Magplex microsphere

Recombinant antigens were covalently coupled to a spectrally distinct microsphere (Magplex Microsphere, Luminex Corp., Austin, TX, USA). Briefly, 2 µg of each antigen was coupled to 1.25 × 10⁶ beads using a BioPlex amine coupling kit (Bio-Rad) according to the manufacturer’s procedure.

The recombinant SARS-CoV-2 Spike protein S1 (RBD, His Tag) containing 319–541 amino acids of the Spike protein was recombinantly produced in Human Embryonic Kidney cells (Innovative Research, Inc, MI). The SARS-CoV-2 nucleocapsid (N) protein (PMC 827, NP2) was produced using suspension cells (ProMab Biotechnologies Inc, CA). Anti-SARS-CoV-2 Spike S1 Antibody (human chimeric, IgG isotype) was purchased from GenScript Biotech Corporation (Piscataway, NJ). PE-conjugated anti-human IgG Fc antibody was purchased from BioLegend (San Diego, CA). MagPlex Microsphere (magnetic beads) were purchased from Luminex (Austin, TX).

Evaluation of vaccine-specific antibodies was performed using previously established microsphere immunoassay (MIA) protocols involving conjugation of carboxylated magnetic microspheres (MagPlex® Microspheres, Luminex, Austin, TX) with FIV glycoprotein (SU_PPR) and soluble CD134 recombinant proteins (rProteins).^{78 (link),79 (link)} Following conjugation protocols, a hemocytometer was used to determine microsphere concentrations, and protein coupling was confirmed via incubation of microspheres with primary antibodies and/or PE-conjugated detection antibodies.^{78 (link)} Successful coupling was defined by a median fluorescence intensity (MFI) of >2000. All samples from FIV-infected and negative control cats were diluted 1:50 in assay buffer and then incubated in duplicate with ~2500 conjugated beads per well. All samples were assayed in conjunction with FIV-A and naïve reference samples diluted 1:50 in assay buffer, as well as four diluent control wells per experiment. The MFI was calculated from ≥100 microspheres per analyte per well (Bio-Plex™ Manager 5.0) and then used for data analysis. All reagent concentrations, volumes, incubation times, acceptable standard recovery, and data analysis were as previously described.^{78 (link),79 (link)}

Two SARS-CoV-2 proteins were coupled to MagPlex microspheres of different regions (Luminex). Nucleocapsid protein expressed from Escherichia coli (N-terminal His6) was obtained from Raybiotech (230-01104-100) and the RBD of spike protein expressed from HEK293 cells was obtained from the laboratory of J. Wrammert63 at Emory University. Coupling was carried out at 22 °C following standard carbodiimide coupling procedures. Concentrations of coupled microspheres were confirmed by Bio-Rad T20 Cell Counter.

Samples were diluted 1:400 in Buffer B (1X PBS [phosphate buffered saline], 0.5% casein, 0.5% polyvinyl alcohol [PVA], 0.8% polyvinylpyrrolidone [PVP], 0.3% Tween-20, 0.02% sodium azide and 3 µg/mL E. coli extract) and tested by MBA as described previously^{3 (link)}. Briefly, Pgp3-coupled MagPlex^® microspheres (Luminex, Austin, TX USA) were incubated with diluted sample for 1.5 h then washed with 0.05% Tween-20 in 1X PBS (PBST). Beads were washed with PBST and incubated with biotinylated mouse anti-human IgG and biotinylated mouse anti-human IgG4 for 45 min to detect anti-Pgp3 antibody bound to beads. After 3 additional washes with PBST, beads were incubated for 30 min with phycoerythrin-labeled streptavidin (SA-PE) to detect bound biotinylated anti-human IgG. After 3 more washes with PBST, loosely bound antibodies were removed with a final 30-min incubation in PBST containing 0.5% BSA and 0.02% sodium azide. Beads were washed one more time, resuspended in 1X PBS, and stored overnight at 4 °C. The next day, plates were read on a Bio-Plex 200 instrument (Bio-Rad, Hercules, CA, USA) equipped with Bio-Plex manager 6.0 software (Bio-Rad). The median fluorescence intensity (MFI) was recorded for each sample.

Antigen-specific antibodies were characterized using the Fc array assay^{74 (link)}. Briefly, antigens were covalently coupled to MagPlex microspheres (Luminex Corporation) using two-step carbodiimide chemistry. Experimental controls included pooled human polyclonal serum IgG (IVIG), S309, an antibody from a SARS-CoV patient that cross-reacts SARS-CoV and SARS-CoV-2, and VRC01, an HIV-specific antibody^{75 (link),76 (link)}. Serum dilutions used in experiments were based on experience from previous work and a small pilot experiment of test concentrations. Final dilutions used in assays varied from 1:250 to 1:5000 depending on the detection reagent (see reporting summary). Antigen-specific antibodies were detected by R-phycoerythrin-conjugated secondary reagents specific to human immunoglobulin isotypes and subclasses and by Fc receptor tetramers as described previously^{77 (link),78 (link)}. Median fluorescent intensity data was acquired on a FlexMap 3D array reader (Luminex Corporation). Samples were run in technical duplicates.

The functionality of the proteins was tested by measuring immunoglobulin G (IgG) antibody response in serum samples from 445 well‐characterized TB suspects from Vietnam and Peru (167 Non‐TB and 278 TB) as described previously 15. Specimens were provided by the Foundation of Innovative New Diagnostics (FIND) and tested at the Natural and Medical Sciences Institute (NMI, University of Tübingen, Reutlingen, Germany). In brief, the purified proteins were covalently coupled to color‐coded beads (MagPlex Microspheres, Luminex Corp, Austin, USA). The bead mixture was incubated with patient sera and protein‐bound human antibodies were detected with PE‐labeled anti‐human IgG on a Luminex FlexMAP3D (Luminex Corp). Median fluorescence intensity was calculated for every sample based on >60 measured beads per bead sort and z‐scores were used to illustrate antibody responses per patient in relationship to the mean of all patients. Receiver operating characteristic (ROC) curves were used to illustrate the discriminating utility of the best performing protein and Mann–Whitney U‐test was used to determine whether the antibody reactivity in the two populations (TB and Non‐TB) is significantly different.