Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity

Abstract

The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.

Immune response to SARS-CoV-2 variants of concern in vaccinated individuals

“…To analyze the humoral response generated by vaccination, SARS-CoV-2 reactive antibody titers in serum samples from vaccinated, convalescent (hereon referred to as “infected”) and un-infected (hereon referred to as “negative”) individuals were measured using MULTICOV-AB 33 ( Fig. 1 ).…”

“…MULTICOV-AB, a multiplex immunoassay which simultaneously analyses 20 antigens was performed as previously described 33 on all samples. In addition to the antigens presently included in MULTICOV-AB ( Extended Data Table 3 ), RBD mutants from variants of concern were also included for measurements of all serum samples.…”

“…Saliva samples were diluted into assay buffer inside a sterile workbench. 25 μL of diluted sample was then added to 25 μL of 1X Bead Mix 33 using a 96-well plate (#3600, Corning). Samples were incubated for 2 hours at 20°C, 750 rpm on a Thermomixer (Eppendorf), after which the unbound antibodies were removed by washing three times with Wash Buffer (1x PBS, 0.05% Tween-20) using a microplate washer (Biotek 405TS, Biotek Instruments GmBH).…”

“…Following another washing step, beads were re-suspended in 100 μL of Wash Buffer and re-shaken for 3 mins at 20°C, 1000 rpm. Plates were then measured using a FLEXMAP3D instrument (Luminex) using the same settings as 33 . Each sample was measured in three independent experiments.…”

“…Plasma samples were diluted 1:50 in assay buffer and then to a final concentration of 1:400 in ACE2 buffer under a sterile workbench, resulting in a final ACE2 concentration of 500 ng/mL in all samples. 25 μL of diluted plasma samples were then added to 25 μL of 1X BeadMix 33 per well of a 96-well plate (#3642, Corning). In addition to the standard bead mix used in MULTICOV-AB, all bead coupled RBD mutants were included.…”