scientific | 02-217-757 | Fisher Scientific |
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, PE | ThermoFisher Scientific | P-2771MP | |
Luminex Magnetic Plate Separator | Luminex Corporation | CN-0269-01 | |
MagPlex beads | Luminex Corporation | MC100XX-ID | |
PE AffiniPure F(ab')2 Goat Anti-Human IgM Fc5μ specific | Jackson ImmunoResearch Laboratories | 109-116-129 | |
Phosphate Buffered Saline | MilliporeSigma | P3813 | Sigma-Aldrich |
SARS Nucleocapsid Protein Antibody | Novos Biologicals | NB100-56683 | Unconjugated, rabbit, polyclonal, Protein G purified |
SARS Nucleocapsid Protein Antibody | Novos Biologicals | NB100-56049 | Unconjugated, rabbit, polyclonal, Protein A purified |
SARS Spike Protein Antibody | Novos Biologicals | NB100-56047 | Unconjugated, rabbit, polyclonal, Protein G purified |
SARS Spike Protein Antibody | Novos Biologicals | NB100-56048 | Unconjugated, rabbit, polyclonal, Protein G purified |
SARS-CoV-2 (2019-nCoV) Spike S1 Antibody, Rabbit MAb | SinoBiological | 40150-R007 | |
Sodium Azide | MilliporeSigma | S2002 | Sigma-Aldrich |
Streptavidin, R-Phycoerythrin Conjugate (SAPE) | ThermoFisher Scientific | S21388 | premium grade |
Tube Revolver/ Rotator | ThermoFisher Scientific | 88881001 | |
TWEEN 20 | MilliporeSigma | P9416 | Sigma-Aldrich |
xMAP Antibody Couplng Kit | Luminex Corporation | 40-50016 | |
xMAP INTELLIFLEX DR-SE | Luminex Corporation | INTELLIFLEX-DRSE-RUO |