## 3 The Results and Discussion
### Optimization of MICaFVi  
Having characterized all of the components of MICaFVi, we optimized the protocol as follows. VM-SPs and AS-MNPs were co-incubated overnight to allow for immuno-capture (Figure 4A). The AS-MNPs were then magnetically captured via a multi-well magnetic separator and then incubated in 4% PFA for 30 min. This step was crucial for the stabilization of the AS-MNPs/VM-SPs complex during the subsequent analysis steps. Moreover, incubation with PFA allows for the neutralization of wt MERS-CoV/SARS-CoV-2, for when camel/human samples will be analyzed in subsequent studies. Following the neutralization/fixation step, the magnetically captured AS-MNPs were washed 3 times in PBS with 0.3% (\(v/v\)) tweet to remove any non-specific bound interactions. The precipitated  
Figure 3: Characterization of anti-spike Ab-coated MNPs (AS-MNPs). (**A**) SEM and TEM images of AS-MNPs showing 500 nm sized MNPs. (**B**) Schematic representation and behavior when analyzed via flow cytometry. Non-stained AS-MNPs were used to set up the zeroing of fluorescence signals (FITC and APC) during flow cytometry analysis. Only singlets were gated for analysis. (**C**) Analysis of the coating efficiency of AS-MNPs via the anti-spike rabbit pAb (blue) using an APC-labeled anti-rabbit secondary Ab (red). (**D**) Analysis of the interaction between the FITC-labeled anti-spike Ab (green) with AS-MB. (**E**) Analysis of the interaction between APC-labeled anti-rabbit secondary Ab (red) and the FITC-labeled anti-spike Ab (green). AS-MNPs were then stained with FITC-labeled mouse monoclonal anti-MERS-CoV spike antibody and APC-labeled secondary anti-rabbit antibody at a dilution of 2/1000 in PBS containing 1% Tween and 50% FBS for 1h. Stained AS-MNPs were magnetically precipitated, then washed 3 times with PBS containing 0.3% (\(v/v\)) tween, and analyzed using flow cytometry (Figure 4B). We followed a very strict gating strategy to eliminate doublets and clumps, as well as false positive and false negative events (see Section 2 and Figure S1). AS-MNPs incubated with PBS (negative control sample) were used to set the zeroing (background) of the fluorescent signal (FITC and APC). The number of double-positive events (representing the complex AS-MNPs/VM-SPs) was quantified and represented as a % of MICaFVi. The results showed that \(\sim\) 60% of AS-MNPs were FITC-positive (Figure 4B), which indicates that they were cross-linked (captured) with the viral particles. To prove that the formation of the AS-MNPs/VMNPs complex was the result of a specific interaction between the spike protein (on the surface of VM-SPs) and its antibody (on the surface of AS-MNPs), and to exclude any possibility of non-specific cross-linking, AS-MNPs were boiled in Laemmli buffer to completely denature the anti-spike antibodies on their surface. These boiled AS-MNPs were used to immuno-capture VM-SPs. Flow cytometry analysis showed that boiled AS-MNPs were still APC-positive to a lesser extent, due to the binding of the APC-labeled secondary antibody to the denatured primary antibody (Figure 4C). No FITC-positive events were recorded, indicating the failure of AS-MNPs/VM-SPs complex formation (Figure 4D). This confirms that the interaction between the spike protein and its antibody on the surface of AS-MNPs is crucial for immune capture (complex formation). To evaluate the specificity of the assay, the VSV-Gpp-pseudotyped lentivirusus expressing the VSV-G protein on its surface was used. We performed MICaFVi using an FITC-labeled primary anti-VSV-G protein antibody in combination with the anti-Rabbit APC-labeled secondary antibody (Figure 5A). Complete cell culture medium (virus-free) was used as a negative control. Flow cytometry analysis of AS-MNPs, incubated with  
Figure 4: MICaFVi detection of VM-SPs. (**A**) Viral capture using AS-MNPs is mediated via a specific interaction between anti-spike antibodies and the spike protein on VM-SPs. Legend and representation of fluorescent antibodies used to stain captured VM-SPs via AS-MNPs.