Plasma samples were obtained from two groups: 100 healthy blood donors and 100 COVID-19 patients, sourced from BioIVT (Hicksville, NY). Table 1 presents the demographic characteristics of both groups. The healthy donor samples were collected prior to the COVID-19 outbreak, with ages ranging from 21 to 77 years (median age 48 years). Among the donors, 59 were female and 41 were male. COVID-19 patients were diagnosed via real-time PCR for SARS-CoV-2 and sampled before October 2020, prior to vaccine availability. Their ages ranged from 21 to 76 years, with a median of 47, including 59 females and 41 males. Plasma samples were stored at -80°C until analysis.
Materials
The study utilized CML microsphere beads (4% w/v, 2 µm), aldehyde/sulfate microsphere beads (4% w/v, 2 µm), FreeStyle 293 expression medium, and FreeStyle 293F cells from Invitrogen (Carlsbad, CA). Human anti-N and anti-S1 IgG and IgM were obtained from GenScript (Piscataway, NJ), while human anti-RBD IgG and IgM were sourced from InvivoGen (San Diego, CA). Human anti-S2 IgG was purchased from Cell Sciences (Newburyport, MA). Rabbit anti-Histag IgG came from Cell Signaling Technology (Danvers, MA). Alexa Fluor 647 AffiniPure donkey anti-human IgG and Alexa Fluor 488 AffiniPure donkey anti-human IgM were acquired from Jackson ImmunoResearch Laboratories Inc (West Grove, PA). Bovine serum albumin fraction V (BSA) was from Sigma-Aldrich, Inc. (St. Louis, MO).
Cell Culture
FreeStyle 293F cells were cultured in FreeStyle 293 expression medium supplemented with penicillin–streptomycin at 37°C in a humidified atmosphere with 8% CO₂ on an orbital shaker at 125 rpm.
Production of Recombinant SARS-CoV-2 Structural Proteins
The coding sequences of SARS-CoV-2 structural proteins from the Wuhan strain were inserted into the pcDNA3.4 vector, generating pcDNA3.4-signal peptide-SARS-CoV2-subunit-8xHis (Biosettia, San Diego, CA). FreeStyle 293F cells were transfected with these vectors using 293fectin. Cells were seeded in Nalgene flasks with FreeStyle medium, and transfection was performed with pcDNA3.4 and 293fectin. Post-transfection, cells were harvested, and supernatants containing recombinant proteins were stored at 4°C for purification.
Purification and Identification of Recombinant Proteins
Proteins were purified using PureCube 100 INDIGO Ni-Agarose (Cube Biotech, Wayne, PA), desalted with Amicon Ultra-15 10 K centrifugal filters, and their concentrations determined via BCA assay. Purity, exceeding 90%, was confirmed by SDS-PAGE with Coomassie blue staining (Figure 2a). MALDI TOF/TOF mass spectrometry identified the proteins as correct with 100% confidence.
Western Blot Analysis
Purified proteins were analyzed by Western blotting. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked, and incubated with antibodies (human IgG, IgM, or rabbit IgG). Blots were detected using chemiluminescence and analyzed with Immobilon Western substrate and FluorChem R System.
Flow Cytometric Assay
SARS-CoV-2 protein-conjugated beads were prepared using Aldehyde/Sulfate or CML beads. Control beads were coupled with BSA. Proteins were coupled according to manufacturer instructions, and beads were stored at 4°C. For antibody analysis, beads were incubated with diluted plasma samples, stained with Alexa Fluor-conjugated secondary antibodies, and analyzed on a Beckman Coulter CytoFlex flow cytometer using CytExpert 1.2 Software. Median fluorescent intensity (MFI) differences between target and control beads determined antibody positivity and levels.
Statistical Analyses
Analyses were performed using GraphPad Prism 6. Data normality was assessed via Kolmogorov–Smirnov test. Chi-square analysis evaluated associations between antibody prevalence and demographic factors. Kruskal–Wallis with Dunn’s test compared antibody levels among groups. All tests were two-tailed with α = 0.05.
