Coronavirus disease 2019 (COVID-19), the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in December 2019 in Wuhan, China, and has spread rapidly around the world [1]). Most people infected with SARS-CoV-2 display an antibody response approximately one week after the infection [2].
Rapid and accurate serological tests detecting anti-SARS-CoV-2 antibodies are essential for estimating SARS-CoV-2 seroprevalence and differentiating immune response to natural infection from vaccination. Indeed, strategies to ease restrictions on human mobility and interaction without provoking a major surge in transmission and mortality will depend on an accurate estimation of SARS-CoV-2 seroprevalence, which can be reliably achieved by massive serological testing. Serology has become indispensable not only for understanding the development and duration of immunity following infection or vaccination, but also for investigating the correlates of protection. Indeed, understanding the duration of protection and risk of reinfection after a natural infection or vaccination is crucial for planning the COVID-19 vaccination, especially with the emergence of more transmissible variants. However, most serologic assays, such as ELISA or Lateral Flow Immunochromatographic assays, either query the recognition of one antigen at a time or several antigens in bulk and provide limited information (IgG or IgM) on the quality of the anti-viral humoral response. Moreover, most of these tests have shown a wide range of performance, with many tests exhibiting inadequate sensitivity and specificity [3]. Yet, the need for accurate, extensive, and broad serological testing is required to understand the correlates of protection and to inform public health decisions.
Methods such as FCMBA immunoassays, which typically use beads with different fluorescent intensities coupled each to a specific antigen, have been used to simultaneously measure multiple viral antigen-specific antibody responses in Human Immunodeficiency Virus [4,5]. This method outperforms ELISA for the detection of Herpes Simplex Virus type-specific immunoglobulins in terms of the number of antigens assayed, sample volume needed, and assay speed [6].
FCMBA technology is a highly sensitive immunoassay whereby different groups of polystyrene carboxylated beads are impregnated with specific fluorescent dyes at varying intensities with the ultimate purpose of exploiting the ability of flow cytometry to detect the dynamic range of these fluorophores. The increasing fluorophore dye intensities should be optimized to enable discrimination of each bead group upon detection by flow cytometry at the specific wavelength of said fluorescent dye. In some examples, up to 11 discriminated bead groups can be identified per fluorescent dye based on intensity (Figure 1A). Each bead group is subsequently coupled with a specific viral antigen against which the humoral immune response will be assessed (Figure 1B). All bead groups are then pooled and added to the serum samples in a single reaction, enabling the antibodies in the serum sample to bind to their corresponding antigens that had been previously coupled to the beads (Figure 2A). Following incubation and wash, a mixture of detection antibodies labeled with different fluorescent dyes and targeting human isotypes such as IgG, IgM or IgA is added to the sample (Figure 2B). Henceforth, the sample can be acquired utilizing a flow cytometry platform (Figure 3A), and specifically bound detection antibodies can be individually distinguished by their respective wavelength channel (Figure 3B). Two discrimination channels can be used to evaluate flow cytometry data to identify different sets of antigen-coupled beads based on their intensity, (Figure 4A) and to distinguish between samples that test positively versus negatively (Figure 4B). This assay can not only quantify multiple specific antibodies bound to their respective antigens but also determine the isotype of these antibodies, thereby allowing for the mapping of the humoral response and the determination of seroconversion status. The versatility of the assay could allow for the detection of large numbers of antibodies as more technical aspects of the FCMBA are taken advantage of, such as using various intensity beads of different dyes coupled to multiple antigens, and using the numerous available channels and specifications of the coupled flow cytometry platform.
Definition of carboxyl particles and their coupling to proteins, antibodies, peptides, oligonucleotides, etc. – (a) beads contain internal dyes of different intensities; (b) chemistry occurs where the primary amine group of the target molecule (a protein, antibody, or peptide) interacts with the microsphere’s carboxyl groups either directly or indirectly to generate a covalent amide bond.
Bead array assay description – (a) distribution of the coupled beads with the targeted biomolecule (in this example, an antigen) in the 96-well microtiter plate, followed by the introduction of the experimental serum or plasma; (b) coupled beads bind and immobilize protein-specific antibodies; (c) antibodies that have been immobilized on the beads are differentiated and identified through the utilization of specific secondary antibodies that possess unique labels, regardless of whether they belong to the same or distinct isotypes.
Executing the analysis – (a) acquisition of the samples using a flow cytometry platform; (b) producing and gathering data and aggregating the outcomes obtained from the various detection antibodies.
Data analysis obtained from flow cytometry using specialized software – (a) identifying individual sets of beads based on their intensity using two discrimination channels; (b) identification and differentiation of samples that test positive versus negative.
During pandemics, the detection of antibody responses using serological tests would allow for the monitoring of disease dissemination and vaccination responses. Highly modulable and flexible serological assays are needed to allow the inclusion of several antigens representing diseases as soon as they manifest. Implementations of FCMBA extended to the global COVID-19 pandemic, with its uses ranging from potential novel screening methods to the determination of disease severity [7,8]. Serological tests based on FCMBA technology are powerful techniques capable of detecting anti-SARS-CoV antibodies at a high throughput scale using only a few microliters of blood samples. Indeed, FCMBA technology was recently used to assess the serological profiles of SARS-CoV-2-naturally-infected or vaccinated individuals reacting to 11 antigens from human coronaviruses by measuring the total IgG, total IgA, and total IgM, as well as the IgA and IgG subtypes [9]. The serological profiling showed increasing levels of antibodies in the plasma of individuals after vaccination, including antibodies specific for the SARS-CoV-2 spike protein, which is targeted by the COVID-19 vaccines. Importantly, the assay also showed some cross-reactivity with the SARS-spike protein. FCMBA has also helped provide insight into the impact of the novel COVID-19 mRNA vaccinations on the immune systems, as it has been used by Takano et al. on vaccine recipients to identify variations in cytokine expression; differences in cytokine and chemokine expression were found to define six unique vaccine-induced immune dynamics in these patients [10]. COVID studies further used the cytokine/chemokine FCMBA as a supplementary means of defining factors associated with the severity of infection [11].
Furthermore, FCMBA technology allows for the testing of cross-neutralizing antibodies, which are antibodies that prevent a pathogen from entering cells, thus preventing the pathogen from replicating and protecting the organism from infection [12]. Detection of such antibodies can aid in passive antibody therapeutics as well as potential active prophylaxis. FCMBA can also be used for investigating the presence of cross-neutralizing antibodies against SAS-CoV-2 in dromedary camels [13]. Using a 5-antigen bead array, including SARS-CoV-2 proteins (S trimer, Membrane, Nucleoprotein, Envelope), and MERS-S protein, and sera of dromedaries that are MERS seropositive but MERS-CoV-free, the authors found that the tested dromedaries had medium-to-high titers of anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins. The discovery of SARS-CoV-2 cross-neutralizing camel antibodies in nonimmunized camels represents a promising venue for the development of SARS-CoV-2 hyperimmune camels, which could be a prominent source of therapeutic agents for the prevention and treatment of COVID-19.
Testing using FCMBA plays a crucial role in the convalescent plasma treatment, where plasma obtained from individuals who have recuperated from severe infections like COVID-19 is utilized. By conducting tests, it becomes possible to identify the specific plasma that can be employed for treatment based on its possession of highly effective neutralizing antibodies [14].
FCMBA technology allows for the timely measurement of various kinds of analytes, including cytokines, chemokines, antibodies, immunoglobulin isotypes, intracellular signaling molecules, and even single nucleotide polymorphisms (SNPs) [15]. The detection and analysis of these analytes have allowed for FCMBA to have broad medical applications on the bench-side and the bedside.
FCMBA has been used to aid in screening for disease in a robust and efficient manner with a small sample size. This has been mainly applied in autoimmune diseases, as was shown in the ability of a multiplex assay to comparatively detect antibodies against autoantigens in patients’ sera [16]. Furthermore, FCMBA’s use in disease screening also extended to allergies, with Zhou et al. observing anti-Poly Ethylene Glycol IgE antibodies developing in response to (PEG) ingestion [17]. FCMBA potential has also been noted in the early detection of some neurodegenerative diseases, as was shown in preclinical data regarding Alzheimer’s disease [18]. The efficiency and practicality provided by FCMBA has also opened the door to allow for screening methods for diseases such as Herpes, Viral Hepatitis, Swine Fever and others in underserved areas [19,20]. The potential for FCMBA applications in diseases is not only limited to screening but also extends to helping identify crucial signatures involved in disease processes, as was shown by Bauer et al., revealing specific inflammatory differences between antibody associated demyelinating diseases and multiple sclerosis [21]. Also revealing cytokine expression levels, Bai et al. have used FCMBA to compare differences in cytokine levels between patients with active tuberculosis, latent tuberculosis, and healthy patients, demonstrating a potential auxiliary efficacy to cytokines as biomarkers in the workup for tuberculosis [22].
Despite being a novel and exciting technology, the FCMBA system does have some disadvantages when compared to its ELISA counterpart. By virtue of being a more recent approach, the standardization and validation of diagnostic algorithms for flow-bead arrays are still not on par with the pre-established ELISA data; however, this will be overcome as more array kits are being approved for clinical and research use moving forward [23,24,25]. Furthermore, inherent to the assay involving multiple analytes and beads, there is a chance for cross-reactivity as the number of analytes increases, limiting the overall possible combinations of antibodies and ligands in the flow-bead microarray method [26,27].
That being said, FCMBA technology also offers multiple advantages. While ELISA-based arrays have the antibodies immobilized to a planar surface, FCMBA-based arrays could enable the binding of ligands to beads in suspension, allowing for a larger reaction area leading to faster binding kinetics [28]. The intrinsic multiplex nature of the FCMBA arrays allows for smaller sample sizes, which is advantageous when sample volume is limited, as is common in animal models and clinical studies. Furthermore, the smoother technical aspect of the suspension FCMBA method allows for faster and more cost-effective analysis when compared to ELISA-based methods [29]. This improved efficiency facilitates the application of FCMBA technology in personalized medicine, be it in allergy testing, autoimmune disease antibody detection, or the assessment of immune response [28]. From a technical standpoint, the suspension FCMBA immunoassay technology overcomes issues faced by the planar ELISA methods, such as mass-transportation limitations and printing and washing artefacts [30]. Through the use of FCMBA technology, the FCMBA arrays allow for enhanced automation in the analysis of the results, enhancing efficiency and reproducibility as compared to ELISA-based methods [31].
Overall, FCMBA provide a faster, more efficient method of detecting various analytes from a small sample, building on the base provided by the ELISA technique while also overcoming many of the limitations that exist in planar surface methods. This novel technique will pave the way for a more personalized approach to medicine, and will aid in screening, passive immune therapy and active prophylaxis against the current and any potential future pandemics.