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 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.
Lyme borreliosis is the most common tick-borne infectious disease in moderate climates of the northern hemisphere1. It is caused by spirochete bacteria of the genus Borrelia, with five known human pathogens that vary in geographical distribution2. The main pathogenic Borrelia species in Europe are B. afzelii and B. garinii, with B. burgdorferi s.s., B. spielmanii and B. bavariensis implicated less frequently. In North America, B. burgdorferi s.s. is the sole causative agent of Lyme borreliosis2,3. Borrelia pathogens are transmitted by members of the tick genus Ixodes, with transmission able to occur within 24 hours of tick bite4.
Lyme borreliosis diagnosis is typically made by clinical symptoms and subsequently confirmed by serology. In both Europe and North America, diagnostic guidelines recommend a two-step testing series consisting of an enzyme-linked immunosorbent assay (ELISA) with a reflex immunoblot to evaluate antibody response against Borrelia specific antigens1,5,6,7,8,9. However, this approach lacks sensitivity and is suboptimal, particularly in the early phase of infection when seroconversion may be incomplete and anti-Borrelia IgG and IgM titers are too low6.
Multiplex immunoassays improve upon traditional immunoassays that measure only one target at a time, and can simultaneously evaluate multiple antibody isotype responses against one or more antigens10,11,12. Assays such as ELISAs are limited to identifying and quantifying a single analyte per reaction, in the current case either circulating IgG or IgM induced against a single bacterial antigen after infection with Borrelia. This report illustrates using bead-based analyte profiling technology for the development of a multiplex immunoassay that simultaneously detects both IgG and IgM antibodies against any of the herein chosen Borrelia antigens in human serum samples. We selected four antigens that together cover the most common pathogenic Borrelia species native in Europe (B. garinii, B. afzelii, B. burgdorferi s.s.) and North America (B. burgdorferi s.s.) (Table 1)2,3. This allows decisive pathogen identification and the ability to discern early IgM and later, more durable, IgG immunoreactivity in patient samples.
Target Isotype | Reporter Channel | Antigen | Borrelia Species | Strain | Antigen Coupling Concentration |
IgM | PE | OspC | B. garinii | 20047 | 5.0 µg/106 beads |
IgG | BV421 | VlsE | B. burgdorferi s.s | B31 | 1.25 µg/106 beads |
IgG | BV421 | DbpA | B. burgdorferi s.s. | ZS7 | 10.0 µg/106 beads |
IgG | BV421 | DbpA | B. afzelii | PKo | 5.0 µg/106 beads |
Table 1: Representative Borrelia antigens used for multiplex assay development.
We initially developed a single-reporter immunoassay that detected either anti-Borrelia antigen IgG or IgM antibodies in two separate reactions and then merged these assays into a dual-reporter multiplex assay that measures both antibody isotypes in the same reaction mixture. Magnetic beads are coupled to a pathogen target antigen of interest, and then incubated with patient serum samples. The bead-coupled antigen gets recognized by, and captures, both circulating IgG and IgM in serum that is generated in an immune response against that pathogen antigen. Assay IgG versus IgM specificity is determined by the selection of a secondary antibody that binds to either IgG or IgM, each with a distinct fluorophore signal associated with the two secondary antibodies. Each fluorescent signal is detected in one of the instrument's two Reporter Channels (i.e., dual-reporter) that has a different excitation laser and emission capture specific for the single fluorophore used to detect either IgG or IgM (here Brilliant Violet 421 or phycoerythrin, respectively). The instrument Classification Channel identifies the color-coded dye that is inherent to different bead sets. Thus, multiple target antigens can be coupled to differently dyed beads, and the bead sets mixed together and used to comprehensively assess diverse anti-pathogen immunoreactivity in serum samples. The Classification Channel identifies each individual bead set (i.e., specific antigen) and measures the fluorescence associated with IgG or IgM against that antigen. The resulting multiplex assay saves time and resources versus less comprehensive classical testing and accurately catalogs Lyme immunoreactivity in limited sample volumes. Whereas a similar dual-reporter approach has been previously used to categorize immune responses in other pathologies such as SARS-CoV-2 infection13, this report details the application of multiplex fluorescent assay technology for characterizing immunoreactivity in Lyme borreliosis.
Appropriate Institutional Review Board/Ethics Committee approval was received for the use of human serum samples in this experimental series. Samples were anonymized residual materials from a German national study of SARS-CoV-2 seroprevalence14. Approval for the use of human samples was granted by the Ethics Committee of the Hannover Medical School, Germany (9086_BO_S_2020). A total of 21 human serum samples were used in the current study.
1. Ethical approval for the use of human samples
2. Reagents and equipment
3. Antigen coupling
4. Assay procedure: Single-reporter IgG or IgM serological assay: 96-well format
5. Assay procedure: Dual-reporter IgG and IgM serological assay: 96-well format
6. Dual reporter IgG and IgM serological assay: 384-well semi-automated format
Experimental overview
The general schemes for the single-reporter and dual-reporter bead-based Borrelia assays are shown in Figure 1. For single antibody targets (i.e., IgG or IgM) generated against a given Borrelia antigen, both antibody classes were evaluated independently in human serum samples using a PE-conjugated anti-isotype antibody for reporting. For the dual-reporter assay with simultaneous analysis of both IgG and IgM immunoreactivity, Borrelia antigen-specific IgG detection used a biotinylated anti-human IgG + BV421-labeled Streptavidin (blue fluorescence) reporter system, while retaining the PE-conjugated reporter (orange fluorescence) for IgM detection. The workflow of the dual-reporter assay is similar to the single-reporter assay, with the exception of an additional 30-minute detection system incubation with the second-channel fluorescence detection reagent BV421-Streptavidin.
Figure 1: Schematic of the single-reporter and dual-reporter bead-based Borrelia assays. (A) The single-reporter instrument was used to develop and initially validate the bead-based assay that characterized either anti-Borrelia IgG or IgM individually, both using a phycoerythritin (PE) fluorescent reporter label that emits signal in the "orange" spectra. Any desired Borrelia antigen can be coupled to the beads and used to capture and quantify either IgG or IgM present in serum samples. Two separate immunoassays are needed to evaluate both IgG and IgM reactivity against a given antigen. (B) The dual-reporter system allowed simultaneous analysis of serum samples for both IgG and IgM antibodies against any individual Borrelia antigen in the same well. This approach uses the same PE-conjugated detection antibody to target anti-Borrelia IgM, but replaces the IgG detection from a PE-based system to using a biotinylated primary detection antibody and BV421-labeled Streptavidin (a "blue" emitter) that needs an additional detection system incubation step of 30 minutes. Please click here to view a larger version of this figure.
Intra-assay precision
Spearman correlation analysis for three representative Borrelia antigens (Figure 2) demonstrated uniform reproducibility of MFI values when detecting anti-Borrelia antibodies present in human sera.
Figure 2: Intra-assay Precision of the dual-reporter bead-based Borrelia assay. Spearman's correlation analysis showed a high intra-assay precision of the dual-reporter assay when a PE detection system was used to detect Borrelia-specific IgM antibodies (A) and a BV421 detection system was used to detect Borrelia specific IgG antibodies (B, C). A total of 21 serum samples were analyzed in duplicates using a semi-automated procedure. In each graph, MFI signals were plotted against each other and analyzed by linear regression. A linear curve (x=y) shown as a red dashed line indicates identical MFI signals for detection systems. Please click here to view a larger version of this figure.
Inter-assay precision
Good assay-to-assay reproducibility was observed with the dual-reporter assay. Levey-Jennings charts (Figure 3) with MFI values of a quality control sample measured over seven independent runs demonstrated the high inter-assay precision for all representative antigens. The average percent Coefficient of Variation (CV% = standard deviation/mean × 100) of the four representative Borrelia antigens was 5.3%.
Dilution linearity
Because the original single-reporter assay and the new dual-reporter assay employ different flow cytometric platforms, we compared median fluorescence outputs of the same antigen immunoassay between the two flow cytometry instruments (Figure 4). Sample dilution series provided linear dilution curves for both IgM and IgG evaluation, with good parallelism of dose-response between instruments through the entire dilution range evaluated (1:100-1:12,800).
Figure 3: Inter-assay precision of the dual-reporter bead-based Borrelia assay. For the evaluation of the inter-assay precision, Borrelia-specific IgM (A) and IgG (B-D) antibody response of a quality control sample was analyzed over seven independent runs, in duplicate wells each time. Assays were performed manually. MFI values were plotted in a Levey-Jennings chart. A green line gives the mean of all values. Two red dashed lines indicate the tolerance range of the inter-assay precision. This was calculated from the mean value ± 2.5 times the standard deviation (2.5 S.D.). Please click here to view a larger version of this figure.
Figure 4: Dilution linearity of the dual-reporter bead-based Borrelia assay. At sample dilutions of 100-fold to 12,800-fold, the dilution curves for both IgM detection (A) and IgG detection (B) were similar when performed manually with the single-reporter instrument and the dual-reporter instrument. At all tested dilutions, MFI values were slightly higher using the single-reporter instrument (red symbols) than the dual-reporter instrument (blue symbols). Shown are dilution curves for 2 representative antigens, with each dilution point representing the average measurement of triplicate wells with standard deviation (SD *) indicated by error bars. Note: Small SDs are not visible at this scale. Please click here to view a larger version of this figure.
Supplemental Materials. Please click here to download this File.
This report highlights the development of a two-reporter bead-based Borrelia immunoassay that reproducibly and sensitively determines anti-Borrelia immunoreactivity in serum samples12. The various pathogenic Borrelia species that cause Lyme borreliosis can be differentiated by variant-specific antigen heterogeneity6,7,8,9. The multiplexed assay concurrently evaluates IgG- and IgM-mediated anti-Borrelia immunoreactivity within the same reaction well, thereby conserving reagents, labor, and sample material otherwise needed to perform two singleplex assays separately. Comparing IgM and IgG responses over time may allow better tracking of disease progression as IgM-to-IgG seroconversion occurs after infection6.
The dual-reporter system uses two different detection antibody systems13,15. Related experiments demonstrated no significant detectable cross-reactivity between Borrelia anti-IgM and anti-IgG detection systems when both antibody classes were analyzed together in the same reaction well12. Considering the lower binding affinity of IgM versus IgG antibodies16,17, we chose a PE-conjugated detection antibody for IgM detection (the first reporter channel of the dual-reporter instrument) because PE is one of the strongest-emitting fluorophores routinely used in immunoassays18. For IgG evaluation, we used a biotinylated detection antibody that was subsequently illuminated with BV421-conjugated streptavidin (the second reporter channel of the instrument)19. Despite the additional 30 min incubation step compared to the single-reporter system, the dual-reporter system yields twice the information per reaction. Overall, the dual-reporter multiplexed assay requires less cumulative time and material inputs than running two single-reporter assays.
Strong performance and stability of the multiplexed Borrelia assay was exemplified by high reproducibility in intra- and inter-assay precision studies, and by demonstration of dilution linearity and dilution parallelism over a wide range of sample concentrations for both IgG and IgM assessment. We observed higher absolute fluorescence emission levels for the same fluorophores using the single-channel instrument versus the dual-channel system (≈1.7× higher with PE), attributable to differences in the optics and calibration settings between the two instruments (Figure 4). Nonetheless, fluorescence emission curves with both fluorophores remained within the linear range of both instruments at high and low sample dilution extremes, and any discrepancy in absolute fluorescence measured did not affect the classification of Borrelia exposure status.12
A major advantage of this bead-based Borrelia multiplex assay is the ease with which the assay can be modified or expanded to evaluate different or additional analytes, e.g., to detect antibodies against antigens of further Borrelia species. xMAP magnetic bead sets contain different dye combinations that can be distinguished in the instrument's Classification Channel and can theoretically be implemented into multiplex assays that can concurrently evaluate up to 500 unique analytes within the same sample. While the current study highlights four representative Borrelia antigens to demonstrate assay functionality and stability and to compare the single- and dual-reporter system, the final assay interrogates eight antigens that together can identify all five clinically-relevant Borrelia pathogens circulating throughout Europe and North America12.
High-throughput performance is possible using standard 384-well microtiter plates in a semi-automated assay format. Assay and instrument compatibility with both 96- and 384-well plates allows the Borrelia multiplex assay to be used as an efficient screening tool for rapidly analyzing large sample sets, such as national studies20. Manual performance of the assay remains possible for smaller sample sets using smaller 96-well plates.
Study limitations include the comparative evaluation of only a few Borrelia immunoreactivity targets in a small number of human serum samples. However, the original study did confirm that assay performance for both IgG and IgM was maintained when analyzing eight antigens from all five known Borrelia species within a larger sample set12. Also, the dual-reporter instrument can only assess two antibody isotypes simultaneously within each reaction, so complete isotype profiling would necessitate performing additional assay reactions13.
In conclusion, this report details the successful merger and conversion of bead-based single-reporter immunoassays into dual-reporter assays that can simultaneously evaluate pathogenic Borrelia-specific IgG and IgM antibodies in human serum samples. This combined approach saves total time, material, and labor inputs to generate the same data volume as two independent single-reporter assays. The multiplex assay can be scaled from 96-well to 384-well microtiter plate format and can be semi-automated by the use of robotic plate and liquid handling instrumentation, making it suitable for high-throughput applications such as large population surveys. Bead-based dual-reporter assay systems have previously demonstrated utility in evaluating, for instance, immune responses to other viral and bacterial pathogens13,21, assessing allogeneic antibody responses against HLA epitopes in organ transplantation22, and exploring mechanisms of autoimmune disease23. The current report detailed the use of multiplex technology to identify exposure to the Borrelia pathogens that cause Lyme disease, as an example of how laboratories can adapt this approach for exploring complex immune mechanisms in diverse pathologies.
The authors have nothing to disclose.
This report was funded by Luminex (Austin, TX). The authors thank Matthew Silverman PhD (Biomedical Publishing Solutions, Panama City, FL; mattsilver@yahoo.com) for analytical and scientific editing assistance. The authors also thank Harald Klein and Christoph von Eichel-Streiber of tgcBIOMICS GmbH (Bingen, Germany) for providing the Borrelia antigens used in the study. Human serum samples for technical assay validation and quality control were obtained from: 1) the Multilocal and Serial Prevalence Study on Antibodies against SARS-CoV-2 in Germany via the Department of Epidemiology, Helmholtz Centre for Infection Research, Braunschweig, Germany; and 2)the Department of Neurology, Sächsisches Krankenhaus Rodewisch (Rodewisch, Germany). Approval for use of human samples was granted by the Ethics Committee of the Hannover Medical School, Germany (9086_BO_S_2020).
Antibodies and Detection Reagents | Source | Catalog Number | |
Biotinylated Goat Anti-Human IgG | Jackson ImmunoResearch (Dianova) | 109-066-098 | |
Brilliant Violet 421-Streptavidin | BD Biosciences | 563259 | |
Donkey Anti-Human IgM | Jackson ImmunoResearch (Dianova) | 709-116-073 | |
Borrelia Antigens | tgcBIOMICS (Bingen, Germany) | ||
Coupling Reagents | |||
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) | Thermo Scientific Pierce | 77149 ProteoChem (100 mg) | |
10x PBS | Fisher Scientific | BP399-4 | |
BSA | Carl Roth | T844.3 | |
MES (2-ethanesulfonic acid; zwitterionic buffer) | Carl Roth | 4256.2 | |
Na2HPO4 | Carl Roth | 4984.1 | |
ProClin300 | Sigma | 48914-U | |
Sulfo-NHS (N-hydroxysulfosuccinimide) | Thermo Scientific Pierce | 24510 (500 mg) | |
Triton X-100 | Thermo Scientific | 85111 | |
Instrumentation and Ancillary Lab Supplies | Source | ||
384-well plate | Corning, Cat# 3570 | ||
96-well deep-well plates | ThermoFisher Scientific, Cat# 95040450 | ||
96-well half-area plates | Corning, Cat# 3690 | ||
BioTek 405 TS Plate Washer | BioTek Instruments/Agilent Technologies, Santa Clara, CA | ||
BioTek MultiFlo FX Plate Washer | BioTek Instruments/Agilent Technologies, Santa Clara, CA | ||
DynaMag Spin Magnet (for isolating beads in microcentrifuge tubes) | ThermoFisher, Cat# 12320D | ||
Flexmap 3D (two-channel, single-reporter instrument) | Luminex Corp., Austin, TX | ||
KingFisher Magnetic Particle Processor (for isolating beads in 96-well plates) | ThermoFisher, Cat# A31508 | ||
MagPlex Microspheres (magnetic, fluorescent, 6.5-µm-diameter beads) | Luminex Corp., Austin, TX | ||
SmartBlock Plates | Eppendorf, Cat# 5363000039 | ||
ThermoMixer C | Eppendorf, Cat# 5382000015 | ||
ThermoTop | Eppendorf, Cat# 5308000003 | ||
xMAP Intelliflex (three-channel, dual-reporter instrument) | Luminex Corp., Austin, TX |