Commentary
In April 2021, researchers from the University of Rochester reported on the methods used to convert multiplex SARS-CoV-2 IgG serology and neutralization assays into novel dual reporter IgG and IgM assays on the Luminex® xMAP® INTELLIFLEX DR-SE (RUO) System [1]. The multiplex SARS-CoV-2 IgG detection assay was developed by Cameron et al. on the Luminex FLEXMAP 3D® System for detecting IgG antibodies against the spike (S), receptor-binding domain (RBD), and nucleocapsid (Nc) antigens of SARS-CoV-2 [2]. The IgG assay demonstrated comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at ≥ 21 days from symptom onset, but had higher sensitivity than the reference assay for samples collected at ≤ 5 days from symptom onset. The assay was also modified to detect potential neutralizing antibodies by including a simple incubation of the antigen coated beads with angiotensin-converting enzyme-2 (ACE2), as a competing reagent, prior to the addition of serum sample.
The conversion and optimization of the single reporter serological assay to a dual reporter format on the INTELLIFLEX DR-SE System was easy, efficient, and seamless. For the dual reporter assay, several different combinations of IgG and IgM detection antibodies and reporter dyes were evaluated in the INTELLIFLEX DR-SE two reporter channels using the standard single reporter assay conditions. In the final dual reporter assay, the same biotin anti-human IgG detection antibody could be used at the same concentration but the streptavidin-Phycoerythrin used in the single reporter FLEXMAP 3D System was replaced with a streptavidin-Super Bright 436 conjugate for use in the second reporter channel of the INTELLIFLEX DR-SE System. To study IgM levels, a directly Phycoerythrin-labeled anti-human IgM was used. The dual reporter serological assay was also converted to a dual reporter neutralization assay by including the ACE2 and microsphere incubation step prior to addition of the serum sample. Using this dual reporter capability, the authors were able to measure anti-SARS-CoV-2 IgG and IgM titers in several hundred samples to more than 180 days from symptom onset, and detect when potentially neutralizing titers were generated (Cameron A, et al., manuscript in preparation).
The dual reporter assays have several added benefits over the single reporter formats. The addition of the second reporter channel provides twice the amount of data in the same reaction for each sample, demonstrating a clear advantage over the single reporter systems. The INTELLIFLEX DR-SE is a new compact, flow-based, multiplex platform that combines the proven performance of xMAP® Technology with modern features and intuitive software to enhance performance, streamline workflow, and improve the user experience.
The work presented by the authors demonstrates how the INTELLIFLEX DR-SE System facilitates monitoring immunoglobulin responses for two isotypes simultaneously, and for multiple antigens, due to the multiplexing capabilities of the xMAP bead-based technology. Thus, the INTELLIFLEX DR-SE can be effectively used to get more information from a single sample compared to conventional singleplex, single reporter serological assays. Furthermore, xMAP multiplex bead technology makes it very easy to expand or contract the number of antigens interrogated in a single reaction. Another example is the multiplexed assay for analysis of antibodies against six different human coronaviruses developed by Trivedi et al. [3] This study provided proof of concept for the feasibility to develop and use a multiplexed microsphere immunoassay as a next generation screening tool for use in large scale seroprevalence studies of human coronaviruses.
The ability to monitor antibody titers and potential neutralizing antibodies of different immunoglobulin types is not limited to IgG, IgM, or coronavirus antigens. With the dual reporter instrument, analysis of virtually any combination of two immunoglobulin types, or two parameters, is possible. The ability of the ACE2 receptor to block antibody binding to S and RBD, but not Nc, in this study suggests that receptor proteins of other viruses may also allow detection of neutralization potential in multiplex reactions. Using this method for measuring inhibition of antigen-specific antibody binding to the receptor binding domains of other key viral antigens could be a significant advantage for monitoring infections, as well as, testing the efficacy of vaccines, convalescent sera, and recombinant antibody treatments in future outbreaks.
The dual reporter capability of this new platform can be extended to other types of serological assays, as well as other assay formats where simultaneous measurement of analyte pairs is of interest. Some examples include autoimmune markers, post-translational modifications, such as phosphorylation and methylation, as well as free versus bound drug forms. As xMAP bead-based technology is well-established for both proteomic and genomic analyses, combining the multiplexing capabilities with the new dual reporter system opens up countless opportunities for novel applications.
References
2. Cameron A, Porterfield CA, Byron L, Wang J, Pearson Z, Bohrhunter JL, et al. A multiplex microsphere IgG assay for SARS-CoV-2 using ACE2-mediated inhibition as a surrogate for neutralization. J Clin Microbiol. 2021 Jan 21;59(2):e02489-20.
3. Trivedi SU, Miao C, Sanchez JE, Caidi H, Tamin A, Haynes L, et al. Development and Evaluation of a Multiplexed Immunoassay for Simultaneous Detection of Serum IgG Antibodies to Six Human Coronaviruses. Sci Rep. 2019 Feb 4;9(1):1390.